In-vitro bioaccessibility of antioxidant properties of bee pollen in Turkey

Abstract

The study aims to determine certain physicochemical properties (moisture, ash, titratable acidity, pH, and color), total phenolic content, antioxidant capacity (CUPRAC, ABTS, DPPH) and bioaccessibility of 20 bee pollen samples (10 unpackaged and 10 trademarked products in their original packages) sold in Turkey. The total phenolic content of the bee pollen samples was detected as 147.10-462.02 mg GAE/g and bioaccessibility ranged from 36.91% to 77.87%. The highest total value for antioxidant capacity was assessed as 83.24-257.27 μmol TE/g by the CUPRAC method. When the bioaccessibility of the antioxidant capacity of bee pollen was compared among the methods, the highest value (95.76%) was obtained for the sample M by the CUPRAC method. Antioxidant capacity and bioaccessibility of the unpackaged bee pollen samples were lower than those of the trademarked products. Based on overall evaluations, it can be deduced that bee pollen known for its potential beneficial effects on health may well be used as a functional food additive in the production of various food products with its high antioxidant and bioaccessibility features.

Keywords:
bee pollen; antioxidant capacity; phenolics; bioaccessibility; functional food

1 Introduction

Pollen, which is a valuable food source for bees and bee larvae, is the product of the male reproductive cells of flowering plants. Flower pollen powder sticks to the whole body of worker bees while strolling (Borycka et al., 2015). Bee pollen is the mixture of flower pollen powder and the digestive enzymes of worker bees, which are collected on their legs and transported to the hive; therefore it contains flower grains, bee enzymes, and nectar sugars. Bee pollens are harvested through pollen traps placed in the entrance of the hives provided that the welfare of worker bees is maintained (Fadzilah et al., 2017; Alicic et al., 2014).

Bee pollen has drawn substantial attention in recent years with its pharmacological properties such as antimicrobial, antioxidant, antifungicidal, antiradiation, anticancer, and anti-inflammatory. Moreover, its preventive effects on arteriosclerosis, gastroenteritis, respiratory diseases, prostatic disorders, and allergic sensitization have been reported. Cardiovascular, gastrointestinal, immune system stimulating effects as well as antiaging properties of bee pollen have also been indicated. It is currently used in the treatment of sniffles, the flu, anemia, allergic diseases and ulcer and enteritis. It is called “the life-giving dust” owing to its extensive therapeutic effects and recognized as one of the functional foods in the food industry (Borycka et al., 2015; Carpes et al., 2007; Karadal et al., 2018; Kacániová et al., 2012; Guiné, 2015; Fadzilah et al., 2017; Karkar et al., 2018; Pascoal et al., 2014).

Certain factors such as unhealthy diet, environmental and industrial pollution and smoking trigger the production of free radicals in the cells and thus inhibit immune defense mechanisms and antioxidant enzymes. Oxidation induced in the body by free radicals produced during this process leads to oxidative stress, which causes lipid peroxidation, enzymatic deactivation, and accelerates the aging process. Demand for natural food products with high antioxidant content to alleviate metabolic problems has increased in recent years. Bee pollen, as well, is one of the high-quality food products with free-radical scavenging activity (Özkök & Silici, 2017; Čeksteryté et al., 2016).

The key components with a potent antioxidant capacity that constitutes the beneficial medical properties of bee pollen are phenolic acids and flavonoids, which are plant-derived polyphenolic substances (Rzepecka-Stojko et al., 2015; Özkök & Silici, 2017). The mean percentage of phenolic content in bee pollen is 1.6%. Phenolic compounds involve flavonoids, leukotriens, catechins and phenolic acids. Flavonoid content of bee pollen is 1.4% and consists primarily of kaempferol, quercetin, and isorhamnetin. Chlorogenic acid is the cardinal of phenolic acids and is found at a level of 0.2% (Komosinska-Vassev et al., 2015). The studies conducted on the antioxidant capacity of bee pollen pointed out a significant diversity in the antioxidant capacity of pollen grains, which is considered to be associated with the multiplicity of geographical area, environmental circumstances, and the plant species that the pollen grains were harvested from (Alicic et al., 2014).

Bee pollen exhibits such a biological diversity that it contains an approximate number of 200 different bioactive substances. The chemical composition of bee pollen includes proteins, amino acids, carbohydrates, lipids, fatty acids, phenolic compounds, enzymes, vitamins and minerals (Rzepecka-Stojko et al., 2015). Bee pollen contains 500-3000 mg mineral materials, 13-55 g carbohydrates, 10-40 g proteins, 1-10 g fat, 0.3-20 g dietary fiber, 20-100 mg vitamins and 40-3000 mg flavonoid glycosides per 100 gram of the sample (Bogdanov, 2006). Furthermore, it contains fat-soluble vitamins such as provitamin A, vitamin E, and vitamin D as well as water-soluble vitamins such as B1, B2, and C at a level of 0.1% and 0.6%, respectively. The amount of mineral in bee pollen is approximately 1.6% which includes both macro and microelements such as calcium, magnesium, sodium, phosphor and potassium, and iron, zinc, manganese, and selenium, respectively (Komosinska-Vassev et al., 2015; Sandıkçı Altunatmaz et al., 2017). It has been underlined in a study investigating the nutritional composition of bee pollen that it is a good nutritional source for the consumers particularly with numerous antioxidant vitamins and bioactive substances in its content (Sattler et al., 2015).

