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Variations of the antimicrobial, antioxidant, sensory attributes and biogenic amines content in Lithuania-derived bee products
Journal Pre-proof Variations of the antimicrobial, antioxidant, sensory attributes and biogenic amines content in Lithuania-derived bee products Elena Bartkiene, Vita Lele, Vytaute Sakiene, Paulina Zavistanaviciute, Egle Zokaityte, Agila Dauksiene, Povilas Jagminas, Dovile Klupsaite, Saulius Bliznikas, Modestas Ruzauskas PII:
S0023-6438(19)31135-1
DOI:
https://doi.org/10.1016/j.lwt.2019.108793
Reference:
YFSTL 108793
To appear in:
LWT - Food Science and Technology
Received Date: 13 May 2019 Revised Date:
27 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Bartkiene, E., Lele, V., Sakiene, V., Zavistanaviciute, P., Zokaityte, E., Dauksiene, A., Jagminas, P., Klupsaite, D., Bliznikas, S., Ruzauskas, M., Variations of the antimicrobial, antioxidant, sensory attributes and biogenic amines content in Lithuania-derived bee products, LWT Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108793. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Variations of the antimicrobial, antioxidant, sensory attributes and biogenic
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amines content in Lithuania-derived bee products
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Elena Bartkiene*, Vita Lele, Vytaute Sakiene, Paulina Zavistanaviciute, Egle Zokaityte, Agila
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Dauksiene, Povilas Jagminas, Dovile Klupsaite, Saulius Bliznikas, Modestas Ruzauskas
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Lithuanian University of Health Sciences, Tilzes str. 18, LT-47181 Kaunas, Lithuania
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*Corresponding author: Elena Bartkiene, Lithuanian University of Health Sciences, Tilzes str.
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18, LT-47181 Kaunas, Lithuania; tel.: +370 37 574565; fax: +370 37 300152,
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[email protected].
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Abstract
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This study examined the antimicrobial and antioxidant properties, overall acceptability (OA),
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including influence of the product-induced emotions, and biogenic amine (BA) content in
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fourteen honey (H1–14), four propolis (P15–18) and four bee bread (BB19–22) samples,
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collected in northwest Lithuania. The all tested bacteria were inhibited by summer honey H13.
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P18 inhibited 10, while bee breads inhibited 10-11 out of the 15 tested bacteria strains. All
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propolis- samples inhibited B.cereus and P.multocida. The highest antioxidant activity and
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content of total phenolic compounds (TPC) were found in bee bread (93% and 394 mg GAE/100
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g, respectively). The TPC had moderate and strong negative correlations with the L* (r=-0.58)
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and b* colour coordinates (r=-0.66), respectively. The each bee products group (BPs) induced
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different intensities of emotions, and the OA showed a moderate positive correlation with the
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“neutral” emotion (r=0.47). A low content of BAs (<15 mg/kg) was identified in six honeys, two
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bee breads and one propolis sample. In sum, Lithuanian bee products possessed valuable
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biological attributes that can be beneficially used in food industry and medicine, although further
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research is needed for the factors, which may contribute to bioactive properties of this region bee
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products and BAs formation.
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Keywords: honey, propolis, bee bread, bioactivity, FaceReader.
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1. Introduction
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Bioactive substances of natural origin attract great interest and are especially appreciated by
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today’s consumers, whose preferences are trending toward natural functional foods (Kieliszek et
52
al., 2018). Among natural products containing bioactive ingredients, honey and other bee
53
products are highly popular as healthy alternatives to synthetic supplements. Bee products are
54
well recognized for their health attributes, particularly, their potential healing properties, which
55
differ according to the region, nectar source, climate, among other factors (Bobiș et al., 2010).
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For this reason, the desire to use these multicomponent natural substances contributes to the
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growth of interest in evaluating their properties. Bee products demonstrate a wide range of
58
desirable characteristics, including antimicrobial and antioxidant properties, and the most
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popular bee products are honey, pollen and their extracts, bee bread, propolis, royal jelly and bee
60
venom. Honey is used not only as a nutritional product but also in traditional medicine and as an
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alternative treatment for clinical conditions because it has antioxidant, anti-inflammatory,
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antibacterial, antidiabetic, respiratory, gastrointestinal, cardiovascular and nervous system
63
protective effects (Samarghandian, Farkhondeh, & Samini, 2017). Another bee product that
64
possesses health benefits is propolis. This product contains a high number of compounds with
65
anti-inflammatory, antioxidant, antiviral and antimicrobial properties (Al-Waili, Al-Ghamdi,
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Ansari, Al-Attal, & Salom, 2012). Bees prepare and use propolis as a sealing material to protect
67
against the entry of microorganisms (fungi and bacteria) into the hive, and create the most sterile
68
environment known in nature (Al-Waili, Al-Ghamdi, Ansari, Al-Attal, & Salom, 2012). The
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main pharmacological activities of propolis are related to the flavonoids and phenolic
70
compounds—the major bioactive constituents of propolis (Banskota et al., 2000; Anthimidou &
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Mossialos, 2013). The properties of the propolis flavonoids to reduce the formation or to remove
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free radicals allow effective regeneration of damaged tissue and the antimicrobial properties of
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propolis prevent from wound infection (Boyanova, Kolarov, Gergova, & Mitov, 2006; Kasiotis,
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Anastasiadou, Papadopoulos, & Machera, 2017). Propolis is very popular and is used in a variety
75
of commercial preparations, including pharmaceutical, nutraceutical and cosmetic products (El-
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Guendouz, Lyoussi, & Miguel, 2019). Being a mixture of many compounds, propolis is not
77
easily subject to extraction and fractionation procedures; therefore, it is usually used as a whole
78
ingredient. The composition of raw propolis may vary, depending on geographical and seasonal
79
factors, because it depends on the flora of the areas from which it is collected (El-Guendouz,
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Lyoussi, & Miguel, 2019).
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Another bee product, bee bread, is characterized by a high nutritional value, good digestibility
82
and rich chemical composition (da Silva, de Souza, Matta, de Andrade, & Vidal, 2006; Habryka,
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Kruczek, & Drygaś, 2016), attributed to the partial fermentation of the bee bread components,
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which, in turn, are more easily assimilated in an organism (Barene, Daberte, & Siksna, 2015).
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Bee bread contains peptides and free amino acids and is an excellent product that could
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supplement nutrient deficiencies in humans to achieve a balanced diet (Nagai, Nagashima,
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Myoda, & Inoue, 2004). Bee bread contains antioxidants (e.g., carotenoids) and natural
88
preservatives (e.g., lactic acid) (Barene, Daberte, & Siksna, 2015). Nonetheless, it should be
89
mentioned that the presence of free amino acids in bee products can lead to the formation of
90
biogenic amines (BAs), which are undesirable compounds in food products (Ruiz-Capillas &
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Herrero, 2019).
92
As stated above, many factors influence bee product characteristics. Since their main quality
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parameters necessary to meet the required regulations for commercialization in the local market
94
of Lithuania have already been investigated, the current research focused on the evaluation of
95
specific characteristics, such as the antioxidant and antimicrobial activities, as well as the BA
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content. Moreover, the overall acceptability, including the influence of the products induced
97
emotions (neutral, happy, sad, angry, surprised, scared, disgusted, contempt, valence, arousal) on
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consumers of the Lithuania-derived honey, propolis and bee bread samples were evaluated.
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2. Materials and Methods
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2.1. Chemicals
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Sodium hydroxide, dansyl chloride, perchloric acid, sodium bicarbonate, ammonium hydroxide,
102
acetonitrile (HPLC grade), ammonium acetate, sodium carbonate, gallic acid and 2,2-difenil-1-
103
picrilhydrazyl (DPPH) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Folin-
104
Ciocalteu reagent was obtained from Scharlau Chemie S. A. (Barcelona, Spain). Ethanol was
105
from FarmaBalt (Riga, Latvia). Tryptamine hydrochloride, 2-phenylethylamine hydrochloride,
106
1,4-diaminobutane dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride,
107
tyramine hydrochloride, spermidine phosphate salt hexahydrate and 1,7-diaminoheptane were
108
obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Spermine diphosphate hexahydrate
109
was from TCI Europe (Tokyo, Japan). All reagents were of analytical grade.
110 111
2.2. Characteristics of the assessed bee products
112
Table 1 provides the farm location and the time of year when the bee products (honey, crude
113
propolis material and bee bread) used in this study were collected. In this experiment, the bee
114
products were obtained from three farms located in northwest Lithuania. Bee bread [BB19–22]
115
and propolis [P15–18] were collected during summer. Most of honey samples [H1, H5, H7–14]
116
were also collected during summer, except H2 and H6, which were obtained in autumn, and H3
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and H4, which were gathered in spring.
118
119
Table 1
120 121
2.3. Evaluation of bee products antimicrobial properties
122
Honey, propolis, and bee bread were investigated for their antimicrobial activities against a
123
variety of pathogenic and opportunistic bacterial strains (A. baumannii 17-380, B. cereus 1801,
124
C. freundii, E. cloacae, E. faecalis 86, Enterococcus faecium 103, Klebsiella pneumoniae,
125
methicillin-resistant Staphylococcus aureus (MRSA) M87fox, P. multocida, P. mirabilis,
126
Pseudomonas aeruginosa 17-331, Salmonella enterica 24 SPn06, S. epidermidis, S.
127
haemolyticus and Streptococcus mutans). Bacterial strains were previously isolated from humans
128
and domestic animals and stored in the Lithuanian University of Health Sciences Collection of
129
Microorganisms. The agar well diffusion assay was used to evaluate the antimicrobial activities
130
of the bee products. For this purpose, a standardized 0.5 McFarland suspension of each
131
pathogenic bacteria strain was inoculated onto the surface of cooled Mueller–Hinton agar (Oxoid
132
Limited, Basingstoke, Hampshire, UK) using sterile cotton swabs. Wells of 6 mm in diameter
133
were punched in the agar, and each well filled with 100 µL of the tested product sample, which
134
was prepared by dissolving 1 g of each bee product in 10 mL of saline solution (9 g/L). The
135
antimicrobial activities against tested bacteria were determined by measuring the inhibition zone
136
(IZ) diameter (mm). The experiments were repeated three times, and the average IZ was
137
calculated.
138 139
2.4. Determination of the total content of phenolic compounds and antioxidant activity of
140
bee products
141
1One gram of bee products was dissolved in 20 mL of aqueous ethanol (500 mL/L), extracted
142
shaking for 1-6 h (1 h for honey and 6 h for bee bread and propolis) at room temperature and
143
then filtered through paper filter. These extracts were further used for total content of phenolic
144
compounds (TPC) and antioxidant activity analysis. The TPC in bee product samples was
145
measured using the Folin-Ciocalteu colorimetric method (Vaher, Matso, Levandi, Helmja, &
146
Kaljurand, 2010 Singleton, Orthofer, & Lamuela-Raventos, 1999). One mL of each extract was
147
mixed with 5 mL of Folin-Ciocalteau (100 mL/L) reagent and 4 mL of 75 g/L NaHCO3. The
148
mixture was allowed to stand at room temperature for 30 min. The absorbance of each sample
149
was measured at 765 nm using a V-1100D spectrophotometer (JP Selecta SA, Barcelona, Spain).
150
The concentration of phenolics was calculated from the calibration curve and the results were
151
expressed as gallic acid equivalent (GAE) in mg/100 gram of raw material.
152
Antioxidant activity of the bee products was evaluated according to the method described by
153
Brand-Williams, Cuvelier, & Berset (1995) with some modifications. DPPH solution (3.6 mL;
154
0.1 mmol/L, in ethanol) was mixed with 0.66 mL of sample dissolved in the aqueous ethanol.
