Algal Research 47 (2020) 101811

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Using spray-dried microalgae in ice cream formulation as a natural colorant: T

Effect on physicochemical and functional properties
⁎
Yaşar Durmaza, , Mahmut Kiliclib, Omer Said Tokerb, Nevzat Konarc, Ibrahim Palabiyikd,
Faruk Tamtürkb
a
Ege University, Faculty of Fisheries, Aquaculture Department, 35100 Izmir, Turkey
b
Yildiz Technical University, Chemical and Metallurgical Engineering Faculty, Food Engineering Department, 34210 Istanbul, Turkey
c
Siirt University, Faculty of Architecture and Engineering, Food Engineering Department, 56100 Siirt, Turkey
d
Namik Kemal University, Agricultural Faculty, Food Engineering Department, 59030 Tekirdağ, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Microalgae can potentially be used as a food ingredient due to its biochemical composition that can enrich the
Microalgae bioactive compound content of the food products. In the present study, spray-dried Nannochloropsis oculata,
Ice cream Porphyridium cruentum and Diacronema vlkianum microalgae species were added to ice cream formulation at
Natural colorant different concentrations (0.10, 0.20 and 0.30 g 100 g−1). The effect of microalgae usage on the color, melting,
Bioactivity
sensory, flow behaviour and functional properties of ice creams was investigated. Ostwal de Waele model de-
Spray-dried
scribed the flow behaviour of the ice cream mixes well and the consistency index increased with P. cruentum
biomass concentration, whereas this value decreased by using N. oculata and D. vlkianum biomasses. Generally,
the color of ice creams was remarkably affected by both microalgae type and concentration, and N. oculata and
D. vlkianum species had a more pronounced effect. Regarding sensory characteristics, ice cream samples pro-
duced with P. cruentum were preferred more than the other samples. The phenolic contents of the ice cream
samples increased with microalgae usage. It can be concluded that microalgae can be used in ice cream to
improve color and functional properties; however, usage level should be optimized carefully to overcome dis-
advantages of the microalgae in terms of sensory characteristics.

1. Introduction
Ice cream is a widely consumed dairy product due to its nutritional
value and cooling effect, especially in summer periods. It is mainly
composed of milk, sweeteners, hydrocolloids, emulsifiers, flavoring and
coloring agents. After the relation between diet and health is realized,
researchers have exerted an effort to investigate natural coloring
agents. Natural food colorants have gained importance in recent years
due to consumers' attitudes and preferences. Also, using natural col-
oring agents may limit health concerns of consumers due to synthetic
constituents of ice cream. In this respect, it is of interest to explore the
addition of natural colorants with health benefits in the ice cream
formulation [1] to improve functional characteristics of the products
since such products are admirably consumed throughout the world by
people of all ages. Microalgae are one of the potential ingredients which
can be added to ice cream formulation for improving the attractiveness
of these products.
Microalgae are valuable natural products due to their nutrient and
bioactive compound contents and used as animal and human food
sources [2]. It is known that microalgae are rich in proteins, amino
acids, vitamins, and various minerals, as well as polysaccharides, sterols
and fatty acids. They have the potential to be used in dairy desserts (e.g.
ice cream) considering their macro- components (polysaccharides and
sulphated polysaccharides as stabilizer) and micro-components (poly-
unsaturated fatty acids as bioactive compounds and pigments as a
coloring agent) due to techno-functional and functional characteristics
[3–7]. In developed countries, people prefer natural foods due to health
concerns such as obesity, diabetes, and cardiovascular diseases. Ac-
cording to the European Commission Joint Research Centre report,
there are some Nannochloropsis oculata originated products (e.g. poly-
unsaturated fatty acids) on the USA and EU market as a food supple-
ment ingredient. This species classified under “no toxins known” status.
Also, Porphyridium cruentum has been given the GRAS (generally re-
cognized as safe) status by the FDA (Food and Drug Administration) of
the USA [8].
The products from microalgae have been widely used as a high-

⁎
Corresponding author.
E-mail address: yasar.durmaz@egu.edu.tr (Y. Durmaz).

