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

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LWT - Food Science and Technology

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The fortification of biscuits with bee pollen and its effect onphysicochemical and antioxidant properties in biscuits

Magdalena Krystyjan a, *, Dorota Gumul a, Rafał Ziobro a, Anna Korus b

a Department of Carbohydrates Technology, Faculty of Food Technology, University of Agriculture in Krakow, Polandb 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

Article history:Received 3 July 2014Received in revised form14 March 2015Accepted 18 March 2015Available online xxx

Keywords:BiscuitsBee pollenPhysicochemical propertiesAntioxidant activitySensory analysis

* Corresponding author. University of AgricultureKrakow, Poland.

E-mail address: m.krystyjan@ur.krakow.pl (M. Kry

http://dx.doi.org/10.1016/j.lwt.2015.03.0750023-6438/© 2015 Elsevier Ltd. All rights reserved.

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a b s t r a c t

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, anattempt was made to find an optimal recipe for biscuits with the addition of pollen, and at the same timeinvestigate the physical, chemical and health-promoting properties in obtained products. Although itwas found that the addition of bee pollen did not affect the fat content in biscuits, it had a statisticallysignificant effect on sugar, protein, ash, fibre, as well as the content of polyphenols and antioxidantpotential. Biscuits that had been improved with bee pollen were characterized by higher penetrationwork and a darker surface when compared to the control. However, only up to 5% of pollen was neededto enable the taste of biscuits to be at the same level as the control.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Pollen is produced by plants as male cells that are needed forpollination and to develop fruit. Each plant species has its owncharacteristics in respect of the shape, size and colour of pollen.Bees pick up pollen from flowers with the help of a comb on theirbodies that allow it to drag the pollen and form pellets (Almeida-Muradian, Pamplona, Coimbra, & Barth, 2005; Snodgrass, 1975).They then bring these pellets to the hive and store them in specialcells, the main use of the pollen being to feed the larvae (Barth &Luz, 1998; Roman, 2006). In order to obtain pollen, apiaristsinstall traps in front of the entrance to the hive, so that whenreturning home the worker-bees lose their pollen pellets. These arethen collected into a container (Campos et al., 2008; Nelson, 1987).

Natural products used as food supplements are recently gainingmore and more attention, with pollen particularly arousing signif-icant interest. In fact, pollen has been used for centuries in tradi-tional medicine (Kroyer & Hegedus, 2001) and as a food in thehuman 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

, Balicka Street 122, 30-149

styjan).

n, M., et al., The fortificationd Science and Technology (20

proteins, sugars, lipids, fibre, mineral salts, amino acids, phenoliccompounds and vitamins. It has been found that at least eighteenamino 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 isrich in polyphenol substances, mainly flavonoids, which make itrelevant to the human diet (Campos, Markham, & Cunha, 1997;Human & Nicolson, 2006). It should be noted, however, that thechemical content of bee pollen varies according to the environ-mental conditions and the plant species from which it is gathered(Campos et al., 2008; Szczesna, Rybak-Chielewska, & Chmielewski,2002). Thanks to its extensive and diversified nutritional signifi-cance of bee pollen, it is considered as food for humans in manycountries and protected by official quality standards and limits:Poland (PN-R-78893 “Obn�o _za pyłkowe” Polish legislation for bee-pollen), Switzerland (Bogdanov et al., 2004) and Brazil (Brazil,2001) (Campos et al., 2008). Nevertheless, pollen cannot beconsumed in substantial amounts within a daily diet, and is usuallyregarded as an antiseptic additive. It is also applied as a para-pharmaceutical in the treatment of various diseases. Previousstudies have shown that bee pollen may be applied as a highlyconcentrated source of energy for athletes (Stanley & Linskens,1974). Others show that pollen has a favourable influence onchronic prostatitis (Estevinho, Rodrigues, Pereira,& Feas, 2012; Hanet al., 2007). However, there is still a constant lack of information as

of biscuits with bee pollen and its effect on physicochemical and15), http://dx.doi.org/10.1016/j.lwt.2015.03.075

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regards the practical applications of bee pollen in food processing.Thus, it seems advisable to undertake research on the enrichmentof confectionery products in bee pollen in order to provide themwith health-promoting substances and phenolic antioxidantswhich is both entirely justified and innovative. Given the fact thatbiscuits are one of the most popular baked sweet goods in theworld, we decided to fortify themwith bee pollen and to develop anoptimal recipe for biscuits with that addition of bee pollen. Suchaction could significantly broaden the supply of pro-health biscuitsas well as widen the possible applications of pollen.