155
After 20 min of shaken shaking in the dark at room temperature, the absorbance at 517 nm was
156
measured. The inhibition of the DPPH radical by the sample was calculated according to the
157
following formula:
158
DPPH (%) = (Acontrol - Asample) / Acontrol x 100,
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where Acontrol was obtained by replacing the sample with ethanol.
160 161
2.5. Evaluation of the bee products colour coordinates and overall acceptability
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The colour coordinates (L*, a*, b*) were assessed using a CIELAB system (Chromameter CR-
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400, Konica Minolta Sensing, Inc., Osaka, Japan). The overall acceptability of the bee products
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was carried out by 50 judges, according to the ISO 11136:2014 method using a 10-point scale,
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ranging from 0 (extremely dislike) to 10 (extremely like). Also, the bee products were tested by
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applying FaceReader 5 software (Noldus Information Technology, Wageningen, The
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Netherlands), scaling the 10 emotion patterns (neutral, happy, sad, angry, surprised, scared,
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disgusted, contempt, valence, arousal). The judges were asked to taste the presented bee products
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one by one in front of a Microsoft LifeCam Studio webcam (Microsoft Corporation, Redmond,
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Washington, USA). The recordings were analysed with FaceReader 5 software and intensity of
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facial expressions was expressed in a scale from 0 to 1. Between the samples, the judges were
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asked to rinse the mouth with water. Figure 1 illustrates the experimental design used to assess
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the emotions induced by different bee products.
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Figure 1
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2.6. Evaluation of biogenic amine (BA) content in bee products
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Sample preparation and determination of the BAs, including cadaverine (CAD), histamine (HIS),
177
phenylethylamine (PHE), putrescine (PUT), spermidine (SPRMD), spermine (SPER), tryptamine
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(TRY) and tyramine (TYR), in bee products was achieved by following the procedure reported
179
by Ben‐Gigirey et al. (1998) with some modifications. The standard BA solutions were
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prepared by dissolving known amounts of each BAs (including internal standard) in 20 mL of
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deionized water Briefly, the extraction of BAs in samples (5 g) was done by using 0.4 mol/L
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perchloric acid. The derivatization of sample extracts and standards was performed using a
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dansyl chloride solution (10 mg/mL) as a reagent. The chromatographic analyses were carried
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out using a Varian ProStar HPLC system (Varian Corp., Palo Alto, California, USA) with two
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ProStar 210 pumps, a ProStar 410 autosampler, a ProStar 325 UV/VIS Detector and Galaxy
186
software (Agilent, Santa Clara, California, USA) for data processing. For the separation of
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amines, a Discovery ® HS C18 column (150 x 4.6 mm, 5 µm; SupelcoTM Analytical,
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Bellefonte, Pennsylvania, USA) was used. The eluents were ammonium acetate (A) and
189
acetonitrile (B) and the elution program consisted of a gradient system with a 0.8 mL/min flow-
190
rate. The detection wavelength was set to 254 nm, the oven temperature was 40°C and samples
191
were injected in 20 µL aliquots. The target compounds were identified based on their retention
192
times in comparison to their corresponding standards.
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2.7. Statistical analysis
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All analyses were undertaken at least in triplicate. Non-parametric Kruskal Wallis test followed
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by Dunn's post hoc test were used for data analysis. P < 0.05 was considered statistically
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significant. Statistics were performed with SPSS for Windows XP V15.0 (SPSS, Inc., Chicago,
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Illinois, USA, 2007).
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3. Results and Discussion
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3.1. Antimicrobial properties of the bee products
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Table 2 presents the IZs of the bee products against the various pathogenic and opportunistic
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strains examined. All the tested pathogenic strains were inhibited by H13 (summer honey). In
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comparison, the other summer honeys showed a narrower inhibition spectrum against the
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pathogens: of the 15 pathogenic strains tested, five (H1), eight (H7), ten (H8), eleven (H9),
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fourteen (H11) and thirteen (H14) were susceptible to the antimicrobial action of the honeys,
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respectively. All of the summer honeys exhibited antimicrobial activity against MRSA. The most
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limited antimicrobial spectrum activity (inhibitory action was shown against only one of the
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tested pathogenic strains) was displayed by the spring honeys (H3 and H4: both showed an
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average IZ for P. multocida of 12.5 mm. Autumn honeys inhibited more pathogens compared
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with the spring honeys, and their average IZ against A. baumannii 17-380, MRSA M87fox, S.
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epidermidis, S. haemolyticus and P. multocida were 12.1, 14.3, 12.4, 12.6 and 20.3 mm,
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respectively. The honey H5 showed inhibitory action against the same pathogens as the autumn
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honeys, and with similar potency, providing average IZ values against A. baumannii 17-380,
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MRSA M87fox, S. epidermidis, S. haemolyticus and P. multocida of 12.1, 15.2, 12.3, 14.5 and
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21.7 mm, respectively. H6 and H12 honeys each inhibited 10 out of the 15 tested pathogenic
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strains. Both of these products (H6 and H12) inhibited K. pneumoniae, A. baumannii 17-380, P.
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mirabilis, MRSA M87fox, C. freundii, S. epidermidis, S. haemolyticus, and P. multocida.
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Additionally, H6 inhibited P. aeruginosa 17-331 and B. cereus 18 01 while H12 also inhibited S.
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enterica 24 SPn06 and E. cloacae.
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Honey, especially medical Manuka honey, has a broad-spectrum of antibacterial activity against
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both Gram-positive and Gram-negative microorganisms, including MRSA and vancomycin-
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resistant Enterococcus, and, also, against some fungi and virus (Gonçalves et al., 2018; Fyfe,
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Okoro, Paterson, Coyle, & McDougall, 2017). Different explanations for the antimicrobial
224
properties of honey have been mentioned in the literature. One suggestion is that the α-
225
glucosidase is critical for decomposing saccharose into fructose and glucose, and glucose
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oxidase catalyses the conversion of glucose to gluconic acid, producing hydrogen peroxide,
227
which is related to honey’s antibacterial activity (Faustino & Pinheiro, 2015). Some other factors
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influencing antimicrobial properties of honey are high osmolarity, low water activity and pH,
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and high viscosity (Morroni et al., 2018; Cianciosi et al., 2018). The antimicrobial activity of
230
honey has also been associated with polyphenols (methyl syringate, phenyllactic acid),
231
flavonoids (quercetin, kaempferol) and synergies with other components (fatty diacids, abscisic
232
acid and methylglyoxal) (Fyfe, Okoro, Paterson, Coyle, & McDougall, 2017; Cianciosi et al.,
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2018). Gonçalves et al. (2018) noted that the bioactivity (antimicrobial and antioxidant) and
234
physicochemical parameters of selected Portuguese monofloral honeys were correlated and
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depended on the honey floral source. Heather honey was not only the darkest of the honeys
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examined but also indicated the greatest antimicrobial and antioxidant properties, which were
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explained by its higher levels of protein, flavonoids and phenolic compounds. Alvarez-Suarez et
238
al. (2018) concluded that different physicochemical parameters can be the reason for the higher
239
antimicrobial activity of M. beecheii honey compared to A. mellifera honey.
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All propolis samples (P15–18) inhibited B. cereus 18 01 and P. multocida. However, these were
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the only strains inhibited by P16 and P17, and P15 only inhibited one other (MRSA M87fox).
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P18 showed the broadest antimicrobial spectrum of the propolis samples, inhibiting 10 of the 15
243
tested pathogenic strains. Besides those inhibited by P15, mentioned above, P18 was active
244
against A. baumannii 17-380, C. freundii, E. cloacae, E. faecalis 86, P. mirabilis, S. epidermidis
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and S. haemolyticus, and the largest IZ against P. multocida.
246
The antimicrobial properties of propolis are mainly afforded by the flavonoids and cinnamic acid
247
derivatives present in this complex mixture of natural substances (Moradkhannejhad, Abdouss,
248
Nikfarjam, Mazinani, & Heydari, 2018), which varies according to the different areas and
249
seasons, as the composition is dictated by the constituents of the plants in the area. However, the
250
composition of two different propolis can be very promising, as exhibited antimicrobial activity
251
against Gram-positive and Gram-negative bacteria, as well as Candida albicans (Bryan, Redden,
252
& Traba, 2016). Przybyłek & Karpiński (2019) summarized that the antibacterial activity of
253
propolis against Gram-positive bacteria was greater than Gram-negative. Moreover, the highest
254
activity of propolis from the Middle East was found, while samples from Germany, Ireland and
255
Korea had the lowest activity. Various mechanisms of action have been proposed for the
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inhibitory effect of propolis against bacteria. One explanation is that propolis physically damages
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the bacterial cell membranes and initiates cell lysis, eventually leading to cell death (Bryan,
258
Redden, & Traba, 2016). In other work, the antimicrobial activity relies on the antibacterial
259
compounds in propolis (phenolic compounds, terpenes, caffeic, ferulic and coumaric acids, esters
260
and flavonoids) (Inui et al., 2014; Veiga et al., 2017). Furthermore, propolis contains flavonoids
261
and phenolic compounds, which have anti-inflammatory action (Funakoshi-Tago et al., 2016).
262
Extracts of propolis have also displayed antibacterial properties based on agar diffusion assays or
263
experiments in liquid broths (Kim & Chung, 2011; Akhir, Adawieah, Bakar, Fadzelly, & Sanusi,
264
2018).
265
All of the tested bee breads (BB19–22) showed antimicrobial activity against A. baumannii 17-
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380, B. cereus 18 01, C. freundii, E, cloacae, E. faecalis 86, MRSA M87fox, P. multocida, P.
267
mirabilis, S. epidermidis and S. haemolyticus. In addition, BB21 inhibited E. faecium 103, and
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BB22 inhibited S. mutans.
269
The study of Bakour et al. (2019) has demonstrated that Moroccan bee bread inhibits the growth
270
of a broad spectrum bacteria and fungi, and this activity is higher against Gram-positive than
271
Gram-negative bacteria. The antimicrobial activity of bee bread can be largely attributed to the
272
high content of phenolic compounds, especially flavonoids and phenolic acids (Bakour et al.,
273
2019).
274
Table 2
275
3.2. Total content of phenolic compounds and antioxidant activity of honey, propolis, and
276
bee bread
277
The TPC and antioxidant activity of the bee products (honey, propolis, bee bread) are shown in
278
Table 3. The average TPC in summer honeys was 243.2 mg GAE/100 g, and H10 possessed the
279
highest TPC among the honey samples while H8 had the lowest. In comparing the average TPC
280
between summer and spring honeys, spring honeys had 25 % less TPC. In H5, H6 and H12
281
honeys, the TPC was 217.6 – 263.5 mg GAE/100 g. Among the propolis samples, P16 had the
282
greatest TPC, and in P15, P17 and P18, the TPC were 17%, 7% and 29% lower, respectively. A
283
comparison of the bee breads revealed the highest TPC in BB21, which was 6%, 22% and 21%,
284
higher than that in BB19, BB20 and BB22, respectively.
285
Summer honey H13 had the lowest antioxidant activity, while the highest one was established in
286
autumn H6. The other autumn honey H2 had an antioxidant activity, which was slightly less than
287
that demonstrated by the spring honeys (on average 6 % lower), and H5 and H6 samples (7 and
288
10 % lower, respectively). Of the propolis samples, P18 recorded the highest antioxidant activity,
289
which was on average 2.3-fold higher than that of the remaining propolis samples. Bee bread
290
antioxidant activity was on average 90 %, with a minimum of BB20 and maximum of BB22.