The research study was aimed to determine the amounts of total phenolic compounds, antioxidant capacities, bioaccessibilities and also certain physicochemical properties (moisture, ash, pH, acidity, and color) of bee pollen used in Turkey as a food supplement.

2 Materials and methods

2.1 Materials

The study material of 20 bee pollen samples was collected from different sales points in Istanbul/Turkey. Ten of them were unpackaged products offered in plastic bags with a minimum of 100 g (Unpackaged (UPBp), A-J). The other ten samples were purchased in their original packages under different trademarks (Packaged (PBp), K-V). Trademarked products were all contained in properly sealed, dark-colored sun-proof packages. All samples were transferred to the laboratory in cold chain and kept at +4 °C until the analyses.

2.2 Methods

All the reagents and chemicals used in the experiments were of analytical grade. The chemicals of antioxidant capacities and total phenolic contents were obtained from Sigma Chemical Co., Aldrich Chemicals Co., USA. All other chemicals used were obtained from the local suppliers. High-quality water obtained using a Milli-Q system (Millipore, Bedford, MA, USA), was used exclusively.

Physico-chemical analysis of bee pollen

Moisture (method no: 925.40), ash (method no: 950.49), acidity (Total) (method no: 935.57) and pH (Method No: 981.12) contents of the bee pollen samples were assessed according to the standard methods of Association of Official Analytical Chemists (2000). The tests were performed at least in triplicate and mean values were reported.

The color measurement of bee pollen samples was carried out by Minolta Spectrophotometer CM-139 3600d (Osaka, Japan) based on CIE L*, a*, b* color system. The results were expressed using the CIELab system. The following parameters were determined: L* (L*= 0 black, L*= 100 white), a* - share of the green colour (a* < 0) or red (a* > 0), b*- share of blue (b* < 0) or yellow (b* > 0). The measurement was repeated three times.

Extraction of extractable, hydrolyzable, and bioaccessible fractions

Three different extraction methods were applied for extractable, hydrolyzable and bioaccessible fractions. These methods were modified from those originally proposed by Vitali et al. (2009) and were used in the analyses of antioxidant capacity and total phenolic content. Briefly, 2 g were taken from each sample and these samples were extracted in 20 mL of HCl (conc)/methanol/water (1:80:10, v/v) mixture at room temperature on an orbital shaker (JB50-D; Shanghai, China) (in 250 rpm at 20 °C) for two hours. The obtained extracts were centrifuged (Sigma 3K 30, Germany) at 3500 rpm for 10 min. The supernatant was used as the extractable fraction in the analyses of total phenolic content and antioxidant capacity. The same procedure was repeated twice with the same solvent on the remaining part of the bee pollen samples. For hydrolyzable fraction, after extractable fraction, the residue was combined with 20 mL of methanol/H₂SO₄conc (10:1) and placed in a water bath at 85 °C for 20 h before being cooled to room temperature. The mixtures were centrifuged at 3500 rpm for 10 min at 4 °C in a centrifuge (Sigma 3K 30, Germany). The supernatants were collected and used as the hydrolyzable fraction for the analysis.

For the determination of bioaccessible fraction, investigated samples were processed by an in vitro digestive enzymatic extraction that mimics the conditions in the gastrointestinal tract according to the procedure of Vitali et al. 2009 with slight modifications. Briefly, 2 g of the samples were mix with 10 mL pure water and 0.5 mL pepsin and set to pH 2 with HCl for the gastric environment and incubated in the shaking water bath at 37 ^oC for 1 h. Afterward, the pH level was increased to 7.2 to mimic the intestinal environment and 2.5 mL NaCl/KCl and 2.5 mL bile/pancreatin were added and then the samples were incubated in the shaking water bath at 37 ^oC for 2.5 h. Finally, the samples were centrifuged at 3500 rpm for 10 min and supernatants were separated. The whole procedure was run in triplicate and all supernatants were stored at 20 °C until used.

Determination of Total Phenolic Contents (TPC)

The extractable, hydrolyzable, and bioaccessible fraction of bee pollens were determined at 760 nm by using Shimadzu UV-1280 UV-VIS spectrophotometer according to the Folin–Ciocalteu method (Naczk & Shahidi, 2004). Gallic acid was used as standard and the results were expressed as mg GAE /g dw. The total phenolic content was calculated as the sum of extractable and hydrolyzable fractions and bioaccessibility was calculated as the percentage of total phenolic content. The procedure was carried out three times for each extract.

Determination of Antioxidant Capacity (AC)

Antioxidant capacities of extractable and hydrolyzable and bioaccessible fraction of the bee pollen samples were determined using radical cation decolorization assay (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Apak et al., 2008), cupric ion reducing antioxidant capacity assay (CUPRAC) (Apak et al., 2004), free radical scavenging assay (2,2-diphenyl-1-picrylhydrazyl) (DPPH) (Brand-Williams et al., 1995), with slight modifications. All assays were repeated three times for each extract collected from the samples and absorbance of samples were measured by using spectrophotometer (Shimadzu UV-1280). A calibration curve was prepared, using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and the results were expressed as µmol TE/g dw for each method.