2. Materials and methods

2.1. Materials

Pastry wheat flour was purchased from the Polish Cereal PlantPZZ (Krakow, Poland) with the following parameters: moisture11.8 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 fibre0.41 g/100 g d.m., insoluble dietary fibre 0.53 g/100 g d.m., totaldietary fibre 0.94 g/100 d.m. and ash 0.31 g/100 g d.m. The totalpolyphenolic content was 1.49 mg catechin/g d.m. and 1.64 mgferulic acid/g d.m., antioxidant activity TEAC e 9.04 mmol Tx/kgd.m. Baking powder, vanilla flavoured sugar, sucrose powder, eggsand margarine were purchased from the local market. Bee pollenwas collected over 2012 from beehives located in the south-easternregions of Poland (Ryman�ow Zdr�oj). This pollen was dried at atemperature of 35 �C to a moisture level of 5 g/100 g. Then it wasground and sieved to obtain its powdered form. It had the followingparameters as determined by the methods given below: total car-bohydrate 60.57 g/100 g d.m., fat 10.07 g/100 g d.m., proteins21.73 g/100 g d.m. (N � 5.7), soluble dietary fibre 1.54 g/100 g d.m.,insoluble dietary fibre 9.47 g/100 g d.m.; total dietary fibre 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 withothers according to the recipe presented in Table 1. The dough wasmixed for 10 min to obtain a homogeneous consistency and thenplaced into the fridge at þ6 �C over a period of 30 min. The doughwas then rolled out and 5mm thick biscuits with a 60mm diameterwere formed and baked at 200 �C for 12 min. The biscuits with thebee pollen substitution in amounts of 2.5, 5, 7.5 and 10% e inrelation to the wheat flour ewere prepared in the same way as thecontrol sample (biscuits without bee pollen). These were thenstored in glass containers for twomonths, protected from light, andat a room temperature of þ21 �C. The results of all the experimentsare given as the average of replicates. Each replicate (biscuit) wasobtained from separately prepared batches of dough.

Table 1Components used for the preparation of biscuit dough.

Components (g) Control Sample B1 B2 B3 B4

Wheat flour 100.0 97.5 95.0 92.5 90.0Margarine 45.0 45.0 45.0 45.0 45.0Sugar powder 26.7 26.7 26.7 26.7 26.7Eggs 10.0 10.0 10.0 10.0 10.0Vanilla-flavoured sugar 5.3 5.3 5.3 5.3 5.3Baking powder 0.7 0.7 0.7 0.7 0.7Bee pollen 0.0 2.5 5.0 7.5 10.0

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

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2.3. Chemical analyses of biscuits

The chemical compositions of fresh biscuits (as prepared on theday of baking) were determined according to the methods of theAssociation of Official Analytical Chemists International (AOAC.,2006): the total protein content by the Kjeldahl procedure (usingthe Büchi B324 extraction system) with a nitrogen to protein con-version factor of 5.7 (method number 950.36); dietary fibre (soluble,insoluble and total dietary fibre) by the enzymaticegravimetricmethod (method number 991.43); fat content by the Soxhlet method[Büchi B811] (method number 935.38) and ash content by carbon-ization (method number 923.03). The total carbohydrate contentwas calculated by subtracting the sum of moisture, protein, fat, andash percentages from 100%. The caloric value was calculated ac-cording to the Atwater system (FAO, 2002): Caloricvalue¼ (4� protein)þ (9� fat)þ (carbohydrate� dietary fibre)� 4.

2.4. The total polyphenol content and antioxidant activity inbiscuits

Extract preparation: 1 g of analysed material was extractedwith 40 mL 0.16 mol/L HCl in 80 mL/100 mL methanol underambient 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 res-idue was re-extracted with 40 mL of 70 mL/100 mL acetone for 2 h.After re-extraction, the sample was centrifuged at the abovementioned conditions. The supernatant was mixed with ethanolextract and stored in a refrigerator.

The total polyphenol content was determined according toSingleton, Orthofer, and Lamuela-Ravent�os (1999). Antioxidantactivity was analysed by a method with free ABTS radical (Re et al.1999), and given as Trolox Equivalent Antioxidant Capacity (TEACe mmol Tx/kg d.m. of sample). The experiment was repeated 3times.

2.5. Colour of biscuits

The measurement of the upper surface colour was carried outwith the use of Konica MINOLTA CM-3500d equipment (KonicaMinolta Inc., Tokyo, Japan), with reference to illuminant D65 and avisual angle of 10�. The results were expressed using the CIELabsystem. The following parameters were determined: L* (L* ¼ 0black, L* ¼ 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 measure-ment was carried out on the day of baking, as well as after one andtwo months of storage. The experiment was repeated 4 times.

