molecules

Article
New Smoothie Products Based on Pumpkin, Banana, and
Purple Carrot as a Source of Bioactive Compounds
Marcin Kidoń 1, * and Pascaline Aimee Uwineza 2

1 Department of Food Technology of Plant Origin, Poznan University of Life Sciences, 60-624 Poznan, Poland
2 Department of Chemistry, Poznan University of Life Sciences, 60-628 Poznan, Poland;
[Link]@[Link]
* Correspondence: [Link]@[Link]

Abstract: Smoothies are fruit- and/or vegetable-based products in form of beverages that are typically
semi-liquid, thick in consistency, and mainly consist of purees and juices. Other ingredients, such
as yogurt, milk, ice cream, sugar, honey, or simply water may also be added. The present study
aimed to elaborate smoothie products based on bananas, pumpkins, and purple carrots. These
fruits and vegetables were chosen due to their high bioactive compounds content, potential health
benefits, and availability to industry. Five smoothie formulations were produced and analyzed for
pH, soluble solids, total phenolic content, anthocyanins, carotenoids, vitamin C, antioxidant activity,
instrumental color, and sensory features. From the analysis, the result showed that the obtained
smoothies were a good source of total phenolic content (39.1 to 55.9 mg/100 g) and anthocyanin
(7.1 to 11.1 mg cyanidin-3-glucoside/100 g), and that they possessed high antioxidant activity (4.3
to 6.2 µM Trolox/g). From sensory evaluation, all the produced smoothies were desirable, but the
formulations with banana were scored higher compared to the pumpkin. In conclusion, smoothies
composed of pumpkin, banana, and purple carrot can be a good new food product that incorporates
nutritional compounds into the human diet.

Keywords: smoothie; phenolic compounds; anthocyanin; carotenoids; vitamin C; antioxidant activity;

Citation: Kidoń, M.; Uwineza, P.A. vegetable; fruit
New Smoothie Products Based on
Pumpkin, Banana, and Purple Carrot
as a Source of Bioactive Compounds.
Molecules 2022, 27, 3049. https:// 1. Introduction
[Link]/10.3390/molecules27103049
Fruits and vegetables are an essential part of the human diet. In particular, they are
Academic Editor: Adele Papetti rich sources of dietary fiber, vitamins, and various phytochemicals. Numerous studies have
Received: 9 April 2022
proved that they play a vital role in health promotion and prevention of certain chronic
Accepted: 6 May 2022
diseases, e.g., hypertension, cancer, coronary heart disease, stroke, etc. [1,2]. In addition
Published: 10 May 2022
to this, they provide a lot of sensory sensations. Fruit and vegetables are rich in colorful,
flavorful, and tasteful compounds.
Publisher’s Note: MDPI stays neutral
Banana is one of the earliest crops cultivated by man and remains a staple food crop for
with regard to jurisdictional claims in
millions of people in the tropical world. Bananas are monocotyledonous plants, belonging
published maps and institutional affil-
to the genus Musa of the family Musaceae in the order Scitamineae. The majority of edible
iations.
cultivars are allopolyploid triploids with a genome constitution of AAA (dessert banana),
AAB (plantains), and ABB (cooking bananas) [3]. This fruit is mostly grown in hot, tropical
climates across the globe, predominantly in Asia, Latin America, and Africa. The biggest
Copyright: © 2022 by the authors.
producers are India, China, Indonesia, the Philippines, Ecuador, and Brazil. Bananas
Licensee MDPI, Basel, Switzerland. production in the world exceeds 100 million tons andit is the second highest produced
This article is an open access article fruit after citrus, accounting for around 16% of global fruit production [4]. Despite having
distributed under the terms and a high calorie content primarily from carbohydrates and dietary fiber, bananas are poor
conditions of the Creative Commons in protein and fat. Vitamins C, A, B1 , B2, and B6 , as well as minerals, such as magnesium,
Attribution (CC BY) license (https:// phosphorus, calcium, and iron could be also found in bananas. Banana consumption
[Link]/licenses/by/ could be associated with a reduction in the risk of gastrointestinal diseases, regulation of
4.0/). carbohydrate metabolism, and weight control [5,6].

Molecules 2022, 27, 3049. [Link] [Link]

