LWT - Food Science and Technology xxx (2015) 1e7

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

The fortication of biscuits with bee pollen and its effect on

physicochemical and antioxidant properties in biscuits
Magdalena Krystyjan a, *, Dorota Gumul a, Rafa Ziobro a, Anna Korus b
a
b

Department of Carbohydrates Technology, Faculty of Food Technology, University of Agriculture in Krakow, Poland
Department of Raw Materials and Processing of Fruit and Vegetables, University of Agriculture in Krakow, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 July 2014
Received in revised form
14 March 2015
Accepted 18 March 2015
Available online xxx

The aim of this study was to indicate the potential application of bee pollen as a dietary supplement.
Biscuits are one of the most consumed confectionery products in the world. With this fact in mind, an
attempt was made to nd an optimal recipe for biscuits with the addition of pollen, and at the same time
investigate the physical, chemical and health-promoting properties in obtained products. Although it
was found that the addition of bee pollen did not affect the fat content in biscuits, it had a statistically
signicant effect on sugar, protein, ash, bre, as well as the content of polyphenols and antioxidant
potential. Biscuits that had been improved with bee pollen were characterized by higher penetration
work and a darker surface when compared to the control. However, only up to 5% of pollen was needed
to enable the taste of biscuits to be at the same level as the control.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Biscuits
Bee pollen
Physicochemical properties
Antioxidant activity
Sensory analysis

1. Introduction
Pollen is produced by plants as male cells that are needed for
pollination and to develop fruit. Each plant species has its own
characteristics in respect of the shape, size and colour of pollen.
Bees pick up pollen from owers with the help of a comb on their
bodies that allow it to drag the pollen and form pellets (AlmeidaMuradian, Pamplona, Coimbra, & Barth, 2005; Snodgrass, 1975).
They then bring these pellets to the hive and store them in special
cells, the main use of the pollen being to feed the larvae (Barth &
Luz, 1998; Roman, 2006). In order to obtain pollen, apiarists
install traps in front of the entrance to the hive, so that when
returning home the worker-bees lose their pollen pellets. These are
then collected into a container (Campos et al., 2008; Nelson, 1987).
Natural products used as food supplements are recently gaining
more and more attention, with pollen particularly arousing significant interest. In fact, pollen has been used for centuries in traditional medicine (Kroyer & Hegedus, 2001) and as a food in the
human diet due to its nutritional and therapeutic properties
(Cocan, Marghitas, Dezmirean, & Laslo, 2005; Linskens & Jorde,
1997). Bee pollen is a rich source of nutrients that includes

* Corresponding author. University of Agriculture, Balicka Street 122, 30-149

Krakow, Poland.
E-mail address: m.krystyjan@ur.krakow.pl (M. Krystyjan).

proteins, sugars, lipids, bre, mineral salts, amino acids, phenolic

compounds and vitamins. It has been found that at least eighteen
amino acids are present in the pollen, among which the proline,
glutamic and aspartic acids, lysine and leucine, are dominant,
constituting approximately 55% of total amino acids. Bee pollen is
rich in polyphenol substances, mainly avonoids, which make it
relevant to the human diet (Campos, Markham, & Cunha, 1997;
Human & Nicolson, 2006). It should be noted, however, that the
chemical content of bee pollen varies according to the environmental conditions and the plant species from which it is gathered
(Campos et al., 2008; Szczesna, Rybak-Chielewska, & Chmielewski,
2002). Thanks to its extensive and diversied nutritional signicance of bee pollen, it is considered as food for humans in many
countries and protected by ofcial quality standards and limits:

za
_ pykowe Polish legislation for beePoland (PN-R-78893 Obno
pollen), Switzerland (Bogdanov et al., 2004) and Brazil (Brazil,
2001) (Campos et al., 2008). Nevertheless, pollen cannot be
consumed in substantial amounts within a daily diet, and is usually
regarded as an antiseptic additive. It is also applied as a parapharmaceutical in the treatment of various diseases. Previous
studies have shown that bee pollen may be applied as a highly
concentrated source of energy for athletes (Stanley & Linskens,
1974). Others show that pollen has a favourable inuence on
chronic prostatitis (Estevinho, Rodrigues, Pereira, & Feas, 2012; Han
et al., 2007). However, there is still a constant lack of information as

http://dx.doi.org/10.1016/j.lwt.2015.03.075
0023-6438/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7

regards the practical applications of bee pollen in food processing.