291
Such kinds of honey as Manuka or Strawberry tree can provide a strong antioxidant activity and
292
even act as chemopreventive agents (Afrin et al., 2017). It is known that phenolic compounds are
293
mainly responsible for the beneficial effect of bee products (Cianciosi et al., 2018). The nectar
294
source, phenolic acids, flavonoids, ascorbic acid, carotenoids, amino acid, protein content and
295
Maillard reaction products, have been described as having a strong impact on the antioxidant
296
activity of bee products (Fernandes, Ferreira, Fonte, Wessel, & Cardoso, 2016). Due to different
297
botanical and geographical sample origin, extraction technique and analysis conditions, various
298
results of TPC and antioxidant activity of bee products can be found in the literature. In our study
299
obtained values of TPC for all tested bee products were higher than values reported for African,
300
Manuka, Strawberry tree and Cuban polifloral honeys (Morroni et al., 2018; Afrin et al., 2017;
301
Alvarez-Suarez et al., 2018). Our results are in the agreement with some studies showing that bee
302
bread presents higher antioxidant activity than propolis (Akhir, Adawieah, Bakar, Fadzelly, &
303
Sanusi, 2018; Suriyatem, Auras, Intipunya, & Rachtanapun, 2017). However, results of this
304
study showed a weak positive correlation between the TPC and antioxidant activity only of the
305
bee bread was found (r = 0.39). A significant linear correlation between TPC and antioxidant
306
activity in honey and honey supplemented with other bee products was mentioned by Juszczak et
307
al. (2016) and by Fernandes et al. (2016) in propolis.
308 309
Table 3
310
3.3. Colour coordinates of the bee products
311
The colour coordinates (L*, a*, b*) of the bee products (honey, propolis and bee bread) are listed
312
in Table 3. The lightness (L*) coordinate ranges from 0 (dark) to 100 (light). Considering the
313
three types of bee products, H4 (spring honey), P15 and B22 were the lightest, and the darkest
314
products in each category were H14 (summer honey), P18 and BB21.
315
The redness–greenness (a* coordinate) of all honeys ranged from 0.10 (H8, summer honey) to
316
5.79 (H3, spring honey). Both spring honeys (H3 and H4) showed significantly higher a*
317
coordinates than the other honey samples tested. Of the propolis samples, P17 and P18 had
318
higher a* coordinates relative to those of P15 and P16. The bee breads had a* coordinates
319
ranging from 3.22 (BB21) to 6.49 (BB20). The b* (blueness–yellowness) coordinates of the
320
honeys ranged from 8.14 (H4) to 29.59 (H8). For the propolis and bee bread, P21 and BB21
321
were found to have the lowest b* coordinates.
322
Correlations of different strengths were identified between each colour coordinate (L*, a*, b*)
323
and the TPC in bee products. A very weak positive correlation between the TPC and a*
324
coordinate was established (r = 0.15) while the TPC had a moderate negative correlation with the
325
L* coordinate (r = -0.58) and a strong negative correlation with the b* coordinate (r = -0.66).
326
Literature data suggest that the darker honeys typically having a higher concentration of
327
polyphenols and higher antioxidant, as well as antimicrobial activities (da Silva, de Souza,
328
Matta, de Andrade, & Vidal, 2006).
329 330
3.4. Overall acceptability and the emotions induced by the tested bee products
331
Overall acceptability and the emotions induced by the bee products are presented in Table 4. The
332
summer honeys scored an average of 6.5 points for overall acceptability, and H1 was assigned
333
the highest overall acceptability. Significant differences existed between the overall acceptability
334
scores given to the spring honeys, as exemplified by H3 and H4. Among H5, H6 and H12, the
335
most acceptable was the latter one, and the H5 and H6 samples scores indicated 26% and 23%,
336
respectively, lower overall acceptability. When comparing all the tested honeys, H4 (spring
337
honey) had the highest overall acceptability. There were no significant differences in the overall
338
acceptability among the propolis samples, as well as between the bee breads.
339
The overall acceptability of the bee products was influenced by their colour characteristics and
340
TPC. Positive strong (for L*) and moderate (for b*) correlations between the bee products
341
overall acceptability and lightness (r = 0.63) and blueness–yellowness (r = 0.57) were observed.
342
Also, negative moderate and weak correlations between the bee products overall acceptability
343
and TPC (r = -0.46) and a* colour coordinate (r = -0.34), respectively, were noted.
344
Usually, the traditional sensory analysis tests are not able to sufficiently predict market
345
performance because most of these test techniques are based on self-reports and, therefore,
346
underlie a conscious/rational decision-making process (Danner, Sidorkina, Joechl, &
347
Duerrschmid, 2014; Köster, 2009). Consequently, the long-term consumer acceptance for special
348
foods is not adequately reflected by using traditional methods (Köster, 2009). In comparison,
349
FaceReader 5 software provides a more accurate acceptability analysis of new products. In the
350
current work, different tendencies of the emotions induced by the different bee products were
351
found, and a moderate positive correlation between the overall acceptability of the bee products
352
and emotion “neutral” was realized (r = 0.47). Between the other evaluated emotions and overall
353
acceptability, weak and very weak correlations were found: negative weak correlations between
354
the overall acceptability and emotions “happy” (r = -0.28), “sad” (r = -0.24), “valence” (r = -
355
0.24); positive weak correlation between the overall acceptability and emotion “scared” (r =
356
0.26); positive very weak correlations between the overall acceptability and emotions “angry” (r
357
= 0.19), “surprised” (r = 0.14), “disgusted” (r = 0.06), “contempt” (r = 0.11) and “arousal” (r =
358
0.19).
359
Table 4
360
3.5. Biogenic amines (BAs) in bee products
361
The BAs content determined in each of the bee product samples is provided in Table 4. A
362
comparison of all the tested bee products (H1–14, P15–18, BB19–22) indicated some of the
363
analysed BAs were present in 6 of the 14 honeys, 2 of the 4 bee breads, and 1 of the 4 propolis
364
samples. In honey samples containing BAs, the highest total BA contents were recorded in two
365
of the summer honeys H8 and H11, with PUT the dominant BA. TRY occurred in three honey
366
samples (H2, H4 and H5). H6 contained small contents of PUT and HIS. TYR was detected in
367
one honey sample. Among the bee breads, PHE and CAD were identified in BB19, and in BB20,
368
the predominant BAs were PUT, CAD and TYR. In propolis samples, only TYR in P17 was
369
found.
370
Honey contains minor concentrations of bioactive compounds, such as phenolic acids,
371
flavonoids, carotenoids, amino acids (mainly proline), proteins (including enzymes) and pollen
372
grains (Gonçalves et al., 2018). BAs can occur in almost all foods that contain proteases or free
373
amino acids and which are subject to the conditions enabling microbial and biochemical activity
374
(Papageorgiou et al., 2018). HIS and TYR are recognized as the most toxic BAs by The
375
European Food Safety Authority (Official Journal of the European Union, 2005). It is known that
376
the toxicity of BAs depends on synergistic effects, for instance, HIS toxicity is enhanced by the
377
presence of CAD, PUT and TYR (Mantis et al., 2005). However, there is not enough data about
378
the toxic dose of each BAs, that is why little legislation exists on BAs risk on human health and
379
only the content of histamine in fish food is controlled by law (Papageorgiou et al., 2018).
380
According to our results, higher amounts of TYR and PUT were detected in propolis P17 and
381
several honey samples (H8, H11), respectively. The information about BAs presence in bee
382
products is scare. So far, only bee venom is described as a rich source of BAs (Eze, Nwodo, &
383
Ogugua, 2016). Therefore, further research on BAs in bee products (especially in bee bread) is
384
needed; particularly on the toxicity and acceptable concentrations, as these products are usually
385
presented as healthy foods.
386 387
4. Conclusion
388
Nowadays, honey and its products are becoming valued natural substances and their
389
physicochemical and biological properties are affected by many factors. Therefore, it is
390
important to expand the information and explore the biological activities of recent products from
391
different geographic regions. This study examined the specific characteristics, such as the
392
antioxidant and antimicrobial activities, as well as the biogenic amines and product-induced
393
emotions of honey, propolis and bee bread from the northwest Lithuania. The findings revealed
394
that all analysed bee products had antimicrobial and antioxidant activities; however, this
395
characteristics varied between samples and product groups. The overall acceptability of the bee
396
products was influenced by the colour characteristics and the TPC. BAs were found in some
397
samples from each product group, however the total content was very low. In conclusion, the
398
tested Lithuanian honey, propolis and bee bread had valuable biological properties that can be
399
beneficially used in food industry and medicine, although further research is needed for the
400
factors, which may contribute to bioactive properties and BAs formation of this region bee
401
products.
402 403 404
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Tables Table 1. Characteristics of the analysed bee products (honey [H1-14], propolis [P15-18], bee bread [BB19-22]). Farm name and location in Lithuania
Sample
P15 P16
P. Jagmino farm, Taurages district, Batakių city
Honey
N. Litvino farm, Kretinga district, Salantų city
G. Puska farm, Mazeikių district, Serksnenų city
Prop olis
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14
P. Jagmino farm, Taurages district, Batakių city G. Puska farm, Mazeikių
Season the bee products were collected Summer Autumn Spring Spring Summer Autumn Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer
P17
BB19 BB20 BB21 BB22
Bee bread
P18
district, Serksnenų city N. Litvino farm, Kretinga district, Salantų city P. Jagmino farm, Taurages district, Batakių city N. Litvino farm, Kretinga district, Salantų city G. Puska farm, Mazeikių district, Serksnenų city
Summer Summer Summer Summer Summer Summer
Table 2. Antimicrobial activity of the bee products (honey [H1—14], propolis [P15–18], bee bread [BB19–22]).
