Chemical composition, bioactive compounds, antioxidant capacity and stability of floral maize (Zea mays L.) pollen

journal of functional foods 10 (2014) 65–74

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Chemical composition, bioactive compounds, antioxidant capacity and stability of floral maize (Zea mays L.) pollen Slad¯ana Žilic´ a,*, Jelena Vancˇetovic´ b, Marijana Jankovic´ a, Vuk Maksimovic´ c a

Department of Food Technology and Biochemistry, Maize Research Institute, Slobodana Bajic´a 1, 11085 Belgrade-Zemun, Serbia b Breeding Department, Maize Research Institute, Slobodana Bajic´a 1, 11085 Belgrade-Zemun, Serbia c Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11000 Belgrade, Serbia

A R T I C L E

I N F O

A B S T R A C T

Article history:

The nutritional composition, the phenolic profiles and antioxidant capacity of the floral pollen

Received 23 February 2014

from maize genotypes were evaluated. In addition, the antioxidant capacity, browning and

Received in revised form 7 May 2014

wavelength spectra of melanoidins were investigated in the pollen samples stored at 4 °C

Accepted 13 May 2014

for 7 days, dried at 40 °C for 6 h and exposed to a temperature of 100 °C during 12 h. The

Available online

results showed that different maize pollen samples had diverse nutritional composition, antioxidant capacity and phenolic compounds were a major contributor to their antioxi-

Keywords:

dant activities. Sweet maize pollen with the highest content of total phenolics and flavo-

Floral maize pollen

noids had the highest antioxidant capacity (104.38 mmol trolox eq/kg). Quercetin diglycoside

Chemical compositions

was the most abundant flavonoid in all pollen samples. Floral maize pollen could be used

Phenolics

as a functional food ingredients, and dietary supplement with therapeutic effects. However,

Flavonoids

pollen is very susceptible to processes of Maillard reaction and phenolics oxidation and these

Antioxidant capacity

factors must be considered when considering its use.

Browning

1.

Introduction

The chemical composition of pollen has gained worldwide research interest covering broad areas, ranging from plant physiology to biochemistry, nutrition and even material science (Schulte, Lingott, Panne, & Kneipp, 2008). Pollen has been used as a “perfect health food” for many centuries due to its abundance of nutrimental constituents and bioactive compounds. Modern research has also shown that pollen mainly possesses the therapeutic effects (Li et al., 2013; Li, Hu, Zhu, & Zheng, 2005). The Pharmacopeia Committee of the People’s

* Corresponding author. Tel.: +381 11 37 56 704; fax: +381 11 37 54 994. E-mail address: [email protected] (S. Žilic´). http://dx.doi.org/10.1016/j.jff.2014.05.007 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. All rights reserved.

Republic of China (1995) and the German Federal Board of Health (Linskens & Jorde, 1997) have officially recognized pollen as a medicine. Results demonstrated that growth of cells derived from the human prostate carcinoma was inhibited by the pollen extract (Habib, Ross, & Buck, 1990). Given that only floral pollen can guarantee a relatively constant concentration of active ingredients, most applications of pollen in modern medicine are pollen preparations of flower pollen. Maize pollen has been studied due to the economic importance of the plant and to ease of obtaining large amounts of pollen. The latter fact makes this pollen particularly useful for chemical analytical studies. Maize pollen can be considered

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journal of functional foods 10 (2014) 65–74

nutritional, since it contains essential components, such as carbohydrates, proteins, amino acids, lipids, vitamins, minerals and trace elements (Malerbo-Souza, 2011). The main biologically active components of maize pollen are the polyphenolic compounds, mostly flavonoid glycosides, with their well known different and important physiological and pharmacological roles. They possess diverse biological properties such as antioxidant, antiaging, anticarcinogen, antiinflammatory, antiatherosclerosis, cardioprotective and they improve the endothelial function (Han, Shen, & Lou, 2007), thus the interest in the impact of flavonoids on human health has been growing. Maize pollen is susceptible to desiccation. Depending mainly on the vapor pressure deficit of the air, the water status of maize pollen can change from being fully hydrated to being nearly dehydrated in 1–4 h (Fonseca & Westgate, 2005). At the same time, the process of rehydration of pollen grains is carried out rapidly when they come in contact with water (Aylor, 2003). Fresh floral maize pollen contains about 40–50% of water. This high moisture is an ideal culture medium for micro-organisms like bacteria and yeast. Taking into account these facts, the adequate methods of preservation and optimal storage conditions of maize pollen should be provided. From the nutritional point of view pollen maintains its quality well at low temperature and reduced moisture content. There are very few reports related to the composition and antioxidant capacity of floral maize pollen. Therefore, in constant search for alternative sources of bioactive compounds, the aim of this study was to investigate the content of phenolic compounds, mainly flavonoids, and to evaluate the antioxidant capacity of floral pollen from different maize genotypes with white, yellow, red, dark red, blue, brown and sweet kernels. To identify potential pollen materials as nutritional ingredients, the contents of oil, proteins and sugars were also determined. Since very little research has been reported on pollen stability to degradation, physical and chemical changes which had occurred under conditions of elevated temperatures and moisture were determined using measurement methods of browning, wavelength spectrum of melanoidins, as well as of antioxidant capacity.