https://doi.org/10.1016/j.algal.2020.101811
Received 12 October 2019; Received in revised form 18 January 2020; Accepted 18 January 2020
2211-9264/ © 2020 Elsevier B.V. All rights reserved.
Y. Durmaz, et al.
protein supplement in human nutrition, aquaculture and nutraceutical
purposes [9]. In recent years, studies about the usage of microalgae in
food matrices have been encountered in the literature. For this aim,
different microalgae types have been used in different products: Iso-
chrysis galbana and Nannochloropsis oculata (0.5 and 1.0%) in chewing
gum [10], Spirulina platensis (0.75 and 1.0%) and Pavlova lutheri (0.25
and 0.50%) in yogurt [3,11], four different microalgae types (Isochrysis
galbana, Tetraselmis suecica, Scenedesmus almeriensis and Nannochloropsis
gaditana with 0.4%) in bread [12] and Isochrysis galbana and Diacro-
nema vlkianum (0.5, 1.0 and 2.0%) in pasta [13]. All the related studies
indicated that microalgae can be used in the food formulations to im-
prove the attractiveness, nutritional and functional characteristics of
the products.
Using dried or encapsulated whole microalgae biomasses may en-
sure multi-functional effects [14,15]. Textural and sensory properties,
melting and flow behaviour, aroma release can be mentioned as the
main ice cream quality parameters [16]. Stabilizers, hydrocolloids, and
some polysaccharides act as stabilizer hydrocolloid have influence on
these quality parameters, especially flow, melting and textural char-
acteristics [17,18]. Various microalgae biomasses include poly-
saccharide and sulphated polysaccharides [19], which may define as
stabilizers, in addition to bioactive compounds. Addition to demand
and expectation of natural colorants for ice cream, these components
should be considered. Therefore, we may achieve multi-functional ef-
fects by using microalgae biomasses in ice cream. In the present study,
spray-dried Nannochloropsis oculata, Diacronema vlkianum and Porphyr-
idium cruentum were used in the ice cream formulation with different
concentrations (0.1, 0.2 and 0.3 g 100 g−1) as a natural colorant to
improve color and nutritional properties. The quality characteristics of
the ice cream mixes (rheology) and end products (fat, dry matter, and
phenolic contents, melting and sensory properties) with respect to mi-
croalgae type and concentrations were investigated.
2. Materials and methods
2.1. Materials
Ultra-high temperature-treated (UHT) milk (Birşah, Istanbul,
Turkey) and vanillin (Pakmaya, Izmit, Turkey) were purchased from a
local market in Istanbul, Turkey. Two different stabilizers as local salep
and emulsifier (mono and diglyceride) and granulated sugar (Orion,
Konya, Turkey) was used as a sweetener in the ice cream formulations.
2.2. Microalgae cultures and dry biomass
Nannochloropsis oculata (CCAP 849/1) and Diacronema vlkianum
(CCAP 914/1) were obtained from The Culture Collection of Algae and
Protozoa (CCAP, Scotland), Porphyridium cruentum (UTEX 161) from
The Culture Collection of University of Texas (Austin, USA). These
species were cultured by using F/2 culture media [20] prepared with
seawater (salinity 35.0 g L−1, pH 7.5, 25 °C, 2% CO2 concentration) and
tubular photobioreactor. The biomasses were harvested and con-
centrated with disc separator (GEA Westfalia Separator, Germany) and
then dried with a spray dryer. Spray drying process was performed with
a laboratory scale spray dryer (B290, Buchi, England), with a nozzle
atomization system including 1.0 μm nozzle diameter. The main para-
meters important for the spray drying process were as inlet air tem-
perature (170 °C) and outlet air temperature (95 °C) and fed rate
(7–9 mL min−1). The residence time of the drying air within the spray
chamber was about 1.5 s.
2.3. Physicochemical analyses of spray-dried algal biomasses
2.3.1. Quantification of the protein, fat and dry matter content
Protein, fat and dry matter content of spray-dried microalgae spe-
cies were performed according to modified procedure of AOAC [21].
2
Algal Research 47 (2020) 101811
Crude protein content (N 6.25) was determined by the Kjeldahl method
after acid digestion. To determine dry matter content, samples were
dried at 105 °C by drying oven (Memmert UF110, Germany) until a
constant weight was achieved. Quantification of the fat content of the
samples was performed by using the Soxhlet method. Fat globules were
extracted with diethyl ether by the extraction unit (Buchi, Switzerland).
2.3.2. Pigment analysis of algal biomasses
Total carotenoid and chlorophyll-a analysis of samples were de-
termined according to the method described by Gouveia et al. [22]. For
this aim, the after addition of 5 mL methanol to 10 mg spray-dried
microalgae samples, the prepared mixture was centrifuged (Hettich,
Universal 320R, Germany) at 2100g for 10 min. Then, the absorbance