Thus, it seems advisable to undertake research on the enrichment
of confectionery products in bee pollen in order to provide them
with health-promoting substances and phenolic antioxidants
which is both entirely justied and innovative. Given the fact that
biscuits are one of the most popular baked sweet goods in the
world, we decided to fortify them with bee pollen and to develop an
optimal recipe for biscuits with that addition of bee pollen. Such
action could signicantly broaden the supply of pro-health biscuits
as well as widen the possible applications of pollen.

2. Materials and methods

2.1. Materials
Pastry wheat our was purchased from the Polish Cereal Plant
PZZ (Krakow, Poland) with the following parameters: moisture
11.8 g/100 g, total carbohydrate 77.19 g/100 g d.m., fat 1.87 g/
100 g d.m., proteins 9.05 g/100 g d.m. (N
5.7), soluble dietary bre
0.41 g/100 g d.m., insoluble dietary bre 0.53 g/100 g d.m., total
dietary bre 0.94 g/100 d.m. and ash 0.31 g/100 g d.m. The total
polyphenolic content was 1.49 mg catechin/g d.m. and 1.64 mg
ferulic acid/g d.m., antioxidant activity TEAC e 9.04 mmol Tx/kg
d.m. Baking powder, vanilla avoured sugar, sucrose powder, eggs
and margarine were purchased from the local market. Bee pollen
was collected over 2012 from beehives located in the south-eastern

w Zdro

j). This pollen was dried at a
regions of Poland (Rymano
temperature of 35  C to a moisture level of 5 g/100 g. Then it was
ground and sieved to obtain its powdered form. It had the following
parameters as determined by the methods given below: total carbohydrate 60.57 g/100 g d.m., fat 10.07 g/100 g d.m., proteins
21.73 g/100 g d.m. (N
5.7), soluble dietary bre 1.54 g/100 g d.m.,
insoluble dietary bre 9.47 g/100 g d.m.; total dietary bre 11.01 g/
100 g d.m. and ash 2.60 g/100 g d.m.

2.2. Preparation of dough and baking biscuits

Dry ingredients were mixed together and then combined with
others according to the recipe presented in Table 1. The dough was
mixed for 10 min to obtain a homogeneous consistency and then
placed into the fridge at 6  C over a period of 30 min. The dough
was then rolled out and 5 mm thick biscuits with a 60 mm diameter
were formed and baked at 200  C for 12 min. The biscuits with the
bee pollen substitution in amounts of 2.5, 5, 7.5 and 10% e in
relation to the wheat our e were prepared in the same way as the
control sample (biscuits without bee pollen). These were then
stored in glass containers for two months, protected from light, and
at a room temperature of 21  C. The results of all the experiments
are given as the average of replicates. Each replicate (biscuit) was
obtained from separately prepared batches of dough.

2.3. Chemical analyses of biscuits

The chemical compositions of fresh biscuits (as prepared on the
day of baking) were determined according to the methods of the
Association of Ofcial Analytical Chemists International (AOAC.,
2006): the total protein content by the Kjeldahl procedure (using
the Bchi B324 extraction system) with a nitrogen to protein conversion factor of 5.7 (method number 950.36); dietary bre (soluble,
insoluble and total dietary bre) by the enzymaticegravimetric
method (method number 991.43); fat content by the Soxhlet method
[Bchi B811] (method number 935.38) and ash content by carbonization (method number 923.03). The total carbohydrate content
was calculated by subtracting the sum of moisture, protein, fat, and
ash percentages from 100%. The caloric value was calculated according to the Atwater system (FAO, 2002): Caloric
value (4
protein) (9
fat) (carbohydrate  dietary bre)
4.
2.4. The total polyphenol content and antioxidant activity in
biscuits
Extract preparation: 1 g of analysed material was extracted
with 40 mL 0.16 mol/L HCl in 80 mL/100 mL methanol under
ambient conditions, for 2 h in a type WB22 water bath (Memmert,
Schwabach, Germany) that was equipped with a shaking device.
Then the extract was separated in a type MPW-350 centrifuge
(MPW MED. INSTRUMENTS, Warsaw, Poland) at 4500 rpm
(1050
g) for 15 min. The supernatant was collected, and the residue was re-extracted with 40 mL of 70 mL/100 mL acetone for 2 h.
After re-extraction, the sample was centrifuged at the above
mentioned conditions. The supernatant was mixed with ethanol
extract and stored in a refrigerator.
The total polyphenol content was determined according to

s (1999). Antioxidant
Singleton, Orthofer, and Lamuela-Ravento
activity was analysed by a method with free ABTS radical (Re et al.
1999), and given as Trolox Equivalent Antioxidant Capacity (TEAC
e mmol Tx/kg d.m. of sample). The experiment was repeated 3
times.
2.5. Colour of biscuits
The measurement of the upper surface colour was carried out
with the use of Konica MINOLTA CM-3500d equipment (Konica
Minolta Inc., Tokyo, Japan), with reference to illuminant D65 and a
visual angle of 10 . The results were expressed using the CIELab
system. The following parameters were determined: L* (L* 0
black, L* 100 white), a* - share of the green colour (a* < 0) or red
(a* > 0), b*- share of blue (b* < 0) or yellow (b* > 0). The measurement was carried out on the day of baking, as well as after one and
two months of storage. The experiment was repeated 4 times.
2.6. The textural properties of biscuits

