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

مجله پژوهشهای علوم و صنایع غذایی ایران

Article 3, Volume 14, Issue 2 - Serial Number 50, May and June 2018, Pages 243-257   

Document Type: Research Article

DOI: 10.22067/ifstrj.v0i0.60468

Authors

Atefe Maqsoudlou^{* 1}; Alireza Sadeghi Mahoonak¹; Mohammad Ghorbani¹; Fidel Toldta²

¹Department of Food Science & Technology, Food Chemistry, Gorgan university of agricultural sciences and natural resources, Iran.

²Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Avenue Agustín Escardino 7, 46980 Paterna, Valencia, Spain.

Abstract

Introduction: 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.

Materials and methods: 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.

Results and discussion: 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

Keywords

ACE; bee pollen; royal jelly; Antioxidant activity; Alcalase; bioactive peptides

References

Almeida, J. F., Reis, A. S., Heldt, L. F. S., Pereira, D., Bianchin, M., Moura, C., Plata-Oviedo, M. V., Haminiuk, C. W.I., Ribeiro, I. S., Luz, C. F. P. And Carpes, S. T. 2016. Lyophilized bee pollen extract: A natural antioxidant source to prevent lipid oxidation in refrigerated sausages. LWT - Food Science and Technology, 71, 1-7.

Arabshahi, S., Ardestani, A., Yazdanparast, R. 2001. Quantitative assessment of antioxidant properties of natural colorants and phytochemicals: carotenoids, flavenois, phenols and indigoids. The role of B-carotene in antioxidants functions. Journal of Science of Food and Agriculture, 81, 559- 568.

Bogdanov, S. 2014. Royal Jelly, Bee Brood: Composition, Health, Medicine: A Review. Bee Product Science, 28 (3), 118-153.

Bougatef.A; Hajji.M; Balti.R. 2009. Antioxidant and free radical – scavenging activities of smoth hound muscle protein hydrolysates obtained by gastro intestinal proteases.journal of food chemistry, 1198-1255.

Coscueta, R. F., Amorim, M. M., Voss, G. B., Nerli, B. B., Pico, G. A., Pintado, M. A. 2016. Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysis. Food Chemistry, 198, 36–44.

Daoud, A., Malika, D., Bakari, S., Hfaiedh, N., Mnafgui, K. Kadri, A., Gharsallah, N. 2015. Assessment of polyphenol composition, antioxidant and antimicrobial properties of various extracts of Date PalmPollen (DPP) from two Tunisian cultivars. Arabian Journal of Chemistry, 48, 437-447.

Deshpande, S., Chryan, M., Salunkhe, D. 1987. Tanin analysis of food products. Critical review in food nutrition, 24, 41- 49.

Guerar, F., Guimas, l., Binet, A. 2002. Production of tuna waste hydrolysates by a commercial neutral protease preparation. Journal of Molecular catalysis B: Enzymatic, 19, 489-498.

Guo, H., Ekusa, A., Iwai, K., Yonekura, M., Takahara, Y., Morimatsu, F. 2009. Royal jelly peptides inhibit lipid peroxidation in vitro and in vivo. Journal of Nutural Science and Vitaminology, 54, 191–195.

Guo, H., Kozuma, Y., & Yonekura, M. 2005. Isolation and properties of antioxidative peptides from water-soluble royal jelly protein hydrolysate. Food Science Technology Research, 11, 222–230.

Hmidet, N., Balti, R., Nasri, R., Sila, A., Bougatef, A., Nasri, M. 2011. Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases. Food Research International, 44, 2703-2711.

Je, J. Y., Lee, M. H., Lee, K. h. Ahn, C. B. 2009. Antioxidant and hypertensive protein hydrolysates produced from tuna liver by enzymatic hydrolysis. Food Research International, 42, 1266-1272.

Kawashima, K., Itoh, H., Miyoshi, M., & Chibata, I. 1979. Antioxidant properties of branched-chain amino acid derivatives.Chemical Pharmacological Bulletin, 27, 1912–1916.

Khantaphant, S., Benjakul, S. 2008. Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity. Comparative Biochemistry and Physiology, 151, 110-115.

Kishimura. H., Benjakul.S. 2011. Antioxidative and ACE inhibitory activities of protein hydrolysates from the muscle nof brownstripe red snapper prepared using pyloric caeca and commercial proteases. Process Biochemistry, 318-327.

Klompong, V., Benjakul. S., Kantachote, D., Shahidi, F. 2007. Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally. Food chemistry, 102 (13), 17-27.

Kroyer. G., Hegedus, N. 2001. Evaluation of bioactive properties of pollen extracts as functional dietary food supplement. Innovative Food Science & Emerging Technologies, 2, 171-174.