Enterobacter cloacae
Citrobacter freundii
Staphylococcus epidermidis
Staphylococcus haemolyticus
-
-
-
-
-
8.6±0.3
13.8±0.4
19.4±0.5
-
-
-
-
-
-
12.4±0.2
12.6±0.2
20.3±0.4
H3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.6±0.3
H4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.4±0.3
H5
-
-
-
12.1±0.4
-
15.2±0.3
-
-
-
-
-
-
12.3±0.2
14.5±0.2
21.7±0.3
H6
8.5±0.2
-
7.2±0.1
11.6±0.2
11.9±0.4
14,6±0.5
-
-
8.5±0.2
-
-
6.7±0.1
17.6±0.2
13.6±0.3
22.8±0.4
H7
9.3±0.2
-
-
10.5±0.1
9.8±0.1
11.2±0.2
-
-
-
-
-
8.6±0.1
12.3±0.4
11.6±0.2
17.6±0.3
H8
-
9.4±0.1
12.3±0.2
11.7±0.2
16.3±0.2
7.6±0.2
7.1±0.2
-
-
-
8.3±0.2
26.8±0.4
14.3±0.2
22.9±0.3
H9
9.2±0.3
8.8±0.2
9.4±0.3
12.8±0.3
11.3±0.2
16.8±0.3
-
-
8.4±0.3
-
-
8.2±0.1
20.1±0.2
14.7±0.3
21.3±0.4
H10
8.1±0.2
8.3±0.2
8.1±0.2
12.0±0.4
11.6±0.3
17.9±0.3
-
7.3±0.1
9.2±0.4
-
-
8.8±0.1
21.5±0.3
16.1±0.2
21.2±0.3
Pasteurella multocida
Streptococcus mutans
-
14.3±0.4
Enterococcus faecium 103
13.5±0.3
-
Enterococcus faecalis 86
-
12.1±0.3
MRSA M87fox
11.2±0.4
-
Proteus mirabilis
-
-
Acinetobacter baumannii 17-380
-
-
Pseudomonas aeruginosa 17-331
-
H2
Salmonella enterica 24 SPn06
H1
Bee products
Klebsiella pneumoniae
Bacillus cereus 18 01
Pathogenic and opportunistic bacterial strains
Inhibition zone, mm
H11
7.6±0.2
7.4±0.1
7.3±0.1
-
11.5±0.3
13.6±0.4
8.2±0.1
9.6±0.2
8.1±0.2
15.7±0.2
8.0±0.2
8.6±0.1
19.6±0.3
15.3±0.4
22.4±0.5
H12
7.8±0.2
7.2±0.2
-
9.2±0.3
8.3±0.1
11.2±0.2
-
-
-
-
7.3±0.1
8.9±0.2
8.3±0.2
11.4±0.3
17.1±0.2
H13
7.3±0.1
8.7±0.3
7.6±0.3
11.3±0.2
11.8±0.2
13.6±0.3
8.3±0.1
8.2±0.3
10.3±0.2
15.6±0.3
7.5±0.3
9.2±0.2
17.4±0.2
16.9±0.3
21.3±0.4
H14
11.2±0.3
8.5±0.2
7.8±0.3
12.5±0.3
-
17.4±0.2
-
8.6±0.2
10.5±0.2
16.8±0.5
7.2±0.2
9.7±0.3
18.9±0.4
17.8±0.4
22.4±0.2
P15
-
-
-
-
-
7.5±0.1
-
-
7.6±0.3
-
-
-
-
-
12.5±0.2
P16
-
-
-
-
-
-
-
7.8±0.4
-
-
-
-
-
14.7±0.4
P17
-
-
-
-
-
-
-
7.4±0.2
-
-
-
-
-
11.6±0.1
P18
-
-
-
9.2±0.1
10.1±0.3
11.9±0.3
13.5±0.4
-
9.30.1
-
13.0±0.2
14.6±0.3
13.2±0.3
11.3±0.2
19.3±0.1
BB19
-
-
-
10.6±0.2
10.2±0.2
11.2±0.1
11.4±0.3
-
11.3±0.4
-
14.0±0.4
15.7±0.4
18.6±0.3
14.5±0.3
21.6±0.4
BB20
-
-
-
11.9±0.3
10.4±0.2
16.8±0.3
13.6±0.2
-
11.2±0.2
-
9.0±0.3
12.6±0.3
15.9±0.2
16.6±0.1
22.7±0.3
BB21
-
-
-
9.5±0.2
9.0±0.3
11.6±0.3
13.4±0.2
11.6±0.2
7.6±0.2
-
13.0±0.4
14.7±0.2
12.6±0.3
13.6±0.2
22.8±0.3
BB22
-
-
-
9.8±0.3
9.5±0.2
11.4±0.3
14.5±0.1
-
7.8±0.1
13.3±0.2
13.0±0.2
15.5±0.3
12.7±0.2
12.1±0.2
19.1±0.3
-
(-) ‒ not inhibited. Data expressed as mean ± standard deviation (n = 3). H - honey, P – propolis, BB - bee bread.
Table 3. The colour coordinates (L*, a*, b*), total content of phenolic compounds (mg GAE/100 g) and DPPH antioxidant activity (%) of the bee products (honey, propolis, bee bread). a* b* TPC, mg GAE/ 100 g NBS H1 56±3 2.9±0.2 21±3 251±6 H2 53±1 2.8±0.2 20±1 237±2 H3 64±3 5.8±0.2 29±1 196±1 H4 71±3 5.3±0.4 24±2 168±5 H5 48±5 0.3±0.1 24±2 242±3 H6 29±2 2.0±0.1 12±1 264±3 H7 54±3 1.5±0.1 22±1 250±3 H8 61±2 0.1±0.0 30±3 215±4 H9 43±2 0.4±0.0 21±2 251±5 H10 38±3 2.1±0.1 18±1 278±2 H11 44±3 0.2±0.1 21±2 237±6 H12 56±2 2.5±0.1 19±1 218±6 H13 59±3 2.6±0.2 26±2 220±6 H14 16±1 0.8±0.1 8±1 245±4 P15 31±2 3.6±0.2 14±1 249±6 P16 30±2 3.8±0.3 13±1 298±5 P17 26±1 5.1±0.1 12±1 276±4 P18 19±2 5.8±0.3 10.0±0.7 211±5 BB19 25±1 4.5±0.3 13.6±0.5 372±4 BB20 27±1 6.5±0.3 15±3 306±4 BB21 18±2 3.2±0.2 9.5±0.2 394±3 BB22 22±2 4.7±0.2 12.1±0.2 311±4 Data expressed as mean ± standard deviation (n = 3). TPC – total phenolic compounds DPPH – 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity. NBS – National Bureau of Standards units H – honey, P – propolis, BB – bee bread. Bee bread
Propolis
Honey
Bee product
L*
DPPH, % 83±3 79±3 84±4 84±4 85±2 88±6 81±4 77±5 74±3 77±5 82±4 76±5 65±5 66±4 32±4 35±2 40±2 80±4 92±3 85±3 91±4 93±4
Table 4. Overall acceptability, emotion response and biogenic amines in the bee products (honey [H1–14], propolis [P15–18], bee bread [BB19–22]). Bee product H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 P15 P16 P17 P18 BB19 BB20 BB21 BB22
Overall acceptability 8±1 8±2 5±2 8±1 6±2 6±2 6±2 6±2 6±2 6±2 7±1 8±2 6±2 7±1 3.1±0.5 2.9±0.7 2.8±0.3 3.1±0.6 3.6±0.5 3.8±0.8 3.3±0.4 3.9±0.9
Neutral 0.30±0.01 0.38±0.03 0.33±0.04 0.25±0.03 0.26±0.04 0.27±0.06 0.33±0.04 0.31±0.03 0.35±0.03 0.27±0.04 0.29±0.01 0.28±0.03 0.26±0.04 0.33±0.06 0.25±0.06 0.27±0.08 0.32±0.06 0.28±0.08 0.23±0.02 0.27±0.04 0.28±0.04 0.25±0.03
Happy 0.04±0.01 0.01±0.001 0.02±0.01 0.01±0.001 0.04±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.01±0.01 0.00±0.00 0.02±0.01 0.02±0.01 0.03±0.01 0.05±0.02 0.03±0.01 0.07±0.02 0.1±0.04 0.03±0.01 0.03±0.01 0.01±0.00 0.01±0.00 0.02±0.01
Sad 0.03±0.01 0.03±0.01 0.02±0.01 0.06±0.02 0.02±0.01 0.02±0.01 0.08±0.01 0.10±0.03 0.05±0.02 0.04±0.01 0.08±0.01 0.09±0.02 0.07±0.02 0.05±0.03 0.05±0.01 0.05±0.01 0.04±0.01 0.10±0.03 0.11±0.01 0.10±0.02 0.07±0.03 0.15±0.02
Emotions induced by the bee products (from 0 to 1) Angry Surprised Scared Disgusted 0.03±0.01 0.35±0.03 0.14±0.02 0.03±0.1 0.07±0.01 0.33±0.04 0.09±0.02 0.01±0.001 0.11±0.02 0.39±0.05 0.09±0.03 0.04±0.01 0.09±0.01 0.39±0.06 0.11±0.01 0.01±0.00 0.05±0.02 0.34±0.05 0.13±0.01 0.02±0.01 0.01±0.00 0.44±0.02 0.09±0.01 0.03±0.01 0.02±0.01 0.24±0.05 0.09±0.02 0.08±0.01 0.00±0.00 0.33±0.04 0.07±0.02 0.06±0.01 0.01±0.00 0.23±0.04 0.16±0.03 0.10±0.01 0.07±0.02 0.40±0.06 0.11±0.03 0.09±0.02 0.02±0.01 0.43±0.02 0.03±0.01 0.06±0.01 0.01±0.00 0.33±0.06 0.14±0.04 0.02±0.01 0.01±0.00 0.47±0.06 0.07±0.02 0.01±0.001 0.02±0.01 0.25±0.04 0.15±0.03 0.02±0.01 0.06±0.02 0.39±0.07 0.19±0.04 0.06±0.02 0.01±0.00 0.41±0.04 0.07±0.02 0.01±0.00 0.01±0.00 0.20±0.05 0.04±0.01 0.05±0.02 0.03±0.01 0.34±0.04 0.07±0.03 0.05±0.01 0.05±0.02 0.32±0.02 0.15±0.01 0.03±0.01 0.01±0.00 0.46±0.02 0.05±0.02 0.02±0.01 0.00±0.00 0.43±0.02 0.13±0.02 0.03±0.01 0.11±0.02 0.33±0.04 0.08±0.02 0.0±0.01 Biogenic amines, mg/kg CAD HIS TYR SPER nd nd 1.8±0.1 nd nd nd nd nd nd nd nd nd nd 2.3±0.2 nd nd nd nd nd nd nd nd nd nd 2.8±0.2 nd nd nd 5.1±0.2 nd 1.8±0.1 2.5±0.3 nd nd 14.5±0.3 nd
Contempt 0.01±0.01 0.03±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.02±0.01 0.03±0.01 0.00±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00
Valence -0.16±0.02 -0.16±0.02 -0.21±0.03 -0.24±0.03 -0.16±0.03 -0.11±0.01 -0.22±0.03 -0.19±0.03 -0.26±0.04 -0.27±0.05 -0.15±0.01 -0.19±0.02 -0.12±0.03 -0.15±0.03 -0.15±0.06 -0.06±0.02 0.06±0.02 -0.19±0.06 -0.28±0.04 -0.15±0.02 -0.20±0.05 -0.27±0.08
Arousal 0.49±0.04 0.41±0.03 0.37±0.04 0.38±0.04 0.33±0.02 0.36±0.04 0.34±0.03 0.29±0.04 0.30±0.05 0.31±0.06 0.34±0.02 0.39±0.03 0.36±0.04 0.35±0.03 0.47±0.07 0.33±0.04 0.27±0.01 0.33±0.04 0.32±0.03 0.37±0.03 0.33±0.06 0.37±0.04
TRY PHE PUT SPRMD Total content H2 2.1±0.2 nd nd nd 3.9 H4 1.8±0.1 nd nd nd 1.8 H5 1.5±0.1 nd nd nd 1.5 H6 nd nd 2.8±0.2 nd 5.1 H8 nd nd 14.4±0.3 nd 14.4 H11 nd nd 13.5±0.2 nd 13.5 BB19 nd 2.2±0.2 nd nd 5.0 BB20 nd nd 1.8±0.1 nd 11.2 P17 nd nd nd nd 14.5 Data expressed as mean ± standard deviation (n = 3). H - honey, P – propolis, BB - bee bread. TRY - tryptamine, PHE - phenylethylamine, PUT - putrescine, CAD - cadaverine, HIS - histamine, TYR - tyramine, SPER – spermine, SPRMD – spermidine; nd – not determined.
Figures Fig. 1. Flowchart for applying FaceReader 5 software (Noldus Information Technology, Wageningen, The Netherlands) to scale the 10 emotion responses (neutral, happy, sad, angry, surprised, scared, disgusted, contempt, valence, arousal) to the bee products.