values of the samples were measured at 475 and 665 nm wavelength
using the spectrophotometer (6305 Model, Jenway, Stafforddshire,
UK). Total chlorophyll-a and total carotenoid concentrations were de-
termined at 665 nm and 475 nm, respectively, and the contents were
calculated using specific absorption coefficients for each pigment [23].
Total anthocyanin content of the spray-dried microalgae samples
according to the pH differential method [24]. First, after addition of
45 mL methanol:water (80:20) to 5 g samples, homogenization step was
carried out using Ultra-Turrax (Daihan, HG-15D, Gang-Won-Do, South
Korea) at 1200 rpm for 2 min followed by holding the mixture in ul-
trasonic bath (Isolab, Istanbul, Turkey) for 1 h at 50 °C. After cen-
trifuging the extracts at 8000 rpm for 10 min, the supernatant was
filtered with filter paper through Whatman No. 1. The extracts were
separately mixed with buffers with pH 1 and pH 4.5 and incubated at
room temperature in a dark place for 30 min. The absorbance of the
extracts was recorded at 510 and 700 nm using a UV–vis spectro-
photometer (UV-1800, Shimadzu, Kyoto, Japan). Total anthocyanin
content was expressed in mg of cyanidin-3-glucoside per kg of spray-
dried microalgae samples.
2.4. Ice cream production
Ice cream formulation and sample preparation method were
adapted from Kurt et al. [25] and Soukoulis and Tzia [26] with some
modifications by using an ice cream maker machine (Delonghi, II Ge-
lataio, ICK5000, China). UHT milk (1000 mL), sugar (250 g), salep as a
stabilizer (9.00 g), emulsifier (4.00 g) and vanillin (0.40 g) were used in
ice cream formulation. Salep which is a traditional milk and orchid
roots drink was used as a stabilizer. Salep composition was previously
determined as 56.1 g 100 g−1 glucomannan and 36.3 g 100 g−1 starch,
4.60 g 100 g−1 protein and 2.07% ash [27]. Meanwhile, the protein, fat
and dry matter and non-fat dry matter content of the milk was 3.0%,
3.3%, 11.0%, and 7.7%, respectively.
The spray-dried microalgae were added to the ice cream mix with
the concentration of 0.1, 0.2 and 0.3 g 100 g−1. The ingredients were
all added to heated milk (85 °C) stirred for 1 min. This step was fol-
lowed by mixing at 1300 rpm for 3 min followed by rapid cooling to
4 °C. The samples were aged for 24 h at 4 °C. The aged ice cream mixes
were whipped at 0 °C for 10 min (Delonghi, II Gelataio, ICK5000,
China, mixing speed approximately 225 rpm) and were frozen, har-
dened using a batch freezer (Arcelik, Gebze, Turkey) at −18 °C for 24 h.
Samples were stored in a deep freezer (Arcelik, Gebze, Turkey) at
freezing conditions of −18 °C prior to analysis.
2.5. Rheological properties of ice cream mix
The rheological properties of the ice cream mixes were determined
using a method performed by Dertli et al. [28] and a stress/strain and
temperature-controlled rheometer (Anton Paar, MCR 302, Austria)
equipped with a parallel plate (50 mm diameter). The gap was adjusted
to 0.5 mm. After loading samples and descending the probe to the de-
sired level, the measurement was started after 2 min for equilibration of
the temperature and relaxation of the samples. The applied shear rate
Y. Durmaz, et al.
range was between 1 and 100 s−1 at 5 °C. Ostwald de Waele model
successfully described the flow behaviour of the samples and the cor-
responding parameters were calculated using the following equation:
σ = K (γ )n (1)
where σ is shear stress (Pa), K is the consistency coefficient (Pa·sn), γ is
shear rate (1/s), and n is flow behaviour index.
2.6. Color properties of ice cream samples
It measured the colors of the ice cream samples according to Akalin,
Karagözlü, & Ünal [29] method by using a colorimeter (CR-400 Chro-
maMeter, Konica Minolta, Japan). Color values were determined as L*,
a* and b*. L* values demonstrate the light intensity (0−100), a* values
demonstrate from red (+) to green (−), and b* values demonstrate
from yellow (+) to blue (−).
2.7. Melting properties of ice cream samples
Differential Calorimetry Scanning (DSC) (TA Instrument Q100, New
Castle, USA) technique was used to determine the melting behaviour of
ice cream samples. 10–15 mg of samples were put into hermetically
sealed aluminum pans and scanned at 10 °C/min over from −40 to
20 °C [30]. The analyses were conducted in duplicate for each sample.
2.8. Sensory evaluation
Hedonic evaluation of ice cream samples was determined based on
protocols described previously by Soukoulis et al. [31] with some
modifications. Basically, 100 mL ice cream samples were presented and
served at certain intervals in odorless, randomly three-digit coded glass
beakers of 100 mL in a covered glass dish. Sensory evaluation was
performed in a room with an appropriate temperature (25 °C) and
lighting in an open sitting condition. To be eligible to participate in this