Table 1
Components used for the preparation of biscuit dough.
Components (g)

Control Sample

B1

B2

B3

B4

Wheat our
Margarine
Sugar powder
Eggs
Vanilla-avoured sugar
Baking powder
Bee pollen

100.0
45.0
26.7
10.0
5.3
0.7
0.0

97.5
45.0
26.7
10.0
5.3
0.7
2.5

95.0
45.0
26.7
10.0
5.3
0.7
5.0

92.5
45.0
26.7
10.0
5.3
0.7
7.5

90.0
45.0
26.7
10.0
5.3
0.7
10.0

Ingredients of the recipe, the number of which changes are marked in bold font.

Penetration proles of biscuits were determined using the TAXT plus texture analyzer (Stable Micro Systems, Haslemere, UK).
Biscuits were placed on a heavy duty platform table with an insert
and 9 mm xing hole, and the penetration test was performed
using a P/6 cylinder probe 6 mm, moving at 1 mm per second at a
distance of 20 mm, so that a hole was made through the biscuit.
Hardness, as the value of maximum force on the acquired plot,
along with penetration work - as the area below the curve - were
calculated. The analyses were carried out on fresh biscuits, i.e. on
those prepared on the day of baking, as well as after one and two
months of storage. The experiment was repeated ten times.

Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7

2.7. Sensory analysis of biscuits

For sensory analyses, the laboratory was equipped with special
boxes, and fullled all the basic requirements stated by the PN-ISO
8589:1998 standard. The evaluation was performed by a panel of 7
women and 6 men that were between the ages of 25 and 55, and of
appropriate sensory sensitivity (PN-ISO 8586-1:1996). The quality
descriptors and weighting coefcients to a ve-point evaluation
scale were chosen by assessors according to the procedure included
in PN-ISO 6658:1998. Finally, the following quality descriptors and
weighting coefcients were chosen: shape (0.1), colour (0.1), surface (0.15), consistency (0.15), fracture (0.1), aroma (0.15) and taste
(0.25). The evaluation was based on the comparison made of these
quality descriptors with those dened in the standard table
(Table 2). The scores (from 5 to 1) for each quality descriptor were
multiplied by the corresponding weighting coefcients, and the
total score is the sum of the multiplied individual quality descriptors. Taking into account the total score, the overall acceptance
was rated as: <2.9 unacceptable, 3.0e3.50 acceptable, 3.51e4.50
good, and 4.51e5.0 very good (Gambu
s et al. 2009). The analyses
were carried out on fresh biscuits, i.e. those on the day of baking, as
well as after one and two months of storage.
2.8. Statistical analysis
The experimental data were subjected to analysis of variance, at
the condence level of a 0.05, using Statistica v. 8.0 software
(Statsoft, Inc., Tulsa, OK, USA). The tests of Fisher were used to
determine statistically signicant differences.
3. Results and discussion
3.1. Chemical composition of biscuits
Table 3 shows the content of essential nutrients and dietary
bre in biscuits. After analysing the data obtained it was found that
the addition of bee pollen did not affect the fat content in biscuits the amount of which oscillated around 21.5%. There was, however, a
small but statistically signicant increase in protein and ash content in those biscuits that had additional bee pollen at 5% or more.
Such an increase comes from the fact that the bee pollen contains
twice as much more protein, and eight times more ash than the
wheat our that was used for baking.