Molecules 2022, 27, 3049 2 of 15

Carrot is a type of root vegetable. This plant is widely cultivated and consumed
throughout the world, such as in America, Europe, Southwest Asia, and Africa. Its popular-
ity is due to its versatile use, taste, and health benefits. The global production of carrots and
turnips in 2019 was about 45 million tons. China produces about 50% of the world’s total
carrots. Other meaningful producers of carrots are Uzbekistan, Russia, the USA, Ukraine,
and Poland [4]. Purple carrot varieties are still popular, and these are traditionally grown in
countries where carrots are originated, such as Turkey, Afghanistan, Egypt, Pakistan, and
India. Cultivated and wild carrots belong to Daucus carota species. Cultivated carrots could
be divided into the following two main groups: the eastern anthocyanin (Daucus carota
ssp. sativus var. atrorubens Alef.), which are yellow or purple in color, and the western
carotene (Daucus carota ssp. sativus var. sativus), which are yellow or orange. Indeed,
carrot roots with purple and yellow colors are the ancestors of today’s more popular or-
ange varieties. Carotenoids and phenolics are the major antioxidant compounds found
in carrots. Alasalvar et al. [7] identified 11 phenolic acids in different colored carrot roots,
but the total concentration of all phenolic acids was the highest in purple carrots. Purple
carrots also contain twice the amount of alpha and beta carotene compared to orange
carrots. The color of purple carrot roots comes from pigments called anthocyanins, which
are a type of flavonoids. Anthocyanins from purple carrot are more stable than pigments
from other sources, even during heating and processing, and also act as very powerful
antioxidants [8–11]. Bioactive compounds found in purple carrot roots could play a vital
role in preventing or delaying cardiovascular disease (CVDs), obesity, diabetes, and cancer.
Anthocyanins and other phenolics are especially successful in reducing metabolic changes
and possessed inflammatory effects, as well as preventing oxidative stress [12].
Pumpkins are defined as fruits of different species classified in the Cucurbitaceae family
and genus Cucurbita, but they are regarded as a vegetable in consumer terms. Pumpkin
originally came from Northeastern Mexico and the Southern United States, but nowadays
this vegetable is spread across every continent. In 2019 global pumpkin production was
about 23 million tons. Asian countries, such as China and India, are the biggest pumpkin
producers in the world. The Russian Federation, Ukraine, and the United States are also
significant pumpkin producers [4]. The most important cultivated species are Cucurbita
pepo, C. maxima, C. moschata, C. mixta, and C. stilbo. Pumpkin fruits have a substantial
variability in shape, size, and color of skin and flesh. Fruits, apart from food usage, also
have decorative and agricultural properties. All parts of the fruit are eatable, but the most
important are the flesh and seeds. It can be consumed raw, but the flesh is also suitable for
the preparation of purees, jams, jellies, and pies, whereas the seeds can be consumed after
drying or roasting, or processed into oil via pressing. Pumpkin fruits are a naturally rich
source of dietary antioxidant vitamins like A, E, and C, carotenoids like β-carotene, lutein,
and zeaxanthin, and phenolic compounds like caffeic acid, gallic acid, 4-hydroxybenzoic
acid, protocatechuic acid, and rutin. Pumpkins can be also recognized as a source of
dietary fiber, minerals, and unsaturated fatty acids. The content of particular compounds
varies, and is affected by genotypic differences and growing conditions. Intake of these
compounds can have a beneficial effect on human health and, as such, pumpkin could be
considered as a “functional food” [13–16]. In the literature, the anti-diabetic, antioxidant,
anti-carcinogenic, anti-inflammatory, and anti-microbial potential of pumpkin ingredients
can be found. Also, anti-kidney stone formation, anti-hypotensive, anti-inflammatory, and
anti-blood-coagulatory effects are well documented [17].Most fruits and vegetables are
consumed fresh. However, many of them are industrially processed into canned, dried,
juiced, frozen, or soup products to extend their shelf life, protect their nutritional value, and
make meal preparation easier. Nowadays, consumer trends are orientated to ready-to-eat
and functional food, and/or the reformulation of typical products to increase nutritional
value. Smoothies could fulfill this consumer demand [1]. Smoothies are new products
on the market, and are potentially a convenient and palatable way to replace at least one
portion of fruit or vegetables from the recommended five portions per day. Smoothies
are usually semi-liquid, thick beverages, obtained by blending fruit, fruit juice, and/or
Molecules 2022, 27, 3049 3 of 15

fruit puree. To increase sensory sensation, water, ice, sugar, sweeteners, spices, seeds,
yogurt, or milk can be added. In order to increase the quantity of bioactive compounds in
smoothies, some researchers proposed incorporating fruit seeds and peels into smoothies,
as these are generally wasted during processing, or selecting raw materials that are rich in
phytochemicals for inclusion in smoothies [18–20]. However, it must be considered that
these products also contain considerable amounts of simple sugars. Excessive consumption
of simple carbohydrates, particularly from low-dietary fiber products like fruit juices, could
be associated with the metabolic syndrome or obesity. Because of this, it is recommended
to consume not more than one portion per day. This is especially important for children
and individuals at risk of obesity or type 2 diabetes, who should limit their consumption of
simple sugar-containing products [21,22].
The phytochemical contents and sensory properties of smoothies can vary substantially
depending on the processing methods utilized, as well as the raw ingredients used during
the preparation [19]. Therefore, it is always advised to carefully select the processing
method and raw materials in order to produce a food product with high nutritional values
and desirable sensory features. The main objectives of this study were the development
of new smoothie formulations based on the available nutritious ingredients, including
bananas, pumpkins, and purple carrots, as well as to investigate bioactive compound
contents, sensory characteristics, antioxidant activity, and other quality parameters of the
obtained products.

2. Results and Discussion

2.1. pH and Soluble Solids
The pH and total soluble solids results of the five produced smoothies, pumpkin and
purple carrot with high sugar (PCH), pumpkin and purple carrot with low sugar (PCL),
banana and purple carrot with low sugar (BCL), banana and purple carrot without sugar
(BCW), and banana, pumpkin, and purple carrot without sugar (BPCW) are presented
in Table 1. The results showed that the pH of the produced smoothies ranged between
3.91 and 4.45, and that soluble solids ranged between 7.9 and 16.7%. The pH and soluble
solids were different due to different ingredient ratios and different ingredient acidity
and sugar content. BCL and BPCW smoothies had the highest pH level of 4.45, while
PCH showed the lowest pH level of 3.91. The pH values of food products are important
for sensory characteristic, as well as for the prevention of microbial spoilage. These pH
values were typical for smoothies and similar to other findings. For example, a smoothie
obtained by mixing juçara, banana, and strawberry pulps has a pH of about 4.0, and a
smoothie consisting of pineapple, watermelon, banana, and coconut milk possessed a pH of
about 4.5 [23,24]. According to other studies, the main ingredients for smoothie production
used in this work showed different acidity. Purple carrot has a pH of about 6.2, bananas
range between 4.8–5.4, pumpkin range between 4.4–4.5, and lemon has a pH of about
2.3 [16,25–28]. It has been claimed that sour fruit juices could successfully replace currently
used artificial acidity regulators, especially in functional food production [28]. Lemon juice
added to smoothies decreased pH effectively.

