The aim of the present investigation was to evaluate the influence of the main operating conditions on the application of an enzymatic hydrolysis on sunflower lecithin at laboratory scale. This study seeks to contribute to the oil industry with useful information for rescaling this modification process, in order to increase the value-added of sunflower lecithins.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Cabezas, Darío Marcelino

Diehl, Bernd W. K.

Madoery, R.

Tomás, Mabel Cristina

Journal of the American Oil Chemists Society

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LETTER TO THE EDITORApplication of Enzymatic Hydrolysis on SunflowerLecithin Using a Pancreatic PLA2D. M. Cabezas • R. Madoery • B. W. K. Diehl •M. C. TomásReceived: 31 May 2010 / Revised: 20 August 2010 / Accepted: 8 September 2010 / Published online: 1 October 2010 AOCS 2010Sir,Lecithins are a mixture of acetone insoluble phos-pholipids, containing mainly phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylinositol (PI),minor compounds such as phosphatidic acid (PA), andother minor substances such as carbohydrates [1]. Theintroduction of changes in the original concentration ofthese phospholipids, by chemical or enzymatic modifica-tion of their structure can lead to lecithins with differentphysicochemical and functional properties, with respect tonative lecithin. These modified compounds can be used inthe development of a variety of food products, such asmargarines, chocolates and bakery products [2, 3]. Enzy-matic hydrolyzed lecithin may present technological andcommercial advantages over native lecithins as O/Wemulsifier and mold- or pan-releasing agent. Consequently,the demand for lysolecithin has been increasing in recentyears [4, 5]. PLA2 is the most employed family of enzymesfor the modification of lecithins [6]. These enzymes play animportant role in biochemical processes such as turnover ofmembrane phospholipids or the regulation of bioactivesubstances, e.g. lysophospholipids, fatty acids, and lipidmediators in inflammatory and digestion processes [7, 8].In Argentina, the production of sunflower oils is ofutmost importance with the consequent economic rele-vance [9]. Sunflower lecithin, i.e. a byproduct of thedegumming process of oils [10–12], is an alternative tosoybean lecithin because it is considered to be a non-GMOproduct, which is currently preferred by certain consumers.The aim of the present investigation was to evaluate theinfluence of the main operating conditions on the applica-tion of an enzymatic hydrolysis on sunflower lecithin atlaboratory scale. This study seeks to contribute to the oilindustry with useful information for rescaling this modifi-cation process, in order to increase the value-added ofsunflower lecithins.The enzymatic hydrolysis process was carried out using anative sunflower lecithin provided by a local oil industry anda porcine pancreatic PLA2 (Lecitase 10 L, 10,580 IU/mL,Novo Nordisk, Denmark). This process was carried out in athermostated reactor at laboratory scale, using 27 g of sun-flower lecithin and 18 ml of CaCl2 solution (0.1 or 0.4 M).The initial pH was adjusted to 7 or 9 by adding 4 N NaOHsolution. Then, the resulting mixture was set at 60 C, andphospholipase A2 solution was incorporated (0.4 or 2.0 ml in100 g of lecithin). The system was kept under continuousagitation (50 rpm) for 40 or 300 min. The evolution of thehydrolysis process was followed by measuring the pH, with apH meter for solid samples (840049 Puncture Tip, SaenzSRL). Products of enzymatic hydrolysis were subjected to asudden decrease in temperature to stop the process ofhydrolysis and then deoiled using acetone, according toAOCS Official Method Ja 4–46, procedures 1–5 [13]. Afterthat, samples were stored at 0 C. The hydrolysis processwas carried out in duplicate.D. M. Cabezas  M. C. Tomás (&)Centro de Investigación y Desarrollo en Criotecnologı́a deAlimentos (CIDCA), CCT La Plata, CONICET,Facultad de Ciencias Exactas,Universidad Nacional de La Plata (FCE, UNLP),47 y 116 (1900), La Plata, Argentinae-mail: mabtom@hotmail.comR. MadoeryCátedra de Quı́mica Orgánica, Facultad de Ciencias Agrarias,Universidad Nacional de Córdoba (FCA, UNC), CiudadUniversitaria S/N, 5000 Córdoba, ArgentinaB. W. K. DiehlSpectral Service GmbH Laboratorium für