It is worth underlining that the caloric value of biscuits was

lowered with the increased amount of bee pollen when compared
to the control sample, and those differences were statistically signicant (Table 3). Such a decreasing tendency was connected with
the lower total carbohydrates content in bee pollen when
compared to wheat our. The sugar present in bee pollen is mainly
fructose, glucose and sucrose (Szczesna et al., 2002); this is a very
important component of biscuits because it shapes their avour,
aroma, colour and texture (Manohar & Rhao, 1997). However, when
introduced in larger quantities, it may increase the spread and
reduce the thickness of biscuits (Kissel, Marshal, & Yamazaki, 1973).
Dietary bre is another important component of pro-health
properties. This does not belong to the bioactive components, but
has a positive effect on human health. According to Esposito et al.
(2005), some fractions of the soluble dietary bre may exhibit
hypocholesterolemic, hypoglycemic and antitumour effects. Signicant differences were observed in the case of both soluble and
insoluble dietary bre. Even introducing the smallest amount of
bee pollen (2.5%) resulted in a 1.4-fold increase in the soluble
fraction, and a 1.6-fold increase in the insoluble fraction of dietary
bre when compared to the control. The 10% addition of bee pollen
contributed to more than a two-fold increase in both of these
fractions. In addition, the content of total dietary bre in biscuits
increased from 50 to 212% with increasing amounts of bee pollen.
While studying the effect of soluble bre on the quality of cakes,
Mudgil, Barak, and Khatkar (2012) also found that the supplement
helped to improve the nutritional quality of the nal product.
The dietary bre incorporated into bakery products prolongs
their freshness due to its ability to retain water, which was
conrmed by Sangnark and Noomhorm (2004). The authors
focused on a dietary bre that was prepared from rice straw, and
which was treated and untreated with an alkaline hydrogen
peroxide solution. They found that, when used as a dietary bre in
bread making, such materials can modify the volume of loaves, as
well as their elasticity and the softness of the bread crumb. They
also conrmed that the direction of the aforementioned changes
depend on the type of bre (Cadden, 1987; Sangnark & Noomhorm,
2004).
3.2. Total polyphenol content and antioxidant activity in biscuits
Pollen is a rich source of phenolic compounds, including cinnamic
acid derivatives, avonoids, avones, isoavones, anthocyanins, and

Table 2
Standard table for the 5 point sensory analysis of biscuits.
Quality
factors

Weighting
coefcients

Score scale
5

Shape

0.1

appropriate, uniform

not correct enough,

with partially broken
edges
not very appropriate,
almost uniform

incorrect, numerous
crushing

0.1

correct, aesthetic,
without a trace of
crushing and curvatures
very appropriate, uniform

correct

Colour

Surface

0.15

even and smooth

sufciently even

Consistency

0.15

highly correct, aesthetic,

without a trace of crushing
and curvatures
very appropriate, from light
cream to light brown, very
uniform
very even and smooth,
without bubbles or cracks
solid, very crispy

solid, crispy

crispy enough

uneven, numerous
bubbles or cracks
too soft or very hard

Fracture

0.1

Aroma

0.15

correct, tiny and uniform

porosity
distinctive, agreeable,
intense, without
strange odours

correct,
uniform porosity
distinctive, agreeable,
without strange odours

not very even, there are

bubbles and cracks
not enough crispy, soft
or hard
not correct enough, few
and irregular pores
not very agreeable, not
much desired

Taste

0.25

typical of biscuits,
pleasant, distinct

typical of biscuits,
pleasant

not very distinctive,

oury

with strange aftertaste, rancid, bitter

highly correct, very tiny and

uniform porosity
very distinctive, very
agreeable, intensive,
noticeable,
without strange odours
very typical of biscuits,
pleasant, very distinct

inappropriate, biscuits
too pale or burnt

faulty, dense, very few

pores
undesired, with strange
odours

Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7

Table 3
The content of nutrients, bre, and caloric value of fresh baked biscuits.
Sample

Control
B1
B2
B3
B4

Water
[g/100 g d.m.]
4.59
5.72
5.69
6.39
6.32

0.17c
0.11b
0.11b
0.09a
0.13a

Fat
[g/100 g d.m.]
21.60
21.55
21.25
21.66
21.70

0.13a
0.07a
0.22a
0.52a
0.14a

Total carbohydrate
[g/100 g d.m.]
66.44
65.35
65.18
64.04
63.84

Protein
[g/100 g d.m.]

0.01a
0.04b
0.05b
0.63c
0.31c

6.96
6.96
7.32
7.32
7.56

0.03c
0.01c
0.05b
0.01b
0.02a

Ash
[g/100 g d.m.]
0.41
0.41
0.55
0.59
0.59

0.01c
0.01c
0.01b
0.01a
0.01a

Dietary bre [g/100 g d.m.]