2.6. The textural properties of biscuits

Penetration profiles of biscuits were determined using the TA-XT plus texture analyzer (Stable Micro Systems, Haslemere, UK).Biscuits were placed on a heavy duty platform table with an insertand 9 mm fixing hole, and the penetration test was performedusing a P/6 cylinder probe 6 mm, moving at 1 mm per second at adistance 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 - werecalculated. The analyses were carried out on fresh biscuits, i.e. onthose prepared on the day of baking, as well as after one and twomonths of storage. The experiment was repeated ten times.

of biscuits with bee pollen and its effect on physicochemical and15), http://dx.doi.org/10.1016/j.lwt.2015.03.075

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7 3

2.7. Sensory analysis of biscuits

For sensory analyses, the laboratory was equipped with specialboxes, and fulfilled all the basic requirements stated by the PN-ISO8589:1998 standard. The evaluation was performed by a panel of 7women and 6 men that were between the ages of 25 and 55, and ofappropriate sensory sensitivity (PN-ISO 8586-1:1996). The qualitydescriptors and weighting coefficients to a five-point evaluationscale were chosen by assessors according to the procedure includedin PN-ISO 6658:1998. Finally, the following quality descriptors andweighting coefficients were chosen: shape (0.1), colour (0.1), sur-face (0.15), consistency (0.15), fracture (0.1), aroma (0.15) and taste(0.25). The evaluation was based on the comparison made of thesequality descriptors with those defined in the standard table(Table 2). The scores (from 5 to 1) for each quality descriptor weremultiplied by the corresponding weighting coefficients, and thetotal score is the sum of the multiplied individual quality de-scriptors. Taking into account the total score, the overall acceptancewas rated as: <2.9 unacceptable, 3.0e3.50 acceptable, 3.51e4.50good, and 4.51e5.0 very good (Gambu�s et al. 2009). The analyseswere carried out on fresh biscuits, i.e. those on the day of baking, aswell as after one and two months of storage.

2.8. Statistical analysis

The experimental data were subjected to analysis of variance, atthe confidence level of a ¼ 0.05, using Statistica v. 8.0 software(Statsoft, Inc., Tulsa, OK, USA). The tests of Fisher were used todetermine statistically significant differences.

3. Results and discussion

3.1. Chemical composition of biscuits

Table 3 shows the content of essential nutrients and dietaryfibre in biscuits. After analysing the data obtained it was found thatthe addition of bee pollen did not affect the fat content in biscuits -the amount of which oscillated around 21.5%. Therewas, however, asmall but statistically significant increase in protein and ash con-tent in those biscuits that had additional bee pollen at 5% or more.Such an increase comes from the fact that the bee pollen containstwice as much more protein, and eight times more ash than thewheat flour that was used for baking.

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

Qualityfactors

Weightingcoefficients

Score scale

5 4

Shape 0.1 highly correct, aesthetic,without a trace of crushingand curvatures

correct, aesthetic,without a trace ofcrushing and curvatures

Colour 0.1 very appropriate, from lightcream to light brown, veryuniform

very appropriate, uniform

Surface 0.15 very even and smooth,without bubbles or cracks

even and smooth

Consistency 0.15 solid, very crispy solid, crispy

Fracture 0.1 highly correct, very tiny anduniform porosity

correct, tiny and uniformporosity

Aroma 0.15 very distinctive, veryagreeable, intensive,noticeable,without strange odours

distinctive, agreeable,intense, withoutstrange odours

Taste 0.25 very typical of biscuits,pleasant, very distinct

typical of biscuits,pleasant, distinct

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It is worth underlining that the calorific value of biscuits waslowered with the increased amount of bee pollen when comparedto the control sample, and those differences were statistically sig-nificant (Table 3). Such a decreasing tendency was connected withthe lower total carbohydrates content in bee pollen whencompared to wheat flour. The sugar present in bee pollen is mainlyfructose, glucose and sucrose (Szczesna et al., 2002); this is a veryimportant component of biscuits because it shapes their flavour,aroma, colour and texture (Manohar& Rhao,1997). However, whenintroduced in larger quantities, it may increase the spread andreduce the thickness of biscuits (Kissel, Marshal,& Yamazaki, 1973).

Dietary fibre is another important component of pro-healthproperties. This does not belong to the bioactive components, buthas a positive effect on human health. According to Esposito et al.(2005), some fractions of the soluble dietary fibre may exhibithypocholesterolemic, hypoglycemic and antitumour effects. Sig-nificant differences were observed in the case of both soluble andinsoluble dietary fibre. Even introducing the smallest amount ofbee pollen (2.5%) resulted in a 1.4-fold increase in the solublefraction, and a 1.6-fold increase in the insoluble fraction of dietaryfibre when compared to the control. The 10% addition of bee pollencontributed to more than a two-fold increase in both of thesefractions. In addition, the content of total dietary fibre in biscuitsincreased from 50 to 212% with increasing amounts of bee pollen.While studying the effect of soluble fibre on the quality of cakes,Mudgil, Barak, and Khatkar (2012) also found that the supplementhelped to improve the nutritional quality of the final product.

The dietary fibre incorporated into bakery products prolongstheir freshness due to its ability to retain water, which wasconfirmed by Sangnark and Noomhorm (2004). The authorsfocused on a dietary fibre that was prepared from rice straw, andwhich was treated and untreated with an alkaline hydrogenperoxide solution. They found that, when used as a dietary fibre inbread making, such materials can modify the volume of loaves, aswell as their elasticity and the softness of the bread crumb. Theyalso confirmed that the direction of the aforementioned changesdepend on the type of fibre (Cadden, 1987; Sangnark& Noomhorm,2004).

3.2. Total polyphenol content and antioxidant activity in biscuits

Pollen is a rich source of phenolic compounds, including cinnamicacid derivatives, flavonoids, flavones, isoflavones, anthocyanins, and

3 2 1

correct not correct enough,with partially brokenedges

incorrect, numerouscrushing

appropriate, uniform not very appropriate,almost uniform

inappropriate, biscuitstoo pale or burnt

sufficiently even not very even, there arebubbles and cracks

uneven, numerousbubbles or cracks

crispy enough not enough crispy, softor hard

too soft or very hard

correct,uniform porosity

not correct enough, fewand irregular pores

faulty, dense, very fewpores

distinctive, agreeable,without strange odours

not very agreeable, notmuch desired

undesired, with strangeodours

typical of biscuits,pleasant

not very distinctive,floury

with strange after-taste, rancid, bitter

of biscuits with bee pollen and its effect on physicochemical and15), http://dx.doi.org/10.1016/j.lwt.2015.03.075

Table 3The content of nutrients, fibre, and calorific value of fresh baked biscuits.

Sample Water[g/100 g d.m.]

Fat[g/100 g d.m.]

Total carbohydrate[g/100 g d.m.]

Protein[g/100 g d.m.]

Ash[g/100 g d.m.]

Dietary fibre [g/100 g d.m.] Calorific value[kcal/100 g]

Insoluble Soluble Total

Control 4.59 ± 0.17c 21.60 ± 0.13a 66.44 ± 0.01a 6.96 ± 0.03c 0.41 ± 0.01c 0.61 ± 0.00e 0.28 ± 0.01d 0.89 ± 0.01e 486 ± 1a

B1 5.72 ± 0.11b 21.55 ± 0.07a 65.35 ± 0.04b 6.96 ± 0.01c 0.41 ± 0.01c 0.87 ± 0.02d 0.47 ± 0.03c 1.34 ± 0.05d 479 ± 1b

B2 5.69 ± 0.11b 21.25 ± 0.22a 65.18 ± 0.05b 7.32 ± 0.05b 0.55 ± 0.01b 0.96 ± 0.03c 0.51 ± 0.01bc 1.47 ± 0.01c 477 ± 2bc

B3 6.39 ± 0.09a 21.66 ± 0.52a 64.04 ± 0.63c 7.32 ± 0.01b 0.59 ± 0.01a 1.02 ± 0.01b 0.56 ± 0.03b 1.58 ± 0.01b 475 ± 2c

B4 6.32 ± 0.13a 21.70 ± 0.14a 63.84 ± 0.31c 7.56 ± 0.02a 0.59 ± 0.01a 1.25 ± 0.01a 0.64 ± 0.03a 1.89 ± 0.05a 474 ± 0c

Parameters in columns denoted with the same letters do not differ statistically at the level of confidence a ¼ 0.05.Number of replications n ¼ 3.

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e74

flavonols (Almaraz-Abarca, Campos, & Avila-Reyes, 2004; Leja,Mareczek, Wy _zgolik, Klepacz-Baniak, & Czeko�nska, 2007; Stanley &Linskens, 1974). Due to the presence of phenolic compounds thatreveal bacteriostatic, anti-inflammatory, anti-allergic, and anticancerproperties, pollen could be regarded as a pro-health dietary sup-plement (Manach, Mazur, & Scalbert, 2005).

The content of total polyphenols in pollenwas 16.9 mg catechin/g d.m., and 38.9 mg of gallic acid/g d.m. The values reported byother authors ranged between 10.5 and 16.8 mg of gallic acid/g d.m.(Morais, Moreira, Feas, & Estevinho, 2011) and 12.93e98 mg ofchlorogenic acid/g d.m (Leja et al., 2007). These discrepancies areobviously due to the high variability in geographical and botanicalorigin of samples (Almaraz-Abarca et al., 2004; Leja et al., 2007).