Auftragsanalytik,Emil Hoffman Str. 33, 50996 Cologne, Germany123J Am Oil Chem Soc (2011) 88:443–446DOI 10.1007/s11746-010-1684-9Phospholipid composition of samples obtained underdifferent conditions of enzymatic hydrolysis, was deter-mined by 31P-NMR analysis using a Bruker Avance300 MHz automatic spectrometer using triphenyl phosphateas internal standard (Spectral Service GmbH, Köln, Ger-many) [14]. Then 100 mg of each hydrolyzed sample wasdiluted in 1 ml of deuterated chloroform, 1 ml of methanoland 1 ml of 0.2 M Cs-EDTA (pH 8.0). The organic layer wasseparated after 15 min shaking, and analyzed by this spec-troscopic technique. Phospholipid composition was expres-sed in terms of molar concentration.The degree of enzymatic hydrolysis associated witheach type of phospholipid was determined using a rela-tionship between the phospholipid concentration in deoiledand hydrolyzed sunflower lecithin, as is shown in Eq. 1:%HPL ¼PLi  PLfPLi  100 ð1Þwhere PL is PC, PI, PE or PA; PLi is the phospholipidconcentration in deoiled lecithin (PCi 37.5%, PIi 37.8%,PEi 15.1%, PAi 4.3%) and PLf is the phospholipid con-centration in the hydrolyzed lecithin.The sunflower lecithin used as starting material pre-sented the following composition: 43.1% phospholipids(PC 36.7%, PI 35.3%, PE 15.0%, PA 5.1%, and 7.9%others phospholipids), 33.4% oil, and 23.5% of othercompounds (glycolipids, complex carbohydrates).The hydrolyzed lecithins present a high concentration ofmajor lysophospholipids, with respect to native (1.1%) anddeoiled sunflower lecithin (2.4%). In this way, Fig. 1shows the 31P-NMR spectra of the deoiled sunflower lec-ithin and two hydrolyzed sunflower lecithin obtained underdifferent processing conditions.The effect of phospholipase A2 on each phospholipid(PC, PE, PI and PA) can be analyzed following the evo-lution of the degree of hydrolysis. In Table 1, it is possibleto observe that PC presented a degree of hydrolysis higherthan 91.4% for all the conditions assayed. When 2.0% ofPLA2 was used, the residual PC concentration in thehydrolyzed lecithin was lower than the detection limit ofthe 31P NMR equipment (\0.1%) (see Fig. 1). Values of%HPE were also high ([80.9%), although slightly lowerthan %HPC, in all experiments. These results can be cor-related with those described by Penci [15] using a very lowamount of substrate (1 mg).Values obtained for the different degrees of hydrolysis(%HPL) and total lysophospholipid content (LPL) wereanalyzed using ANOVA, and the influence of each variablewas associated with the percentage of the explained variance(Table 2).Phospholipase A2 content and reaction time were sig-nificant for all the analyzed parameters. A rise of thesevariables showed a highly significant increase (p \ 0.01)of the different degrees of hydrolysis obtained for the mainphospholipids (see Table 1). Phospholipase content was thevariable that presented the highest effect on the explainedFig. 1 31P-NMR spectra at 300 MHz of different modified sunflowerlecithins444 J Am Oil Chem Soc (2011) 88:443–446123variance of %HPL values ([47.3%), especially for %HPCand %HPI.Calcium chloride concentration and pH were not sig-nificant variables on the hydrolysis of PC, PI and PE(p [ 0.05). However, a significant increase of %HPA val-ues when decrease the levels of both variables wasrecorded.On the other hand, a highly significant effect of thePLA2 9 time interaction on the hydrolysis of PC, PE andPI was observed. At high concentrations of PLA2, thereaction time did not present an important effect on theparameters %HPC and %HPI, due to the high degree ofhydrolysis exhibited by both phospholipids ([96.0%). Inaddition, a wide range of %HPE values was determined fora major level of PLA2 in the period studied.The hydrolyzed sunflower lecithins presented a LPLcontent ranging from 61.9–93.8% (Table 1). The influenceof the different variables on the total lysophospholipidconcentration of the hydrolyzed sunflower lecithin wasaccording to the degree of hydrolysis discussed above. Ahighly significant increase (p \ 0.01) of LPL content wasrecorded as a function of the rise of the variables PLA2content and reaction time which represent 90.1 and 7.1% ofthe explained variance for this parameter, respectively.Acknowledgments This work was supported by grants fromCONICET (PIP 