Table 1. pH and total soluble solids of smoothies.

Sample pH Soluble Solids [%]

PCH 3.91 ± 0.01 10.5 ± 0.2
PCL 4.37 ± 0.01 7.9 ± 0.1
BCL 4.45 ± 0.01 16.7 ± 0.2
BCW 4.35 ± 0.01 12.7 ± 0.2
BPCW 4.45 ± 0.01 12.2 ± 0.1

The total soluble solids are related mainly to the sugar concentration in the product.
The BCL smoothie had the highest total soluble solids (16.7%), followed by the BCW
(12.7%) while the PCL smoothie had the lowest total soluble solids (7.9%, Table 1). The
Molecules 2022, 27, 3049 4 of 15

result showed that there was a large difference in soluble solids values in the produced
smoothies. The formulations which contained banana had almost two times the soluble
solids values of those with pumpkins and carrots only. Aditionally, Uzodinma et. al. [24]
noticed that an increased amount of bananas in smoothie formula decreased moisture
content. According to different research, banana itself has a higher total soluble solid
content compared to pumpkins and purple carrots. Ribeiro et al. [29] reported 23.1 Brix
in banana pulp, and Wang et al. [5] reported 14.73 Brix in banana smoothies. Witrowa-
Rajchert et al. [30] reported 15.2% dry matter content in fresh purple carrots cv. Deep
Purple. Additionally, Quit“ao-Teixeira et al. [31] reported 8.21 Brix in carrot juice, and
Zinash et al. [32] reported a range from 4.1 to 10.3 Brix in pumpkin. However, the previous
research on soluble solids content of different smoothie formulations or products available
on the market indicated similar results in the range from 8.6 to 15.4 [19,29,33].

2.2. Total Phenolic Content (tpc) in the Smoothies

The result showed that the total phenolic content (TPC) in produced smoothies ranged
from 39.2 to 55.8 mg/100 g. The BCL smoothie had the highest total phenolic content
followed by the BCW, the BPCW, and the PCH, while the PCL had the lowest total phenolic
(Table 2). It could be concluded that total phenolic content in smoothies composed of
banana and carrot was higher than in those composed of pumpkin and carrot. All raw
materials used for smoothie production were a source of phenolics but, according to
the literature, they contained different levels. Thus, the differences in total phenolic
content could be attributed to the different ingredients used for smoothie formulation.
Bashmil et al. [34] presented the total phenolic content from 38 to 128 mg of GAE/100 g
for banana pulps depending on cultivars and maturity stage. Priori et al. [14] reported
that the total phenolic content found in different evaluated pumpkins cultivars ranged
from 26.31 to 79.89 mg/100 g. The richest source of phenolics could be the purple carrot.
Leja et al. [35] found 311.5 mg/100 g of total phenolic content in its roots.

Table 2. Total phenolic content, carotenoids, vitamin C content, and antioxidant activity of
different smoothies.

Total Phenolic Sum of

Vitamin C Antioxidant
Sample Content (TPC) Carotenoids
(mg/100 g) Activity (µM TE/g)
(mg/100 g) (µg/100 g)
PCH 44.9 ± 2.9 ab 62 ± 8 b 0.34 ± 0.02 b 4.3 ± 0.2 a
PCL 39.2 ± 0.6 a 72 ± 16 b 0.41 ± 0.01 c 4.3 ± 0.1 a
BCL 55.8 ± 1.1 c 11 ± 1 a 0.27 ± 0.01 a 6.2 ± 0.2 c
BCW 47.4 ± 0.6 b 45 ± 12 b 0.31 ± 0.01 b 5.2 ± 0.1 b
BPCW 46.6 ± 2.1 b 108 ± 10 c 0.25 ± 0.01 a 4.9 ± 0.1 b
a–c —different letters in the same column indicate significant differences between data p < 0.05.

Smoothie products could be considered as a very rich source of phenolic compounds.

For example, smoothies composed from orange juice, papaya juice, melon juice, carrot
puree, and skimmed milk contained 45.6 mg GAE/100 mL [36], those composed from
apple juice, orange juice, strawberry, whole apple (pulp+juice), and banana contained
44 mg GAE/100 mL [19], while 148.7 mg chlorogenic acid/100 g was found in the case of
a smoothie consisting of orange carrot and water [37].
Phenolic compounds that came from smoothies could be easily available for biological
action. As found by Ribeiro et al. [38] bioaccessibility of the smoothie phenolic compounds
varied from 20 to 47% between gastric and intestinal digests. More than that, processing
typically applied during smoothie preparation, such as heating and grinding, could increase
the bioavailability of phenolic compounds. Unfortunately, up to now, there have been
no recommendation for the amount of phenolic compounds consumptions. But some
researchers suggest that a long–term intake of phenolics from diets could reduce the risk
of certain diseases such as cancers, cardiovascular diseases, type 2 diabetes, osteoporosis,
pancreatitis, etc. [39].
Molecules 2022, 27, 3049 5 of 15