2.

Material and methods

2.1.

Chemical and reagents

All chemicals and solvents were of HPLC or analytical grade. Potassium persulphate (dipotassium peroxdisulphate) and sodium chloride were purchased from Fluka Chemie AG (Buchs, Switzerland). Acetone, hydrochloric acid, formic acid, ethanol, sodium carbonate, sodium nitrite and aluminum chloride were purchased from Merck (Darmstad, Germany). 6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (trolox), Folin–Ciocalteu reagent, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), gallic acid, (+)-catechin, quercetin, trichloroacetic acid, acetonitrile and sugar standards were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium hydroxide was provided by J. T. Baker (Deventer, Holland). Ultrapure water was used throughout the experiments.

2.2.

Pollen samples

Seven floral pollen samples were manually collected from different maize genotypes with white, yellow, red, dark red, blue, brown and sweet kernels. Each sample consisted of pollen collected from 30 plants. Maize genotypes with different colors of kernels were grown during 2013 in the field of the Maize Research Institute (Belgrade, Serbia), in a randomized complete block design (RCB) with two replications. Standard cropping practices were applied to provide adequate nutrition and to keep the disease-free plots. When two-thirds of the tassel central branch has started pollinating, paper bags were placed on it. After 24 h, tassels were cut off from plants, sent to the laboratory and each sample was shaken in the bag and separately sieved to separate the pollen from anthers and other impurities. All chemical analyses or extractions were performed during the day when pollen was collected from plants. The extracts were kept at −30 °C prior to spectrophotometric, HPLC and LC–MS analyses. In addition, for the analysis of antioxidant capacity, browning and wavelength spectra of melanodins, samples with an average of about 45% moisture were stored in a fridge at 4 °C for 7 days, dried in a ventilated oven at 40 °C for 6 h and exposed to a temperature of 100 °C in an oven during 12 h.

2.3.

Analytical procedures

2.3.1.

Measurement of oil and total protein content

The standard AOAC (1995) chemical methods were applied to determine the content of oil and total proteins. The results are given as percentage of dry matter (d.m.).

2.3.2. Extraction of protein fractions and measurement of protein fraction content Pollen (0.5 g) was successively extracted by the Osborne procedure described by Lookhart and Bean (1995) with some modifications. The salt soluble protein fraction was extracted with 0.5 M NaCl. Supernatants of three extractions were collected, transferred to the volumetric flask and the corresponding extraction solutions were added to 25 mL. Albumins–globulins were separated from non protein nitrogen by precipitation from the salt soluble fraction with 10% trichloroacetic acid. The nitrogen content was determined in extracts by the micro Kjeldahl method and the protein content was calculated by using the conversion factor of 6.25. The results are given as percentage of total proteins.

2.3.3.

Measurement of sugar content

Pollen (0.1 g) was extracted by mixing with 5 mL of 40% (v/v) ethanol and shaking for 30 min at ambient temperature. Extracts were centrifuged at 10,000 × g for 10 min and clear supernatants were used. High pressure liquid chromatography (HPLC) analyses were performed on a Waters Breeze chromatographic system (Waters, Milford, MA, USA) connected to Waters 2465 electrochemical detector with 3 mm gold working electrode and hydrogen reference electrode. Separation of sugars was performed on CarboPac PA1 (Dionex, Sunnyvale, CA, USA) 250 × 4 mm column equipped with corresponding CarboPac PA1 guard column using 0.2 M NaOH as mobile phase. Sugars were

journal of functional foods 10 (2014) 65–74

isocratically eluted for 20 min at a flow rate of 1.0 mL/min at constant temperature of 30 °C. Signals were detected in pulse mode using following waveform: E 1 = +0.15 V for 300 ms; E2 = +0.75 V for 150 ms; E3 = −0.80 V for 150 ms and within 100 ms of integration time. Filter timescale was of 0.2 s and range was 1 μA for the full mV scale. For the preparation of 0.2 M NaOH solution, 10.5 mL of sodium hydroxide solution (50% w/w, low carbonate, J.T. Baker, Deventer, Holland) was diluted to final volume of 1 L with vacuum degassed deionized water. Quantitative analysis was performed by the external standard method using pure standard compounds as references for concentration and retention time. Data acquisition and quantification were carried out by the Waters Empower 2 Software (Waters). All sugar standards were obtained from Sigma (Sigma Co. St. Louis, MO, USA). The sugar content is expressed as mg/ gram of d.m. The content of reducing and total detected sugars was calculated on the basis of the results of the HPLC analysis for mono- and disaccharides.

2.3.4.