Insoluble
0.61
0.87
0.96
1.02
1.25

0.00e
0.02d
0.03c
0.01b
0.01a

Soluble
0.28
0.47
0.51
0.56
0.64

0.01d
0.03c
0.01bc
0.03b
0.03a

Caloric value
[kcal/100 g]

Total
0.89
1.34
1.47
1.58
1.89

0.01e
0.05d
0.01c
0.01b
0.05a

486
479
477
475
474

1a
1b
2bc
2c
0c

Parameters in columns denoted with the same letters do not differ statistically at the level of condence a 0.05.
Number of replications n 3.

avonols (Almaraz-Abarca, Campos, & Avila-Reyes, 2004; Leja,

_

ska, 2007; Stanley &
Mareczek, Wyzgolik,
Klepacz-Baniak, & Czekon
Linskens, 1974). Due to the presence of phenolic compounds that
reveal bacteriostatic, anti-inammatory, anti-allergic, and anticancer
properties, pollen could be regarded as a pro-health dietary supplement (Manach, Mazur, & Scalbert, 2005).
The content of total polyphenols in pollen was 16.9 mg catechin/
g d.m., and 38.9 mg of gallic acid/g d.m. The values reported by
other authors ranged between 10.5 and 16.8 mg of gallic acid/g d.m.
(Morais, Moreira, Feas, & Estevinho, 2011) and 12.93e98 mg of
chlorogenic acid/g d.m (Leja et al., 2007). These discrepancies are
obviously due to the high variability in geographical and botanical
origin of samples (Almaraz-Abarca et al., 2004; Leja et al., 2007).
It seems that the trial to supplement biscuits with pollen is fully
justied. Baked products have drastically reduced levels of phenolic
compounds (Alvarez-Jubete et al., 2010) because of the depolymerization of polyphenols and decarboxylation of phenolic acids
that occur during thermal treatment. Enrichment of biscuits with
pollen - which is rich in these substances - should have a signicant
inuence on their content in the nal products. The total phenolic
content in biscuits, with 2.5e10% pollen, increased in comparison to
the control in the 51e192% range if they were expressed as catechin,
and 59e240% when they were reported as gallic acid (Table 4). Based
on the results it can be observed that the content of total polyphenols in biscuits - along with the share of pollen - increased in
parallel with the level of their addition (Table 4). It should also be
noted that due to the high content of phenolic compounds, the
antioxidant activity of pollen was also high and equalled
72.27 mmolTx/kg d.m, when measured with ABTS radical according
to the method of Re et al. (1999) e and expressed as TEAC. Such high
antioxidant activity was caused mostly by phenolic compounds,
especially avonoids and cinnamic acid derivatives (Natella,
Nardini, Di Felice, & Saccini, 1999). The antioxidant activity of biscuits with a 2.5e10% addition of pollen increased in the range
86.4e230.4% when compared to the control, which is the consequence of rising amounts of polyphenols in biscuits (Table 4). This is
also conrmed by the high correlation between TPC and antioxidant

activity. The values of correlation coefcients for the total polyphenol content and antioxidant activity equalled R 0.9907 when
TPC was expressed as catechin, and R 0.9901 when it was
expressed as gallic acid. Antioxidant activity was also reported in the

, No
^

, and Kada

kova

(2014) concerning
studies of Solgajova
zkova
cookies, in which 16 and 32% of wheat our was replaced with rape

Za

mky and Lena

rtovce.
bee pollen derived from two localities: Nove
In the study it was observed that cookies with rape bee pollen were
characterized by stronger scavenging of DPPH, in a range between
32.66 and 66.91%, in comparison to the control (6.2%). Based on the

et al. (2014), and the data presented here, it
results of Solgajova
could be observed that larger amounts of pollen have resulted in
higher antioxidant activity of the nal product.
Summarizing the results shown above, it should be suggested
that a 10% addition of pollen is the most effective in terms of the
enrichment of biscuits in phenolic compounds, and thus improvement of their antioxidant activity. It could be observed that even a
2.5% addition of pollen caused an increase in the polyphenol content by approximately 50%, and a rise in antioxidant activity by
86.4% (Table 4). At a 10% addition of pollen the content of polyphenols, as well as antioxidant activity, were 3 times higher in
comparison to the control sample, so in order to provide maximum
nutritional benets, this level of supplementation seems to be
optimal.
Readily available published data on bio-active compounds in
pollen show only the aspect of differences in their levels between
various locations, but there is little or no information as regards
reports about bakery or pastry products which could be prepared

et al. (2014)
with the addition of this ingredient. Recently, Solgajova
analysed selected chemical compounds and antioxidant activity
(but not the contents of antioxidants) of cookies with rape bee
pollen. It seems, however, that the results presented here are more
complete and give a better view of the chemical and physical
changes in pastry products caused by pollen fortication. This
seems necessary to interest the producers in extending their
product portfolio with biscuits that have been fortied with pollen,
which have high potential as a source of polyphenols, and antioxidant activity in the human diet.

Table 4
The total polyphenol content and antioxidant activity in fresh baked biscuits.

