Comparative analysis of functionality of spray dried whey protein hydrolysates obtained by enzymatic and microbial hydrolysis

Salem M. Embiriekah, Maja Lj. Bulatović, Marija Lj. Gnjatović, Maja S. Vukašinović-Sekulić, Tanja Ž. Krunić, Danica B. Zarić, Marica B. Rakin


The aim of this study was to examine the bioactive potential of hydrolysate powders produced by enzymatic and microbial hydrolysis of whey proteins followed by spray drying, in order to reveal which one of these processes result in a product with significantly improved functional properties. Hydrolysate powders produced by the two different biotechnological processes were compared based on their antioxidant (DPPH and FTC), antibacterial as well as erythrocyte membrane stabilizing activities. The performed tests revealed that the concentration of at least 178.4 mg mL-1 of the whey protein hydrolysate powder, produced by tryptic digestion, could inhibit the process of lipid peroxidation by 50 %, suppress the microbial contamination caused by S. aureus ATCC25923, B. cereus ATCC 11778 and L. monocytogenes, and provide the antioxidant and membrane stabilizing activities greater than 50 %. On the other hand, the hydrolysate powder obtained by whey fermentation at the concentration of at least 811.5 mg mL-1 achieved 50 % of all tested bioactivities, with the emphasis on the significantly more pronounced antibacterial activity against all tested strains. In that sense, tryptic hydrolysis could be highlighted as an optimal process that provides production of the whey hydrolysate with pronounced bioactive properties that could be considered as a very promising natural food supplement.


whey; bioactivity; hydrolysis; fermentation; food supplement

Full Text:

PDF (1,445 kB)


Midmore P, Naspetti S, Sherwood AM, Vairo D, Wier M, Zanoli R. Consumers Attitudes to Quality and Safety of Organic and Low input Foods; A review. Aberystwyth: University of Wales; 2005: 24-29

Papas AM. Diet and antioxidant status. Food Chem Toxicol. 1999; 37: 999–1007.

Moskovitz J, Yim KA, Choke PB. Free radicals and disease. Arch Biochem Biophys. 2002; 397: 354-359.

Gad AS, Khadrawy YA, El-Nekeety AA, Mohamed SR, Hassan NS, AbdelWahhab MA. Antioxidant activity and hepatoprotective effects of whey protein and Spirulina in rats. Nutrition. 2011; 27: 582-589.

Peng X, Xiong YL, Kong B. Antioxidant activity of peptide fractions from whey protein hydrolysates as measured by electron spin resonance. Food Chem. 2009; 113: 196–201.

Vavrusova M, Pindstrup H, Johansen LB, Andersen ML, Andersen HJ, Skibsted LH. Characterisation of a whey protein hydrolysate as antioxidant. Int Dairy J. 2015; 47: 86-93.

Önay-Uçar E, Arda N, Pekmez M, Yilmaz AM, Böke-Sarikahya N, Kirmizigül S, Yalçin AS. Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions. Food Chem. 2014; 150: 34-40.

Zhang Q-X, Wu H, Ling Yu-F, Lu R-R. Isolation and identification of antioxidant peptides derived from whey protein enzymatic hydrolysate by consecutive chromatography and Q-TOF MS. J Dairy Res. 2013; 80: 367–373.

Lin S, Tian W, Li H, Cao J, Jiang W. Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. Int J Food Sci Tech. 2012; 47: 2045–2051.

Bayram T, Pekmez M, Arda N, Yalçin AS. Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment. Talanta. 2008; 75: 705–709.

Hernández-Ledesma B, Dávalos A, Bartolomé B, Amigo L. Preparation of antioxidant enzymatic hydrolysates from α-lactalbumin and β-lactoglobulin. Identification of active peptides by HPLC–MS/MS. J Agric Food Chem. 2005; 53: 588–593.

Peña-Ramos E A, Xiong YL. Antioxidant activity of whey protein hydrolysates in a liposomal system. J Dairy Sci. 2001; 84: 2577–2583.

Peña-Ramos EA, Xiong YL, Arteaga GE. Fractionation and characterisation for antioxidant activity of hydrolysed whey protein. J Sci Food Agric. 2004; 84: 1908–1918.

Peña-Ramos EA, Xiong YL. Whey and soy protein hydrolysates inhibit lipid oxidation in cooked pork patties. Meat Sci. 2003; 64: 259–263.

