III Symposium on Agricultural and Agroindustrial Waste Management March 12-14, 2013 - Sao Pedro, SP, Brazil CHICKEN SKIN FAT AS RAW-MATERIAL FOR MODIFYING LIPIDS TO HIGH VALUE DIACYLGLYCEROL RICH IN PUFA Vivian Feddern1*; Leonor Almeida de Souza-Soares2; Xuebing Xu3 1Embrapa Suínos e Aves, Concórdia-SC-Brasil. * [email protected] 2Universidade Federal do Rio Grande, FURG, Rio Grande-RS-Brasil [email protected] 3University of Aarhus, Aarhus-Denmark [email protected] ABSTRACT: As one of the main chicken meat producers and exporters, Brazil has also high waste generation, which includes chicken skin, a low value residue rarely utilized or underutilized. Skin fat may be extracted and reacted with different sources of lipids, acids or alcohols through interesterification reactions. Chicken skin in rich in oleic and palmitic fatty acids, the former may be kept or increased through this reaction in the modified lipids obtained, while the last may be decreased, turning this source more applicable nutritionally or technologically. In the present work, chicken skin fat and branched-chain fatty acids undergone lipase-catalyzed interesterification reaction through Central Composite Rotatable Design (CCDR), varying enzyme concentration (5-15%), water addition (0-10%), substrate molar ratio (2:1-6:1 branched fatty acids: chicken skin fat) and time (8-24 h) in order to produce modified lipids rich in polyunsaturated fatty acids. The interesterified samples were applied on thin layer chromatography plates, extracted, methylated and injected into gas chromatograph. Different lipids as diacylglycerols (DAGs) rich in oleic and linoleic fatty acids were obtained. In modified DAG, around 6% of oleic acid was achieved when water was added in the reactional medium and also 2.5% of essential linolenic acid was observed when branched compounds were increased. Thus, chicken skin interesterification may be an alternative to increase unsaturated fatty acids with application onto technological purposes with improved nutritional value. Keywords: branched-chain fatty acids, interesterification, lipid profile INTRODUCTION Brazil is the world leader in chicken meat exports (3.9 million tons) and the third large producer comprising 13.0 million tons of poultry in 2011 (UBABEF, 2012). Thus, high amount of residues, including chicken skin are being generating, knowing that each broiler carcass contains around 15% skin (Hayse & Marion, 1973, cited by Sheu & Chen, 2002), a low value residue rich in oleic and palmitic fatty acids (FAs) is being lost or underutilized. The former FA is known for its benefits by lowering blood cholesterol levels, reducing the risk of cardiovascular diseases (Lopez-Huertas, 2010), while the last is usually referred as a risk factor to cause this types of diseases (Labarthe, 2011). The nutritional, technological and physical properties of triacylglycerols can be modified by lipase-catalyzed interesterification (Kourist et al., 2010; Xu, 2003). Through this type of reaction, low-grade skin fat can be transformed into high-grade modified lipid, with specific and desired properties. In a given oil/fat is possible to maintain desire monounsaturated fatty acids as oleic and reduce saturated ones using sn-1,3 specific lipase. This enzyme is known to be regiospecific, acting only in sn-1 and sn-3 positions in the triacylglycerol backbone, preserving sn-2 position where usually the essential and other unsaturated fatty acids are. These reactions are carried out under milder conditions (temperature lower than 70 ºC, III Symposium on Agricultural and Agroindustrial Waste Management March 12-14, 2013 - Sao Pedro, SP, Brazil atmospheric pressure) with higher selectivity, while chemical reactions are performed around 180 ºC and originate dark products (Teng et al., 2009; Xu, 2003). The specific distribution of FAs in the triacylglycerol backbone plays a key role in lipid digestion and absorption. Branched fatty compounds are of commercial significance because the branching may cause changes in physical properties. For instance, a broader liquidity range and low surface tension is interesting for low-temperature applications and spreadability. These compounds are used in surfactants, lubricants, cosmetics, and as polymers additives, defoamers and wood protecting agents (Behr & Laufenberg, 1991, cited by Knothe et al., 2007). As far as we know, there are no reports regarding interesterification reaction combining chicken skin fat with branched-chain fatty acids. Thus, the purpose of this work was to react these substrates in order to produce modified lipids rich in polyunsaturated fatty acids and lower the saturated ones in the final products. MATERIAL AND METHODS Raw-material Ckicken skin was donated by a chicken-processing industry of the south region of Rio Grande do Sul state, Brazil, and a mixture of branched fatty acids, named 18-MEA (18- Methyl Eicosanoic Acid), a