2.3 Statistical analysis

Data obtained from the analyses were statistically evaluated with a computer-based program JMP IN 7.0.0 (Statistical Discovery from SAS Institute Inc. (2007), the LSD (Least Significant Difference) test was performed to determine the significant difference between the mean values at the p ≤ 0.05 level.

3 Results and discussion

3.1 Physicochemical properties

The results of moisture, ash, pH, titratable acidity and the color analyses of the bee pollen samples were shown in Table 1. The mean moisture, ash, pH, titratable acidity levels of the unpackaged (UPBp) products were determined as 20.98%, 3.61%, 4.55, and 3.67%, while those of the originally packaged samples (PBp) were 21.36%, 3.89%, 4.80, and 3.64%, respectively (Table 1). No statistically significant difference was noted between the packaged and unpackaged bee pollen products in terms of physicochemical properties except for the titratable acidity (p < 0.05). The ash contents of bee pollen samples found in this study were similar to those reported by Isık et al. (2019), which ranged from 2.14-2.18 g/100 g in hot air dried bee pollen samples.

Table 1
Physicochemical compositions of bee pollens.

BPs	Moisture (%)	Ash (%)	pH	Acidity (%)	L*	a*	b*
A *	20.74 ± 0.06	3.21 ± 0.03	4.84 ± 0.05	3.94 ± 0.00	57.91 ± 0.97	3.53 ± 0.05	23.43 ± 0.71
B	20.62 ± 0.00	3.17 ± 0.01	4.35 ± 0.04	2.63 ± 0.00	57.94 ± 0.53	3.89 ± 0.43	24.55 ± 0.65
C	20.65 ± 0.42	3.15 ± 0.09	4.71 ± 0.03	4.34 ± 0.00	60.01 ± 0.55	3.59 ± 0.20	25.94 ± 0.43
D	21.64 ± 0.00	3.41 ± 0.02	4.44 ± 0.06	4.57 ± 0.00	59.84 ± 0.48	3.47 ± 0.30	25.69 ± 0.55
E	20.80 ± 0.41	4.21 ± 0.03	4.37 ± 0.08	3.05 ± 0.00	57.77 ± 0.53	2.62 ± 0.10	22.23 ± 0.45
F	19.27 ± 0.39	2.93 ± 0.06	4.82 ± 0.03	5.23 ± 0.00	55.75 ± 0.84	4.67 ± 0.13	24.70 ± 0.56
G	22.40 ± 0.07	4.31 ± 0.01	4.55 ± 0.04	3.21 ± 0.00	52.21 ± 1.25	4.97 ± 0.38	22.57 ± 0.89
H	22.35 ± 0.15	4.47 ± 0.04	4.30 ± 0.04	3.33 ± 0.00	47.73 ± 0.47	6.67 ± 0.34	19.92 ± 0.30
I	20.94 ± 0.39	4.09 ± 0.04	4.57 ± 0.03	3.22 ± 0.00	57.48 ± 1.16	4.66 ± 0.37	23.73 ± 0.46
J	19.93 ± 0.06	3.18 ± 0.04	4.55 ± 0.04	3.15 ± 0.00	57.56 ± 0.74	4.15 ± 0.39	26.64 ± 0.40
K **	22.17 ± 0.01	3.80 ± 0.01	4.78 ± 0.03	2.51 ± 0.00	55.48 ± 0.43	3.73 ± 0.45	22.15 ± 0.19
L	21.40 ± 0.20	3.52 ± 0.03	4.67 ± 0.04	3.25 ± 0.00	56.03 ± 0.76	2.89 ± 0.13	23.86 ± 0.45
M	22.45 ± 0.33	4.14 ± 0.01	4.48 ± 0.04	5.82 ± 0.00	53.01 ± 0.71	5.08 ± 0.05	22.68 ± 0.41
N	20.03 ± 0.05	3.67 ± 0.01	4.83 ± 0.04	5.34 ± 0.00	63.65 ± 0.57	1.54 ± 0.24	28.81 ± 0.31
O	25.93 ± 0.19	4.87 ± 0.01	4.60 ± 0.07	3.26 ± 0.00	59.69 ± 0.20	4.50 ± 0.12	30.39 ± 0.07
P	20.48 ± 0.24	5.03 ± 0.01	4.86 ± 0.03	4.65 ± 0.00	56.93 ± 1.42	3.80 ± 0.18	27.84 ± 1.15
R	20.97 ± 0.92	3.82 ± 0.06	4.81 ± 0.03	2.51 ± 0.00	53.67 ± 0.51	6.30 ± 0.31	26.59 ± 0.46
S	20.26 ± 0.42	3.37 ± 0.04	4.81 ± 0.03	2.51 ± 0.00	52.96 ± 0.34	5.47 ± 0.16	23.31 ± 0.22
T	17.90 ± 0.28	2.92 ± 0.01	5.41 ± 0.05	3.99 ± 0.00	53.66 ± 0.41	7.19 ± 0.39	26.99 ± 0.32
V	22.04 ± 1.13	3.73 ± 0.06	4.78 ± 0.04	2.61 ± 0.00	57.62 ± 1.02	3.40 ± 0.08	24.55 ± 0.63
Min-Max	17.90-25.93	2.92-5.03	4.30-5.41	2.51-5.82	47.73-63.65	1.54-7.19	19.92-30.39
Mean ± SD
UPBp	20.98 ± 0.97a	3.61 ± 0.59a	4.55 ± 0.19a	3.67 ± 0.82b	56.42 ± 3.76a	4.22 ± 1.11a	23.94 ± 2.02a
PBp	21.36 ± 2.09a	3.89 ± 0.65a	4.80 ± 0.25a	3.64 ± 1.25a	56.27 ± 3.40a	4.39 ± 1.68a	25.72 ± 2.81a

