Optimization of Milk Cholesterol Reduction Using Beta-cyclodextrin and Catechin hydrate

Introduction: Cholesterol with the formula of C27H45OH is a requisite component in cell membrane and cell growth (Nataf et al 1948). The compound has a major role in human heart health. It has been proved that high cholesterol is a risk factor for human cardiovascular diseases and coronary heart diseases. Cholesterol is synthesized in mammalian cells (Ma and Shieh 2006). It is mostly found within the milk fat globule membrane and asymmetrically distributed in the both layer of the outer bilayer and it is found in triglyceride core in less amount (Lopez and Menard 2011). Beta-cyclodextrin (βCD) is known as a safe food ingredient which is a cyclic oligosaccharide consisting of seven glucopyranose molecules that are linked together with α 1- 4 bonds. The size and geometry of the βCD hydrophobic internal cavity allows good complexing with free and esterified cholesterol (Chafic Awad & Gray 1999). Absorption of cholesterol in human intestine decreases by consumption of green tea catechins. Phosphatidylcholine (PC) is an important substance for cholesterol solubility in micelles containing of bile salts, PC and cholesterol. This effect is probably due to the interaction of catechins and PC in bile salts micelles (Ogawa et al 2016). It is assumed that catechin hydrate can help cholesterol removal from fat globules with similar mechanism. Then, separated cholesterol molecules can be easily entrapped in βCD cavity. The aim of this study is evaluating the effect of using catechin hydrate to decrease the amount of necessary beta- cyclodextrin for achieving maximum cholesterol reduction in homogenized and sterilized milk. Material and Methods: Sterilized and homogenized milk with 3% fat was purchased from Pegah Dairy Co (Isfahan, Iran). Beta-cyclodextrin was purchased from Merk and catechin hydrate was purchased from Sigma Chemical CO (St. Louis, MO, USA). All reagents and solvents were of analytical grade. Response Surface Methodology (RSM) was used to optimize conditions that affect milk cholesterol reduction. Central composite design with 20 experimental points (6 center points, 6 axial points, 8 corner points, 3 blocks, 3 factors and 5 levels) was used. 70 ml of milk with different catechin hydrate concentrations was stirred (Alfa D 500 stirrer) at 750 rpm for 30 min at 25℃. Then, samples were stirred with different beta-cyclodextrin concentrations at various mixing time (according to RSM design) and centrifuged at 1000 × g, 25℃, for 10 min. The supernatant was separated and used for cholesterol determination by gas chromatography (GC)(Alonso et al 1995). Protein and fat content of milk were assayed at central points of experimental design using milkoscan. Radical scavenging and total anti- oxidant capacity were measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Balakrishnan and Agrawal 2014) and ascorbic acid methods (Taj khan et al 2017), respectively. Sensory evaluation of samples was carried out with 10 panelists (5 females and 5 males, ages 25 to 40 years old). A five- point hedonic scale was provided to the panelists (Keshtkaran et al 2012). The amount of fat, protein, radical scavenging DPPH and total antioxidant capacity were measured in central points. The statistical analyses were based on the mean ± standard deviation (SD) in experimental samples. Mean value of control and treated samples were compared using studentʹs t- test (P ≤ 0.05). Sensory evaluation data was analyzed by SPSS software. Results and Discussion: The interaction between βCD and mixing time at constant catechin hydrate concentration (0.01%) showed that maximum cholesterol reduction (96.96%) was obtained at βCD concentration of 0.12% and mixing time of 38 min. The reason for this result is due to the interaction of catechin hydrate with some of fat globule membrane components such as PC and casein in homogenized milk that decreases cholesterol solubility. So cholesterol can be easily separated from milk fat globules. Then, maximum cholesterol reduction was achieved at low concentration of βCD (βCD: cholesterol molar ratio of 4:1). The interaction between catechin hydrate and mixing time at constant concentration of βCD 0.12% showed that by increasing catechin hydrate concentration to more than 0.03%, cholesterol reduction decreased. This effect is due to the competition between cholesterol and catechin hydrate for entrapping in βCD cavity. The interaction between catechin hydrate concentrations and mixing time at constant βCD concentration (1.3%) showed reverse correlation between these two factors. Maximum cholesterol reduction was obtained at catechin hydrate concentration from 0.01% to 0.08% and mixing time from 27.5 to 49 min; or catechin hydrate concentration from 0.08% to 0.12% and mixing time from 6 to 27.5 min. Comparison between control and treated milk at central points showed that fat content decreased in treated samples. Because cholesterol molecules are a part of milk lipids that is separated from treated milk. On the other hand, some of milk free fatty acids could be entrapped in βCD cavity. Also, the amount of protein in treated samples decreased because of entrapping of amino acids in βCD cavity and absorption of negative charge of proteins on the outer surface of βCD. Therefore, part of milk proteins together with βCD– cholesterol complex leaves the environment during centrifugation process (Maskooki et al 2013). While, the amount of antioxidant activity measured by ascorbic acid and DPPH methods increased which was due to the residual catechin hydrate in treated milk. Sensory evaluation results showed that no significant difference was observed between control and treated sample. Conclusion: According to results, it seems that catechin hydrate has dual effects on cholesterol reduction from sterilized and homogenized milk. A positive effect is observed at the low concentration of catechin hydrate due to the separation of cholesterol from fat globules that decreases the needed amount of βCD and negative effect is due to the competition between catechin hydrate and cholesterol for entrapping in βCD cavity. Overall, the positive effect dominates the negative one at low concentration of catechin hydrate. It should be mentioned that the reasonable βCD: cholesterol molar ratio for cholesterol separation is 1:1 but practically this molar ratio is not enough, because cholesterol is placed in fat globule membranes. In previous studies βCD: cholesterol molar ratio was 34:1 while in this study the molar ratio decreased to 4:1, because catechin hydrate separates cholesterol from fat globule membranes and cholesterol easily entraps in βCD cavity. Therefore, by decreasing the amount of needed βCD for milk cholesterol removal, this process can be done in industrial scale.

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