Measurement of total phenolics content

For the detection of total phenolics, extracts were prepared by continuous shaking of 0.2 g of pollen in 10 mL of 70% (v/v) aqueous acetone for 30 min at room temperature. After centrifugation (10 min at 10,000 × g) the supernatant was used for experiments. The total phenolic content was determined according to the Folin–Ciocalteu procedure (Singleton, Orthofer, & Lamuela-Raventos, 1999). Aliquots (0.1 mL) of aqueous acetonic extracts were transferred into test tubes and their volumes were adjusted to 0.5 mL with distilled water. After addition of the Folin–Ciocalteu reagent (0.25 mL) and 20% aqueous sodium carbonate solution (1.25 mL), tubes were vortexed. After 40 min, the absorbance was recorded at 725 nm against a blank containing an extraction solvent instead of a sample. The total phenolic content of each sample was determined by means of a calibration curve prepared using gallic acid and expressed as milligrams of gallic acid equivalents (GAE) per kilogram of d.m. Stock solution of gallic acid was prepared in ethanol at a concentration of 1.0 mg/mL. All calibration solutions (0.5, 1.0, 5.0, 10.0, 20.0, 30.0 and 50 μg/mL) were prepared by serial dilution of the stock solution with ethanol. The correlation coefficients were obtained using the linear regression model in Excel® (Microsoft) (R2 = 0.996).

2.3.5.

Measurement of total flavonoid content

For the detection of total flavonoids, 0.1 g of pollen was extracted in 5 mL of 40% (v/v) ethanol for 30 min at room temperature. The supernatant was centrifuged for 10 min at 10,000 × g and then used in experiments. The total flavonoid content was determined according to Zhishen, Mengcheng, and Jianming (1999). Briefly, 50 μL of 5% NaNO2 was mixed with 100 μL of the appropriate extracts. After 6 min, 500 μL of a 10% AlCl 3 solution was added to form a flavanoid–aluminum complex. After 7 min, 250 μL of 1 M NaOH was added, and the mixture was centrifuged at 5000 × g for 5 min. The absorbance of the supernatant was read at 510 nm against the blank containing the extraction solvent instead of a sample. The total flavonoid content was expressed as milligrams of catechin equivalents (CE) per kilogram of d.m. All calibration solutions of catechin (1.0, 5.0, 10.0, 20.0, 30.0, 50.0, 60.0 and 80.0 μg/mL) were prepared by serial dilution of the stock solutions (1.0 mg/

67

mL) with ethanol. The correlation coefficients were obtained using the linear regression model in Excel ® (Microsoft) (R2 = 0.989).

2.3.6.

Analysis of individual flavonoids

Prepared ethanolic solutions of flavonoids were used for the analyses of individual compounds. After filtering through a 0.45 μm nylon filter, samples were kept at −70 °C prior to the LC–MS analysis. Quantification of individual flavonoid compounds was done by the reversed phase HPLC analysis. Samples were injected in the Waters HPLC system consisted of 1525 binary pumps, thermostat and 717+ autosampler connected to the Waters 2996 diode array and EMD 1000 single quadropole detector with the ESI probe (Waters). The separation of flavonoids was performed on a Symmetry C-18 RP column 125 × 4 mm size with a 5-μm particle diameter (Waters) connected to appropriate guard column. Two mobile phases, A (0.1% formic acid) and B (acetonitrile), were used at a flow of 1 mL/min with the following gradient profile: the first 20 min from 10 to 22% B; next 20 min of linear rise up to 40% B, followed by 5 min reverse to 10% B and additional 5 min of the equilibration time. The post column flow splitter (ASI, Richmond, CA, USA) with a 5/1 split ratio was used to obtain optimal mobile phase inflow for ESI probe. For the LC–MS analysis, signals for each compound were detected in a negative ESI scan mode with the following parameters: capillary voltage 3.0 kV, cone voltage −30 V, extractor and RF lens voltages were 2.0 and 0.2 V respectively. Source and desolvation temperatures were 120 and 380 °C respectively, with the N2 gas flow of 450 L/h. Detected compounds were qualitatively analyzed through a comparison of literature data for their retention times, as well as characteristic UV and MS spectra with the ones recorded from our pollen samples. Due to the lack of specific standards, values are presented as minimal quercetin equivalents, obtained by normalization of the HPLC peak area for each compound with peak area of the lowest detected amount of quercetin (yellow maize pollen). In addition, quercetin was analyzed quantitatively by the external standard method using pure standard compound as a reference for the concentration, retention time and characteristic UV/MS spectra. The data acquisition and spectral evaluation for peak confirmation were carried out by the Waters Empower 2 Software (Waters).

2.3.7.

Analysis of total antioxidant capacity (TAC)

The antioxidant capacity of fresh, stored and heat-treated maize pollen samples were measured according to the QUENCHER method described by Serpen, Gökmen, Pellegrini, and Fogliano (2008), using 7 mM aqueous solution of ABTS (2,2-azino-bis/ 3-ethil-benothiazoline-6-sulphonic acid) with 2.45 mM K2O8S2 as the stock solution. The working solution of ABTS•+ was obtained by diluting the stock solution in water/ethanol (50:50, v/v). The pollen samples (5 mg) were mixed with 20 mL of ABTS•+ working solution, and the mixture was rigorously shaken at 4 °C for 25 min. After centrifugation at 9200 × g for 5 min (10 °C) the absorbance measurement was performed at 734 nm. The total antioxidant capacity was expressed as Trolox equivalent antioxidant capacity (TEAC) in mmol of trolox per kilogram of d.m.

