Influence of β-Cyclodextrin on Reduction of Cholesterol Content in Dairy, Egg and Meat Products L. Alonso1 and J. Fontecha2 1Instituto de Productos Lácteos de Asturias (CSIC) 2Instituto de Investigación en Ciencias de la Alimentación (CSIC-UAM) INTRODUCTION For the past 20 years, consumers have been reducing their fat consumption due to health concerns surrounding animal high fat diets. This trend has affected several food products. Dairy, egg and meat products have been considered to increase the risk for cardiovascular diseases in humans because, in comparison to other lipid sources, they contain a high proportion of cholesterol. Studies describing the association between dairy, egg and meat food consumption and the risk of cardiovascular disease (CVD) have been inconsistent. In large epidemiological studies, consumption of high cholesterol fat products has been associated with an increased CVD risk, while intake of low cholesterol fat foods has not. Methods for reducing cholesterol in foods have been developed, including between other, adsorption with saponin and digitonin and removal by supercritical carbon dioxide extraction. Nowadays, a number of studies have indicated that cholesterol removal from food products was most effectively achieved by beta-cyclodextrin (β-CD) (1). OBJECTIVE The aim of this work was the use of β-CD with pasteurized cow’s and ewe´s whole milk for making butter and cheese and its influence in cristal, natural eggs and pates with the purpose of manufacturing low cholesterol food products. Analysis of cholesterol and fatty acids by gas chromatography  The technique chosen for cholesterol determination was as described by Alonso et al. (2) using methylated fat by capillary gas chromatography (GC) using a HP-5 fused silica capillary column 30 m x 0.32 mm i.d. x 0.25 m film thickness. Approximately 30 mg anhydrous milk fat and 0.1 ml 5-α-cholestane as internal standard (3.5 mg/mL in hexane) was dissolved in 1 mL of hexane; 0.5 μL of the resulting solution was injected for GC analysis. The GC analysis was on an Agilent Technology 6890 chromatograph (Palo Alto, CA) equipped with flame ionization detector (FID). Fatty acids were determined. as fatty acid methyl esters on a Agilent Technology 6890 chromatograph (Palo Alto, CA) with FID detector. Fatty acids were separated using CP-Sil 88 fused-silica capillary column (3). Statistical analysis  Experimental data were treated by analysis of variance (ANOVA) using the statistical software SAS (version 8.02, SAS Institute Inc, Cary, NC, USA). Differences among treatments were determined by statistical analysis using a Student t-test where (P > 0.05) was considered statistically significant. MATERIAL AND METHODS Sampling Raw cow´s and ewe´s pasteurized milk obtained from Central Lechera Asturiana (Oviedo) and Montes de Toledo (Toledo) respectively, was treated with β-CD (purity 99.5 %, Shandong Xinda Fine Chemical Co., Ltd. Qingdao, China) as proposed by (1) for obtaining butter and cheese. Cream was churned to butter using a continuous butter churn machine of 1500 Kg/h of butter (Contimab, Westfalia, Denmark) and cheese was made with the protocol as Manchego cheese. Crystal and natural egg were supplied by the company Huevos Maryper (Murcia) and patés by Malvasia, S.A. (Soria).   Lipid extraction Lipids were extracted from samples following the International Standard Method. RESULTS AND DISCUSION The cholesterol content (in %, g/100g fat) from control butter and β-CD butter varied between 0.286 ± 0.02% and 0.015 ± 0.008% respectively with a reduction of cholesterol after churning the cream for production of commercial butter of 95.53 ± 2.8% (Figure 1). In cheese the cholesterol reduction in three months Manchego cheese was 91.31 ± 2.4% (Figure 2) comparing with the control cheese. Table 1. Polyunsaturated fatty acids composition (g/100g fat) of control milk and milk with β-cyclodextrin added. Fatty acid Control milk ß-CD milk Linoleic(n-6) C18:2-cis-9, trans-13 0.182 ± 0.05 0.174 ± 0.05 C18:2-trans-8, cis-13 0.053 ± 0.01 0.050 ± 0.01 C18:2-cis-9, trans-12 0.081 ± 0.01 0.083 ± 0.01 C18:2-trans-11, cis-15 0.433 ± 0.08 0.412 ± 0.07 C18:2-cis-9,cis-15 0.024 ± 0.01 0.021 ± 0.01 C18:2-cis-9,cis-12 1.836 ± 0.07 1.819 ± 0.06 Linoleic conjugated (CLA) C18:2 cis-9, trans-11 0.672 ± 0.08 0.663 ± 0.07 C18:2 cis-11, trans-13 0.009 ± 0.00 0.007 ± 0.01 C18:2 trans-10, cis-12 0.021 ± 0.01 0.019 ± 0.01 Linolenic C18:3 (n-6) 0.152 ± 0.01 0.167 ± 0.01 C18:3 (n-3) 0.315 ± 0.01 0.332 ± 0.09 Total Conjugated linoleic 0.702 ± 0.08 0.689 ± 0.07 Total n-3 Polyunsaturated 0.441 ± 0.01 0.403 ± 0.02 Total n-6 Polyunsaturated 2.156 ± 0.10 2.142 ± 0.08 Figure 1 Figure 2 Cholesterol reduction in natural and crystal eggs ranged from 80 to 82 ± 2.6% (Figure 3) and in paté (mouse) about 82.5 ± 2.3% (Figure 4) comparing with control samples. Figure 3 Figure 4 CONCLUSION Results from the present study suggest that the treatment with β-cyclodextrin can be applied to dairy, egg and meat products for making low cholesterol food products without altering the nutritional fatty acid properties. Control With b-CD Control With b-CD No difference (P < 0.05) for the linoleic acid C18:2 (n-6) for control milk (1.836 ± 0.07) and treated β-CD milk (1.819 ± 0.06) was observed. In this study, the three conjugated linoleic acid isomers analysed were C18:2-cis-9 trans-11, C18:2-cis-11 trans-13 and C18:2-trans 10 cis-12 (Table 1). The main CLA isomer biologically active C18:2-cis-9 trans-11 (rumenic acid) did not show differences (P < 0.05) between control milk (0.672 ± 0.08) and β-CD milk (0.663 ± 0.07). The same patterns were observed for the total CLA in control milk (0.702 ± 0.08) and treated milk (0.689 ± 0.07). References 1. Alonso L, Cuesta P, Fontecha J, Juárez M and Gilliland SE. 2009. Use of β-cyclodextrin to decrease the level of cholesterol in milk fat. J. Dairy Sci. 92: 863-860 2. Alonso, L., L. Lozada, J. Fontecha, and M. Juarez. 1995. Determination of cholesterol in milk fat by gas chromatography with direct injection and sample saponification. Chromatographia 41: 23-25 3. Alonso, L., J. Fontecha, L. Lozada, M.J.Fraga, and M. Juárez. 1999. Fatty acid composition of caprine milk: major, branched-chain and trans fatty acids. J. Dairy Sci. 82: 878-884