2.3. Anthocyanins Content

Anthocyanins belong to flavonoids. These compounds are present in many fruits and
vegetables, and their products which give these fruits and vegetables their red, purple, and
orange colors. In human nutrition, they play an important role as antioxidants, and they
are used in the food industry as natural colorants. Anthocyanins are widely distributed in
the human diet and, in the United States, 12.5 mg/day [40] are estimated to be consumed,
while according to a European survey, anthocyanin consumption varies between 19.8 and
64.9 mg/day [41].
Total anthocyanin content ranged from 7.1 to 11.13 mg/100 g in all produced smoothies
(Table 3). The BCL sample had the highest amount of total anthocyanin at 11.13 mg/100 g,
followed by PCL and PCH (at about 10 mg/100 g), while the smoothies produced from ba-
nana and carrots without sugar had the lowest amount of total anthocyanins (BCW sample
at 7.1 mg/100 g). González-Tejedor et al. [42] detected only 3.74 mg/100 g of anthocyanin
in the purple smoothie that was composed of purple seedless grapes, cucumber, beet, and
broccoli. The amount of anthocyanins in a smoothie produced by Keenan et al. [43] from
strawberries, apples, apple juice, bananas, and oranges was 34.67 mg/100 g of dry weight.

Table 3. Individual and the sum of anthocyanin content of different smoothies (mg/100 g; compound
1-cyanidin 3-xylosylglucosylgalactoside, compound 2-cyanidin 3-xylosylgalactoside, compound 3-
sinapic acid derivative of cyanidin 3-xylosylglucosylgalactoside, compound 4-ferulic acid derivative
of cyanidin 3-xylosylglucosylgalactoside).

Sum of
Sample Compound 1 Compound 2 Compound 3 Compound 4
Anthocyanins
PCH 0.63 ± 0.01 0.87 ± 0.02 1.01 ± 0.02 7.69 ± 0.17 10.2 ± 0.5 bc
PCL 0.69 ± 0.01 0.93 ± 0.01 1.02 ± 0.01 7.75 ± 0.01 10.4 ± 0.3 cd
BCL 0.64 ± 0.01 0.74 ± 0.01 0.70 ± 0.01 9.02 ± 0.02 11.1 ± 0.3 d
BCW 0.47 ± 0.01 0.61 ± 0.01 0.45 ± 0.01 5.57 ± 0.08 7.1 ± 0.3 a
BPCW 0.44 ± 0.01 0.54 ± 0.02 0.71 ± 0.01 6.31 ± 0.17 8.0 ± 0.5 ab
a–d —different letters in the same column indicate significant differences between data p < 0.05.

Purple carrots have been reported as very good sources of anthocyanins, including
acylated forms [10,11,44,45]. Anhocyanins were chromatogram recorded at 520 nm. Four
different anthocyanins in were found in the examined smoothies. According to retention
time, spectra characteristic, and data in the literature, four cyanidin derivatives are detected
and two had acylated form with sinapic acid (compound 3) and ferulic acid (compound 4).
The predominant compound 4 corresponds to cyanidin 3-O-feruloyl-(xylosyl-glucosyl-
galactoside), which represents approximately 75–80% of all anthocyanin (Table 3).
In the present work, the only source of anthocyanins in smoothies was the purple
carrot. Additionally, the amount of purple carrot used in the recipe was the same in all
produced smoothies. Other factors, such as the presence of sugar or pH and heating could
also affect anthocyanin content in the final product.
The most probable effect on anthocyanin content could be caused by the addition of
sucrose. In the present study, the result showed that the smoothies produced with the
addition of some sucrose (white sugar) had higher values of total anthocyanin content
compared to those smoothies without sugar added. This is because the addition of sucrose
could have had a significant protective effect on anthocyanins, as has been reported by
Tsai et al. [46]. Additionally, Nikkhah et al. [47] reported a protective effect of sugar (sucrose
20%) on anthocyanin stability in berries, and they showed that the effect of added sugar on
anthocyanin stability depends on its structure, concentration, and type of sugar.
Other studies suggest that acylated anthocyanins from purple carrot possess unusual
stability when compared to unacylated compounds. The study of Sadilova et al. [48]
observed that elderberry anthocyaninswere were sensitive to thermal treatment. After 3 h
of heating, only 50% of elderberry pigments were retained at 95 ◦ C. On the other hand,
Molecules 2022, 27, 3049 6 of 15

Kirca et al. [8] reported that half of initial content of anthocyanins from purple carrots juice
degraded after 5 h of heating at 90 ◦ C.

2.4. Carotenoids
The result of total carotenoids (Table 2) showed that BPCW smoothie produced from
banana, pumpkin, and carrots had the highest amount of total carotenoids (108 µg/100 g),
followed by PCL (72 µg/100 g) while BCL smoothie had the lowest amount of total
carotenoids (11 µg/100 g).
According to chromatographic data, only α- and β-carotene were detected in produced
smoothies despite other findings. In fresh banana, pumpkin, and purple carrot also lutein,
lycopene, β-cryptoxanthin, violaxanthin, astaxanthin, antheraxanthin, and zeaxanthin were
detected [49,50].
The smoothies which had pumpkins in their formulation showed at least two times
higher content of total carotenoids than smoothies that had banana and carrots only. The
value of total carotenoids obtained in this study was low compared to the result obtained
by Nawirska-Olszanska et al. [51] who got the range from 4.9 to 7.4 mg/100 g fresh weight
in pumpkin puree enriched with Japanese quince, cornelian cherry, strawberry, and apples.
Balaswamy et al. [52] reported a range from 0.14 to 1.54 mg/100 g of total carotenoids in
the smoothies made from mango, pineapple, sapota, pomegranate, and papaya.