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2.3.8.

Measurement of browning

The samples were dissolved in dH2O and filtrated through a 0.22-μm nylon filter. Browning indices of Maillard reaction product (MRPs) samples before and after storage and heating were recorded by their absorbance at 420 nm on a spectrophotometer (Agilent 8453, Santa Clara, CA, USA) using a 1 cm pathlength cell (Kim & Lee, 2008).

2.3.9.

Wavelength spectra of melanodins

Wavelength spectra of melanoidins from aqueous extracts were recorded by a UV-vis spectrophotometer (Agilent 8453) with the wavelength ranging from 200 to 700 nm (Kim & Lee, 2008). The wavelength spectra of melanoidins of all pollen samples from maize with red kernel that were very similar to the respective spectra of the other maize pollen samples, as well as a drastically different wavelength spectrum of melanoidins of heattreated pollen from maize with blue kernel are presented.

2.4.

Statistical analysis

The analytical data are reported as mean ± standard deviation of at the least two independent extractions. Significance of differences between pollen samples were analyzed by Fisher’s least significant differences test, after the analysis of variance for trials set up according to the randomized complete block design. Differences with p < 0.05 were considered significant. The coefficient of variation (CV) was determined for each trait.

3.

Results and discussion

3.1.

Chemical composition

Maize pollen contains members of all of the major classes of organic and inorganic nutrients. However, different varieties of maize apparently produce pollen which varies greatly in its composition. In this study, the contents of moisture, oil and proteins of floral pollen from different colored maize are shown in Table 1. The relative water content of maize pollen plays an important role both in the viability and in its chemical stability and microbiological safety that are necessary conditions for its use in the human diet. Fresh pollen moisture levels in our

study (43.07–50.88%) were similar to moisture level indicated in other researches (Barnabas, Kovacs, Abranyi, & Pfahler, 1988). Such high moisture content requires the application of preventive methods, such as quick drying at max 40 °C, lyophilization, and freeze drying (Dominguez-Valhondo, Gil, Hernandez, & Gonzalez-Gomez, 2011), in order to preserve the nutritional quality of pollen during storage. According to our study, the oil content in seven maize pollen samples was high and ranged from 9.87 to 17.66%. Pollen from blue-seeded maize had the highest content of oil followed by pollen from white-, red-, dark red-, yellow-, brown- and sweet-seeded maize. Considerable variation for oil (CV = 17.68%) among floral pollen was found. Pollen oil contains important therapeutic and nutritive substances. Alkanes, alkenes, fatty acids, triterpene esters and triacylglycerols were found as the main constituents of the maize pollen lipid extracts wherein the free fatty acids constitute 8–22% of the total (Bianchi, Murelli, & Ottavino, 1990). The protein content of pollen is believed to be one of the best indicators for nutritive quality. Maize pollen can be suspected to contain low amounts of protein (about 15%) and to be deficient in some essential amino acids (Somerville & Nicol, 2006). However, this study reports relatively high concentration of protein in maize pollen (22.69–24.84% d.m.) which is congruent to the data reported by Lundgren and Wiedenmann (2004). The same authors have reported that, contrary to expectation, maize pollen was not deficient in essential amino acids. According to Linskens and Pfahler (1977), maize pollen have the large amounts of free amino acids that represent a valuable reserve material. It should be emphasized that the free amino acid-diet was absorbed with a 3.5-times-higher transfer rate from the gut compared with the protein-diet. Also, the free amino acid-diet was assimilated with greater efficiency than the protein-diet (80% versus 58%) (Rønnestad, Conceição, Aragão, & Dinis, 2000). In our study the content of non protein nitrogen, as an indicator of free amino acid content, was analyzed. In pollen samples, the content of non protein nitrogen was higher by 3.8- to 28.8-fold than the albumin–globulin protein fraction. Pollen from maize with blue color of kernels had the highest non protein nitrogen content (57.63% of total proteins) and the lowest content of the albumin–globulin protein fraction (2.00% of total proteins), and vice versa the pollen from dark red maize. It should be noted that the low level of variation that was observed in the content of total proteins

Table 1 – The contents of moisture, oil and proteins in the floral pollen from maize with different colored kernels. Pollen samples

Moisture (%)

Oil (% d.m.)

Total proteins (% d.m.)