Figure 1 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561
HIGHLIGHTS
All the tested bee products showed antimicrobial activities The highest content of total phenolic compounds (TPC) was found in polyfloral bee bread The acceptability of bee products was influenced by the colour and the TPC The emotional evaluation of bee products with FaceReader was performed. Biogenic amines were identified in six honeys, two bee breads and one propolis
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0023-6438(19)31135-1
DOI:
https://doi.org/10.1016/j.lwt.2019.108793
Reference:
YFSTL 108793
To appear in:
LWT - Food Science and Technology
Received Date: 13 May 2019 Revised Date:
27 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Bartkiene, E., Lele, V., Sakiene, V., Zavistanaviciute, P., Zokaityte, E., Dauksiene, A., Jagminas, P., Klupsaite, D., Bliznikas, S., Ruzauskas, M., Variations of the antimicrobial, antioxidant, sensory attributes and biogenic amines content in Lithuania-derived bee products, LWT Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108793. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Variations of the antimicrobial, antioxidant, sensory attributes and biogenic
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amines content in Lithuania-derived bee products
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Elena Bartkiene*, Vita Lele, Vytaute Sakiene, Paulina Zavistanaviciute, Egle Zokaityte, Agila
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Dauksiene, Povilas Jagminas, Dovile Klupsaite, Saulius Bliznikas, Modestas Ruzauskas
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Lithuanian University of Health Sciences, Tilzes str. 18, LT-47181 Kaunas, Lithuania
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*Corresponding author: Elena Bartkiene, Lithuanian University of Health Sciences, Tilzes str.
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18, LT-47181 Kaunas, Lithuania; tel.: +370 37 574565; fax: +370 37 300152,
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[email protected].
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Abstract
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This study examined the antimicrobial and antioxidant properties, overall acceptability (OA),
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including influence of the product-induced emotions, and biogenic amine (BA) content in
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fourteen honey (H1–14), four propolis (P15–18) and four bee bread (BB19–22) samples,
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collected in northwest Lithuania. The all tested bacteria were inhibited by summer honey H13.
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P18 inhibited 10, while bee breads inhibited 10-11 out of the 15 tested bacteria strains. All
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propolis- samples inhibited B.cereus and P.multocida. The highest antioxidant activity and
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content of total phenolic compounds (TPC) were found in bee bread (93% and 394 mg GAE/100
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g, respectively). The TPC had moderate and strong negative correlations with the L* (r=-0.58)
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and b* colour coordinates (r=-0.66), respectively. The each bee products group (BPs) induced
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different intensities of emotions, and the OA showed a moderate positive correlation with the
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“neutral” emotion (r=0.47). A low content of BAs (<15 mg/kg) was identified in six honeys, two
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bee breads and one propolis sample. In sum, Lithuanian bee products possessed valuable
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biological attributes that can be beneficially used in food industry and medicine, although further
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research is needed for the factors, which may contribute to bioactive properties of this region bee
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products and BAs formation.
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Keywords: honey, propolis, bee bread, bioactivity, FaceReader.
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1. Introduction
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Bioactive substances of natural origin attract great interest and are especially appreciated by
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today’s consumers, whose preferences are trending toward natural functional foods (Kieliszek et
52
al., 2018). Among natural products containing bioactive ingredients, honey and other bee
53
products are highly popular as healthy alternatives to synthetic supplements. Bee products are
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well recognized for their health attributes, particularly, their potential healing properties, which
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differ according to the region, nectar source, climate, among other factors (Bobiș et al., 2010).
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For this reason, the desire to use these multicomponent natural substances contributes to the
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growth of interest in evaluating their properties. Bee products demonstrate a wide range of
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desirable characteristics, including antimicrobial and antioxidant properties, and the most
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popular bee products are honey, pollen and their extracts, bee bread, propolis, royal jelly and bee
60
venom. Honey is used not only as a nutritional product but also in traditional medicine and as an
61
alternative treatment for clinical conditions because it has antioxidant, anti-inflammatory,
62
antibacterial, antidiabetic, respiratory, gastrointestinal, cardiovascular and nervous system
63
protective effects (Samarghandian, Farkhondeh, & Samini, 2017). Another bee product that
64
possesses health benefits is propolis. This product contains a high number of compounds with
65
anti-inflammatory, antioxidant, antiviral and antimicrobial properties (Al-Waili, Al-Ghamdi,
66
Ansari, Al-Attal, & Salom, 2012). Bees prepare and use propolis as a sealing material to protect
67
against the entry of microorganisms (fungi and bacteria) into the hive, and create the most sterile
68
environment known in nature (Al-Waili, Al-Ghamdi, Ansari, Al-Attal, & Salom, 2012). The
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main pharmacological activities of propolis are related to the flavonoids and phenolic
70
compounds—the major bioactive constituents of propolis (Banskota et al., 2000; Anthimidou &
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Mossialos, 2013). The properties of the propolis flavonoids to reduce the formation or to remove
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free radicals allow effective regeneration of damaged tissue and the antimicrobial properties of
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propolis prevent from wound infection (Boyanova, Kolarov, Gergova, & Mitov, 2006; Kasiotis,
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Anastasiadou, Papadopoulos, & Machera, 2017). Propolis is very popular and is used in a variety
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of commercial preparations, including pharmaceutical, nutraceutical and cosmetic products (El-
76
Guendouz, Lyoussi, & Miguel, 2019). Being a mixture of many compounds, propolis is not
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easily subject to extraction and fractionation procedures; therefore, it is usually used as a whole
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ingredient. The composition of raw propolis may vary, depending on geographical and seasonal
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factors, because it depends on the flora of the areas from which it is collected (El-Guendouz,
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Lyoussi, & Miguel, 2019).
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Another bee product, bee bread, is characterized by a high nutritional value, good digestibility
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and rich chemical composition (da Silva, de Souza, Matta, de Andrade, & Vidal, 2006; Habryka,
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Kruczek, & Drygaś, 2016), attributed to the partial fermentation of the bee bread components,
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which, in turn, are more easily assimilated in an organism (Barene, Daberte, & Siksna, 2015).
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Bee bread contains peptides and free amino acids and is an excellent product that could
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supplement nutrient deficiencies in humans to achieve a balanced diet (Nagai, Nagashima,
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Myoda, & Inoue, 2004). Bee bread contains antioxidants (e.g., carotenoids) and natural
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preservatives (e.g., lactic acid) (Barene, Daberte, & Siksna, 2015). Nonetheless, it should be
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mentioned that the presence of free amino acids in bee products can lead to the formation of
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biogenic amines (BAs), which are undesirable compounds in food products (Ruiz-Capillas &
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Herrero, 2019).
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As stated above, many factors influence bee product characteristics. Since their main quality
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parameters necessary to meet the required regulations for commercialization in the local market
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of Lithuania have already been investigated, the current research focused on the evaluation of
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specific characteristics, such as the antioxidant and antimicrobial activities, as well as the BA
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content. Moreover, the overall acceptability, including the influence of the products induced
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emotions (neutral, happy, sad, angry, surprised, scared, disgusted, contempt, valence, arousal) on
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consumers of the Lithuania-derived honey, propolis and bee bread samples were evaluated.
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2. Materials and Methods
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2.1. Chemicals
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Sodium hydroxide, dansyl chloride, perchloric acid, sodium bicarbonate, ammonium hydroxide,
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acetonitrile (HPLC grade), ammonium acetate, sodium carbonate, gallic acid and 2,2-difenil-1-
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picrilhydrazyl (DPPH) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Folin-
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Ciocalteu reagent was obtained from Scharlau Chemie S. A. (Barcelona, Spain). Ethanol was
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from FarmaBalt (Riga, Latvia). Tryptamine hydrochloride, 2-phenylethylamine hydrochloride,
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1,4-diaminobutane dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride,
107
tyramine hydrochloride, spermidine phosphate salt hexahydrate and 1,7-diaminoheptane were
108
obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Spermine diphosphate hexahydrate
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was from TCI Europe (Tokyo, Japan). All reagents were of analytical grade.
110 111
2.2. Characteristics of the assessed bee products
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Table 1 provides the farm location and the time of year when the bee products (honey, crude
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propolis material and bee bread) used in this study were collected. In this experiment, the bee
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products were obtained from three farms located in northwest Lithuania. Bee bread [BB19–22]
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and propolis [P15–18] were collected during summer. Most of honey samples [H1, H5, H7–14]
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were also collected during summer, except H2 and H6, which were obtained in autumn, and H3
117
and H4, which were gathered in spring.
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119
Table 1
120 121
2.3. Evaluation of bee products antimicrobial properties
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Honey, propolis, and bee bread were investigated for their antimicrobial activities against a
123
variety of pathogenic and opportunistic bacterial strains (A. baumannii 17-380, B. cereus 1801,
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C. freundii, E. cloacae, E. faecalis 86, Enterococcus faecium 103, Klebsiella pneumoniae,
125
methicillin-resistant Staphylococcus aureus (MRSA) M87fox, P. multocida, P. mirabilis,
126
Pseudomonas aeruginosa 17-331, Salmonella enterica 24 SPn06, S. epidermidis, S.
127
haemolyticus and Streptococcus mutans). Bacterial strains were previously isolated from humans
128
and domestic animals and stored in the Lithuanian University of Health Sciences Collection of
129
Microorganisms. The agar well diffusion assay was used to evaluate the antimicrobial activities
130
of the bee products. For this purpose, a standardized 0.5 McFarland suspension of each
131
pathogenic bacteria strain was inoculated onto the surface of cooled Mueller–Hinton agar (Oxoid
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Limited, Basingstoke, Hampshire, UK) using sterile cotton swabs. Wells of 6 mm in diameter
133
were punched in the agar, and each well filled with 100 µL of the tested product sample, which
134
was prepared by dissolving 1 g of each bee product in 10 mL of saline solution (9 g/L). The
135
antimicrobial activities against tested bacteria were determined by measuring the inhibition zone
136
(IZ) diameter (mm). The experiments were repeated three times, and the average IZ was
137
calculated.
138 139
2.4. Determination of the total content of phenolic compounds and antioxidant activity of
140
bee products
141
1One gram of bee products was dissolved in 20 mL of aqueous ethanol (500 mL/L), extracted
142
shaking for 1-6 h (1 h for honey and 6 h for bee bread and propolis) at room temperature and
143
then filtered through paper filter. These extracts were further used for total content of phenolic
144
compounds (TPC) and antioxidant activity analysis. The TPC in bee product samples was
145
measured using the Folin-Ciocalteu colorimetric method (Vaher, Matso, Levandi, Helmja, &
146
Kaljurand, 2010 Singleton, Orthofer, & Lamuela-Raventos, 1999). One mL of each extract was
147
mixed with 5 mL of Folin-Ciocalteau (100 mL/L) reagent and 4 mL of 75 g/L NaHCO3. The
148
mixture was allowed to stand at room temperature for 30 min. The absorbance of each sample
149
was measured at 765 nm using a V-1100D spectrophotometer (JP Selecta SA, Barcelona, Spain).
150
The concentration of phenolics was calculated from the calibration curve and the results were
151
expressed as gallic acid equivalent (GAE) in mg/100 gram of raw material.
152
Antioxidant activity of the bee products was evaluated according to the method described by
153
Brand-Williams, Cuvelier, & Berset (1995) with some modifications. DPPH solution (3.6 mL;
154
0.1 mmol/L, in ethanol) was mixed with 0.66 mL of sample dissolved in the aqueous ethanol.
155
After 20 min of shaken shaking in the dark at room temperature, the absorbance at 517 nm was
156
measured. The inhibition of the DPPH radical by the sample was calculated according to the
157
following formula:
158
DPPH (%) = (Acontrol - Asample) / Acontrol x 100,
159
where Acontrol was obtained by replacing the sample with ethanol.