study, panelists had to be over the age of 20 years, had to have the
capacity to understand the intent of the study, and had to make an
informed decision regarding consent. The age composition of the pa-
nelists was 64.5% (20–25) and 35.5% (35–52) whereas the gender
composition was % 23 male and 77% female. The purpose of the study
was explained to the panelists through a letter of information. The
panelists signed an informed consent form if they were in agreement
with the purpose of the study. Each panelist was trained before eva-
luation in order to familiarize with the sensory analysis, samples, and
methodology. They were asked to cleanse their palates with water be-
tween each sample and without a break. Degree of liking of samples
were evaluated for color, structure, taste, odor, melting, texture,
strange taste and overall acceptability in a scale ranging from 1 to 5
points (1: very bad, 2: dislike, 3: not too bad, 4: good and 5: very good).
Panelists evaluated all samples in 6 sessions consecutively in five days.
The manner in which the treatment combinations were divided be-
tween the sessions and the order in which the samples were presented
was randomized to minimize the carryover effects.
Table 1
Physicochemical properties of the dried algae samples.
Micro-algae Dry matter (%) Protein content (mg g−1 dw) Fat content (%)
P. cruentum 96.6 ± 0.05a 130.0 ± 21.0b 0.37 ± 0.02b
N. oculata 92.3 ± 0.25b 314.0 ± 12.0a 7.73 ± 0.79a
D. vlkianum 96.1 ± 0.09a 288.0 ± 4.90a 8.86 ± 0.63a
Different letters in the same column are showed statistically significant by Tukey test (p < 0.05). Data are the means ± SD of three replicates.
⁎
mg cyanidin-3-glucoside kg−1.
3
Algal Research 47 (2020) 101811
2.9. Bioactivity measurements of ice cream samples
2.9.1. Extraction
Five grams of each sample was weighed and put into a 100 mL glass
bottle. 45 mL acetone solution (99%) as extraction solvent was added to
the bottle and homogenization was performed by using Ultra-Turrax
(Daihan, HG-15D, Gang-Won-Do, South Korea) at 1200 rpm for 2 min.
The mixtures were shaken for 24 h at room temperature in the darkness
for efficient extraction of bioactive compounds. After that, extracts
were centrifuged (Hettich, Universal 320R, Germany) at 8000 rpm for
10 min. The supernatant was filtered with filter paper through
(Whatman No. 1) and put into a 50 mL falcon tubes. Then, cen-
trifugation and filtering processes of samples were performed.
2.9.2. Total phenolic content
Total phenolic contents of samples prepared according to Section
2.9.1 and in vitro digested ice cream samples were determined by the
method by Singleton and Rossi [32] after modification. 0.5 mL extract
put into a test tube and added 0.2 N 2.5 mL Folin-Ciocalteu reagent.
After 3 min 2 mL 7.5% Na2CO3 added and waiting 30 min in a dark
medium. At the end of the holding time, the absorbance was measured
at 760 nm using a UV–VIS spectrophotometer (UV-1800; Shimadzu,
Japan). Total phenolic contents were calculated as gallic acid equiva-
lent (mg GAE/kg).
2.10. Statistical analysis
The quantitative data were expressed as the mean ± standard
deviation in the tables. The results were analyzed using analysis of
variance (ANOVA). Tukey's test was applied to determine if the dif-
ferences between the parameters of the samples were significant. The
statistical analyses were performed using the MINITAB-Express
(Minitab Inc., State College, PA, USA) and MSTAT statistical packages
(Michigan State University, East Lansing, MI, USA). Differences with p
values < 0.05 were considered as a statistically significant effect of the
corresponding factor on the parameters.
3. Results and discussion
3.1. Physicochemical properties of spray-dried microalgae
Dry matter, fat, protein, total carotenoid, and chlorophyll-a contents
of the spray-dried microalgae samples were presented in Table 1. The
dry matter content of the samples was found above 95% after drying
process and it was significantly lower for dried N. oculata (p < 0.05).
This can be resulted from structure of the cell wall which may affect
water removal from the samples during the drying process [20,33]. The
fat content of the samples changed between 0.37 for P. cruentum to
8.86% for D. vlkianum. Crude protein contents of dried biomasses of P.
cruentum, N. oculata and D. vlkianum were determined as 130.0, 314.0
and 288 mg g−1 in dry weight, respectively. P. cruentum had the lowest
crude protein content among the microalgae types analyzed
(p < 0.05).
As in this study, pigment levels should be taken into consideration
when investigating the potentials of microalgae as colorants.
Total carotenoid (mg g−1 dw) Chlorophyll-a (mg g−1 dw) Anthocyanin content⁎
0.40 ± 0.12b 1.06 ± 0.21b 23.96 ± 0.12a
0.77 ± 0.14a 4.76 ± 0.14a 2.34 ± 0.24c
0.65 ± 0.09a 4.22 ± 0.09a 13.19 ± 0.47b
Y. Durmaz, et al. Algal Research 47 (2020) 101811