Albumins–globulins (% of total proteins)

Non protein nitrogen (% of total proteins)

Red maize White maize Yellow maize Blue maize Dark red maize Brown maize Sweet maize Mean CV (%)

44.34 ± 0.50c 43.66 ± 0.24c,d 43.68 ± 0.16c,d 48.86 ± 0.57b 50.88 ± 0.22a 44.64 ± 0.32c 43.07 ± 0.41d 45.30 ± 3.02 5.56

14.88 ± 0.10b 15.39 ± 0.13b 13.46 ± 0.16c 17.66 ± 0.08a 13.85 ± 0.21c 12.30 ± 0.42d 9.87 ± 0.10e 13.92 ± 2.46 17.68

23.43 ± 0.41b 24.60 ± 0.41a 23.24 ± 0.13b 22.69 ± 0.09c 23.29 ± 0.55b 24.84 ± 0.07a 24.83 ± 0.33a 23.80 ± 0.95 4.01

2.14 ± 0.24d 5.66 ± 0.23b 4.11 ± 0.49c 2.00 ± 0.25d 10.22 ± 0.05a 2.09 ± 0.05d 5.18 ± 0.34b 4.48 ± 2.95 65.87

53.52 ± 0.59c 47.03 ± 0.56e 49.57 ± 0.89d 57.63 ± 0.30a 37.78 ± 0.29 55.20 ± 0.02b 47.17 ± 0.27e 49.70 ± 6.62 13.33

a–e

Mean of pollen samples followed by the same letter within the same column are not significantly different (p > 0.05); CV, coefficient of variation.

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Table 2 – The contents of sugars in the floral pollen from maize with different colored kernels. Pollen samples

Glucose (mg/g d.m.)

Fructose (mg/g d.m.)

Sucrose (mg/g d.m.)

Total detected reduced sugars (% d.m.)

Total detected sugars (% d.m.)

Red maize White maize Yellow maize Blue maize Dark red maize Brown maize Sweet maize Mean CV (%)

103.34 ± 9.67a 47.95 ± 3.12d 65.41 ± 3.65b 44.66 ± 1.59d 49.69 ± 3.87d 52.63 ± 3.21c,d 61.01 ± 4.28b,c 60.68 ± 20.21 33.31

193.30 ± 11.40a 103.48 ± 6.56d 149.81 ± 9.34b 148.40 ± 7.13b 120.35 ± 5.55c 139.82 ± 7.62b 126.50 ± 8.54c 140.24 ± 28.61 20.40

0.53 ± 0.03f 50.26 ± 2.63a 42.93 ± 1.51b 28.71 ± 0.98c 8.30 ± 0.45d 1.42 ± 0.08f 4.26 ± 0.23e 19.48 ± 20.91 107.32

29.66 ± 2.36a 15.14 ± 1.21d 21.52 ± 1.07b 19.31 ± 0.77b,c 17.01 ± 1.10c,d 19.24 ± 1.25b,c 18.75 ± 1.50c 20.09 ± 4.27 23.24

29.71 ± 1.96a 20.17 ± 1.41c 25.81 ± 1.36b 22.18 ± 1.02d 17.83 ± 0.94e 19.38 ± 1.41de 19.18 ± 1.34de 22.04 ± 4.67 19.39

a–f

Mean of pollen samples followed by the same letter within the same column are not significantly different (p > 0.05); CV, coefficient of variation.

(CV = 4.01%) does not necessarily mean that protein constituents are not highly variable among genotypes. The content of albumin–globulin protein fraction and non protein nitrogen varied considerably among pollen samples (CV = 65.87% and 13.33%, respectively). The albumin–globulin protein fraction mainly consists of enzymes that are present in certain strata of the walls of the pollen grains. Kalinowski, Radłowski, and Bartkowiak (2002) have separated 12 enzymes from mature pollen grains of maize, while Li, Suen, Huang, Kung, and Huang (2012) reported that in maize pollen, three enzymes, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease, are predominant.

3.2.

Sugar content

Substantial differences in sugar content were found for pollen collected from the flowers of different colored maize genotypes (Table 2). Fructose was detected as the major sugar species in maize pollen samples. The content of fructose ranged from 103.48 to 193.30 mg/g. Compared with the content of fructose the content of glucose was lower by 1.85–3.32 times. This finding indicates that pollen of different maize genotypes would tend to have no constant proportion of sugars. In addition, considerable high variation for sucrose content (CV = 107.32%) was found. The highest sucrose content was detected in pollen from white-seeded maize followed by pollen from yellowseeded maize having the values of 50.26 and 42.93 mg/g d.m., respectively. Pollen from red-seeded maize had the lowest content of sucrose (0.53 mg/g d.m.). To our knowledge, no results of sugar content in floral maize pollen are available. According to our study, the content of reducing sugars was higher (on average 20.09% d.m.) than that of non-reducing sugars. Chantarudee et al. (2012) reported that the average contents of total and reducing sugars in bee maize pollen from Thailand were 14.71 and 14.11 g/100 g, respectively. In these samples the fructose and glucose were the predominant sugar species.

3.3.