2.5. Ascorbic Acid

Vitamin C (ascorbic acid) it is a water-soluble vitamin. Humans are not able to
synthesize their own vitamin C, and it must be supplied from external sources, such as
foods or supplements.
The result presented in Table 2 showed that the vitamin C content in the produced
smoothies ranged from 0.25 to 0.41 mg/100 g. The PCL smoothie had the highest vitamin
C content (0.41 mg/100 g), while the PBCW smoothie had the lowest vitamin C content
(0.25 mg/100 g).
It should be admitted, however, that smoothies obtained in this study are not a
significant source of vitamin C. A reason for this could be the low ascorbic acid content in
the fruits and vegetables selected for smoothie formulation. Carrots have been reported to
have only 4 to 29 mg of ascorbic acid per 100 g [53,54]. The ascorbic acid content of bananas
varied from 2.5 to 17.5 mg/100 g [49]. Research on pumpkins revealed that the vitamin C
content of this fruit ranged from 3 to 15 mg/100 g, depending on the variety and maturity
stage [15].
Vitamin C is well known as the less stable nutrient during processing, and it is
significantly affected by many factors, such as heat, oxygen, light, storage temperature,
and storage time [55].Yadav [56] reported a significant reduction of ascorbic acid content
in carrot and other fruit juice-blended nectar processed under a temperature of 80 ◦ C
for 5 min. Patras et al. [57] showed a significant reduction in ascorbic acid levels in the
thermal processing of carrots purees that resulted in a 46% reduction, as compared to
un-processed samples. During smoothie production, fruits were crushed (which could
release endogenous enzymes) and/or heated, and these proccesses could strongly affect
vitamin C content in the final product.

2.6. Antioxidant Activity

An antioxidant is any substance that delays or inhibits oxidative damage to a target
molecule. The main property of an antioxidant is its ability to trap free radicals. An-
tioxidant activity, then, is the cumulative capacity of food components to scavenge free
radicals [58]. In this study, free radical scavenging activity against ABTS•+ (2,20 -azinobis(3-
ethylbenzthiazoline)-6-sulfonic acid was used.
The result showed that the antioxidant activity in the produced smoothies ranged
from 4.3 to 6.2 µM TE/g (Table 2). The BCL smoothie had the highest antioxidant activity,
Molecules 2022, 27, 3049 7 of 15

followed by the BCW sample. The smoothies which contained pumpkin had the lowest
antioxidant activity.
In this present study, the antioxidant activity values were quite high, and the use of
different raw materials had an affect on antioxidant activity. In addition, the smoothies
which had bananas in their formulation showed higher antioxidant activity than the
pumpkin with carrots only. In our work, correlation between antioxidant activity and the
total phenolic content (TPC) was calculated. The results obtained showed a high positive
linear correlation between these parameters (Pearson correlation coefficient R = 0.937).
Similar results have been well documented in other studies, and it has been reported that
the antioxidant activity of many fruits was more closely correlated to total phenolic contents
than to ascorbic acid and other compound content [59,60]. Jiratanan and Liu [61], in their
research on antioxidant activity, concluded that depending on the particular product,
processing parameters, and methods, thermal processing may enhance, reduce, or cause no
change in total antioxidant activity.
It has been suggested that food processing such as cooking or grinding might improve
the extractability of antioxidant compounds by breaking down the cell walls. Further-
more, the thermally processed smoothies had a high level of antioxidants after processing
when compared to fresh or high hydrostatic pressure (HHP) processed smoothies. The
antioxidant capacity of HHP processed smoothies decreased during storage, and the level
of reduction was greater than in thermally treated samples. This could suggest that the
enzymatic degradation systems play important role in antioxidant capacity deterioration.
Thermal treatment could lead to the heat denaturation of enzymes, while HHP conditions
could not inactivate them [62].

2.7. Color Attributes of Smoothies

Color is an important quality attribute of fresh and processed food products that de-
termines the first impression and influences consumers’ choices, perceptions, and purchase
behavior. The results of the instrumental color determination of the produced smoothies
are presented in Table 4. The results showed that L* values of produced smoothies ranged
from 12.9 to 26.5. The highest value of lightness (L*) was found in the BCW smoothie,
and the darkest sample was the PCL smoothie. From the results obtained, the lightness of
banana/carrot smoothies were about two times higher than the pumpkin/carrot smoothies.
Positive a* values means that the hue of color is red, and these values ranged from 20.7 to
25.0. The red color of produced smoothies was attributed to the anthocyanin that comes
from purple carrots. b* values of smoothies ranged from 2.9 to 11.7. The highest value
was found in the PCH smoothie, and the lowest was found in the BCL smoothie. The
presence of pumpkin in the smoothie formula increased the value of b* parameters in
smoothies. Also, Buniowska et al. [63] observed a 16% higher value of the b* parameter in
the smoothie which contained carrot juice, pumpkin, and mango, compared with a sample
where papaya was used instead of pumpkin. Smoothies containing juçara, banana, and
strawberry produced by Ribeiro et al. [23] were a little darker, but the intensity of red was
at least two times lower compared to samples from our study.

Table 4. Color parameters of smoothies.