160 161
2.5. Evaluation of the bee products colour coordinates and overall acceptability
162
The colour coordinates (L*, a*, b*) were assessed using a CIELAB system (Chromameter CR-
163
400, Konica Minolta Sensing, Inc., Osaka, Japan). The overall acceptability of the bee products
164
was carried out by 50 judges, according to the ISO 11136:2014 method using a 10-point scale,
165
ranging from 0 (extremely dislike) to 10 (extremely like). Also, the bee products were tested by
166
applying FaceReader 5 software (Noldus Information Technology, Wageningen, The
167
Netherlands), scaling the 10 emotion patterns (neutral, happy, sad, angry, surprised, scared,
168
disgusted, contempt, valence, arousal). The judges were asked to taste the presented bee products
169
one by one in front of a Microsoft LifeCam Studio webcam (Microsoft Corporation, Redmond,
170
Washington, USA). The recordings were analysed with FaceReader 5 software and intensity of
171
facial expressions was expressed in a scale from 0 to 1. Between the samples, the judges were
172
asked to rinse the mouth with water. Figure 1 illustrates the experimental design used to assess
173
the emotions induced by different bee products.
174
Figure 1
175
2.6. Evaluation of biogenic amine (BA) content in bee products
176
Sample preparation and determination of the BAs, including cadaverine (CAD), histamine (HIS),
177
phenylethylamine (PHE), putrescine (PUT), spermidine (SPRMD), spermine (SPER), tryptamine
178
(TRY) and tyramine (TYR), in bee products was achieved by following the procedure reported
179
by Ben‐Gigirey et al. (1998) with some modifications. The standard BA solutions were
180
prepared by dissolving known amounts of each BAs (including internal standard) in 20 mL of
181
deionized water Briefly, the extraction of BAs in samples (5 g) was done by using 0.4 mol/L
182
perchloric acid. The derivatization of sample extracts and standards was performed using a
183
dansyl chloride solution (10 mg/mL) as a reagent. The chromatographic analyses were carried
184
out using a Varian ProStar HPLC system (Varian Corp., Palo Alto, California, USA) with two
185
ProStar 210 pumps, a ProStar 410 autosampler, a ProStar 325 UV/VIS Detector and Galaxy
186
software (Agilent, Santa Clara, California, USA) for data processing. For the separation of
187
amines, a Discovery ® HS C18 column (150 x 4.6 mm, 5 µm; SupelcoTM Analytical,
188
Bellefonte, Pennsylvania, USA) was used. The eluents were ammonium acetate (A) and
189
acetonitrile (B) and the elution program consisted of a gradient system with a 0.8 mL/min flow-
190
rate. The detection wavelength was set to 254 nm, the oven temperature was 40°C and samples
191
were injected in 20 µL aliquots. The target compounds were identified based on their retention
192
times in comparison to their corresponding standards.
193
2.7. Statistical analysis
194
All analyses were undertaken at least in triplicate. Non-parametric Kruskal Wallis test followed
195
by Dunn's post hoc test were used for data analysis. P < 0.05 was considered statistically
196
significant. Statistics were performed with SPSS for Windows XP V15.0 (SPSS, Inc., Chicago,
197
Illinois, USA, 2007).
198 199
3. Results and Discussion
200
3.1. Antimicrobial properties of the bee products
201
Table 2 presents the IZs of the bee products against the various pathogenic and opportunistic
202
strains examined. All the tested pathogenic strains were inhibited by H13 (summer honey). In
203
comparison, the other summer honeys showed a narrower inhibition spectrum against the
204
pathogens: of the 15 pathogenic strains tested, five (H1), eight (H7), ten (H8), eleven (H9),
205
fourteen (H11) and thirteen (H14) were susceptible to the antimicrobial action of the honeys,
206
respectively. All of the summer honeys exhibited antimicrobial activity against MRSA. The most
207
limited antimicrobial spectrum activity (inhibitory action was shown against only one of the
208
tested pathogenic strains) was displayed by the spring honeys (H3 and H4: both showed an
209
average IZ for P. multocida of 12.5 mm. Autumn honeys inhibited more pathogens compared
210
with the spring honeys, and their average IZ against A. baumannii 17-380, MRSA M87fox, S.
211
epidermidis, S. haemolyticus and P. multocida were 12.1, 14.3, 12.4, 12.6 and 20.3 mm,
212
respectively. The honey H5 showed inhibitory action against the same pathogens as the autumn
213
honeys, and with similar potency, providing average IZ values against A. baumannii 17-380,
214
MRSA M87fox, S. epidermidis, S. haemolyticus and P. multocida of 12.1, 15.2, 12.3, 14.5 and
215
21.7 mm, respectively. H6 and H12 honeys each inhibited 10 out of the 15 tested pathogenic
216
strains. Both of these products (H6 and H12) inhibited K. pneumoniae, A. baumannii 17-380, P.
217
mirabilis, MRSA M87fox, C. freundii, S. epidermidis, S. haemolyticus, and P. multocida.
218
Additionally, H6 inhibited P. aeruginosa 17-331 and B. cereus 18 01 while H12 also inhibited S.
219
enterica 24 SPn06 and E. cloacae.
220
Honey, especially medical Manuka honey, has a broad-spectrum of antibacterial activity against
221
both Gram-positive and Gram-negative microorganisms, including MRSA and vancomycin-
222
resistant Enterococcus, and, also, against some fungi and virus (Gonçalves et al., 2018; Fyfe,
223
Okoro, Paterson, Coyle, & McDougall, 2017). Different explanations for the antimicrobial
224
properties of honey have been mentioned in the literature. One suggestion is that the α-
225
glucosidase is critical for decomposing saccharose into fructose and glucose, and glucose
226
oxidase catalyses the conversion of glucose to gluconic acid, producing hydrogen peroxide,
227
which is related to honey’s antibacterial activity (Faustino & Pinheiro, 2015). Some other factors
228
influencing antimicrobial properties of honey are high osmolarity, low water activity and pH,
229
and high viscosity (Morroni et al., 2018; Cianciosi et al., 2018). The antimicrobial activity of
230
honey has also been associated with polyphenols (methyl syringate, phenyllactic acid),
231
flavonoids (quercetin, kaempferol) and synergies with other components (fatty diacids, abscisic
232
acid and methylglyoxal) (Fyfe, Okoro, Paterson, Coyle, & McDougall, 2017; Cianciosi et al.,
233
2018). Gonçalves et al. (2018) noted that the bioactivity (antimicrobial and antioxidant) and
234
physicochemical parameters of selected Portuguese monofloral honeys were correlated and
235
depended on the honey floral source. Heather honey was not only the darkest of the honeys
236
examined but also indicated the greatest antimicrobial and antioxidant properties, which were
237
explained by its higher levels of protein, flavonoids and phenolic compounds. Alvarez-Suarez et
238
al. (2018) concluded that different physicochemical parameters can be the reason for the higher
239
antimicrobial activity of M. beecheii honey compared to A. mellifera honey.
240
All propolis samples (P15–18) inhibited B. cereus 18 01 and P. multocida. However, these were
241
the only strains inhibited by P16 and P17, and P15 only inhibited one other (MRSA M87fox).
242
P18 showed the broadest antimicrobial spectrum of the propolis samples, inhibiting 10 of the 15
243
tested pathogenic strains. Besides those inhibited by P15, mentioned above, P18 was active
244
against A. baumannii 17-380, C. freundii, E. cloacae, E. faecalis 86, P. mirabilis, S. epidermidis
245
and S. haemolyticus, and the largest IZ against P. multocida.
246
The antimicrobial properties of propolis are mainly afforded by the flavonoids and cinnamic acid
247
derivatives present in this complex mixture of natural substances (Moradkhannejhad, Abdouss,
248
Nikfarjam, Mazinani, & Heydari, 2018), which varies according to the different areas and
249
seasons, as the composition is dictated by the constituents of the plants in the area. However, the
250
composition of two different propolis can be very promising, as exhibited antimicrobial activity
251
against Gram-positive and Gram-negative bacteria, as well as Candida albicans (Bryan, Redden,
252
& Traba, 2016). Przybyłek & Karpiński (2019) summarized that the antibacterial activity of
253
propolis against Gram-positive bacteria was greater than Gram-negative. Moreover, the highest
254
activity of propolis from the Middle East was found, while samples from Germany, Ireland and
255
Korea had the lowest activity. Various mechanisms of action have been proposed for the
256
inhibitory effect of propolis against bacteria. One explanation is that propolis physically damages
257
the bacterial cell membranes and initiates cell lysis, eventually leading to cell death (Bryan,
258
Redden, & Traba, 2016). In other work, the antimicrobial activity relies on the antibacterial
259
compounds in propolis (phenolic compounds, terpenes, caffeic, ferulic and coumaric acids, esters
260
and flavonoids) (Inui et al., 2014; Veiga et al., 2017). Furthermore, propolis contains flavonoids
261
and phenolic compounds, which have anti-inflammatory action (Funakoshi-Tago et al., 2016).
262
Extracts of propolis have also displayed antibacterial properties based on agar diffusion assays or
263
experiments in liquid broths (Kim & Chung, 2011; Akhir, Adawieah, Bakar, Fadzelly, & Sanusi,
264
2018).
265
All of the tested bee breads (BB19–22) showed antimicrobial activity against A. baumannii 17-
266
380, B. cereus 18 01, C. freundii, E, cloacae, E. faecalis 86, MRSA M87fox, P. multocida, P.
267
mirabilis, S. epidermidis and S. haemolyticus. In addition, BB21 inhibited E. faecium 103, and
268
BB22 inhibited S. mutans.
269
The study of Bakour et al. (2019) has demonstrated that Moroccan bee bread inhibits the growth
270
of a broad spectrum bacteria and fungi, and this activity is higher against Gram-positive than
271
Gram-negative bacteria. The antimicrobial activity of bee bread can be largely attributed to the
272
high content of phenolic compounds, especially flavonoids and phenolic acids (Bakour et al.,
273
2019).
274
Table 2
275
3.2. Total content of phenolic compounds and antioxidant activity of honey, propolis, and
276
bee bread
277
The TPC and antioxidant activity of the bee products (honey, propolis, bee bread) are shown in
278
Table 3. The average TPC in summer honeys was 243.2 mg GAE/100 g, and H10 possessed the
279
highest TPC among the honey samples while H8 had the lowest. In comparing the average TPC
280
between summer and spring honeys, spring honeys had 25 % less TPC. In H5, H6 and H12
281
honeys, the TPC was 217.6 – 263.5 mg GAE/100 g. Among the propolis samples, P16 had the
282
greatest TPC, and in P15, P17 and P18, the TPC were 17%, 7% and 29% lower, respectively. A
283
comparison of the bee breads revealed the highest TPC in BB21, which was 6%, 22% and 21%,
284
higher than that in BB19, BB20 and BB22, respectively.
285
Summer honey H13 had the lowest antioxidant activity, while the highest one was established in
286
autumn H6. The other autumn honey H2 had an antioxidant activity, which was slightly less than
287
that demonstrated by the spring honeys (on average 6 % lower), and H5 and H6 samples (7 and
288
10 % lower, respectively). Of the propolis samples, P18 recorded the highest antioxidant activity,
289
which was on average 2.3-fold higher than that of the remaining propolis samples. Bee bread
290
antioxidant activity was on average 90 %, with a minimum of BB20 and maximum of BB22.