derivative of lanolin was produced by Croda, Malmo, Sweden. Lipozyme® RM IM, a 1,3 specific lipase from Rhizomucor miehei was donated by Novozymes A/S (Bagsværd, Denmark). Standards of fatty acids, as well as mono, di and TAG were purchased from Sigma-Aldrich. All chemical solvents were of analytical grade. Fat was extracted from chicken skin according to Bligh & Dyer (1959) method, following some modifications (Christie, 1982; Smedes & Thomasen, 1996). The mixture of branched fatty acids had the following lipid profile: C14 1.8%, C16 6.7%, C18 3.8%, C18:1 0.9%, C18:2 11.9%, C18:3 15.9%, C20 2%, C20:2 12%, C24 0.8%. Chicken skin fat lipid profile: C14 0.53%, C16 23.5%, C18 6.1%, C18:1 34.8%, C18:2 28.3%, C18:3, 2.3%. Interesterification Reaction Interesterification reactions type acidolysis were carried out at 60 ºC in water-jacket reactors using hexane as solvent and Lipozyme® RM IM as biocatalyst. Four variables at three levels were studied: enzyme concentration (5-15%), water addition (0-10%), substrate molar ratio (2:1-6:1 branched fatty acids: chicken skin fat) and time (8-24 h), according to a Central Composite Design (CCD) with 27 experimental runs. Thin layer chromatography (TLC), Methylation and Gas Chromatography Interesterified samples were isolated and applied on Silica gel 60 TLC plates (20x20 cm, Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid 70/30/1(v/v/v) as developing solvents in a closed chamber for 90 min. Following spraying with 1% ethanolic solution of 2,7-dichlorofluorescein, the bands were visualized under UV light. The bands containing diacylglycerols (DAGs) were scraped off and extracted with chloroform: methanol (2:1), evaporated under nitrogen and subjected to methylation. The fatty acid methyl esters (FAME) were prepared according to Feddern et al. (2010). A gas chromatograph (Thermo Scientific DSQ II - TRACE GC ULTRA, USA) with an auto sampler injection system (Thermo Scientific triplus ™ Autosampler), a flame ionization detector (FID) and a fused silica capillary column (30 m x 0.25 mm x 0.25 µm) were used to separate DAG fraction. Carrier gas and conditions used followed the same abovementioned authors. The peaks were identified by comparing the samples retention times with the standards, using Xcalibur software (version 2.0.7) of Thermo Fisher Scientific Inc. (Waltham, MA, USA). III Symposium on Agricultural and Agroindustrial Waste Management March 12-14, 2013 - Sao Pedro, SP, Brazil RESULTS AND DISCUSSION Modified DAGs obtained through enzymatic reactions presented different FAs, but the main are shown in Table 1, which are of interest, namely palmitic (C16:0), oleic (C18:1), linoleic (C18:2) and linolenic FAs (C18:3), being the last two essential ones. Although saturated FAs are referred as risk causing factor and should be low in the diet, Cintra et al. (2006) pointed out that negative effects of high saturated FAs content present in chicken skin diet can be counter balanced by the positive effects of its MUFA content. Table 2 depicts the main effects of the significant variables (P<0.05), namely C18:1 and C18:3. For C18:1, the more water was added in the reactional medium, more monounsaturated fatty acids (MUFA), up to 6%, were incorporated into DAG. On the opposite, when increasing branching compounds, less MUFA were incorporated (-6% for substrate alone and -7.4% for substrate x time interaction). Regarding C18:3, increasing branching compounds (from 2:1 to 6:1) into the system, an increment (2.5%) of this essential FA in DAG was observed. It depends of what property or nutrient the industry or professional is looking for, it’s possible to direct the formation of different structured lipids. CONCLUSION The research presents an alternative to reuse chicken skin and utilize it for technological and nutritional purposes, depending on the desire property or fatty acid to be incorporated through enzyme interesterification. ACKNOWLEDGMENTS Aarhus University staff where the work was developed, CAPES/PDEE Scholarship (process number 1104-09-8) given to the first author and Novozymes are thanked. REFERENCES Bligh, E. G., Dyer, W. J. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, v.37, n.8, p.911-917, 1959. Christie, W. W. Lipid analysis. Oxford: Pergamon Press. Chromatographic and spectroscopic analysis of lipids: general principles. Ch.3, p. 25-49, 1982. Cintra, D. E. C., Costa, A. G. V., et al. Lipid profile of rats fed high-fat diets based on flaxseed, peanut, trout, or chicken skin. Nutrition, v.22, n.2, p.197-205, 2006. Feddern, V., Xu, X., Souza-Soares, L. A., Badiale-Furlong, E. Modified triacylglycerol production through acidolysis from chicken skin fat and branched chain fatty acids. In: 4º Congresso Internacional de Bioprocessos na Indústria de Alimentos. Curitiba, PR. São Paulo: TecArt, 2010. Knothe, G., Kenar, J. A., Gunstone, F. D. 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