1735), ANPCyT (PICT 1085), and UNLP (11/X502).Sunflower lecithin was provided by Vicentin SAIC; Thorsten Buchenand Sandro Goñi are acknowledged for their technical assistance.References1. Cabezas DM, Diehl B, Tomás MC (2009) Sunflower lecithin:application of a fractionation process with absolute ethanol. J AmOil Chem Soc 86:189–1962. Schneider M (1989) Fractionation and purification of lecithin. In:Szuhaj BF (eds) Lecithin: sources, manufacture & uses. Chapter7. AOCS Press, Champaign, pp. 109–1303. van Nieuwenhuyzen W, Tomás MC (2008) Update on vegetablelecithin and phospholipid technologies. Eur J Lipid Sci Technol110:472–4864. Erickson MC (2008) Chemistry and function of phospholipids.In: Akoh CC, Min DB (eds) Food lipids: chemistry, nutrition andbiotechnology, Chapter 2, 3rd edn. CRC Press, Boca Raton,pp 39–625. Hirai H, Sono R, Koh H (1998). Process for manufacturingvegetable lysolecithins. European Patent EP08708406. Schmitt H, Heirman M (2007) Enzymatic modification of leci-thin. US Patent US7189544B27. De Marı́a L, Vind L, Oxenbøll KM, Svendsen A (2007) Phos-pholipases and their industrial application. Appl Microbiol Bio-technol 74:290–3008. Wilton DC (2005) Phospholipases A2: structure and function. EurJ Lipid Sci Technol 107:193–2059. Ministerio de Economı́a y Producción Argentina (acceded Agost2010). Informe de Cadenas Alimentarias: Sector Oleaginoso.http://www.alimentosargentinos.gov.ar/0-3/revistas/r_41/cadenas/Oleaginosos_Oleaginosas.htm10. Cabezas DM, Diehl B, Tomás MC (2009) Effect of processingparameters on sunflower PC enriched fractions extracted withaqueous-ethanol. Eur J Lipid Sci Technol 111:993–100211. Pan LG, Campana A, Tomás MC, Añón MC (2000) A kineticstudy of phospholipid extraction by degumming process in sun-flower seed oil. J Am Oil Chem Soc 77:1273–127612. Pan LG, Noli A, Campana A, Barrera M, Tomás MC, Añón MC(2001) Influence on the processing conditions on degummingprocess in sunflower seed oil. J Am Oil Chem Soc 78:553–55413. CS AO (1994) Official methods and recommended practices ofthe American Oil Chemists’ Society, 4th edn. AOCS Press,ChampaignTable 1 Degree of hydrolysis of main phospholipids (%HPL) andtotal lysophospholipid content (LPL) of hydrolyzed sunflower lecit-hins under different operating conditionsPLA2a Time(min)CaCl2(M)InitialpH%HPCb %HPIb %HPEb %HPAb LPLb0.4 40 0.1 7 91.7 44.7 80.9 69.8 63.20.4 40 0.1 9 91.4 44.8 81.6 44.8 61.90.4 40 0.4 7 93.3 45.1 82.0 58.8 65.20.4 40 0.4 9 95.5 46.4 84.5 39.9 66.50.4 300 0.1 7 94.9 62.0 85.4 91.9 74.90.4 300 0.1 9 94.4 61.6 86.1 60.2 69.40.4 300 0.4 7 97.9 63.6 85.7 80.3 74.30.4 300 0.4 9 97.6 65.0 86.5 53.8 72.62.0 40 0.1 7 100.0 92.4 88.8 100.0 87.82.0 40 0.1 9 100.0 93.4 85.8 83.0 88.12.0 40 0.4 7 100.0 100.0 88.1 90.8 91.42.0 40 0.4 9 100.0 97.2 90.1 60.5 88.72.0 300 0.1 7 100.0 100.0 96.7 100.0 93.82.0 300 0.1 9 100.0 96.0 97.7 100.0 92.02.0 300 0.4 7 100.0 100.0 97.1 100.0 93.92.0 300 0.4 9 100.0 100.0 96.2 88.4 92.5a ml PLA2 solution in 100 g of lecithinb Average values (n = 2). The coefficient of variation was lowerthan 6%Table 2 Percentage of explained variance according to ANOVA forthe degree of hydrolysis of main phospholipids (%HPL) and totallysophospholipid content (LPL)Variablesa Explained variance (%)%HPC %HPI %HPE %HPA LPLPLA2 74.0 91.3 59.2 47.3 90.1Time 6.6 5.4 31.7 15.3 7.1CaCl2 nsv nsv nsv 5.7 nsvpH nsv nsv nsv 24.6 0.5PLA2 9 time 6.6 2.6 5.3 nsv 0.9PLA2 9 CaCl2 5.6 nsv nsv nsv nsvPLA2 9 pH nsv nsv nsv 1.8 nsvnsv not significantly variable (p \ 0.05)a time 9 CaCl2, time 9 pH, and CaCl2 9 pH interactions were nsvfor all parameters analyzedJ Am Oil Chem Soc (2011) 88:443–446 44512314. Diehl BWK (2008) NMR spectroscopy of natural substances. In:Holzgrabe U, Wawer I, Diehl B (eds) NMR spectroscopy inpharmaceutical analysis, Chapter 5. Elsevier, Jordan Hill,pp 194–19615. Penci MC, Constenla DT, Carelli AA (2010) Free-fatty acidprofile obtained by enzymatic solvent-free hydrolysis of sun-flower and soybean lecithins. Food Chem 120:332–338446 J Am Oil Chem Soc (2011) 88:443–446123

Application of Enzymatic Hydrolysis on Sunflower Lecithin Using a Pancreatic PLA2

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Application of Enzymatic Hydrolysis on Sunflower Lecithin Using a Pancreatic PLA2

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