Sample L* a* b*
b 25.0 ± 0.2 c 11.7 ± 0.2 c
PCH 14.7 ± 0.1
PCL 12.9 ± 0.1 a 20.7 ± 0.2 a 7.7 ± 0.2 b
BCL 22.9 ± 0.2 c 24.5 ± 0.1 c 2.9 ± 0.3 a
BCW 26.5 ± 0.1 e 22.3 ± 0.1 b 3.4 ± 0.2 a
BPCW 24.5 ± 0.1 d 22.4 ± 0.1 b 7.3 ± 0.2 b
a–e —different letters in the same column indicate significant differences between data p < 0.05.
 2.8. Sensory Analysis
In Figure 1, sensory evaluation scores were presented. The studies sh
products possessed good sensory attributes. The highest sensory score in
Molecules 2022, 27, 3049 8 of 15
consistency, and overall acceptance was found in smoothies composed o
purple carrots (samples BCL and BCW). The smoothies produced from pum
pleSensory
2.8. carrots (samples PCH and PCL) had higher scores in color than others,
Analysis
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In Figure scored
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The studies obtained
showed that all the lo
products possessed good sensory attributes. The highest sensory score in terms of taste,
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consistency, and overall acceptance was found in smoothies composed of bananas and
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sidered
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ceptance score (7.9), followed by the BPCW smoothie (7.7), while the PCL
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gested that soluble solids and the pH of smoothies had a strong effect on
the lowest score (6.6). These results showed that the materials incorporated into smoothie
[Link]
production Smoothie products
a great influence characterized
on consumer by Other
acceptability. aboutresearch
13 Brixhasdegrees
suggestedof solu
with
that pH values
soluble solids andbetween 3.7 and 4.2,
the pH of smoothies had afit very
strong well
effect on with consumers’
consumer acceptance. senso
Smoothie products characterized by about 13 Brix degrees of soluble solids, and with
[33]. Sugars and acid ratio mainly influence the taste of the smoothie. Despit
pH values between 3.7 and 4.2, fit very well with consumers’ sensory preferences [33].
resultsand
Sugars ofacid
Uzodinma
ratio mainlyet influence
al. [64], the
who had
taste high
of the scoresDespite
smoothie. in thethis,
sensory
and theattribut
ple, watermelon,
results of Uzodinma et [Link],
[64], who and coconut
had high scores insmoothies without
the sensory attributes forthe addition o
pineapple,
watermelon, banana, and coconut smoothies without the addition of any external sweetener,
sweetener, low soluble solids smoothies (PCL and PCH) produced in our s
low soluble solids smoothies (PCL and PCH) produced in our study obtained lower scores
lower
for [Link] for taste.

Figure 1. Sensory scores of different smoothies.

Figure 1. Sensory scores of different smoothies.
3. Materials and Methods
3.1. Materials
3. Materials and Methods
Fresh pumpkin (Cucurbita maxima cv. Bambino) and purple carrots (Daucus carota ssp.
3.1. Materials
sativus var. atrorubens Alef. cv. Purple Sun) were cultivated on a private farm located in
the center of Poland (Sieradz district, Łódź province, Poland). Vegetables were harvested
Fresh pumpkin (Cucurbita maxima cv. Bambino) and purple carrots (Dau
in October 2018. Unblemished and similar-sized pumpkins and carrots were selected and
sativus
stored notvar.
longeratrorubens
than 4 weeksAlef. cv. Purple
in a storeroom Sun)4 ◦were
at about C untilcultivated on a private f
smoothie production.
Ripe bananas,of
the center lemons,
Polandand(Sieradz
white sugar were purchased
district, from the local
Łódź province, supermarket
Poland). in
Vegetables w
Poznan, Poland.
in October 2018. Unblemished and similar-sized pumpkins and carrots wer
Molecules 2022, 27, 3049 9 of 15

All chemicals used were analytical or gradient grade (for HPLC) purity and provided
by Poch (Polish Chemical Reagents, Gliwice, Poland) or Merck Kgaa (Darmstadt, Germany).

3.2. Smoothies Preparation

The smoothies’ production included two main steps, which were the pretreatment
of raw materials, and blending. The steps for the pretreatment of raw materials were as
follows: all ingredients were washed with tap water; purple carrots roots were peeled and
chopped into about 2 cm pieces with a kitchen knife; pumpkin was peeled, and the seeds
and the soft inner flesh parts were removed, before the pumpkin was chopped into about
2 cm cubic pieces; bananas were peeled by hand and chopped into about 2 cm pieces with a
kitchen knife; the lemons were cut in half and the juice was squeezed using a portable citrus
juicer. The second step was formulation and blending. The pumpkin, banana, purple carrot,
lemon juice, water, and sugar were weighed according to the recipes presented in Table 5,
before being placed in the Thermomix TM 3 device (Vorwerk SE & Co. KG, Wuppertal,
Germany). The raw materials were ground and heated at 90 ◦ C for 10 min to pasteurize the
smoothie, and to produce a good, smooth texture. Five different products were obtained,
as follows: pumpkin and purple carrot with high sugar (PCH); pumpkin and purple carrot
with low sugar (PCL); banana and purple carrot with low sugar (BCL); banana and purple
carrot without sugar (BCW); banana, pumpkin, and purple carrot without sugar (BPCW).

Table 5. Smoothie compositions (g/100 g).