It seems that the trial to supplement biscuits with pollen is fullyjustified. Baked products have drastically reduced levels of phenoliccompounds (Alvarez-Jubete et al., 2010) because of the depoly-merization of polyphenols and decarboxylation of phenolic acidsthat occur during thermal treatment. Enrichment of biscuits withpollen - which is rich in these substances - should have a significantinfluence on their content in the final products. The total phenoliccontent in biscuits, with 2.5e10% pollen, increased in comparison tothe control in the 51e192% range if theywere expressed as catechin,and59e240%when theywere reported as gallic acid (Table 4). Basedon the results it can be observed that the content of total poly-phenols in biscuits - along with the share of pollen - increased inparallel with the level of their addition (Table 4). It should also benoted that due to the high content of phenolic compounds, theantioxidant activity of pollen was also high and equalled72.27mmolTx/kg d.m, whenmeasuredwith ABTS radical accordingto themethod of Re et al. (1999)e and expressed as TEAC. Such highantioxidant activity was caused mostly by phenolic compounds,especially flavonoids and cinnamic acid derivatives (Natella,Nardini, Di Felice, & Saccini, 1999). The antioxidant activity of bis-cuits with a 2.5e10% addition of pollen increased in the range86.4e230.4% when compared to the control, which is the conse-quence of rising amounts of polyphenols in biscuits (Table 4). This isalso confirmed by the high correlation betweenTPC and antioxidant

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

Samples Total polyphenoliccontent(TPC)(mg catechin/g d.m.)

Total polyphenoliccontent(TPC)(mg gallic acid/g d.m.)

TEAC(mmolTx/kg d.m.)

Control 0.79 ± 0.10e 1.42 ± 0.17e 12.86 ± 0.81e

B1 1.19 ± 0.11d 2.26 ± 0.21d 23.97 ± 0.75d

B2 1.54 ± 0.04c 3.11 ± 0.14c 29.26 ± 0.32c

B3 1.93 ± 0.14b 4.03 ± 0.09b 38.12 ± 0.11b

B4 2.31 ± 0.16a 4.84 ± 0.23a 42.49 ± 0.63a

Parameters in columns denotedwith the same letters do not differ statistically at thelevel of confidence a ¼ 0.05.Number of replications n ¼ 3.

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activity. The values of correlation coefficients for the total poly-phenol content and antioxidant activity equalled R ¼ 0.9907 whenTPC was expressed as catechin, and R ¼ 0.9901 when it wasexpressed as gallic acid. Antioxidant activitywas also reported in thestudies of Solgajov�a, No�zkov�a, and Kad�akov�a (2014) concerningcookies, in which 16 and 32% of wheat flour was replaced with rapebee pollen derived from two localities: Nov�e Z�amky and Len�artovce.In the study it was observed that cookies with rape bee pollenwerecharacterized by stronger scavenging of DPPH, in a range between32.66 and 66.91%, in comparison to the control (6.2%). Based on theresults of Solgajov�a et al. (2014), and the data presented here, itcould be observed that larger amounts of pollen have resulted inhigher antioxidant activity of the final product.

Summarizing the results shown above, it should be suggestedthat a 10% addition of pollen is the most effective in terms of theenrichment of biscuits in phenolic compounds, and thus improve-ment of their antioxidant activity. It could be observed that even a2.5% addition of pollen caused an increase in the polyphenol con-tent by approximately 50%, and a rise in antioxidant activity by86.4% (Table 4). At a 10% addition of pollen the content of poly-phenols, as well as antioxidant activity, were 3 times higher incomparison to the control sample, so in order to provide maximumnutritional benefits, this level of supplementation seems to beoptimal.

Readily available published data on bio-active compounds inpollen show only the aspect of differences in their levels betweenvarious locations, but there is little or no information as regardsreports about bakery or pastry products which could be preparedwith the addition of this ingredient. Recently, Solgajov�a et al. (2014)analysed selected chemical compounds and antioxidant activity(but not the contents of antioxidants) of cookies with rape beepollen. It seems, however, that the results presented here are morecomplete and give a better view of the chemical and physicalchanges in pastry products caused by pollen fortification. Thisseems necessary to interest the producers in extending theirproduct portfolio with biscuits that have been fortified with pollen,which have high potential as a source of polyphenols, and antiox-idant activity in the human diet.

3.3. Physical properties of biscuits

3.3.1. ColourColour 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 canbe used to judge the completion of the baking process (Mamat, AbuHardan, & Hill, 2010; Wade, 1988). Changes in food colour duringstorage also provide information about its freshness, and in turn itsshelf life.

