Utilisation Of Spirulinasp. And Chlorellapyrenoidosa Biomass For The Productionof Enzymatic Protein Hydrolysates

This aim of this study was to assess the hydrolysis reaction of the biomass of Chlorella pyrenoidosaandSpirulinasp. LEB 18,using commercial proteases that act in different pH ranges, to obtain protein hydrolysates with promising application in food or food supplement, improving functional and nutritional food properties. Threecentral composite study designs were carried out for each microalga (Chlorella and Spirulina). The 2 3 type central composite design was utilized with three replications at the central point, varying the enzyme concentration (5 to 10 U.mL -1 ), the concentrationof substrate (5 to 10 %) and reaction time (60 to 240 min), for a total of 11 experiments per planning. The highestdegrees of hydrolysis (52.9% and 55.31%) forSpirulinaand Chlorella,respectively, were obtained with 4 h of reaction. The results show that it is possible to obtain enzymatic protein hydrolysates with different DH from microalgae biomass. Keywordsexperimental design; enzymatic hydrolysis,microalgae, proteases I. 1.INTRODUCTION Microalgae have been the subject of biotechnology research due to their nutritional, economic and ecological importance. Many microalgae are used in food production because they provide various useful substances, such as vitamins, minerals, pigments, fatty acids and proteins [1]. SpirulinaandChlorellaare microalgae with biomass that is rich in proteins, with values above 50%,biocompounds with high added value can be extracted from them.These microalgae are notable as theypossess a GRAS(Generally Recognized as Safe)certification from the FDA(Food and Drug Administration),which ensures they can be used as a food and drug [2]. The enzymatic hydrolysis of polymers in foods is an important process, used to improve the physical, chemical and functional properties of foods without damaging their nutritional value. It improves the absorption characteristics of proteins. 3 Protein hydrolysates have been consistently reported as a suitable source of protein for human nutrition because they are absorbed more effectivelyin the gastrointestinal tract when compared with intact protein or free amino acids[4], [5] and [6]. Many scientific studies have been carried out under different experimental conditions to obtain hydrolysates from different protein sources such as: whey [7] and [8], fish [9] and [10]and chicken [5]. Recently, proteins from microalgae have been recommended as an alternative source due to their abundant protein content and amino acid profile [11].The microalgaeSpirulinasp.LEB 18 andChlorella pyreinodosahave a high protein concentration in their biomass and are promising sources of protein for protein hydrolysates. The enzymatic hydrolysis of cellular proteins fromgreen algae has been described as a highly promising method of improving the digestibility of protein and obtaining a balanced protein for human consumption [4] and [12].However, there are hardly any data available on the use of proteinfrom Chlorellaand Spirulinaasprotein hydrolysates for human nutrition. The aim of this study was to assess the influence of variables of the process involved in enzymatic reactions to obtain protein hydrolysates, using three different commercial proteases that act in different pH ranges (alkaline, neutral and acid) with amicroalgal biomasssubstrate of Spirulina and Chlorella. II. MATERIALS AND METHODS 2.1 Microalgae The microalgae used for obtaining protein hydrolysateswere Chlorella pyrenoidosa,in powder form, produced by Fuqing King Dnarmsa Co. Ltd., China and Spirulinasp.LEB 18, isolated from Mangueira Lagoon 13 and produced in the pilot plant of the Laboratory of Biochemical Engineering, RESEARCH ARTICLE OPEN ACCESS Jorge A. V. Costa et al Int. Journal of Engineering Research and Applications www.ijera.com ISSN : 2248-9622, Vol. 4, Issue 5( Version 1), May 2014, pp.29-38 www.ijera.com 30 | P a g e atMangueira Lagoon (33° 30’ 13’’S e 53° 08’ 59’’ W) in Santa Vitória do Palmar, Brazil.The biomass ofSpirulinasp.LEB 18 was ground in a ball mill (Model Q298, QUIMIS) and sieved. 2.2 Enzyme The enzymes used wereProtemax 580Lof Bacillus lichenformis, ProtemaxN200 of Bacillus subtilis(Prozyn, São Paulo, Brazil) and pepsin derived from pig stomach (VetecQuímicaFina LTDA, Rio de Janeiro, Brazil). Different proteases were used in order to assess the behavior of hydrolysis at different pH (alkaline, acid and neutral) and to enable a comparison between proteases that do not exist in the human gastrointestinal tract (GIT),such as pepsin, to obtain protein hydrolysates. 