Ingredient PCH PCL BCL BCW BPCW

pumpkin 50 50 0 0 30
banana 0 0 40 44 40
purple carrot 10 10 10 10 10
lemon juice 4 2 2 3 3
sugar 6 4 4 0 0
water 30 34 44 43 17

Smoothies were hot transferred to jars, closed with caps, and cooled in tap water. The
sensory analysis and analysis of the color of the smoothies were performed a day after
processing. For other measurements, smoothie samples were freeze-dried and ground into
powder. Freeze drying was performed as follows: first of all, the samples were frozen in
a low-temperature freezer (Arktico A/S, Esbjerg, Denmark) at −50 ◦ C for 24 h, and then
were subjected to freeze-drying in a freeze-dryer system, the FreeZone 6 dryer (Labconco,
Kansas City, MO, USA), for 48 h. For the first 24 h, the temperature of the bulk tray dryer
shelves was set at 4 ◦ C for primary drying and then, for the next 24 h, it was set to 30 ◦ C for
secondary drying.

3.3. Determination of pH and Soluble Solids

The pH determination was conducted using a pH-meter Orion model 710A (Thermo
Fisher Scientific, Waltham, MA, USA). The total soluble solids were determined in an
optical refractometer, model HI96801 (Hanna Instruments, Woonsocket, RI, USA). The
mean value was calculated from three measurements of each sample.

3.4. Total Phenolic Compounds (TPC) Extraction and Analysis

Phenolic compounds were extracted from freeze-dried samples with a mixture of
methanol/water/acetic acid (25/24/1; v/v/v). The powdered sample (1 g) was mixed with
extraction solvent (22 mL), and shaken for 1 h in a laboratory shaker. Then, the liquid was
transferred to a 25 mL volumetric flask and filled up with the extraction solvent, which
was followed by centrifugation for 5 min at 5000× g. The supernatant was collected in the
tube for further analysis.
Total phenolic content (TPC) was analyzed using the Folin–Ciocalteu method [65].
A test tube was obtained, and 0.2 mL of the extract, 0.8 mL of distilled water, and 5 mL
Molecules 2022, 27, 3049 10 of 15

of Folin–Ciocalteu reagent (0.1 M) were added. The solution was roughly mixed for
2 min, then 4 mL of Na2 CO3 solution (75 g/L) was added, and the reaction was carried
out for 1 h at room temperature in darkness. Absorbance was read at 765 nm using a
spectrophotometer UV-VIS 830 plus Metertech (Metertech Inc., Taiwan). All measurements
were performed in triplicate, and the results were expressed as mg gallic acid equivalent
(GAE) per 100 g of fresh weight of the sample.

3.5. Anthocyanins Analysis

The anthocyanin content of produced smoothies was determined by using the HPLC
method described by Oszmiański & Sapis [66]. Phenolic extract was used for analysis. The
HPLC system Agilent 1260 Infinity (Agilent Technologies Inc., Santa Clara, CA, USA) was
used, equipped with a degasser, binary pump, autosampler holder, column holder, and a
diode array detector (DAD). For chromatographic resolution, a Zorbax SB C-18 column
with a diameter of 4.6 × 150 nm and 5 µm granulation was used. The chromatographic
conditions were as follows: injection volume 20 µL; flow rate 1.0 mL/min; solvent A formic
acid/water (1/9; v/v); solvent B: formic acid/acetonitrile/water (1/3/6; v/v/v). The
elution gradient was linear as follows: from 0 to 15 min solvent B increased from 20 to
50%, from 16 to 20 min solvent B increased from 50 to 100%, from 20 to 21 min solvent B
remained 100% and from 21 to 23 min solvent B decreased to 20%. The detector was set
for scanning in the range of 400 to 700 nm. Quantification was performed at 520 nm and
calculated as mg cyanidin-3-glucoside per 100 g of fresh weight of the sample.

3.6. Extraction and Analysis of Carotenoids

The carotenoids from each freeze-dried smoothie sample were extracted by using the
accelerated solvent extractor Dionex ASE 350 (Thermo Fisher Scientific, Waltham, MA,
USA) with acetone as the solvent. The extraction process was as follows: 1 g of each sample
was weighed into extraction cells (34 mL), about 4 g of sand was added and mixed roughly.
Pure acetone was used as the extraction solvent, with 3 cycles of 10 min for each sample.
The pressure was 1500 psi and, after each cycle, the cells were washed with acetone. The
process occurred at room temperature (23 ◦ C). The obtained extract was evaporated and
filled with acetone in a 10 mL volumetric flask.
The same HPLC system as for anthocyanin was used. The chromatographic condi-
tions were as follows: injection volume 10 µL; flow rate: 0.5 mL/min; solvent A acetoni-
trile/0.05% triethylamine (v/v); solvent B methanol/ethyl acetate (11/9; v/v). The elution
gradient was linear as follows: from 0 to 50 min solvent B increased from 5 to 40%, from 50
to 60 min solvent B increased from 40 to 80%, and from 60 to 75 min solvent B decreased
from 80 to 5%. The detector was set for scanning in the range of 400 to 700 nm, while
carotenoid quantification was performed at 454 nm. All measurements were performed
in duplicate, and the results were expressed as µg of β-carotene/100 g of fresh weight of
the sample.