Analysing the colour of biscuits (Table 5), it was observed thatthe addition of bee pollen significantly darkened the surface ofbiscuits when compared to the control. The values of the L*

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

Sample Time Colour Texture

L*(D65) a*(D65) b*(D65) Hardness [N] Penetration work [N$mm]

Control Before storage 76.00 ± 0.11a 3.90 ± 0.15de 29.82 ± 0.59i 33.99 ± 2.64bc 33.15 ± 3.80g

After 1 mth 75.45 ± 2.40a 3.37 ± 0.21d 27.49 ± 0.89j 31.41 ± 1.55cde 36.96 ± 2.37fg

After 2 mths 74.66 ± 3.42a 3.38 ± 0.10d 27.34 ± 1.04j 33.35 ± 2.15bcd 41.35 ± 2.76efg

B1 Before storage 71.34 ± 0.22b 5.46 ± 0.17d 33.84 ± 0.85fg 26.85 ± 0.99ef 56.25 ± 3.80bcd

After 1 mth 70.73 ± 1.14b 4.88 ± 0.37d 31.64 ± 0.70h 27.12 ± 1.74ef 45.36 ± 4.86defg

After 2 mths 71.27 ± 1.06b 4.77 ± 0.21de 31.17 ± 0.64h 21.07 ± 1.89g 35.00 ± 2.26g

B2 Before storage 65.97 ± 1.71cd 8.69 ± 1.21a 36.82 ± 0.40cd 27.32 ± 1.26ef 62.12 ± 3.40bc

After 1 mth 67.20 ± 2.29c 7.59 ± 1.00bc 34.45 ± 0.16ef 28.34 ± 0.96def 34.39 ± 2.08g

After 2 mths 65.43 ± 3.25cd 7.10 ± 1.01c 32.97 ± 1.60g 36.02 ± 4.32a 42.29 ± 7.42def

B3 Before storage 65.21 ± 0.62cd 9.05 ± 0.59a 39.18 ± 0.28b 25.99 ± 1.30efg 63.23 ± 5.49b

After 1 mth 66.77 ± 0.30c 7.49 ± 0.22bc 36.72 ± 0.25d 25.52 ± 1.30fg 42.81 ± 5.33efg

After 2 mths 64.83 ± 2.45cd 7.52 ± 0.28bc 35.49 ± 1.14e 26.31 ± 3.78efg 45.43 ± 6.70defg

B4 Before storage 63.93 ± 1.49d 8.99 ± 0.93a 40.42 ± 0.30a 26.74 ± 0.78ef 79.89 ± 2.30a

After 1 mth 64.82 ± 0.97cd 8.32 ± 0.66ab 37.87 ± 0.30c 37.51 ± 2.88ab 49.97 ± 5.42cde

After 2 mths 64.96 ± 0.84cd 8.24 ± 0.52ab 37.72 ± 0.39cd 34.32 ± 3.31ab 37.52 ± 3.28efg

Parameters in columns denoted with the same letters do not differ statistically at the level of confidence a ¼ 0.05.Number of replications n ¼ 4 (for colour) and n ¼ 10 (for texture).

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e7 5

parameter remained at a lower level when compared to the controlsample. This is, of course, unsurprising since bee pollen is charac-terized by a definitely darker colour when compared towheat flour.There were not, however, any changes observed resulting from thestorage 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 wasadded to the biscuits. During storage the values of these parametersslightly decreased, both for the control and the biscuits with beepollen. For the a* parameter, the differences were generally notstatistically significant, while within the b* parameter the differ-ence between the fresh samples and those stored for one and twomonths proved to be statistically significant (Table 5).

One of the factors that contribute to the colour of biscuits wascaused by Maillard reactions between reducing sugar and aminoacids (K€oksel & G€okmen, 2008; Mundt & Wedzicha, 2007). As aresult of this non-enzymatic reaction, high-molecular-weightmacromolecule materials known as melanoidins were formed dur-ing baking. Thus, the colour of biscuits develops with increasingtemperature and baking time (K€oksel & G€okmen, 2008), and de-pends on the amount of sugars and proteins present in the in-gredients used for baking. Because bee pollen contained moreproteins than substituted wheat flour and the reducing sugars in thedough came from pollen, the amount of the melanoidins formed infortified biscuits was significantly higher than those in the control.This resulted in a lower L* parameter. Gallagher, Kenny, and Arendt(2005) and O’Brien, Chapmen, Neville, Keogh, and Arendt (2003)confirmed that the protein content has a negative correlation withan L* value, indicating that the increase of protein content in biscuitreduces the L* value. The darker colour of biscuits enriched with beepollenwas also affected by the pollen itself, which has amuch darkercolour thanwheat flour. The colour of pollen pellets ranged frompaleyellow, through to orange, and up to yellow-brown, due to thepresence of flavonoids and carotenoids. The colour of bee pollendepends onmany factors, including display to sunlight, the humidityof the pollen, dust and pollution from soot, or dark fungal spores.Furthermore, the colour may depend on the type and amount ofliquid, sugar or nectar mixed with the pollen by the bees in theformation of pellets (Stanley & Linskens, 1974).