2.3 Enzymatic activity The enzymatic activity of commercial proteases was determined according to the method described by Ma and colleagues [14].The tyrosine content of the supernatant was determined by colorimetry at 650nm usingFolin phenol reagent [15].The enzyme was inactivated by the addition of 10% trichloroacetic acid (TCA) and the activity defined as the amount of enzyme that releases 1μg of tyrosine per minute, under the conditions used in the study. 2.4 Percentual Composition Total protein, ash, moisture and lipids were determined according to methods described by AOAC [16]. To determine the amount of protein, the total nitrogen micro-Kjeldahlmethod was used with a conversion factor of 6.25. Ash was determined by the gravimetric method in a muffle (550-600 °C) and moisture content by the gravimetric method in an oven (105 °C). The lipids were extracted with 2:1 (v/v) chloroform/methanol, purified with 0.9% (w/v) NaCl and 2:1 (v/v) methanol/water mixed according to Folch and Lees [17]and transferred to a rotary evaporator; the solvent was removed at approximately 37°C, the content of lipids was determined gravimetrically. The remainder was considered to be carbohydrates. 2.5 Experimental design In this study, the factorial planning was used to determine which variables had significant effects on the degree of hydrolysis. Thus, the study assessed the influence of the enzyme concentration, substrate concentration and reaction time. The initial content of the substrate, the concentration of added enzyme and reaction time were the variables studied througha factorial design. Six2 3 type factorial planningswere carried out with three repetitions at the central point; the degree of hydrolysis of proteins was the dependent variable.Analyses were carried out in duplicate and the datawere statistically analyzed.Table 1 presentsthe matrix of the 2 3 central composite design with the levels and values of the independent variables used in the factorial designs.The proposed levels for each variable were based on preliminary experiments (data not shown). Table 1. Matrix ofthe2 3 centralcomposite designwithactualandcodedvariables Assay CE (U.mL -1 ) CS (%) t (min) 1 -1 (5) -1 (5) -1 (60) 2 +1 (10) -1 (5) -1 (60) 3 -1 (5) +1 (10) -1 (60) 4 +1 (10) +1 (10) -1 (60) 5 -1 (5) -1 (5) +1 (240) 6 +1 (10) -1 (5) +1 (240) 7 -1 (5) +1 (10) +1 (240) 8 +1(10) +1 (10) +1 (240) 9 0 (7.5) 0 (7.5) 0 (150) 10 0 (7.5) 0 (7.5) 0 (150) 11 0 (7.5) 0 (7.5) 0 (150) a CE = enzyme concentration; CS =substrate concentration; t = time The influences of the enzyme concentration, substrate concentration and reaction time on the degree of hydrolysis response were statistically analyzedin order to assess the effects and verify the empirical models through regression coefficients and analysis of variance (ANOVA) with a significance of 95%. 2.6 Enzymatic Hydrolysis Process Six experimental designswerecarried out, namely C1, C2 and C3, for hydrolysis using Chlorella pyrenoidosa as a protein source,and S1, S2 and S3 for reactions usingSpirulinasp.LEB 18. Thenumbers represent the use of different enzymes in the study (1: Protemax 580L, 2: Protemax N200, and 3: Pepsin); the biomasses were previously Jorge A. V. Costa et al Int. Journal of Engineering Research and Applications www.ijera.com ISSN : 2248-9622, Vol. 4, Issue 5( Version 1), May 2014, pp.29-38 www.ijera.com 31 | P a g e characterized regarding their percent composition. 16 The assaysC1 and S1 were carried out withProtemax 580L,in bicarbonate-sodium carbonate buffer pH 9.5 at the optimum enzyme activity temperature of 60 °C; C2 and S2 were carried out with Protemax N200, in sodium phosphate buffer 0.2M pH 7.0 at the optimum enzyme activity temperature of 55 °C;and C3 and S3 were carried out the enzyme pepsin, in 0.1 M KCl-HClbuffer pH 2.3 at the optimum temperature of 37°C. All experiments were carried out with agitation of 180 rpm in a "Shaker" (Certomat BS-1), in aqueous solution with a total volume corresponding to 100mL in erlenmeyertype reactors.The amounts of enzyme and substrate added corresponded to the values established during the factorial design. The hydrolysis reactions were accompanied for 4 h. 2.7 Determination of the Degree of Hydrolysis The analysis of the degree of hydrolysis (DH) was carried out at 0h, 1h, 2.5h and 4h. Aftereach elapsed time, 1 mL aliquots of hydrolysate were inactivated by the addition of 9 mL of trichloroacetic acid (TCA) 6.25% and maintainedat rest for 10 min. They were subsequently centrifuged for 5 min at 5000 rpm to remove the insoluble material precipitated by the TCA. The content of soluble proteins in the filtrate was determined using the Folin-Lowrymethod, expressed as mg of albumin.The degree of hydrolysis was estimated according to the method described by Hoyle and Marrit [18]and expressed as the percentage of soluble proteins in the TCA compared with the total initial protein amount, and calculated according to Equation (1):