3.7. Ascorbic Acid Extraction and Analysis

Extraction of ascorbic acid was prepared by mixing 1 g of each freeze-dried smoothie
sample with about 22 mL of 1% meta-phosphoric acid. The mixture was shaken for 15 min
in a laboratory shaker at room temperature. The solution was centrifuged at 4000× g for
10 min, and the supernatant was collected in a 50 mL volumetric flask and filled up with
1% meta-phosphoric acid. The obtained supernatant of 2 mL was mixed with 1 mL of
5% dithiothreitol and filled up to 10 mL with 1% meta-phosphoric acid. The sample was
filtered through a 0.45 µm PTFE filter into the amber vials and analyzed by HPLC [67].
The same HPLC system as for anthocyanin was used. The chromatographic conditions
were as follows: injection volume 10 µL; flow rate, 0.7 mL/min; solvent A 1 mM potassium
dihydrogen phosphate in water; solvent B methanol. The elution gradient was linear as
follows: from 0 to 6 min, solvent B increased from 5% to 22%, and from 6 to 15 min solvent
Molecules 2022, 27, 3049 11 of 15

B decreased from 22% to 5%. The detector was set for scanning in the range of 200 to
380 nm. Quantification was performed at 245 nm [67].

3.8. Antioxidant Activity

The antioxidant activity was determined using the ABTS•+ method, as described
by Re et al. [68]. Phenolic extract was used for analysis. Absorbance of ABTS•+ solution
in PBS pH 7.4 buffer was read after 6 min of incubation with the added sample or PBS
buffer (control) at 30 ◦ C at 734 nm, using a spectrophotometer Heλios α (Thermo Fisher
Scientific, Waltham, MA, USA). From the absorbance measurement, the percentage of
radical reduction of the extract was calculated from the following formula:

Ac − As
% o f radical reduction = ·100% (1)
Ac

where Ac —absorbance ABTS•+ solution of the control, As —absorbance of ABTS•+ incu-

bated with the sample. The percentage of radical reduction was plotted as a function of
sample concentration or Trolox as a standard. The results were expressed as µM Trolox
equivalents/g of fresh weight sample.

3.9. Instrumental Color Measurement

The color of the smoothie samples was measured using a spectrophotometer Konica
Minolta 3600 d (Konica Minolta Co., Chiyoda, Japan). Color was expressed in CIE L*a*b*
system coordinates. The smoothie samples were filled into a glass cuvette with an optical
length of 10 mm, and this was placed over the aperture of the spectrophotometer. Then,
reflectance color from the surface was measured using illuminant D65, at a 10◦ observer
angle, with the specular component excluded. Three measurements for each sample
were performed.

3.10. Sensory Evaluation

The sensory evaluation was conducted by a group of 7 panelists (4 women and
3 men, aged 20–45) with formal classroom training in sensory evaluation. To qualify,
panelists had to be non-smokers, and had to have no allergy to any of the smoothie
ingredients. All panelists work or study at Poznań University of Life Sciences and were
previously involved in the sensory evaluation of fruits, vegetables, and their products.
Samples were evaluated busing the 9-degree hedonic scale (9-extremely like; 1-extremely
dislike). The assessment included the following quality attributes: smell, taste, color,
consistency, and overall acceptance. All sensory tests were conducted in the room intended
for sensory analysis, equipped with individual booths illuminated with normal lighting.
All conditions in the testing area were controlled with minimum distractions to ensure
panelists’ comfortable judgment.

3.11. Statistical Analysis

The analysis of variance (ANOVA) was used to determine the significance of the main
effects. Tukey’s post-hoc test was used to determine differences between the mean values
of multiple groups. Correlations were analyzed with Pearson’s test. Statistical significance
was set at p < 0.05. The Statistica 13.1 software (TIBCO Software Inc., Palo Alto, CA, USA)
and Excel 2010 (Microsoft Corporation, Redmond, WA, USA) were used for the calculations.

4. Conclusions
Smoothies produced from pumpkins, bananas, and purple carrots can be considered
valuable products as sources of bioactive compounds and from sensory points of view. The
findings revealed that the combination of these three ingredients contained a significant
amount of total phenolic content (TPC), which ranged from 39.2 to 55.8 mg/100 g. The
strong positive correlation between total phenolic content (TPC) and antioxidant activity
was observed. The smoothies with pumpkin and purple carrots had higher total carotenoid
Molecules 2022, 27, 3049 12 of 15

content compared to those with bananas and purple carrots, whereas the smoothies that
contained bananas in their formulation showed higher antioxidant activity compared to
those with pumpkins and purple carrots only. The sensory panelists approved of all of
the smoothies formulated. However, the smoothies produced from banana and purple
carrots received excellent scores for smell, taste, consistency, and overall acceptance, while
pumpkin and purple carrot smoothies received better scores for color. As a result, making
smoothies from the selected ingredients could be convenient and an alternative to their
commonly known products. However, more research is needed to check changes of
bioactive compound content during the storage of smoothies. And to minimize the level of
sugar added to the product, since excessive sugar consumption should be avoided from
the nutritional point of view.

Author Contributions: M.K.: conceptualization, methodology, investigation, formal analysis, writing-

original draft, writing-review and editing, supervision, funding acquisition. P.A.U.: methodology,
investigation, writing-original draft, writing-review, and editing. All authors have read and agreed
to the published version of the manuscript.
Funding: Publication was financed within the framework of the Polish Ministry of Science and Higher
Education’s program: “Regional Initiative Excellence” in the years 2019–2022 (No. 005/RID/2018/19),
financing amount 12,000,000 PLN.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from the authors on request.
Acknowledgments: The authors would like to thank the colleagues in the Department of Food
Technology of Plant Origin and the Department of Chemistry and anonymous reviewers for their
comments that helped to improve this paper.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

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