Fig. 1. Flow behaviour of ice cream mixes.

(K: control ice cream, P: samples containing Porphyridium cruentum, N: samples containing Nannochloropsis oculata, D: samples containing Diacronema vlkianum, 1:
0.10 g 100 g−1, 2: 0.20 g 100 g−1, 3: 0.30 g 100 g−1). Data are the means ± SD of three replicates.

Carotenoid, chlorophyll-a, and anthocyanin contents of the dried mi- Table 2

croalgae samples were presented in Table 1 and they changed between Rheological parameters of the Ostwald de Waele model determined for the ice
0.40 and 0.77 mg g−1, 1.06 and 4.76 mg g−1, and 2.34 and cream samples.
23.96 mg cyaniding-3-glucoside kg−1 in dry weight, respectively. P. Samples K (Pa·sn) n R2
cruentum had the lowest pigment contents among the microalgae types
analyzed. No significant difference was observed between carotenoid Control 8.80 ± 0.10d 0.361 ± 0.001e 0.9981
P1 11.9 ± 0.12c 0.324 ± 0.001f 0.9997
and chlorophyll-a content of the N. oculata and D. vlkianum; however,
P2 13.6 ± 0.59b 0.310 ± 0.006fg 0.9995
regarding anthocyanin content N. oculata was the richest. Pigment P3 14.7 ± 0.84a 0.298 ± 0.012g 0.9997
compositions can be modified using different drying techniques and N1 8.87 ± 0.13d 0.367 ± 0.004e 0.9998
post-harvest processes. In addition, encapsulation can be applied to N2 6.99 ± 0.42ef 0.399 ± 0.007cd 0.9998
improve the stability of the pigments against extrinsic factors such as N3 6.12 ± 0.33f 0.416 ± 0.006ab 0.9996
D1 7.81 ± 0.08de 0.384 ± 0.001d 0.9998
temperature, pH, light and mechanical processes etc. D2 6.07 ± 0.12f 0.409 ± 0.003bc 0.9998
D3 4.93 ± 0.16g 0.432 ± 0.006a 0.9997