Phenolics composition and quantification

Total phenolic and flavonoid contents of tested maize pollen samples are presented in Table 3. The total phenolic and flavonoid contents in pollen have not been greater with the maize having more kernel pigment. For example, pollen from sweet

maize with light yellow color of kernel had the highest level of total phenolic and flavonoid contents of 9933.01 mg GAE/ kg and 15,001.09 mg CE/kg, respectively, while pollen from blueseeded maize had the second lowest contents. In comparison to the other pollen samples, pollen from yellow maize had the lowest content of analyzed bioactive compounds (7779.34 mg GAE/kg and 8928.25 mg CE/kg, respectively). According to our results, maize pollen is a rich source of phenolic compounds, especially flavonoids. However, little has been published on the flavonoid derivatives from maize pollen. Ceska and Styles (1984) reported that the flavonoid pattern of maize pollen is characterized by the accumulation of quercetin and isorhamnetin diglycosides and by the absence of flavones, which are common in other maize tissues. According to early research of Goss (1968), maize pollen is yellow because of the presence of the flavonoid pigment quercetin and its derivatives. Our results are in well accordance with these data. The quantitative determination of individual flavonoid glycosides is difficult because most of their pure standards are not commercially available. In this study, quercetin is quantified and expressed in μg/g of pollen dry matter, but normalized amounts are given for other identified flavonoids (Table 4). The glycosides of quercetin and isorhamnetin were isolated from seven floral maize pollen samples. In addition to the quercetin aglycone, its eight

Table 3 – The contents of total phenolics and flavonoids in the floral pollen from maize with different colored kernels. Pollen samples

Total phenolics (mg GAE/kg d.m.)

Total flavonoids (mg CE/kg d.m.)

Red maize White maize Yellow maize Blue maize Dark red maize Brown maize Sweet maize Mean CV (%)

9050.51 ± 132.86b 8232.64 ± 151.77d 7779.34 ± 21.70e 8635.80 ± 48.08c 8897.62 ± 397.88b,c 9757.26 ± 176.34a 9933.01 ± 85.65a 8898.02 ± 774.13 8.70

10,876.96 ± 516.73b 9376.30 ± 409.31c,d 8928.25 ± 122.45d 9163.63 ± 166.43d 10,216.58 ± 264.68b 10,562.05 ± 800.42b 15,001.09 ± 912.69a 10,589.27 ± 2079.63 19.63

a–e Mean of pollen samples followed by the same letter within the same column are not significantly different (p > 0.05); CV, coefficient of variation.

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1.11 5.54 1.27 5.69 1.45 7.04 10.05

5.87 2.73 1.00 1.62 1.01 3.00 2.56

466.82 ± 36.75a* 215.53 ± 17.51b* 81.61 ± 5.28e* 141.32 ± 10.56d* 91.85 ± 6.13e* 239.75 ± 18.01b* 198.47 ± 15.63c*

derivatives were identified, as well as two derivatives of isorhamnetin (Table 4, Fig. 1). Quercetine diglycoside (625 m/ z) was found to be the most dominant form of flavonols in analyzed maize pollen, while rutin and isorhamnetin glycoside were detected only in trace amounts. Quercetin in maize pollen exhibited broad ranges of variation from 81.61 to 466.82 μg/g. According to Lundgren and Wiedenmann (2004), the average value of field maize pollen samples for the quercetin content was 324.16 μg/g. Flavonoids, as exemplified by quercetin, have been shown to have many effects on cells in vitro. Quercetin has antioxidant activities, inhibits protein kinases, inhibits DNA topoisomerases and regulates gene expression related to oxidative stress and the antioxidant defence system (Moskaug, Carlsen, Myhrstad, & Blomhoff, 2004).

0.87 0.58 0.14 0.65 0.21 0.70 1.29

3.4. Antioxidant capacity of fresh, heat-treated and stored floral maize pollen

a–e Mean of pollen samples followed by the same letter within the same column are not significantly different (p > 0.05). * Amount of quercetin (in μg/g d.m.).

8.97 6.38 5.82 6.55 5.37 7.26 8.50 2.13 2.58 1.48 3.47 1.61 3.29 3.59 1.86 0.75 0.67 0.13 1.10 0.55 0.15 6.56 4.53 5.37 4.69 5.44 5.72 3.65 6.54 5.18 7.09 7.46 8.74 7.11 12.99 34.53 31.22 38.21 32.20 45.49 33.33 42.30 3.93 5.18 9.60 10.12 9.62 6.38 8.65 Red maize White maize Yellow maize Blue maize Dark red maize Brown maize Sweet maize

2.36 1.69 2.47 1.99 2.80 1.71 2.40

Isorhamnetin glycoside (477 m/z) Hyperoside (463 m/z) Isohyperoside (463 m/z) Rutin (609 m/z) Rutin derivative (609 m/z) Isorhamnetin diglycoside (639 m/z) Quercetin diglycoside (625 m/z) Quercetin sophoroside glycoside (787 m/z) Hyperoside glycoside (625 m/z) Pollen samples

Table 4 – Normalized amounts of different flavonoids and their corresponding glycosides present in different pollen ethanolic extracts.