291
Such kinds of honey as Manuka or Strawberry tree can provide a strong antioxidant activity and
292
even act as chemopreventive agents (Afrin et al., 2017). It is known that phenolic compounds are
293
mainly responsible for the beneficial effect of bee products (Cianciosi et al., 2018). The nectar
294
source, phenolic acids, flavonoids, ascorbic acid, carotenoids, amino acid, protein content and
295
Maillard reaction products, have been described as having a strong impact on the antioxidant
296
activity of bee products (Fernandes, Ferreira, Fonte, Wessel, & Cardoso, 2016). Due to different
297
botanical and geographical sample origin, extraction technique and analysis conditions, various
298
results of TPC and antioxidant activity of bee products can be found in the literature. In our study
299
obtained values of TPC for all tested bee products were higher than values reported for African,
300
Manuka, Strawberry tree and Cuban polifloral honeys (Morroni et al., 2018; Afrin et al., 2017;
301
Alvarez-Suarez et al., 2018). Our results are in the agreement with some studies showing that bee
302
bread presents higher antioxidant activity than propolis (Akhir, Adawieah, Bakar, Fadzelly, &
303
Sanusi, 2018; Suriyatem, Auras, Intipunya, & Rachtanapun, 2017). However, results of this
304
study showed a weak positive correlation between the TPC and antioxidant activity only of the
305
bee bread was found (r = 0.39). A significant linear correlation between TPC and antioxidant
306
activity in honey and honey supplemented with other bee products was mentioned by Juszczak et
307
al. (2016) and by Fernandes et al. (2016) in propolis.
308 309
Table 3
310
3.3. Colour coordinates of the bee products
311
The colour coordinates (L*, a*, b*) of the bee products (honey, propolis and bee bread) are listed
312
in Table 3. The lightness (L*) coordinate ranges from 0 (dark) to 100 (light). Considering the
313
three types of bee products, H4 (spring honey), P15 and B22 were the lightest, and the darkest
314
products in each category were H14 (summer honey), P18 and BB21.
315
The redness–greenness (a* coordinate) of all honeys ranged from 0.10 (H8, summer honey) to
316
5.79 (H3, spring honey). Both spring honeys (H3 and H4) showed significantly higher a*
317
coordinates than the other honey samples tested. Of the propolis samples, P17 and P18 had
318
higher a* coordinates relative to those of P15 and P16. The bee breads had a* coordinates
319
ranging from 3.22 (BB21) to 6.49 (BB20). The b* (blueness–yellowness) coordinates of the
320
honeys ranged from 8.14 (H4) to 29.59 (H8). For the propolis and bee bread, P21 and BB21
321
were found to have the lowest b* coordinates.
322
Correlations of different strengths were identified between each colour coordinate (L*, a*, b*)
323
and the TPC in bee products. A very weak positive correlation between the TPC and a*
324
coordinate was established (r = 0.15) while the TPC had a moderate negative correlation with the
325
L* coordinate (r = -0.58) and a strong negative correlation with the b* coordinate (r = -0.66).
326
Literature data suggest that the darker honeys typically having a higher concentration of
327
polyphenols and higher antioxidant, as well as antimicrobial activities (da Silva, de Souza,
328
Matta, de Andrade, & Vidal, 2006).
329 330
3.4. Overall acceptability and the emotions induced by the tested bee products
331
Overall acceptability and the emotions induced by the bee products are presented in Table 4. The
332
summer honeys scored an average of 6.5 points for overall acceptability, and H1 was assigned
333
the highest overall acceptability. Significant differences existed between the overall acceptability
334
scores given to the spring honeys, as exemplified by H3 and H4. Among H5, H6 and H12, the
335
most acceptable was the latter one, and the H5 and H6 samples scores indicated 26% and 23%,
336
respectively, lower overall acceptability. When comparing all the tested honeys, H4 (spring
337
honey) had the highest overall acceptability. There were no significant differences in the overall
338
acceptability among the propolis samples, as well as between the bee breads.
339
The overall acceptability of the bee products was influenced by their colour characteristics and
340
TPC. Positive strong (for L*) and moderate (for b*) correlations between the bee products
341
overall acceptability and lightness (r = 0.63) and blueness–yellowness (r = 0.57) were observed.
342
Also, negative moderate and weak correlations between the bee products overall acceptability
343
and TPC (r = -0.46) and a* colour coordinate (r = -0.34), respectively, were noted.
344
Usually, the traditional sensory analysis tests are not able to sufficiently predict market
345
performance because most of these test techniques are based on self-reports and, therefore,
346
underlie a conscious/rational decision-making process (Danner, Sidorkina, Joechl, &
347
Duerrschmid, 2014; Köster, 2009). Consequently, the long-term consumer acceptance for special
348
foods is not adequately reflected by using traditional methods (Köster, 2009). In comparison,
349
FaceReader 5 software provides a more accurate acceptability analysis of new products. In the
350
current work, different tendencies of the emotions induced by the different bee products were
351
found, and a moderate positive correlation between the overall acceptability of the bee products
352
and emotion “neutral” was realized (r = 0.47). Between the other evaluated emotions and overall
353
acceptability, weak and very weak correlations were found: negative weak correlations between
354
the overall acceptability and emotions “happy” (r = -0.28), “sad” (r = -0.24), “valence” (r = -
355
0.24); positive weak correlation between the overall acceptability and emotion “scared” (r =
356
0.26); positive very weak correlations between the overall acceptability and emotions “angry” (r
357
= 0.19), “surprised” (r = 0.14), “disgusted” (r = 0.06), “contempt” (r = 0.11) and “arousal” (r =
358
0.19).
359
Table 4
360
3.5. Biogenic amines (BAs) in bee products
361
The BAs content determined in each of the bee product samples is provided in Table 4. A
362
comparison of all the tested bee products (H1–14, P15–18, BB19–22) indicated some of the
363
analysed BAs were present in 6 of the 14 honeys, 2 of the 4 bee breads, and 1 of the 4 propolis
364
samples. In honey samples containing BAs, the highest total BA contents were recorded in two
365
of the summer honeys H8 and H11, with PUT the dominant BA. TRY occurred in three honey
366
samples (H2, H4 and H5). H6 contained small contents of PUT and HIS. TYR was detected in
367
one honey sample. Among the bee breads, PHE and CAD were identified in BB19, and in BB20,
368
the predominant BAs were PUT, CAD and TYR. In propolis samples, only TYR in P17 was
369
found.
370
Honey contains minor concentrations of bioactive compounds, such as phenolic acids,
371
flavonoids, carotenoids, amino acids (mainly proline), proteins (including enzymes) and pollen
372
grains (Gonçalves et al., 2018). BAs can occur in almost all foods that contain proteases or free
373
amino acids and which are subject to the conditions enabling microbial and biochemical activity
374
(Papageorgiou et al., 2018). HIS and TYR are recognized as the most toxic BAs by The
375
European Food Safety Authority (Official Journal of the European Union, 2005). It is known that
376
the toxicity of BAs depends on synergistic effects, for instance, HIS toxicity is enhanced by the
377
presence of CAD, PUT and TYR (Mantis et al., 2005). However, there is not enough data about
378
the toxic dose of each BAs, that is why little legislation exists on BAs risk on human health and
379
only the content of histamine in fish food is controlled by law (Papageorgiou et al., 2018).
380
According to our results, higher amounts of TYR and PUT were detected in propolis P17 and
381
several honey samples (H8, H11), respectively. The information about BAs presence in bee
382
products is scare. So far, only bee venom is described as a rich source of BAs (Eze, Nwodo, &
383
Ogugua, 2016). Therefore, further research on BAs in bee products (especially in bee bread) is
384
needed; particularly on the toxicity and acceptable concentrations, as these products are usually
385
presented as healthy foods.
386 387
4. Conclusion
388
Nowadays, honey and its products are becoming valued natural substances and their
389
physicochemical and biological properties are affected by many factors. Therefore, it is
390
important to expand the information and explore the biological activities of recent products from
391
different geographic regions. This study examined the specific characteristics, such as the
392
antioxidant and antimicrobial activities, as well as the biogenic amines and product-induced
393
emotions of honey, propolis and bee bread from the northwest Lithuania. The findings revealed
394
that all analysed bee products had antimicrobial and antioxidant activities; however, this
395
characteristics varied between samples and product groups. The overall acceptability of the bee
396
products was influenced by the colour characteristics and the TPC. BAs were found in some
397
samples from each product group, however the total content was very low. In conclusion, the
398
tested Lithuanian honey, propolis and bee bread had valuable biological properties that can be
399
beneficially used in food industry and medicine, although further research is needed for the
400
factors, which may contribute to bioactive properties and BAs formation of this region bee
401
products.
402 403 404
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Tables Table 1. Characteristics of the analysed bee products (honey [H1-14], propolis [P15-18], bee bread [BB19-22]). Farm name and location in Lithuania
Sample
P15 P16
P. Jagmino farm, Taurages district, Batakių city
Honey
N. Litvino farm, Kretinga district, Salantų city
G. Puska farm, Mazeikių district, Serksnenų city
Prop olis
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14
P. Jagmino farm, Taurages district, Batakių city G. Puska farm, Mazeikių
Season the bee products were collected Summer Autumn Spring Spring Summer Autumn Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer
P17
BB19 BB20 BB21 BB22
Bee bread
P18
district, Serksnenų city N. Litvino farm, Kretinga district, Salantų city P. Jagmino farm, Taurages district, Batakių city N. Litvino farm, Kretinga district, Salantų city G. Puska farm, Mazeikių district, Serksnenų city
Summer Summer Summer Summer Summer Summer
Table 2. Antimicrobial activity of the bee products (honey [H1—14], propolis [P15–18], bee bread [BB19–22]).