3.2. Flow behaviour of ice cream mix
Flow behaviour of ice cream mixes was shown in Fig. 1. As shown in
the figure, shear rate increased higher than the shear stress values,
indicating that apparent viscosity decreased with increasing shear rate.
This type of flow was known as shear thinning behaviour. Entangled
network can be broken by the shear stress applied during measurement
and polymeric materials can be aligned in the flow direction as a result
of shear stress; therefore, the apparent viscosity of the mixes decreased
[18]. The obtained experimental shear rate versus shear stress data was
fitted to the Ostwald de Waele model with R2 values close to unity
(R2 > 0.99). Flow behaviour index (n) of ice cream mixes changed
between 0.298 and 0.432, implying also shear thinning behaviour
(Table 2). Higher n values showed the resemblance of the Newtonian
flow behaviour, in other words, lower shear thinning attitude. Shear
thinning level of the ice cream mix is important for processability and
sensory properties of the end products. Lower shear thinning behaviour
showed the stability of the mix samples against forces applied during
pumping and mixing of the products. The ice cream mix sample with
higher shear thinning behaviour was pumped and mixed easily.
Therefore, energy requirement decreased, and end products prepared
from this type of mix can achieve the desired texture and mouthfeel
[34]. The use of P. cruentum for flow behaviour index (n) can be stated
as more advantageous than the use of other microalgae species. For this
type of microalgae, a significant decrease in n values was detected with
a concentration increase. However, the increase in the use of other
microalgae species (N. oculata and D. vlkianum) led to an increase in n
Different letters in the same column indicate significant differences (p < 0.05).
(P: samples containing Porphyridium cruentum, N: samples containing
Nannochloropsis oculata, D: samples containing Diacronema vlkianum, 1:
0.10 g 100 g−1, 2: 0.20 g 100 g−1, 3: 0.30 g 100 g−1.) Data are the
means ± SD of three replicates.
value. This result suggested that not only the pigment or bioactive
components in the microalgae, but also the macromolecules in their
structure can be effective in regulating the product flow behaviours.
Consistency coefficient content of the ice cream mix samples changed
with the addition of microalgae. Consistency coefficient values of ice
cream mixes enriched with N. oculata were found to be very close to
that of the control sample. While addition of P. cruentum resulted in
increasing of K value, usage of D. vlkianum and N. oculata in the for-
mulation caused decrease in K value. Physicochemical composition of
the micro-algae type, dry matter, fat and protein contents significantly
affected rheological characteristics of the ice cream mixes differently.
The major components in microalgae biomasses, such as proteins,
polysaccharides and fat may affect flow behaviour of ice cream. For
instance, P. cruentum biomass contains various carbohydrates; (i) cell
storage polymers (starch derivatives); (ii) lipopolysaccharides; and (iii)
extracellular polysaccharides (EPS) [35,36]. Sulphated polysaccharides
related with P. cruentum were also identified in previous studies [14].
Sulphated polysaccharides have the potential to act as a gelling agent
and stabilizer in the food composition [37]. Polysaccharides found in P.
cruentum biomass have been determined to form viscous solutions at
low concentrations and gelation properties [38]. D. vlkianum is rich in

4
Y. Durmaz, et al.
Fig. 2. Changes of color values of ice cream samples.
(K: control ice cream, P: samples containing Porphyridium cruentum, N: samples containing Nannochloropsis oculata, D: samples containing Diacronema vlkianum, 1:
0.10 g 100 g−1, 2: 0.20 g 100 g−1, 3: 0.30 g 100 g−1). Data are the means ± SD of three replicates.
lipids and can be used to improve the PUFA content of various foods
[13]. N. oculata is a remarkable type of microalgae as lipid content
[20]. The results obtained in this study are also consistent with the
previous findings for fat content (Table 1). The effect of N. oculata and
D. vlkianum on K value was thought to be a result of the fat levels of
these microalgae. Rheological properties of the ice cream mixes are
important for the quality of end product as well as machinability of the
mixes. Therefore, microalgae biomass addition might affect flow be-
haviour of ice cream and improve quality as a multi-functional in-
gredient due to its biochemical composition.
3.3. Color properties of ice cream samples
In recent years, usage possibilities of microalgae as a natural col-
orant in different food materials including biscuits, mayonnaises, pud-
dings, chewing gum, yogurt, bread, and pasta were investigated by
several researchers [3,10–13,39–43]. Fig. 2 showed the color properties
of ice cream including various species and levels of spray-dried mi-
croalgae. Ice cream samples enriched with P. cruentum were in pinkish
color and the other two algae species were greenish color. Color
properties of ice creams were significantly affected by microalgae ad-
dition. L*, a* and b* values of control ice cream prepared without
microalgae (control sample) were found to be 84.38, −1.02 and 8.09,
respectively. They changed to 80.78, 11.41 and 8.73 by addition of
0.3 g 100 g−1 P. cruentum and to 78.13, −5.62 and 21.68 by the ad-
dition of 0.3 g 100 g−1 N. oculata and to 72.49, −6.17 and 19.94, when
D. vlkianum was used in the formulation at 0.3 g 100 g−1, respectively.
Microalgae type and concentration affected the color characteristics of
the ice cream at different levels. As L* values of ice cream were not
significantly affected by type and concentration, b* values significantly
5
Algal Research 47 (2020) 101811
increased with increasing microalgae concentration in the formulation.
All microalgae types also resulted in increased a* values in a different
manner. As ice cream samples including P. cruentum had positive va-
lues, the other samples had negative values, indicating an increase of
green color. Contribution of microalgae to the color of ice cream sam-
ples was related to pigment composition of microalgae. Chlorophyll
content of N. oculata and D. vlkianum was found to be higher than that
of P. cruentum; therefore, green color was dominant in the samples
prepared with the former species. P. cruentum is a source of water-so-
luble and red colorant phycophiliproteins, including fucocyanin, which
is important for food applications [14]. Phycoerythrin is another
component of this microalgae composition that is important for red
color [35]. Also, according to our results, anthocyanin content of the P.
cruentum was highest, which resulted in observation of red/pink color
in these samples. As shown in also Fig. 2, all the ice cream samples have
attractive color properties.
3.4. Melting behaviour of ice cream samples
Melting behaviour of ice cream samples can affect both shelf-life
stability of the sample and consumer perception such as mouthfeel and
flavor release. Effect of usage of microalgae in formulation on melting
characteristics (Tonset: temperature at which melting starts, Tend: tem-
perature at which melting is completed, ΔH: energy required for com-
plete melting) is presented in Table 3. Tonset, Tend and ΔH values of the
control ice cream were found to be −14.1 °C, 11.0 °C and 132.8 J/ g,
respectively. Microalgae addition did not remarkably influence the
melting behaviour, providing an advantage for using microalgae in ice
cream formulation. Although increasing melting parameters (Tonset, Tend
and ΔH) with the addition of microalgae can provide an advantage in
Y. Durmaz, et al. Algal Research 47 (2020) 101811

Table 3 properties, P. cruentum had a positive effect on the color of ice cream
Melting properties of ice cream samples. due to its pink color. There are insignificant differences among samples
Samples Tonset (°C) Tend (°C) ΔH (J/g) comparing with control sample in terms of general sensory character-
istics (p < 0.05), which might have resulted from usage of microalgae
Control −14.1 ± 0.49abc
11.0 ± 1.03b
132.8 ± 1.06a at very low amount in the formulation since an excessive quantity of
P1 −13.0 ± 1.03ab 11.5 ± 1.30b 136.9 ± 1.70a
dried algae adversely affected general sensory quality especially taste,
P2 −12.9 ± 0.63a 11.3 ± 2.64b 135.6 ± 19.0a
P3 −14.1 ± 0.54abc 11.3 ± 1.48b 140.3 ± 15.4a
odor and overall acceptability. These results were under accordance
N1 −13.4 ± 1.07ab 8.73 ± 2.64a 136.7 ± 13.6a with previous results conducted various microalgae and food [3,11,13].
N2 −15.3 ± 0.13abc 9.52 ± 0.09a 143.8 ± 3.54a They might be due to their unique off-flavor properties of microalgae.
N3 −13.7 ± 0.04abc 9.11 ± 0.76a 132.2 ± 14.6a In hedonic evaluation, it was thought that the panelist perception of off-
D1 −13.7 ± 0.49abc 11.0 ± 0.85b 143.6 ± 2.62a
flavor factors caused not only the smell but also the decrease in the taste
D2 −15.8 ± 0.04c 9.49 ± 1.30a 134.8 ± 6.72a
D3 −15.3 ± 0.32bc 7.58 ± 1.84a 139.8 ± 7.00a and overall acceptability parameters. Increasing microalgae con-
centration in the formulation negatively affected taste of the ice cream
Different letters in the same column indicate significant differences (p < 0.05). samples and N. oculata and D. vlkianum provided a bitter taste after
(P: samples containing Porphyridium cruentum, N: samples containing consumption of the samples. Also, in previous studies, increasing mi-
Nannochloropsis oculata, D: samples containing Diacronema vlkianum, 1: croalgae concentration in pasta [13] and yogurt [3] resulted decrease in
0.10 g 100 g−1, 2: 0.20 g 100 g−1, 3: 0.30 g 100 g−1.) Data are the taste, odor and general appreciation scores of panelists. Enrichment of
means ± SD of three replicates.
ice cream by using dried P. cruentum is more acceptable in terms of taste
parameter. Undesirable taste of N. oculata and D. vlkianum could be
terms of stability or resistance of melting during consumption, it can
improved with different processes and drying conditions. The sensory
negatively affect sensory characteristics. Regarding the decreasing
scores related with ice cream samples indicated that microalgae types
trend with microalgae addition, the stability of the ice cream decreased,
especially P. cruentum can be used to improve the color of the ice cream
which might negatively affect the consumption preference of the ice
samples; however, further studies are required to eliminate dis-
cream. Therefore, using microalgae in ice cream matrix may cause
advantages of them. In particular, the deodorization process to be ap-
problems when considering the results of our study. However, these
plied at low temperature under vacuum before or after drying may
potential problems may be solved by further optimization studies; e.g.
result in positive changes in sensory results. Another approach may be
composition and contents of sugars and bulking agents and over-run
the use of aromatic components to mask off-flavor molecules of mi-
value [44,45].
croalgae added in related foods.