3.3. Physical properties of biscuits

Samples

Total polyphenolic
content
(TPC)
(mg catechin/g d.m.)

Control
B1
B2
B3
B4

0.79
1.19
1.54
1.93
2.31

0.10e
0.11d
0.04c
0.14b
0.16a

Total polyphenolic
content
(TPC)
(mg gallic acid/g d.m.)
1.42
2.26
3.11
4.03
4.84

0.17e
0.21d
0.14c
0.09b
0.23a

TEAC
(mmolTx/kg d.m.)

12.86
23.97
29.26
38.12
42.49

0.81e
0.75d
0.32c
0.11b
0.63a

Parameters in columns denoted with the same letters do not differ statistically at the
level of condence a 0.05.
Number of replications n 3.

3.3.1. Colour
Colour is important in the selection and the purchasing of food
products (Calvo, Salvador, & Fiszman, 2001), including biscuits.
Moreover, as colour develops during the later stage of baking, it can
be used to judge the completion of the baking process (Mamat, Abu
Hardan, & Hill, 2010; Wade, 1988). Changes in food colour during
storage also provide information about its freshness, and in turn its
shelf life.
Analysing the colour of biscuits (Table 5), it was observed that
the addition of bee pollen signicantly darkened the surface of
biscuits when compared to the control. The values of the L*

Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7

Table 5
Physical parameters (colour and texture) of biscuits.
Sample

Time

Colour

Texture

L*(D65)
Control

B1

B2

B3

B4

Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths

76.00
75.45
74.66
71.34
70.73
71.27
65.97
67.20
65.43
65.21
66.77
64.83
63.93
64.82
64.96

a*(D65)
a

0.11
2.40a
3.42a
0.22b
1.14b
1.06b
1.71cd
2.29c
3.25cd
0.62cd
0.30c
2.45cd
1.49d
0.97cd
0.84cd

3.90
3.37
3.38
5.46
4.88
4.77
8.69
7.59
7.10
9.05
7.49
7.52
8.99
8.32
8.24

b*(D65)
de

0.15
0.21d
0.10d
0.17d
0.37d
0.21de
1.21a
1.00bc
1.01c
0.59a
0.22bc
0.28bc
0.93a
0.66ab
0.52ab

29.82
27.49
27.34
33.84
31.64
31.17
36.82
34.45
32.97
39.18
36.72
35.49
40.42
37.87
37.72

Hardness [N]
i

0.59
0.89j
1.04j
0.85fg
0.70h
0.64h
0.40cd
0.16ef
1.60g
0.28b
0.25d
1.14e
0.30a
0.30c
0.39cd

33.99
31.41
33.35
26.85
27.12
21.07
27.32
28.34
36.02
25.99
25.52
26.31
26.74
37.51
34.32

bc

2.64
1.55cde
2.15bcd
0.99ef
1.74ef
1.89g
1.26ef
0.96def
4.32a
1.30efg
1.30fg
3.78efg
0.78ef
2.88ab
3.31ab

Penetration work [N$mm]

33.15
36.96
41.35
56.25
45.36
35.00
62.12
34.39
42.29
63.23
42.81
45.43
79.89
49.97
37.52

3.80g
2.37fg
2.76efg
3.80bcd
4.86defg
2.26g
3.40bc
2.08g
7.42def
5.49b
5.33efg
6.70defg
2.30a
5.42cde
3.28efg

Parameters in columns denoted with the same letters do not differ statistically at the level of condence a 0.05.
Number of replications n 4 (for colour) and n 10 (for texture).