Ktari, N., Nasri, R., Mnafgui, K., Hamden, K., Belguith, O., Boudaouara, T., Feki, A., Nasri, M. 2014. Antioxidative and ACE inhibitory activities of protein hydrolysates from zebra blenny (Salaria basilisca) in alloxan-induced diabetic rats, 5113 (14), 73-77.

Lassoued, I., Mora, L., Nasri, R., Aydi, M., Toldra, F., Aristoy, M., C., Barkia, A., Nasri, M. 2015. Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from thornback ray (Raja clavata) muscle. Journal of Proteomics.

Leblanc, B. W., Davis, O. K., Boue, S., DeLucca, A., Deeby, A. 2009. Antioxidant activity of Sonoran Desert bee pollen. Food Chemistry, 115, 1299–1305.

Liu, J. R., Yang, Y. C., Shi, L.S., Peng, C. C. 2008. Antioxidant properties of royal jelly associated with larval age and time of harvest. Journal of Agriculture and Food Chemistry, 56 (23), 11447-11452.

Mao, W. Y., Cheng, X., Wang, X., Wu, S. I. 2011. Free-radical-scavenging and anti-inflammatory effect of yak milk casein before and after enzymatic hydrolysis. Food Chemistry, 126 (2011), 484–490.

Marghitas, L. A., Stanciu, O. G., Dezmirean, D. S., Bobis, O., Popescu, O., Bogdanov, S., Campos, M. S., 2009. In vitro antioxidant capacity of honeybee-collected pollen of selected floral origin harvested from Romania. Food Chemistry, 115, 878–883.

Marinova, M., Tchorbanov, P. 2010. Preparation of Antioxidant Enzymatic Hydrolysates from Honeybee-Collected Pollen Using Plant Enzymes. Enzyme Research, 41, 5949-50.

Matsuoka,T., Kawashima,T., Nakamura, T.,Kanamaru,Y., Yabe, T. 2012. Isolation and characterization of proteases that hydrolyze royal jelly proteins from queen bee larvae of the honeybee, Apis mellifera. Apidologie, 43, 685–697.

Morais, M., Moreira, L., Feas, X., Estevinho, L.M. 2011. Honeybee-collected pollen from five Portuguese natural parks: Palynological origin phenolic content antioxidant properties and antimicrobial activity. Food and Chemical Toxicology, 49, 1096-1101.

Nagai, T., Inoue, R., Suzuki, N., Myoda, T., Nagashima, T. 2005. Antioxidative ability in a linoleic acid oxidation system and scavenging abilities against active oxygen species of enzymatic hydrolysates from pollen Cistus ladaniferus. International Journal of Molecular Medicine, 15(2), 259-63.

Nagai, T., & Inoue, R. 2004. Preparation and the functional properties of water extract and alkaline extract from royal jelly.Food Chemistry, 84, 181–186.

Nasri, R., Younes, I., Jridi, M., Trigui, M., Bougatef, A., Arroume, N., Dhulster, P., Nasri. M., Châabouni, M.K., 2013. ACE inhibitory and antioxidative activities of Goby (Zosterissessor ophiocephalus) fish protein hydrolysates: Effect on meat lipid oxidation. Food Research International, 54, 552–561.

Pascoal, A., Rodrigues, S., Teixeira, A., Feas, X., L, M. Estevinho. 2013. Biological activities of commercial bee pollens: antimicrobial, antimutagenic, antioxidant and anti-inflammatory. Food and Chemical Toxicology.

Salampessy, J., Reddy, N., Kailasapathy, K., Phillipsa, M. 2015. Functional and potential therapeutic ACE-inhibitory peptides derived from bromelain hydrolysis of trevally proteins. Journal of Functional Foods, 14, 716-725.

Sun, L., Powers, J. R., and Tang, J. 2007. Evaluation of antioxidant activity of asparagus, broccoli and their juices. Food Chemistry, 105, 101- 106.

Villanueva, A., Vioque, J., Sanchez-Vioque, R., Clemente, A., Pedroche, J., Bautista, J., Millan, F. 1999. Peptide Characteristics of Sunflower Protein Hydrolysates. Journal of the American Oil Chemists Society, 76, 1455-1460.

Wiriyaphan, C., Chitsomboon, B., Yongsawadigul, J. 2012. Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts. Food Chemistry, 132, 104-111.

Zhang, M., Mu, T.-H., & Sun, M.-J. (2014). Purification and identification of antioxidant peptides from sweet potato protein hydrolysates by Alcalase. Journal of Functional Foods, 7, 191–200.