Peng X, Xiong YL, Kong B. Antioxidant activity of peptide fractions from whey protein hydrolysates as measured by electron spin resonance. Food Chem. 2009; 113: 196–201.

Lee NK, Yun CW, Kim SW, Chang HI, Kang CW, Paik HD. Screening of lactobacilli derived from chicken feces and partial characterization of Lactobacillus acidophilus A12 as an animal probiotics. J Microbiol Biotechnol. 2008; 18: 338–342.

Ou CC, Lu TM, Tsai JT, Yen JH, Chen HW, Lin MY. Antioxidative effects of lactic acid bacteria: Intact cells vs. intracellular extracts. J Food Drug Anal. 2009; 17: 209–216.

Sun J, Hu XL, Le GW, Shi YH. Lactobacilli prevent hydroxyl radical production and inhibit Escherichia coli and Enterococcus growth in system mimicking colon fermentation. Lett Appl Microbiol. 2010; 50: 264–269.

Soleymanzadeh N, Mirdamadi S, Kianirad M. Antioxidant activity of camel and bovine milk fermented by lactic acid bacteria isolated from traditional fermented camel milk (Chal). Dairy Sci Technol. 2016; 96: 443–457.

Virtanen T, Pihlanto A, Akkanen S, Korhonen H. Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. J Appl Microbiol. 2007; 102: 106–115.

Foegeding EA, Davis JP, Doucet D, McGuffey MK. Advances in modifying and understanding whey protein functionally. Trends Food Sci Tech. 2002 ;13: 151–159.

Nathalie C, Gruppen H. Hydrolysis of whey protein isolate with bacillus licheniformis protease: fractionation and identification of aggregating peptides. J Agric Food Chem. 2007; 55: 9241−9250.

Sinha R, Radha C, Prakash J, Kaul P. Whey protein hydrolysate: Functional properties, nutritional quality and utilization in beverage formulation, Food Chem. 2007; 101: 1484–1491.

Webb KE. Intestinal absorption of protein hydrolysis products: a review. J Anim Sci. 1990; 68: 3011−3022.

Embiriekah S, Bulatović M, Borić M, Zarić D, Rakin M. Antioxidant activity, functional properties and bioaccessibility of whey protein hydrolysates. Int J Dairy Tech. 2018; 71: 243-252.

Bulatović M, Zarić D, Rakin M, Krunić T, Lončarević I, Pajin B. Chocolate as a Carrier for Cocoa’s Functional Ingredients, The Diversified Benefits of Cocoa and Chocolate, 1st Edition, Chapter 3, Ed. Bonifacia Zayas Espinal, Nova Science Publishers, New York, 2017.

Nnanna IA, Wu C. Diary protein hydrolysates. Handbook of Food Products Manufacturing; Hui, Y. H., Ed.; Wiley: Hoboken, NJ, 2007; Vol. 2, pp 537−556.

Gauthier SF, Pouliot Y. Functional and biological properties of peptides obtained by enzymatic hydrolysis of whey proteins. J Dairy Sci. 2003; 86: 78−87.

Isolauri E, Sutas Y, Turjanmaa K. Efficacy and safety of hydrolyzed cow milk and amino acid-derived formulas in infants with cow milk allergy. J Pediatr. 1995; 127: 550−557.

Rango V, Giampietro PG, Bruno G, Businco L. Allergenicity of milk protein hydrolysate formulae in children with cow’s milk allergy. Eur J Pediatr. 1993; 152: 760−762.

Anandharamakrishnan C, Rielly CD, Stapley AGF. Effects of Process Variables on the Denaturation of Whey Proteins during Spray Drying. Drying Technol. 2007; 25: 799-807.

Ma J-J, Mao X-Y, Wang Q, Yang S, Zhang D, Chen S-W, Li Y-H. Effect of spray drying and freeze drying on the immunomodulatory activity, bitter taste and hygroscopicity of hydrolysate derived from whey protein concentrate. LWT - Food Sci Technol. 2014; 56: 296-302.

McCue PP, Shetty K. Phenolic antioxidant mobilization during yogurt production from soymilk using Kefir cultures. Process Biochem. 2005; 40: 1791-1797.

Azuma K, Nakayama N, Koshioka M, Ippoushi K, Yamaguchi Y, Kohata K, Yamauchi Y, Ito H, Higashio H. Phenolic antioxidants from the leaves of Corchorus olitorius L. J Agric Food Chem. 1999; 47: 3963-3966.