3.3.2. TextureBiscuits, baked with the addition of bee pollen, were signifi-

cantly softer than the control sample, but the amount of additionseemed not to be important on the day of baking as all the samples

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were characterized by comparable hardness, and the differencesobserved were not statistically significant (Table 5). The changes asregards storage were generally small, and only in the case ofproducts obtained with a 7.5 and 10% of pollen, a slight increase inhardness over the initial values could be observed.

In the case of the second analysed texture parameter e pene-tration work e the influence as regards the change in formulationwas much more pronounced (Table 5). The increase in its valueobserved at the lowest applied level of pollen (2.5%) was 70%, whilewith the addition of 10% it reached 141%, with intermediate valuesfor other concentrations of pollen. The changes in penetrationworkover the storage period highly depended on the presence of pollen.For the control sample, a growing trend could be observed, andafter the second month of storage, the increase in penetrationworkwas 25%. Reverse changes could be seen in the case of pollensupplemented biscuits, which revealed higher values of penetra-tion work after storage. Final values, determined after 2 months,ranged from 35 (B1 formulation) to 45.43 (B3) and were compa-rable with the control sample (41.35).

In general it could be observed that the presence of bee pollen inbiscuit formulation does not exert any negative impact on thetexture of biscuits. Both a slight decrease in the maximum breakingforce (hardness), and an increase in penetration work, could beregarded as beneficial. However, the advantage of bee pollen sup-plemented biscuits could only be observed with fresh products, asduring storage their texture becomes more and more similar to thecontrol, probably due to water sorption/redistribution. As it wasearlier observed by Baltsavias, Jurgens, and van Vliet (1999), themechanical properties of biscuits may change during storage due toglass transition, which in turn depends on the moisture contentand sorption properties of the product.

3.4. Sensory parameters of biscuits

The sensory assessment is shown in Table 6. According to thedata, those biscuits supplemented with bee pollen obtained ahigher total score. Supplementation at a level of 5% kept the sen-sory parameters close to the control sample, both in fresh biscuitsas well as those that had been stored. It should be also noted that atall concentrations of pollen, the overall assessment allowed theclassification of the product to be acceptable (totalscore¼ 3.0e3.50) in the case of themaximum additive (sample B4),and as good (total score ¼ 3.51e4.50) in other concentrations andthe control sample. The high scores were obtained from the control

of biscuits with bee pollen and its effect on physicochemical and15), http://dx.doi.org/10.1016/j.lwt.2015.03.075

Table 6Total score of sensory analysis of biscuits.

Sample Time Shape �(0.1) Colour �(0.1) Surface �(0.15) Consistency �(0.15) Fracture �(0.1) Aroma �(0.15) Taste �(0.25) Total score

Control Before storage 0.46 ± 0.04ab 0.40 ± 0.07abc 0.64 ± 0.09bc 0.69 ± 0.07a 0.44 ± 0.04ab 0.44 ± 0.04ef 1.04 ± 0.24a 4.11 ± 0.13ab

After 1 mth 0.42 ± 0.08ab 0.37 ± 0.11bc 0.63 ± 0.09bc 0.62 ± 0.15abc 0.41 ± 0.10abc 0.60 ± 0.14abc 1.03 ± 0.22a 4.07 ± 0.68ab

After 2 mths 0.40 ± 0.08b 0.36 ± 0.12bc 0.62 ± 0.11bc 0.60 ± 0.10abcd 0.43 ± 0.08ab 0.57 ± 0.14abcd 1.00 ± 0.24ab 3,98 ± 0.65abc

B1 Before storage 0.46 ± 0.09ab 0.41 ± 0.06abc 0.62 ± 0.05bc 0.64 ± 0.09ab 0.41 ± 0.06abc 0.47 ± 0.04def 1.07 ± 0.23a 4.09 ± 0.33ab

After 1 mth 0.42 ± 0.09ab 0.40 ± 0.07abc 0.65 ± 0.07bc 0.59 ± 0.11bcd 0.41 ± 0.09abc 0.65 ± 0.10a 1.00 ± 0.12ab 4.11 ± 0.49ab

After 2 mths 0.43 ± 0.09ab 0.43 ± 0.07ab 0.60 ± 0.14c 0.62 ± 0.11abc 0.39 ± 0.07abcd 0.62 ± 0.15abc 0.83 ± 0.24bc 3.91 ± 0.57bc

B2 Before storage 0.49 ± 0.03a 0.47 ± 0.04a 0.75 ± 0.00a 0.60 ± 0.12abcd 0.46 ± 0.04a 0.47 ± 0.04def 1.11 ± 0.20a 4.34 ± 0.33a

After 1 mth 0.42 ± 0.09ab 0.41 ± 0.09abc 0.69 ± 0.08ab 0.60 ± 0.10abcd 0.38 ± 0.10bcd 0.63 ± 0.12ab 0.98 ± 0.22ab 4.11 ± 0.57ab

After 2 mths 0.42 ± 0.08ab 0.42 ± 0.06abc 0.63 ± 0.09bc 0.56 ± 0.12bcde 0.39 ± 0.06abcd 0.57 ± 0.15abcd 0.83 ± 0.26bc 3.81 ± 0.59bc

B3 Before storage 0.47 ± 0.04a 0.47 ± 0.04a 0.75 ± 0.00a 0.47 ± 0.05e 0.34 ± 0.09cd 0.46 ± 0.04ef 0.64 ± 0.20cd 3.61 ± 0.24cd

After 1 mth 0.44 ± 0.05ab 0.41 ± 0.10abc 0.65 ± 0.07bc 0.51 ± 0.08de 0.34 ± 0.07cd 0.54 ± 0.13bcde 0.73 ± 0.14cd 3.61 ± 0.47cd

After 2 mths 0.43 ± 0.08ab 0.39 ± 0.09bc 0.65 ± 0.07bc 0.51 ± 0.16de 0.35 ± 0.10cd 0.53 ± 0.15cde 0.75 ± 0.24cd 3.60 ± 0.64cd

B4 Before storage 0.49 ± 0.03a 0.41 ± 0.06abc 0.73 ± 0.05a 0.54 ± 0.10cde 0.37 ± 0.08bcd 0.39 ± 0.07e 0.39 ± 0.16e 3.31 ± 0.19d

After 1 mth 0.42 ± 0.09ab 0.37 ± 0.09bc 0.63 ± 0.06bc 0.48 ± 0.09e 0.33 ± 0.12d 0.50 ± 0.12de 0.63 ± 0.27d 3.35 ± 0.55d

After 2 mths 0.40 ± 0.08b 0.35 ± 0.13c 0.57 ± 0.15c 0.51 ± 0.13de 0.33 ± 0.13d 0.47 ± 0.18def 0.60 ± 0.27d 3.23 ± 0.54d

Parameters in columns denoted with the same letters do not differ statistically at the level of confidence a ¼ 0.05.Number of replications n ¼ 13.

M. Krystyjan et al. / LWT - Food Science and Technology xxx (2015) 1e76

sample, and comparable results were received from the B1 and B2recipe, where biscuits were supplemented with bee pollen inamounts of 2.5 and 5% in relation to thewheat flour. Biscuits B3 andB4 gained lower scores where substitution was at a level of 7.5 and10% respectively (Table 6).

When comparing particular sensory characteristics of biscuitsdirectly after their baking and after storage, no significant differ-ences were observed between the control and the sample with the2.5 and 5% additions of bee pollen (Table 6). Even after storage,there were either no differences or they were statistically insig-nificant. On this basis it can be concluded that the applied fortifi-cation of biscuits with bee pollenwas possible and gave the desiredresults, at a level of 5%. Slight differences were noticed between thecontrol and those biscuits with a 7.5 and 10% supplementation ofpollen. In particular, such characteristics as taste, consistency andfracture deteriorated. One of the main factors as regards the dete-rioration of the taste of biscuits was bee pollen; when used insignificant quantities, the biscuits were leaving a delicate bittertaste on the tongue. In addition, the colour of the product dependson the chemical composition of bee pollen, which in turn dependson the plant species and environmental conditions (Szczesna et al.,2002), as well as other factors that were discussed previously. Thus,the flavonoids and carotenoids as well as the reducing sugarsintroduced to the product with bee pollen affected its colour.Curiously, the darkening of colour caused by the addition of beepollen was not observed by the assessors.

4. Conclusions

The possibility of fortifying biscuits with bee pollen appears tobe justified. This is not only due to the enrichment of confectioneryproducts in the antioxidant compounds having health promotingproperties, but also due to the possibility of having introduced beepollen itself - which is rich in these bioactive substances - into thehuman diet. Based on the data obtained, it was concluded that theaddition of bee pollen did not affect the fat content in biscuits.However, it did have a statistically significant effect on the increasein content of sugar, protein, ash, fibre and polyphenols, as well asthe antioxidant potential of the final product. Biscuits improvedwith bee pollenwere characterized by higher penetrationwork andhaving a darker surface when compared to the control. Applyingbee pollen to fortify the biscuits was possible, and even at the stagewhen the highest amount was applied, this addition enabled thedesired results. However, only 5% of pollen was needed to enablethe taste of biscuits to be at the same level as the control.

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Acknowledgements

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

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