Quercetin glycoside derivative (463 m/z)

Quercetin (301 m/z)

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Polyphenols are excellent scavengers of radicals and the number of hydroxyl groups on the phenyl ring seems to enhance the antioxidant capacity of a polyphenolic molecule (Wettasinghe & Shahidi, 2000). The QUENCHER method with ABTS reagents was used to determine the antioxidant capacity of fresh floral maize pollen samples (Table 5), and these values were compared to measured amounts of total phenolics and flavonoids. As expected, a higher content of phenolic compounds in the maize pollen samples contributed to their higher antioxidant capacity being the highest in sweet maize pollen (104.38 mmol trolox eq/kg). This level was higher by about 24% than that found in yellow-seeded maize pollen. The fresh pollen had ABTS radical scavenging activities with the following descending order: sweet > brown > red > dark red > white > blue > yellow. By comparison, the maximum of ABTS radical cation scavenging activity of different colored maize kernels was 35.66 mmol trolox eq/kg in blue popping maize (Žilic´, Serpen, Akıllıog˘lu, Gökmen, & Vancˇetovic´, 2012). According to Chantarudee et al. (2012), the methanolic extract of floral maize pollen had higher antioxidant activity than methanolic extract of bee maize pollen with EC50 values of 365.2 and 428.6 μg/mL, respectively. It is not clear yet how honey bees modify the physical or chemical structure of the pollen by the functional combination of nectar, enzymes and bee secretions when they pack the maize pollen. However, the derivatized or modified bee pollen form or active compounds may have different extraction preferences, stabilities to degradation or bioactivities. On the other hand, it is well known that floral maize pollen is sensitive to dehydration, as well as rehydration (Aylor, 2003). Deterioration of pollen during storage and drying involves many physical and chemical changes, such as disrupted intracellular integrity, decreased activities of enzymes, lipid peroxidation, phenolic oxidation, and Maillard reactions, as well as the change in odor, taste, color and shape (Van Bilsen, Hoekstra, Crowe, & Crowe, 1994). In order to determine the extent of changes, antioxidant capacity of stored and heat-treated floral maize pollen was evaluated. After storage at 4 °C for 7 days antioxidant capacity of pollen was decreased by 2% (in white maize pollen) to 34% (in blue-seeded maize pollen), in relation to that in fresh samples (Table 5). Our results also showed that total antioxidant capacity was decreased in pollen samples heat-treated at 40 °C for 6 h, except

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Fig. 1 – Base peak LC-MS chromatogram of different pollen extracts. Peaks are noted as follows: 1-hyperoside glycoside; 2-quercetine sophoroside glycoside; 3-quercetin diglycoside; 4-isorhamnetin diglycoside; 5-rutin derivative; 6-rutin; 7-isohyperoside; 8-hyperoside; 9-isorhamnetin glycoside; 10-quercetine glycoside derivative; 11-quercetin.

in pollen of blue-seeded maize (Table 5). However, although heating affected the reduction of phenolic compounds (Žilic´ et al., 2013), the total antioxidant capacity was increased in pollen samples heat-treated at 100 °C for 12 h. The average value was 145.71 mmol trolox eq/kg. It is evident that the depletion of the natural antioxidants in maize pollen could be balanced by the formation of new compounds with antioxidant activities. The formation of MRPs (Maillard reaction products) could be the reason of enhanced total antioxidant capacity.

3.5. Browning development and wavelength spectra of melanodins With the aim to investigate the level of MRP formation in pollen samples, the formation of colored matter over the storage and thermal treatments was monitored. The data are shown in Figs. 2 and 3. The colored compounds can be grouped into two general classes both having the antioxidant activity: low molecular weight compounds and melanoidins which are brown polymers and possess molecular weights of several thousand daltons and discrete chromophore groups (Serpen, Capuano, Fogliano, & Gökmen, 2007). In this study the ad-

vanced stage of the browning reaction was monitored by the increase in absorbance at 420 nm that corresponds with research of Kim and Lee (2008). The browning development was drastically increased in pollen samples heat-treated at 100 °C for 12 h (Fig. 2). The colored compound concentration was the highest in the sample of red-seeded maize pollen (Figs. 2 and 3) that had the highest antioxidant capacity (201.51 mmol trolox eq/kg) (Table 5). On the other hand, blue-seeded maize pollen with the lowest concentration of colored compounds (Figs. 2 and 3) had the lowest antioxidant capacity (70.89 mmol trolox eq/kg) (Table 5). However, in addition to the health promoting, potentially harmful compounds have been associated with the Maillard reaction. Recently, few heat induced contaminants have gained much interest because of their toxicological and carcinogenic potential: acrylamide, furan, chloropropanols, CML (Nε-carboxymethillisine) and HMF (5hydroxymethyl-2-furaldehyde) (Capuano & Fogliano, 2011; Wenzl, Lachenmeier, & Gökmen, 2007). The components of MRPs varied with concentration of reactants such as sugars and amino acids. According to our study, the rate of browning color development in floral maize pollen samples was dependent not only on temperature and moisture but also on glucose and fructose concentration. Since fructose exists predominantly in the

Table 5 – ABTS radical scavenging capacity (mmol trolox eq/kg of d.m.) of fresh, stored and heat-treated maize pollen. Pollen samples

Fresh

Stored (7 days/4 °C)

Heated (6 h/40 °C)

Heated (12 h/100 °C)

Red maize White maize Yellow maize Blue maize Dark red maize Brown maize Sweet maize Mean CV (%)

95.38 ± 1.48b 84.98 ± 2.37c,d 79.94 ± 2.34d 84.49 ± 2.63c,d 86.65 ± 8.13c 96.35 ± 0.54b 104.38 ± 5.22a 90.21 ± 8.60 9.52

68.60 ± 3.09c 82.59 ± 6.82b 64.50 ± 2.17c 55.35 ± 3.36d 82.16 ± 1.35b 93.22 ± 7.56a 84.01 ± 3.65b 75.76 ± 13.26 17.50

61.37 ± 5.78b,c 57.23 ± 3.56c,d 49.44 ± 1.63d 70.15 ± 3.81a 49.51 ± 4.42d 60.26 ± 0.10b,c 54.46 ± 0.36c,d 57.49 ± 7.31 12.72

201.51 ± 3.07a 186.90 ± 15.48a,b 145.24 ± 4.30c 70.89 ± 2.19f 107.99 ± 8.24e 178.03 ± 15.69b 129.43 ± 12.45d 145.71 ± 46.80 32.12

a–f

Mean of pollen samples followed by the same letter within the same column are not significantly different (p > 0.05); CV, coefficient of variation.

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Fresh/~45% moisture

Stored (7 days at 4ºC/~45% moisture)

Heated (6h/40ºC)

Heated (12h/100ºC)

2,5

Abs 420 nm

2 1,5 1 0,5 0 RMP

WMP

YMP

BlMP

DRMP

BwMP

SMP

Maize samples

Fig. 2 – Development of browning (as measured by absorbance at 420 nm) of melanoidins before and after storage and heating of maize pollen. RMP-pollen of red maize; WMP-pollen of white maize; YMP-pollen of yellow maize; BlMP-pollen of blue maize; DRMP-pollen of dark red maize; BwMP-pollen of brown maize; SMP-pollen of sweet maize. The vertical bars represent the standard deviation of each data point.

open-chain form, causing more rapid progress of the initial stages of the Maillard reaction than glucose (Dills, 1993), it could be concluded that a high pollen susceptibility to deterioration was caused primarily by its amply fructose abundance (on average 140.24 mg/g). Also, fructose readily dehydrates to give HMF (Liu, Tang, Wu, Bi, & Cui, 2012). Although the browning development was slightly increased in pollen samples stored at 4 °C for 7 days, the formed colored compounds may be result of the Maillard reaction, as well as the phenolic oxidation (Figs. 2 and 3). Absorption spectra shown in Fig. 3 clearly indicate the extent of the Maillard reaction in stored and heat-treated floral maize pollen. The compounds formed early in the Maillard reaction absorb in the UV, e.g. HMF, but do not absorb in the visible region of the spectrum. After crossing the UV region the absorption curve progressively came into the blue-absorbing region of the visible spectrum and yellow, orange and brown colors appeared (MacDougall & Granov, 1998). Although the performed measurements do not define the color of the yellow to brown pigments or how they related to the concentration of MRPs, a high correlation between HMF, as well as acrylamide

contents and the browning development has been previously reported (Serpen & Gökmen, 2009; Žilic´ et al., 2013).

4.

Conclusion

The obtained data highlight the high nutritional value of maize pollen, as well as abundance of its bioactive compounds with high antioxidant capacity. The results of this study contribute to expanding the knowledge about possible health benefits of floral maize pollen. Due to the exceptionally high content of phenolic compounds, floral maize pollen could be used as a dietary supplement with therapeutic effects. Further, floral maize pollen could be used as ingredient of functional foods such as bread, cookies, noodles, instant soup, etc., as well as healthy drinks such as pollen beverage, tea and pollen yoghurt. However, a high moisture content of floral maize pollen and its susceptibility to the process of the Maillard reaction and phenolics oxidation requires the application of preventive

4,5

red fresh red stored 7 days at 4ºC red (40ºC/6h) red (100ºC/12h) blue (100ºC/12h)

4 3,5 3

Abs

2,5 2 1,5 1 0,5 0 200

250

300

350

400

450 500 550 Wavelenth (nm)

600

650

700

750

Fig. 3 – Comparison of the UV-vis spectra of melanoidins before and after storage and heating of pollen from maize with red kernels, as well as UV-vis spectra of melanoidins after heating of pollen from maize with blue kernels.

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methods in order to preserve the nutritional quality of pollen during storage.

Acknowledgements This work was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant nos. TR-31069 and 173040) and supported by the COST action FA1005 (Infogest). REFERENCES

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