Enterobacter cloacae
Citrobacter freundii
Staphylococcus epidermidis
Staphylococcus haemolyticus
-
-
-
-
-
8.6±0.3
13.8±0.4
19.4±0.5
-
-
-
-
-
-
12.4±0.2
12.6±0.2
20.3±0.4
H3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.6±0.3
H4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.4±0.3
H5
-
-
-
12.1±0.4
-
15.2±0.3
-
-
-
-
-
-
12.3±0.2
14.5±0.2
21.7±0.3
H6
8.5±0.2
-
7.2±0.1
11.6±0.2
11.9±0.4
14,6±0.5
-
-
8.5±0.2
-
-
6.7±0.1
17.6±0.2
13.6±0.3
22.8±0.4
H7
9.3±0.2
-
-
10.5±0.1
9.8±0.1
11.2±0.2
-
-
-
-
-
8.6±0.1
12.3±0.4
11.6±0.2
17.6±0.3
H8
-
9.4±0.1
12.3±0.2
11.7±0.2
16.3±0.2
7.6±0.2
7.1±0.2
-
-
-
8.3±0.2
26.8±0.4
14.3±0.2
22.9±0.3
H9
9.2±0.3
8.8±0.2
9.4±0.3
12.8±0.3
11.3±0.2
16.8±0.3
-
-
8.4±0.3
-
-
8.2±0.1
20.1±0.2
14.7±0.3
21.3±0.4
H10
8.1±0.2
8.3±0.2
8.1±0.2
12.0±0.4
11.6±0.3
17.9±0.3
-
7.3±0.1
9.2±0.4
-
-
8.8±0.1
21.5±0.3
16.1±0.2
21.2±0.3
Pasteurella multocida
Streptococcus mutans
-
14.3±0.4
Enterococcus faecium 103
13.5±0.3
-
Enterococcus faecalis 86
-
12.1±0.3
MRSA M87fox
11.2±0.4
-
Proteus mirabilis
-
-
Acinetobacter baumannii 17-380
-
-
Pseudomonas aeruginosa 17-331
-
H2
Salmonella enterica 24 SPn06
H1
Bee products
Klebsiella pneumoniae
Bacillus cereus 18 01
Pathogenic and opportunistic bacterial strains
Inhibition zone, mm
H11
7.6±0.2
7.4±0.1
7.3±0.1
-
11.5±0.3
13.6±0.4
8.2±0.1
9.6±0.2
8.1±0.2
15.7±0.2
8.0±0.2
8.6±0.1
19.6±0.3
15.3±0.4
22.4±0.5
H12
7.8±0.2
7.2±0.2
-
9.2±0.3
8.3±0.1
11.2±0.2
-
-
-
-
7.3±0.1
8.9±0.2
8.3±0.2
11.4±0.3
17.1±0.2
H13
7.3±0.1
8.7±0.3
7.6±0.3
11.3±0.2
11.8±0.2
13.6±0.3
8.3±0.1
8.2±0.3
10.3±0.2
15.6±0.3
7.5±0.3
9.2±0.2
17.4±0.2
16.9±0.3
21.3±0.4
H14
11.2±0.3
8.5±0.2
7.8±0.3
12.5±0.3
-
17.4±0.2
-
8.6±0.2
10.5±0.2
16.8±0.5
7.2±0.2
9.7±0.3
18.9±0.4
17.8±0.4
22.4±0.2
P15
-
-
-
-
-
7.5±0.1
-
-
7.6±0.3
-
-
-
-
-
12.5±0.2
P16
-
-
-
-
-
-
-
7.8±0.4
-
-
-
-
-
14.7±0.4
P17
-
-
-
-
-
-
-
7.4±0.2
-
-
-
-
-
11.6±0.1
P18
-
-
-
9.2±0.1
10.1±0.3
11.9±0.3
13.5±0.4
-
9.30.1
-
13.0±0.2
14.6±0.3
13.2±0.3
11.3±0.2
19.3±0.1
BB19
-
-
-
10.6±0.2
10.2±0.2
11.2±0.1
11.4±0.3
-
11.3±0.4
-
14.0±0.4
15.7±0.4
18.6±0.3
14.5±0.3
21.6±0.4
BB20
-
-
-
11.9±0.3
10.4±0.2
16.8±0.3
13.6±0.2
-
11.2±0.2
-
9.0±0.3
12.6±0.3
15.9±0.2
16.6±0.1
22.7±0.3
BB21
-
-
-
9.5±0.2
9.0±0.3
11.6±0.3
13.4±0.2
11.6±0.2
7.6±0.2
-
13.0±0.4
14.7±0.2
12.6±0.3
13.6±0.2
22.8±0.3
BB22
-
-
-
9.8±0.3
9.5±0.2
11.4±0.3
14.5±0.1
-
7.8±0.1
13.3±0.2
13.0±0.2
15.5±0.3
12.7±0.2
12.1±0.2
19.1±0.3
-
(-) ‒ not inhibited. Data expressed as mean ± standard deviation (n = 3). H - honey, P – propolis, BB - bee bread.
Table 3. The colour coordinates (L*, a*, b*), total content of phenolic compounds (mg GAE/100 g) and DPPH antioxidant activity (%) of the bee products (honey, propolis, bee bread). a* b* TPC, mg GAE/ 100 g NBS H1 56±3 2.9±0.2 21±3 251±6 H2 53±1 2.8±0.2 20±1 237±2 H3 64±3 5.8±0.2 29±1 196±1 H4 71±3 5.3±0.4 24±2 168±5 H5 48±5 0.3±0.1 24±2 242±3 H6 29±2 2.0±0.1 12±1 264±3 H7 54±3 1.5±0.1 22±1 250±3 H8 61±2 0.1±0.0 30±3 215±4 H9 43±2 0.4±0.0 21±2 251±5 H10 38±3 2.1±0.1 18±1 278±2 H11 44±3 0.2±0.1 21±2 237±6 H12 56±2 2.5±0.1 19±1 218±6 H13 59±3 2.6±0.2 26±2 220±6 H14 16±1 0.8±0.1 8±1 245±4 P15 31±2 3.6±0.2 14±1 249±6 P16 30±2 3.8±0.3 13±1 298±5 P17 26±1 5.1±0.1 12±1 276±4 P18 19±2 5.8±0.3 10.0±0.7 211±5 BB19 25±1 4.5±0.3 13.6±0.5 372±4 BB20 27±1 6.5±0.3 15±3 306±4 BB21 18±2 3.2±0.2 9.5±0.2 394±3 BB22 22±2 4.7±0.2 12.1±0.2 311±4 Data expressed as mean ± standard deviation (n = 3). TPC – total phenolic compounds DPPH – 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity. NBS – National Bureau of Standards units H – honey, P – propolis, BB – bee bread. Bee bread
Propolis
Honey
Bee product
L*
DPPH, % 83±3 79±3 84±4 84±4 85±2 88±6 81±4 77±5 74±3 77±5 82±4 76±5 65±5 66±4 32±4 35±2 40±2 80±4 92±3 85±3 91±4 93±4
Table 4. Overall acceptability, emotion response and biogenic amines in the bee products (honey [H1–14], propolis [P15–18], bee bread [BB19–22]). Bee product H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 P15 P16 P17 P18 BB19 BB20 BB21 BB22
Overall acceptability 8±1 8±2 5±2 8±1 6±2 6±2 6±2 6±2 6±2 6±2 7±1 8±2 6±2 7±1 3.1±0.5 2.9±0.7 2.8±0.3 3.1±0.6 3.6±0.5 3.8±0.8 3.3±0.4 3.9±0.9
Neutral 0.30±0.01 0.38±0.03 0.33±0.04 0.25±0.03 0.26±0.04 0.27±0.06 0.33±0.04 0.31±0.03 0.35±0.03 0.27±0.04 0.29±0.01 0.28±0.03 0.26±0.04 0.33±0.06 0.25±0.06 0.27±0.08 0.32±0.06 0.28±0.08 0.23±0.02 0.27±0.04 0.28±0.04 0.25±0.03
Happy 0.04±0.01 0.01±0.001 0.02±0.01 0.01±0.001 0.04±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.01±0.01 0.00±0.00 0.02±0.01 0.02±0.01 0.03±0.01 0.05±0.02 0.03±0.01 0.07±0.02 0.1±0.04 0.03±0.01 0.03±0.01 0.01±0.00 0.01±0.00 0.02±0.01
Sad 0.03±0.01 0.03±0.01 0.02±0.01 0.06±0.02 0.02±0.01 0.02±0.01 0.08±0.01 0.10±0.03 0.05±0.02 0.04±0.01 0.08±0.01 0.09±0.02 0.07±0.02 0.05±0.03 0.05±0.01 0.05±0.01 0.04±0.01 0.10±0.03 0.11±0.01 0.10±0.02 0.07±0.03 0.15±0.02
Emotions induced by the bee products (from 0 to 1) Angry Surprised Scared Disgusted 0.03±0.01 0.35±0.03 0.14±0.02 0.03±0.1 0.07±0.01 0.33±0.04 0.09±0.02 0.01±0.001 0.11±0.02 0.39±0.05 0.09±0.03 0.04±0.01 0.09±0.01 0.39±0.06 0.11±0.01 0.01±0.00 0.05±0.02 0.34±0.05 0.13±0.01 0.02±0.01 0.01±0.00 0.44±0.02 0.09±0.01 0.03±0.01 0.02±0.01 0.24±0.05 0.09±0.02 0.08±0.01 0.00±0.00 0.33±0.04 0.07±0.02 0.06±0.01 0.01±0.00 0.23±0.04 0.16±0.03 0.10±0.01 0.07±0.02 0.40±0.06 0.11±0.03 0.09±0.02 0.02±0.01 0.43±0.02 0.03±0.01 0.06±0.01 0.01±0.00 0.33±0.06 0.14±0.04 0.02±0.01 0.01±0.00 0.47±0.06 0.07±0.02 0.01±0.001 0.02±0.01 0.25±0.04 0.15±0.03 0.02±0.01 0.06±0.02 0.39±0.07 0.19±0.04 0.06±0.02 0.01±0.00 0.41±0.04 0.07±0.02 0.01±0.00 0.01±0.00 0.20±0.05 0.04±0.01 0.05±0.02 0.03±0.01 0.34±0.04 0.07±0.03 0.05±0.01 0.05±0.02 0.32±0.02 0.15±0.01 0.03±0.01 0.01±0.00 0.46±0.02 0.05±0.02 0.02±0.01 0.00±0.00 0.43±0.02 0.13±0.02 0.03±0.01 0.11±0.02 0.33±0.04 0.08±0.02 0.0±0.01 Biogenic amines, mg/kg CAD HIS TYR SPER nd nd 1.8±0.1 nd nd nd nd nd nd nd nd nd nd 2.3±0.2 nd nd nd nd nd nd nd nd nd nd 2.8±0.2 nd nd nd 5.1±0.2 nd 1.8±0.1 2.5±0.3 nd nd 14.5±0.3 nd
Contempt 0.01±0.01 0.03±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.02±0.01 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.02±0.01 0.03±0.01 0.00±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00
Valence -0.16±0.02 -0.16±0.02 -0.21±0.03 -0.24±0.03 -0.16±0.03 -0.11±0.01 -0.22±0.03 -0.19±0.03 -0.26±0.04 -0.27±0.05 -0.15±0.01 -0.19±0.02 -0.12±0.03 -0.15±0.03 -0.15±0.06 -0.06±0.02 0.06±0.02 -0.19±0.06 -0.28±0.04 -0.15±0.02 -0.20±0.05 -0.27±0.08
Arousal 0.49±0.04 0.41±0.03 0.37±0.04 0.38±0.04 0.33±0.02 0.36±0.04 0.34±0.03 0.29±0.04 0.30±0.05 0.31±0.06 0.34±0.02 0.39±0.03 0.36±0.04 0.35±0.03 0.47±0.07 0.33±0.04 0.27±0.01 0.33±0.04 0.32±0.03 0.37±0.03 0.33±0.06 0.37±0.04
TRY PHE PUT SPRMD Total content H2 2.1±0.2 nd nd nd 3.9 H4 1.8±0.1 nd nd nd 1.8 H5 1.5±0.1 nd nd nd 1.5 H6 nd nd 2.8±0.2 nd 5.1 H8 nd nd 14.4±0.3 nd 14.4 H11 nd nd 13.5±0.2 nd 13.5 BB19 nd 2.2±0.2 nd nd 5.0 BB20 nd nd 1.8±0.1 nd 11.2 P17 nd nd nd nd 14.5 Data expressed as mean ± standard deviation (n = 3). H - honey, P – propolis, BB - bee bread. TRY - tryptamine, PHE - phenylethylamine, PUT - putrescine, CAD - cadaverine, HIS - histamine, TYR - tyramine, SPER – spermine, SPRMD – spermidine; nd – not determined.
Figures Fig. 1. Flowchart for applying FaceReader 5 software (Noldus Information Technology, Wageningen, The Netherlands) to scale the 10 emotion responses (neutral, happy, sad, angry, surprised, scared, disgusted, contempt, valence, arousal) to the bee products.
Figure 1 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561
HIGHLIGHTS
All the tested bee products showed antimicrobial activities The highest content of total phenolic compounds (TPC) was found in polyfloral bee bread The acceptability of bee products was influenced by the colour and the TPC The emotional evaluation of bee products with FaceReader was performed. Biogenic amines were identified in six honeys, two bee breads and one propolis
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: