Optimization of production of bioactive peptides through the enzymatic hydrolysis of bee pollen protein by Alcalase and comparison with the royal jelly

Bee pollen, commonly referred as the ‘‘life-giving dust’’, results from the agglutination of flower pollens with nectar using salivary substances of the honeybees (Almeida-Muradian et al., 2005). Pollen contains 10 to 40% protein, 1 to 13% lipid, 13 to 55% carbohydrates and 2 to 6% minerals. Royal Jelly is produced by enzymatic digesting of bee pollen by proteases and other natural enzymes. Based on dry weight, it contains 27-41% protein, 30% carbohydrates, 8-19% lipids, minerals, trace elements and some vitamins (Sabatini et al., 2009; Wytrychowski et al., 2013). The antioxidant properties of royal jelly and bee pollen, are related to main proteins and phenolic compounds and flavonoids (Nagai and Inue, 2004). The antioxidant activity of peptides can be evaluated using DPPH, radicals scavenging activity, Ferric reducing, Ferrous chelating activity (Khantaphant et al., 2011). Antioxidant and ACE inhibitory activity of pollen, royal jelly and peptides were investigated by different researchers (Bogdanov, 2014; Morais et al., 2011; Salampessy et al., 2015; Marinova and Tchorbanov, 2010; Wiriyaphan et al., 2012). The objective of present research was optimization of enzymatic hydrolysis of bee pollen protein by Alcalase according to its antioxidant and ACE inhibitory activity compared to royal jelly.
The preparation of the bee pollen extract was performed by mixing the bee pollen with water (1:10) (w/v). The macerates were filtered and centrifuged at 12000 g. The obtained supernatant was lyophilized. The royal jelly extract were prepared using method described by Liu et al., 2008. The total phenolic content of the extracts was recorded using the Folin–Ciocalteu method (Moreira et al., 2008). DPPH radical-scavenging activity was determined as described by Bersuder, Hole, and Smith (1998). The ability of the hydrolysate to reduce iron (III) was determined according to the method of Bougatef et al. (2008). Bee pollen was added and homogenized with 5 volumes of distilled water. pH and temperature of the solution were adjusted to pH=8 and 50◦C. Alcalase in the concentration range of 1 to 2% w/w were added to the pollen protein solution. Enzymatic hydrolysis performed during different times 2 to 5 hours. Hydrolysis was stopped by heating at 80˚C for 10 min. The hydrolysats were centrifuged at 4000x g for 30 min to remove the residue. The supernatants were pooled and then lyophilized (Matsuoka et al., 2012). DPPH radical scavenging ability and reducing power of pollen hydrolysates of pollen hydrolysates were measured. Also ACE-inhibitory activity of pollen hydrolysates was measured was assayed by method reported by Nakamura et al. (1995). Statistical analysis of results before hydrolysis was done by SPSS. Optimization of enzymatic hydrolysis was done by Response Surface Methodology (RSM) in Design Expert software.
Total phenol value measured for pollen ranged between 48.15 to 174 mg Gallic acid/g for royal jelly ranged from 9.24 to 87.261 mg Gallic acid/g. Considering that royal jelly is obtained by direct digestion of pollen, the amounts of their phenolic compounds were comparable (Bogdanov, 2014). Phenolic compounds increased by increasing concentration royal jelly and pollen extract in dose dependent manner. Increasing concentrations of royal jelly in range of 300 to 1000 mg/l was more effective than pollen (p