• Means with different superscripts in the columns indicate significant difference (p < 0.05). Data are expressed as means ± standard deviations (SD). Min: Minimum value of means, Max: Maximum values of means. BPs: Bee pollen samples.
• ^*
  UPBp: Unpackaged bee pollen samples A-J;
• ^**
  PBp: Packaged bee pollen samples K-V.

Bárbara et al. (2015) reported similar results indicating that the mean moisture, ash, pH, titratable acidity levels of 21 bee pollen (Melipona mandacaia) samples collected from two different regions of Bahia and Brazil were 36.0%, 4.9%, 3.49, and 146 meq·kg⁻¹, respectively. Duarte et al. (2018) found similar pH values for bee pollen produced in the Alagoas, Northeast region of Brazil by M. subnitida and T. clavipes respectively 4.9 and 5.9.

Dried bee pollen moisture content was limited 6-10% established by the local food standards by Argentina, Poland, Bulgaria, and Switzerland. However, in Brazil food standards moisture content was 30% in bee pollen and 4% in dehydrated pollen (Melo & Almeida-Muradian, 2011). Almeida-Muradian et al. (2005) detected a mean level of 7.4% and 2.2% for moisture and ash, respectively in the study investigating the chemical composition of 10 different dried bee pollen products collected from the southern part of Brazil. According to the legal regulations in Brazil, the maximum permissible ash level in dried bee pollen is 4%. (Brasil, 2001). In another study carried out in Brazil, bee pollen samples had 2.61-11.06% moisture, 1.58-3.61% ash contents and pH values of 4.60-5.90 (Marchini et al., 2006). Similar findings were reported in some other studies (Almeida-Muradian et al., 2005; Feás et al., 2012; Coronel et al., 2004; Vit & Santiago, 2008). The chemical composition of bee pollen varies extensively depending on the plant species and growing conditions, agricultural practice, climate, botanical features and also harvesting and the storage conditions (Anjos et al., 2019a, b; Bonvehí et al., 2001; Almaraz-Abarca et al., 2004).

According to CIE color measurement system based on a three-coordinate diagram (L*, a*, b*) colors in numeric values located in different spaces of the diagram are evaluated as different colors as follows: L*=100 white, L*=0 black; positive a* red, negative a* green; positive b* yellow and negative b* blue. CIA measurement values for the bee pollen samples were shown in Table 1. Mean L*, a*, and b* values were 56.42, 4.22, 23.94 and 56.27, 4.39, 25.72 for the unpackaged and packaged bee pollen samples, respectively. Color measurement values revealed no statistically significant difference among the groups.

3.2 Total phenolic contents and in vitro bioaccessibilities of bee pollen samples

Total phenolic contents and in vitro bioaccessibilities of bee pollen samples were shown in Table 2. The phenolic contents of the extractable and hydrolyzable fractions and the total phenolic contents of the bee pollen samples were detected to be 16.13-74.98 mg/g GAE, 73.48-395.41 mg/g GAE, and 147.10-462.02 mg/g GAE, respectively. As expected, the phenolic contents of hydrolyzable fractions of all samples were higher than those of the extractable fractions. The mean values for total phenolic content (TPC) were quite high at a level of statistical significance (p < 0.05) in the samples indicated by sample I (462.02 mg/g GAE) and J (437.55 mg /g GAE).

Table 2
Extractable, hydrolyzable, bioaccessible fraction and bioaccessibility (%) of bee pollens.

Total phenolic contents (mg GAE /g)					Bioaccessibility (%)
BPs	Extractable fraction	Hydrolyzable fraction	TPC a	Bioaccessible fraction
A *	47.88 ± 2.02fg	244.84 ± 11.95d	292.73 ± 9.93d	118.08 ± 1.79jk	40.37 ± 1.98g
B	45.55 ± 3.05gh	239.60 ± 21.29d	285.15 ± 18.24de	146.07 ± 5.37hıjk	51.27 ± 1.40def
C	57.52 ± 0.58c	284.78 ± 14.30c	342.30 ± 13.72c	183.32 ± 9.72cdefg	53.65 ± 4.99de
D	42.00 ± 1.31hı	282.37 ± 19.14c	324.41 ± 20.46c	169.74 ± 7.21fgh	52.50 ± 5.53de
E	41.85 ± 1.46hı	189.00 ± 10.19e	230.86 ± 11.66f	172.10 ± 12.74efgh	74.50 ± 1.76ab
F	50.2 ± 1.13ef	297.84 ± 7.23c	348.04 ± 8.37c	208.85 ± 0.92cd	60.03 ± 1.71cd
G	25.87 ± 1.52k	363.59 ± 12.22b	389.46 ± 13.75b	250.81 ± 8.52ab	64.40 ± 0.08bc
H	55.17 ± 2.65cd	241.48 ± 11.10d	296.65 ± 8.45d	151.88 ± 6.69ghıj	51.19 ± 0.80def
I	66.6 ± 0.35b	395.41 ± 13.38a	462.02 ± 13.72a	268.32 ± 2.03a	58.09 ± 1.28cd
J	74.08 ± 2.04a	363.47 ± 13.75b	437.55 ± 11.71a	206.23 ± 67.11cde	46.94 ± 14.08efg
K **	53.23 ± 3.32de	246.44 ± 7.46d	299.67 ± 4.14d	123.50 ± 8.23ıjk	41.20 ± 2.18fg
L	56.14 ± 0.49cd	233.40 ± 1.31d	289.55 ± 1.80d	153.50 ± 9.48ghı	53.03 ± 3.60de
M	34.44 ± 3.44j	191.37 ± 5.83e	225.82 ± 2.40fg	175.78 ± 8.15defgh	77.87 ± 4.44a
N	73.62 ± 0.71a	73.48 ± 19.02f	147.10 ± 19.74h	53.15 ± 9.74l	36.91 ± 11.57g
O	74.98 ± 0.96a	89.74 ± 5.49f	164.73 ± 6.44h	112.40 ± 8.32k	68.38 ± 7.73abc
P	38.53 ± 2.95ı	225.93 ± 9.21d	264.47 ± 12.17e	197.87 ± 4.09cdef	74.86 ± 1.90ab
R	41.77 ± 1.04hı	167.12 ± 9.56e	208.89 ± 8.52fg	153.38 ± 9.30ghı	73.39 ± 1.46ab
S	16.13 ± 1.48l	186.14 ± 14.62e	202.27 ± 13.14g	156.87 ± 11.34ghıj	77.53 ± 0.57a
T	42.72 ± 0.40h	248.23 ± 7.07d	290.95 ± 6.67d	216.26 ± 8.51bc	74.38 ± 4.63ab
V	42.38 ± 1.63hı	243.32 ± 6.27d	285.70 ± 7.90de	204.76 ± 9.81cdef	71.65 ± 1.45ab
Min-Max	16.13-74.98	73.48-395.41	147.10-462.02	53.15-268.32	36.91-77.87
Mean ± SD
UPBp	50.68 ± 13.32a	290.24 ± 65.04a	340.92 ± 70.42a	187.54 ± 48.35a	55.30 ± 10.01b
PBp	47.40 ± 17.42a	190.52 ± 62.61b	237.91 ± 54.89b	154.75 ± 48.21b	64.92 ± 15.44a

• Mean values (Mean) ± standard deviation (SD) (N = 3 × 2) with different lowercase (A-K) in the same column are significantly different (p < 0.05) according to different bee polen samples. Min: Minimum value of means, Max: Maximum values of means; BPs: Bee pollen samples; GAE: gallic acid equivalent.
• ^*
  UPBp: Unpackaged bee pollen samples A-J;
• ^**
  PBp: Packaged bee pollen samples K-V;
• ^a
  TPC: Total phenol content was calculated as the sum of extractable and hydrolyzable fraction.

The total phenolic contents of bioaccessible fractions of the bee pollen samples ranged between 53.15 and 268.32 mg/g GAE. The highest value of 268.32 mg/g GAE belonged to the sample I (Table 2). The bioaccessibilities of total phenolic content were detected 36.91-77.87% for the samples. The total phenolic content bioaccessibility of the samples M (77.87%) and S(77.53%), which sold in their original trademarked packages were significantly higher (p < 0.05) than other samples, and followed by the other trademarked packaged products P (74.86%), T (74.38%), R (73.39%), and V (71.65%) (Table 2).

Despite the insignificant differences between the packaged and unpackaged bee pollen products in terms of physicochemical properties, the total phenolic contents, antioxidant capacities, and their bioaccessibilities were quite higher at a level of statistical significance (p < 0.05). These differences might be explained by the packaged samples that are protected against the factors affecting the food quality such as oxidation, light, and moisture, as expected. It is known that the difference in the total phenolic contents of the bee pollen samples might have resulted from a variety of parameters such as the growing area of plant flora, the climate, pollen harvesting procedures, methods and also the storage conditions. (Parejo et al., 2002; Žiaková et al., 2003).

3.3 Antioxidant capacities and in vitro bioaccessibilities of the bee pollen samples

The physiological role of antioxidants is to prevent tissue injury caused by free radicals occurring during biochemical reactions in the body (Alhan & Şan, 2002). Antioxidant activity emerges through a series of mechanisms in which a variety of bioactive substances participate, therefore any measurement method focusing on a single group of active substances in the assessment of the antioxidant capacity of foods fails to reflect the present antioxidant capacity of the relevant food (Frankel & Meyer, 2000; Karadag et al., 2009). Furthermore, antioxidant capacity measurements may vary actually depending on the chemical composition of food, class of the active compounds of the food, interaction of food components and active compound as well as the sample preparation technique and conditions of the antioxidant capacity method used (Sariburun et al., 2010). Therefore, the ABTS, CUPRAC, and DPPH methods were applied to assess the antioxidant capacity of the bee pollen samples taking into account the selectivity and feasibility of these methods and the data were evaluated in this study. The antioxidant capacities of extractable, hydrolyzable and bioaccessible fractions according to these three methods were given in Table 3.

Table 3
Extractable, hydrolyzable and bioaccessible antioxidant capacities of bee pollens.

Antioxidant capacity (µmol TE/g)
BPs	ABTS			CUPRAC			DPPH
	Extractable fraction	Hyrolysable fraction	Total AC	Extractable fraction	Hydrolysable fraction	Total AC	Extractable fraction	Hydrolysable fraction	Total AC
A *	26.31 ± 0.48d	45.88 ± 3.61ı	72.19 ± 3.13hı	20.79 ± 2.01ef	75.54 ± 0.46jk	96.34 ± 1.55k	12.92 ± 0.31hı	37.30 ± 0.45ıj	50.22 ± 0.14h
B	16.56 ± 0.28h	61.30 ± 3.64cde	77.87 ± 3.36fg	21.51 ± 0.55ef	83.55 ± 1.49hı	105.07 ± 0.98ıjk	10.83 ± 0.13j	62.37 ± 0.68a	73.20 ± 0.55c
C	35.21 ± 0.26b	64.18 ± 1.74c	99.40 ± 2.01b	15.65 ± 4.92fg	93.76 ± 0.76g	109.41 ± 4.16ı	16.26 ± 0.18d	50.61 ± 0.79d	66.87 ± 0.61d
D	30.90 ± 0.30c	80.49 ± 3.30a	111.40 ± 3.61a	47.96 ± 0.48b	79.25 ± 0.79ıj	127.22 ± 0.30h	13.70 ± 0.28fg	41.63 ± 1.39	55.33 ± 1.12g
E	37.95 ± 0.12a	42.48 ± 2.76ı	80.44 ± 2.88e	38.88 ± 1.50c	69.16 ± 0.72k	108.05 ± 0.79ı	13.19 ± 0.03gh	41.25 ± 0.89fg	54.45 ± 0.92g
F	37.99 ± 0.30a	72.22 ± 1.28b	110.22 ± 0.98a	26.08 ± 2.95de	136.91 ± 2.77e	163.01 ± 5.73e	20.69 ± 0.70b	54.11 ± 0.47c	74.80 ± 1.17c
G	16.62 ± 0.63h	57.95 ± 0.90ef	74.58 ± 0.27gh	8.32 ± 1.46gh	139.62 ± 0.88e	147.95 ± 0.58g	12.47 ± 0.24ı	42.21 ± 1.42f	54.68 ± 1.66g
H	37.87 ± 0.13a	50.69 ± 1.99gh	88.57 ± 1.85cde	8.68 ± 1.96gh	183.90 ± 4.26b	192.58 ± 6.23d	16.50 ± 0.18d	44.31 ± 0.94e	60.81 ± 0.76ef
I	38.20 ± 0.10a	53.12 ± 0.54gh	91.33 ± 0.45cd	6.59 ± 3.93h	152.50 ± 1.64d	159.10 ± 5.57ef	19.44 ± 0.18c	40.39 ± 0.91fgh	59.83 ± 1.08f
J	35.32 ± 0.40b	49.98 ± 1.70h	85.32 ± 1.30e	11.13 ± 3.44gh	137.23 ± 1.51e	148.37 ± 1.94g	15.65 ± 0.24e	39.16 ± 0.33hı	54.81 ± 0.57g
K **	17.30 ± 0.75h	51.78 ± 0.08gh	69.09 ± 0.67ı	14.57 ± 2.47fg	138.82 ± 4.48e	153.41 ± 6.95fg	2.76 ± 0.09l	41.68 ± 0.57fg	44.43 ± 0.66ı
L	22.00 ± 0.71e	70.21 ± 1.94b	92.22 ± 2.66c	64.88 ± 4.00a	137.95 ± 2.97e	202.84 ± 1.04c	6.39 ± 0.32k	55.83 ± 0.99bc	62.21 ± 0.68e
M	26.20 ± 0.33d	60.76 ± 0.79de	86.96 ± 1.13e	61.48 ± 3.49a	136.72 ± 2.14e	198.21 ± 1.71cd	6.10 ± 0.18k	44.55 ± 0.37e	50.65 ± 0.19h
N	19.66 ± 0.63g	57.64 ± 0.10ef	77.32 ± 0.74fg	64.30 ± 7.98a	192.96 ± 1.16a	257.27 ± 6.37a	5.91 ± 0.38k	44.46 ± 0.85e	50.37 ± 1.23h
O	20.76 ± 0.75f	58.68 ± 0.65e	79.45 ± 0.10f	33.32 ± 3.47cd	174.10 ± 7.25c	207.43 ± 10.72c	0.44 ± 0.05m	35.25 ± 1.12jk	35.69 ± 1.16j
P	14.67 ± 0.23ı	72.68 ± 1.03b	87.36 ± 1.27de	57.70 ± 0.39a	184.66 ± 3.05b	242.37 ± 2.66b	22.45 ± 0.38a	61.39 ± 2.06a	83.84 ± 2.44a
R	10.07 ± 0.55k	43.01 ± 1.43ı	53.09 ± 1.99k	19.45 ± 3.44ef	86.99 ± 2.36h	106.44 ± 5.80ıj	13.40 ± 0.14fgh	33.21 ± 1.23k	46.61 ± 1.37ı
S	6.20 ± 0.78l	54.02 ± 2.22fg	60.23 ± 3.01j	38.18 ± 2.50c	116.34 ± 2.52f	154.53 ± 5.03efg	13.91 ± 0.25f	54.76 ± 1.31c	68.67 ± 1.56d
T	22.28 ± 0.29e	64.93 ± 0.75c	87.22 ± 0.46de	24.35 ± 8.35e	73.22 ± 5.54jk	97.58 ± 2.81jk	20.18 ± 0.04b	57.31 ± 0.65b	77.48 ± 0.61b
V	11.32 ± 0.48j	37.63 ± 1.18j	48.96 ± 1.67k	6.25 ± 3.45h	76.98 ± 4.49j	83.24 ± 1.04l	15.50 ± 0.55e	40.08 ± 0.58gh	55.58 ± 1.13g
Min-Max	6.20-38.20	37.63-80.49	48.96-111.40	6.25-64.88	69.16-192.96	83.24-257.27	0.44-22.45	33.21-62.37	35.69-83.84
Mean ± SD
UPBp	31.30 ± 8.40a	57.83 ± 11.76a	89.13 ± 13.74a	20.56 ± 13.59a	115.14 ± 38.56a	135.71 ± 30.64b	15.17 ± 3.07a	45.33 ± 7.73a	60.50 ± 8.25a
PBp	17.05 ± 6.18b	57.14 ± 10.83a	74.19 ± 15.17b	38.45 ± 21.93b	131.88 ± 42.55a	170.33 ± 59.47a	10.70 ± 7.25b	46.85 ± 9.59a	57.55 ± 14.92a

• Mean values represented by the same letters within the same column are not significantly different at p ≤ 0.05. Data are expressed as means ± standard deviations (SD). Min: Minimum value of means, Max: Maximum values of means. BPs: Bee pollen samples; TE: Trolox Equivalent.
• ^*
  UPBp: Unpackaged bee pollen samples A-J;
• ^**
  PBp: Packaged bee pollen samples K-V.

According to the results of the ABTS method, antioxidant capacities of extractable fractions of the bee pollen samples ranged between 6.20 and 38.20 μmol TE/g, whereas those of hydrolyzable fractions were 37.63-80.49 μmol TE/g (Table 3). The highest value (111.40 μmol TE/g) for antioxidant capacity belonged to the sample D, followed by the samples F (110.22 μmol TE/g) and C (99.40 μmol TE/g). The mean value of total antioxidant capacity (TAC) of the unpackaged bee pollen samples, which was determined to be 89.13 μmol TE/g was higher than packaged products (74.19 μmol TE/g), unexpected. According to the CUPRAC method, the highest TAC value of 257.27 μmol TE/g was detected in the sample N, followed by the samples P (242.37 μmol TE/g), O (207.43 μmol TE/g), and L (202.84 μmol TE/g), respectively. The highest values in terms of the total antioxidant capacities of both fractions were obtained with the CUPRAC method in comparison to the other methods used. This situation might be explained by CUPRAC method have sensitivity and selectivity for both hydrophilic and lipophilic materials and also the feasibility of the method for the bioactive compounds in bee pollen samples (Apak et al., 2004).

When the antioxidant capacities of the bee pollen samples were measured by the DPPH method the hydrolyzable fractions (33.21-62.37 μmol TE/g) exhibited significantly higher values than those of extractable fractions (0.44-22.45 μmol TE/g). No significant differences in total antioxidant capacity levels measured by the DPPH method were found between packaged and unpackaged bee pollen samples (p > 0.05). The lowest antioxidant capacity value has obtained in this method when compared to the other methods used. Therefore, it can be concluded that the DPPH method is not suitable for the assessment of the antioxidant capacity of bee pollen.

Bee pollen is a biological substance covered with a double-layered wall. The outer layer called exine is composed of sporopollenin and the inner layer intine has a semipermeable membrane-like structure. The exine is a complex solid substance that is found to have survived on the surfaces of fossils of millions of years of age. It is rather hard and even impossible to digest the material on most occasions (Frenguelli, 2003). Therefore, the extractable fractions have lower antioxidant capacity than the hydrolyzable fractions in all of the antioxidant capacity methods used.

Previous research showed that bee pollen had a high antioxidant capacity (Almedia et al., 2017; Campos et al., 2003; Suriyatem et al., 2017; Sun et al., 2017; Mărghitaş et al. 2009; Carpes et al., 2007) that varies due to the ecological diversity and predominance of the plant species that it was collected from. However, there is no direct correlation available between the total phenolic content (TPC) and the antioxidant capacity (Özkök & Silici, 2017; Dorman et al., 2003; Miliauskas et al., 2004). TPC of 12 bee pollen samples were determined to be 691.67-1383.67 mg/kg in a study conducted in Slovakia (Fatrcová-Šramková et al., 2013) Özkök & Silici (2017) reported in a study performed with 20 bee pollens that the mean total phenolic content of samples was 2340.07 mg GAE/100 g and the mean antioxidant capacities were 42.37 mg AAE/g. Yesiltas et al. (2014) indicated that total phenolic content of 9 bee pollen samples collected from different regions of Turkey was 12.0-36.7 mg GAE/g) and antioxidant capacities of the samples according to ABTS, CUPRAC, DPPH, and FRAP methods were 15.2-33.6 mg TE/g, 20.7-89.4 mg TE/g, 5.7-15.2 mg TE/g, and 5.2-15.7 mg TE/g, respectively. Karkar et al. (2018) reported that antioxidant capacities of chestnut bee pollen samples were 3.70-34.18 mg TE/g, 3.15-20.24 mM TE/g by CHROMAC ve FRAP methods, respectively. A previous study by Anjos et al. (2019a) average total phenolic content of 6 bee pollen samples which found as predominant pollen Cistus ladanifer (42.6%) were detected 6.81 mg QE/g and average antioxidant capacity was EC50: 2.62 mg/mL by DPPH methods. On the other hand, the total phenolic content value (6.9-21 mg GAE/g) in bee pollens were lower than the values in this study reported by Duarte et al. (2018). Also, Duarte et al. (2018) declared that bee pollen contains T. clavipes (123.4 mg GAE/100) were the highest antioxidant capacity using FRAP assay and the highest correlation (0.98) between FRAP assay and total phenolic content.

The widely ranging results of the above-mentioned studies were linked with the climate changes, the origin of the harvested plant as well as the storage conditions of the products (Anjos et al., 2019b; Choi et al., 2006; Kumazawa et al., 2004; Marcucci et al., 2001; Almaraz-Abarca et al., 2004). Also, the chemical and nutritional quality of bee pollens depend on the harvest and transport methods, storage conditions and packaging properties (Sattler et al., 2015; Arruda et al., 2013).

The absorbed amount of a nutrient in the gastrointestinal tract known as bioaccessibility may vary depending on numerous factors such as the physical structure and chemical composition of the food, interactions of the food ingredients, and the processing methods (Sandström, 2001). In vitro bioaccessibilities of the bee pollen samples included in the study, which were assessed by three different antioxidant capacity measurement methods (ABTS, CUPRAC and DPPH) were shown in Figure 1. Bioaccessibility values were detected to be 7.54-95.76%, 22.47-65.28, and 4.59-26.07 by CUPRAC, ABTS, and DPPH methods, respectively. These differences between antioxidant methods might be the diversity in the chemical composition of the bee pollen samples and the sensibility of each method toward different bioactive compounds (Al-İsmail & Aburjai, 2004; Almeida-Muradian et al., 2005; Morais et al., 2011). In the presented study, CUPRAC method proved to be the most appropriate method for bioaccessibility, which was considered to have resulted from the high sensibility of the active compounds with high antioxidant capacities to the relevant method.

Figure 1
Bioaccessibility (%) of antioxidant capacities of bee pollen.

Based on overall evaluations, average values for AC bioaccessibilities of the unpackaged products (A-J) were determined to be 57.56%, 50.33% and 11.04%, according to CUPRAC, ABTS, and DPPH methods, whereas those of the packaged bee pollen products (K-V) were 30.48%, 32.49%, and 14.41%, respectively (Figure 1).

4 Conclusion

Bee pollen is considered a functional food product with its high total phenolic content and antioxidant capacity. Besides, the bioaccessibilities of the antioxidant capacity was determined to be fairly high. Based on the results of all antioxidant capacity assays used in the study, it was demonstrated that the antioxidant capacities of the hydrolyzable fractions were higher than those of the extractable fractions. Furthermore, the CUPRAC method proved to be significantly superior (p < 0.05) to ABTS and DPPH methods based on the data obtained through the assessments of TAC and their bioaccessibilities. The difference between the packaged and unpackaged bee pollen products in terms of antioxidant properties is considered highly likely to be associated with the chemical composition, harvested plant species, geographical features and the climate of the harvesting area, and certain environmental circumstances as well as the storage and sales conditions of the bee pollen products. Preservation of the chemical compounds such as antioxidants present in bee pollens depends on good storage conditions. Antioxidant properties because of phenolics are decreased based on storage conditions contained high heat, light, oxygen or moisture during shelf life. For this reason, food packaging of bee pollen positively affected antioxidant properties in this study. Therefore, bee pollen should be stored in a cool, dry place and in well packaging materials. Consequently, it can be deduced that bee pollen with its general chemical features has emerged as a natural antioxidant source in a healthy diet both solely and as a supplement contained in different food products.

• Practical Application: As a functional food component with high antioxidant properties and bioaccessibility, bee pollen can be used in the production of various foods and as a dietary supplement in nutrition.

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Publication Dates

• Publication in this collection
  31 July 2020
• Date of issue
  June 2021

History

• Received
  13 Mar 2020
• Accepted
  26 Apr 2020