3.5. Bioactive properties of ice cream samples 4. Conclusion

The total phenolic content (TPC) of ice cream samples is shown in
Fig. 3. TPC of the ice cream increased with increasing microalgae
amount in the formulations. Antioxidant characteristics of polyphenols
can protect the human body against damage caused by reactive oxygen
species, which can result in some health problems such as cancer,
diabetes mellitus, neurodegenerative and inflammatory diseases [46].
Therefore, enrichment of ice cream, widely used dairy-based products,
with polyphenols is important for human health since antioxidants
improve the health of people by fighting with oxidative stress.
3.6. Sensory evaluation
In this study sensory properties (color, texture, taste, odor, re-
sistance to melting, mouthfeel, strange taste, overall acceptability) of
ice cream samples including various spray-dried microalgae were in-
vestigated (Fig. 4). While the species of P. cruentum algae showed
pinkish color, the others were showed greenish color. Regarding color
In this study, usage possibility of various microalgae as a natural
colorant in ice cream formulation was investigated and it can be con-
cluded that species and concentration levels should be considered
during usage of microalgae in the food formulations. In addition, when
the results of this study are taken into consideration, the direct use of
microalgae biomasses rich in pigment contents without separating the
related pigments has been found to cause changes in sensory and
technological properties. Significant differences were found in these
changes. Because the biochemical compositions of microalgae have
significant differences on a species basis. The importance of biomass
variety on flow and melting behaviour, which is important on ice cream
quality characteristics, was found remarkable. Therefore, in microalgae
culture, it may be beneficial to reach different biochemical composi-
tions for target foodstuff by creating different stress and growing con-
ditions. In addition, it can be concluded that bioactive compounds from
biomasses other than pigments may have the potential to be used as
natural food additives in food production to control technological
properties. Aquatic food sources are generally disadvantageous for
sensory properties due to some flavor and odor characteristics. In the
present study, similar findings were observed for some species. These
negative effects can be eliminated/reduced by encapsulation and post-
harvest processes (e.g. deodorization). In this study, the potential of
using spray-dried microalgae as a natural colorant was investigated.
Spray drying technique is a common drying technique that is suitable
for multi-purposes usage due to its relatively low cost and high effi-
ciency. However, it may be possible to improve the level and stability of
microalgae colorants with the application of different drying techniques
and/or the development of encapsulation techniques.

Statement of informed consent, human/animal rights

Fig. 3. Total phenolic content of ice cream samples enriched with microalgae at No conflicts, informed consent, human or animal rights applicable.
different concentrations. All sensory research procedures followed were in accordance with the
Error bars show SD. Data are the means of three replicates, 0.1: 0.10 g 100 g−1, ethical standards of the committee responsible for human experi-
0.2: 0.20 g 100 g−1, 0.3: 0.30 g 100 g−1. mentation (institutional and national), and with the Helsinki

6
Y. Durmaz, et al.
Fig. 4. Sensory scores of ice cream incorporated with dried microalgae.
C = color; S = structure (texture); T = taste; O = odor; M = resistance to melting; CS = mouthfeel; ST = strange taste; OA overall acceptability. (K: control ice
cream, P: samples containing Porphyridium cruentum, N: samples containing Nannochloropsis oculata, D: samples containing Diacronema vlkianum, 1: 0.10 g 100 g−1, 2:
0.20 g 100 g−1, 3: 0.30 g 100 g−1). Error bars show SD. Data are the means of three replicates.
Declaration of 1975, as revised in 2013.
CRediT authorship contribution statement
Yaşar Durmaz: Methodology, Formal analysis. Mahmut Kilicli:
Methodology, Formal analysis. Omer Said Toker: Conceptualization,
Writing - original draft. Nevzat Konar: Conceptualization, Writing -
original draft. İbrahim Palabiyik: Conceptualization, Writing - original
draft. Faruk Tamtürk: Methodology, Formal analysis.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank Ege University; Fisheries Faculty, where culture
of algae have been carried out. The authors would like to thank the
technical staff at the Yildiz Technical University; Food Engineering
Department Laboratory for some analyses of ice-cream.
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