Haraguchi H, Ishikawa H, Sanchez Y, Ogura T, Kubo Y, Kubo I. Antioxidative constituents in Heterotheca inuloides. Bioorg Med Chem. 1997; 5: 865-871.

Dam Arawwawalaa LDAM, Arambewelaa LSR, Ratnasooriya WD. Alpinia calcarata Roscoe: A potent antiinflammatory agent. J Ethnopharmacol. 2012; 139: 889-892.

Zhang Q, Wu H, Ling Y, Lu R. Isolation and identification of antioxidant peptides derived from whey protein enzymatic hydrolysate by consecutive chromatography and Q-TOF MS. J Dairy Res. 2013; 80: 367-373.

Mojica L, de Mejía EG. Optimization of enzymatic production of anti-diabetic peptides from black bean (Phaseolus vulgaris L.) proteins, their characterization and biological potential. Food Funct. 2016; 7: 713-727.

Daliri EB-M, Oh DH, Lee BH. Bioactive Peptides. Foods. 2017; 6:32.

Virtanen T, Pihlanto A, Akkanen S, Korhonen H. Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. J Appl Microbiol. 2007; 102: 106-115.

Niki E, Yoshida Y, Saito Y, Noguchi N. Lipid peroxidation: mechanisms, inhibition, and biological effects. Biochem Biophys Res Commun. 2005; 338: 668-676.

Peng X, Kong B, Xia X, Liu Q. Reducing and radical-scavenging activities of whey protein hydrolysates prepared with Alcalase. Int Dairy J. 2010; 20 360-365.

Breiteneder H, Mills ENC. Molecular properties of food allergens. J Allergy Clin Immunol Pract. 2005; 115: 14-23.

Tsabouri S, Douros K, Priftis, KN. Cow Milk Allergenicity. Endocr Metab Immune Disord Drug Targets. 2014; 14: 16–26.

Vane JR, Bolting RM. New insights into the mode of action of anti-inflammatory drugs. Inflam Res. 1995; 44: 1-10.

Chou CT. The Antiinflammatory Effect of an Extract of Tripterygium wilfordii Hook F on Adjuvant‐induced Paw Oedema in Rats and Inflammatory Mediators Release. Phytother Res. 1997; 11: 152-154.

Iskandar MM, Dauletbaev N, Kubow S, Mawji N, Lands LC. Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4. Br J Nutr. 2013; 110: 58-68.

Piccolomini AF, Iskandar MM, Lands LC, Kubow S. High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells. Food Nutr Res. 2012; 56: doi: 10.3402/fnr.v56i0.17549.

Théolier J, Hammami R, Labelle P, Fliss I, Jean J. Isolation and identification of antimicrobial peptides derived by peptic cleavage of whey protein isolate. J Funct Foods 2013; 5: 706-714.

Pellegrini A, Dettling C, Thomas U, Hunziker P. Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochim Biophys Acta 2001; 1526: 131-140.

Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloubes R, Postle K, Riley M, Slatin S, Cavard D. Colicin biology. Microbiol Mol Biol Rev. 2007; 71: 158-229.

Abriouel H, Franz CM, Ben Omar N, Galvez A. Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev. 2011; 35: 201-232.

Tenovuo J, Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral dis. 2002; 8: 23-29.

Gilliland SE; Speck ML. Antagonistic action of Lactobacillus acidophilus toward intestinal and foodborne pathogens in associative cultures. J Food Prot. 1977; 40: 820-823.

Warny M, Fatimi A, Bostwick EF, Laine DC, Lebel F, LaMont JT, Pothoulakis C, Kelly CP. Bovine immunoglobulin concentrate-clostridium difficile retains C difficile toxin neutralising activity after passage through the human stomach and small intestine. Gut. 1999; 44: 212-217.

Olorunfemi OB, Adebolu TT, Adetuyi FC. Antibacterial Activities of Micrococcus lactis strain isolated from Nigerian fermented cheese whey against Diarrhoea causing organisms. Res Journalf Biol Sci. 2006; 1: 24-27.

Sagong HG, Lee SY, Chang PS, Heu S, Ryu S, Choi YJ, Kang DH. Combined effect of ultrasound and organic acids to reduce Escherichia coli O157: H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh lettuce. Int J Food Microbiol. 2011; 145: 287-292.


Copyright (c) 2018 IHTM - Centar za elektrohemiju

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.