parameter remained at a lower level when compared to the control

sample. This is, of course, unsurprising since bee pollen is characterized by a denitely darker colour when compared to wheat our.
There were not, however, any changes observed resulting from the
storage of biscuits, regardless of the amount of the additive.
All samples had higher share of red (a* > 0) and yellow (b* > 0)
colour than the control, and was greater the more bee pollen was
added to the biscuits. During storage the values of these parameters
slightly decreased, both for the control and the biscuits with bee
pollen. For the a* parameter, the differences were generally not
statistically signicant, while within the b* parameter the difference between the fresh samples and those stored for one and two
months proved to be statistically signicant (Table 5).
One of the factors that contribute to the colour of biscuits was
caused by Maillard reactions between reducing sugar and amino
ksel & Go
kmen, 2008; Mundt & Wedzicha, 2007). As a
acids (Ko
result of this non-enzymatic reaction, high-molecular-weight
macromolecule materials known as melanoidins were formed during baking. Thus, the colour of biscuits develops with increasing
ksel & Go
kmen, 2008), and detemperature and baking time (Ko
pends on the amount of sugars and proteins present in the ingredients used for baking. Because bee pollen contained more
proteins than substituted wheat our and the reducing sugars in the
dough came from pollen, the amount of the melanoidins formed in
fortied biscuits was signicantly higher than those in the control.
This resulted in a lower L* parameter. Gallagher, Kenny, and Arendt
(2005) and OBrien, Chapmen, Neville, Keogh, and Arendt (2003)
conrmed that the protein content has a negative correlation with
an L* value, indicating that the increase of protein content in biscuit
reduces the L* value. The darker colour of biscuits enriched with bee
pollen was also affected by the pollen itself, which has a much darker
colour than wheat our. The colour of pollen pellets ranged from pale
yellow, through to orange, and up to yellow-brown, due to the
presence of avonoids and carotenoids. The colour of bee pollen
depends on many factors, including display to sunlight, the humidity
of the pollen, dust and pollution from soot, or dark fungal spores.
Furthermore, the colour may depend on the type and amount of
liquid, sugar or nectar mixed with the pollen by the bees in the
formation of pellets (Stanley & Linskens, 1974).
3.3.2. Texture
Biscuits, baked with the addition of bee pollen, were signicantly softer than the control sample, but the amount of addition
seemed not to be important on the day of baking as all the samples

were characterized by comparable hardness, and the differences

observed were not statistically signicant (Table 5). The changes as
regards storage were generally small, and only in the case of
products obtained with a 7.5 and 10% of pollen, a slight increase in
hardness over the initial values could be observed.
In the case of the second analysed texture parameter e penetration work e the inuence as regards the change in formulation
was much more pronounced (Table 5). The increase in its value
observed at the lowest applied level of pollen (2.5%) was 70%, while
with the addition of 10% it reached 141%, with intermediate values
for other concentrations of pollen. The changes in penetration work
over the storage period highly depended on the presence of pollen.
For the control sample, a growing trend could be observed, and
after the second month of storage, the increase in penetration work
was 25%. Reverse changes could be seen in the case of pollen
supplemented biscuits, which revealed higher values of penetration work after storage. Final values, determined after 2 months,
ranged from 35 (B1 formulation) to 45.43 (B3) and were comparable with the control sample (41.35).
In general it could be observed that the presence of bee pollen in
biscuit formulation does not exert any negative impact on the
texture of biscuits. Both a slight decrease in the maximum breaking
force (hardness), and an increase in penetration work, could be
regarded as benecial. However, the advantage of bee pollen supplemented biscuits could only be observed with fresh products, as
during storage their texture becomes more and more similar to the
control, probably due to water sorption/redistribution. As it was
earlier observed by Baltsavias, Jurgens, and van Vliet (1999), the
mechanical properties of biscuits may change during storage due to
glass transition, which in turn depends on the moisture content
and sorption properties of the product.
3.4. Sensory parameters of biscuits
The sensory assessment is shown in Table 6. According to the
data, those biscuits supplemented with bee pollen obtained a
higher total score. Supplementation at a level of 5% kept the sensory parameters close to the control sample, both in fresh biscuits
as well as those that had been stored. It should be also noted that at
all concentrations of pollen, the overall assessment allowed the
classication of the product to be acceptable (total
score 3.0e3.50) in the case of the maximum additive (sample B4),
and as good (total score 3.51e4.50) in other concentrations and
the control sample. The high scores were obtained from the control

Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7

Table 6
Total score of sensory analysis of biscuits.
Sample

Time

Shape
(0.1)

Control

Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths
Before storage
After 1 mth
After 2 mths

0.46
0.42
0.40
0.46
0.42
0.43
0.49
0.42
0.42
0.47
0.44
0.43
0.49
0.42
0.40

B1

B2

B3

B4

0.04ab
0.08ab
0.08b
0.09ab
0.09ab
0.09ab
0.03a
0.09ab
0.08ab
0.04a
0.05ab
0.08ab
0.03a
0.09ab
0.08b

Colour
(0.1)
0.40
0.37
0.36
0.41
0.40
0.43
0.47
0.41
0.42
0.47
0.41
0.39
0.41
0.37
0.35

0.07abc
0.11bc
0.12bc
0.06abc
0.07abc
0.07ab
0.04a
0.09abc
0.06abc
0.04a
0.10abc
0.09bc
0.06abc
0.09bc
0.13c

Surface
(0.15)
0.64
0.63
0.62
0.62
0.65
0.60
0.75
0.69
0.63
0.75
0.65
0.65
0.73
0.63
0.57

0.09bc
0.09bc
0.11bc
0.05bc
0.07bc
0.14c
0.00a
0.08ab
0.09bc
0.00a
0.07bc
0.07bc
0.05a
0.06bc
0.15c

Consistency
(0.15)
0.69
0.62
0.60
0.64
0.59
0.62
0.60
0.60
0.56
0.47
0.51
0.51
0.54
0.48
0.51

0.07a
0.15abc
0.10abcd
0.09ab
0.11bcd
0.11abc
0.12abcd
0.10abcd
0.12bcde
0.05e
0.08de
0.16de
0.10cde
0.09e
0.13de

Fracture
(0.1)
0.44
0.41
0.43
0.41
0.41
0.39
0.46
0.38
0.39
0.34
0.34
0.35
0.37
0.33
0.33

0.04ab
0.10abc
0.08ab
0.06abc
0.09abc
0.07abcd
0.04a
0.10bcd
0.06abcd
0.09cd
0.07cd
0.10cd
0.08bcd
0.12d
0.13d

Aroma
(0.15)
0.44
0.60
0.57
0.47
0.65
0.62
0.47
0.63
0.57
0.46
0.54
0.53
0.39
0.50
0.47

0.04ef
0.14abc
0.14abcd
0.04def
0.10a
0.15abc
0.04def
0.12ab
0.15abcd
0.04ef
0.13bcde
0.15cde
0.07e
0.12de
0.18def

Taste
(0.25)
1.04
1.03
1.00
1.07
1.00
0.83
1.11
0.98
0.83
0.64
0.73
0.75
0.39
0.63
0.60

0.24a
0.22a
0.24ab
0.23a
0.12ab
0.24bc
0.20a
0.22ab
0.26bc
0.20cd
0.14cd
0.24cd
0.16e
0.27d
0.27d

Total score
4.11
4.07
3,98
4.09
4.11
3.91
4.34
4.11
3.81
3.61
3.61
3.60
3.31
3.35
3.23

0.13ab
0.68ab
0.65abc
0.33ab
0.49ab
0.57bc
0.33a
0.57ab
0.59bc
0.24cd
0.47cd
0.64cd
0.19d
0.55d
0.54d

Parameters in columns denoted with the same letters do not differ statistically at the level of condence a 0.05.
Number of replications n 13.

sample, and comparable results were received from the B1 and B2

recipe, where biscuits were supplemented with bee pollen in
amounts of 2.5 and 5% in relation to the wheat our. Biscuits B3 and
B4 gained lower scores where substitution was at a level of 7.5 and
10% respectively (Table 6).
When comparing particular sensory characteristics of biscuits
directly after their baking and after storage, no signicant differences were observed between the control and the sample with the
2.5 and 5% additions of bee pollen (Table 6). Even after storage,
there were either no differences or they were statistically insignicant. On this basis it can be concluded that the applied fortication of biscuits with bee pollen was possible and gave the desired
results, at a level of 5%. Slight differences were noticed between the
control and those biscuits with a 7.5 and 10% supplementation of
pollen. In particular, such characteristics as taste, consistency and
fracture deteriorated. One of the main factors as regards the deterioration of the taste of biscuits was bee pollen; when used in
signicant quantities, the biscuits were leaving a delicate bitter
taste on the tongue. In addition, the colour of the product depends
on the chemical composition of bee pollen, which in turn depends
on the plant species and environmental conditions (Szczesna et al.,
2002), as well as other factors that were discussed previously. Thus,
the avonoids and carotenoids as well as the reducing sugars
introduced to the product with bee pollen affected its colour.
Curiously, the darkening of colour caused by the addition of bee
pollen was not observed by the assessors.

4. Conclusions
The possibility of fortifying biscuits with bee pollen appears to
be justied. This is not only due to the enrichment of confectionery
products in the antioxidant compounds having health promoting
properties, but also due to the possibility of having introduced bee
pollen itself - which is rich in these bioactive substances - into the
human diet. Based on the data obtained, it was concluded that the
addition of bee pollen did not affect the fat content in biscuits.
However, it did have a statistically signicant effect on the increase
in content of sugar, protein, ash, bre and polyphenols, as well as
the antioxidant potential of the nal product. Biscuits improved
with bee pollen were characterized by higher penetration work and
having a darker surface when compared to the control. Applying
bee pollen to fortify the biscuits was possible, and even at the stage
when the highest amount was applied, this addition enabled the
desired results. However, only 5% of pollen was needed to enable
the taste of biscuits to be at the same level as the control.

Acknowledgements
This project was supported by a special grant, BM-4770/KTW/
2014.

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Please cite this article in press as: Krystyjan, M., et al., The fortication of biscuits with bee pollen and its effect on physicochemical and
antioxidant properties in biscuits, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.075