Article pubs.acs.org/IECR
Concentration of Omega‑3 Polyunsaturated Fatty Acids from Oil of Schizochytrium limacinum by Molecular Distillation: Optimization of Technological Conditions GuiYu Zhang,† Jing Liu,‡ and YuanFa Liu*,† †
School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China Beijing Research Institute for Nutrition Resources, Beijing 100069 China
‡
ABSTRACT: Production of omega-3 polyunsaturated fatty acids concentrates from oil of Schizochytrium limacinum was optimized to obtain the highest omega-3 polyunsaturated fatty acid (ω-3PUFA) enrichment ratio. In this process, the content of docosapentenoic acid (DPA), the content of docosahexenoic acid (DHA) in the final product, the percentage recovery of the heavy phase, and the ω-3PUFA enrichment ratio were optimized. Single-factor experiments were carried out to study the effects of evaporator temperature, feeding rate, feeding temperature, and roller speed. A central composite design was used to optimize technological conditions. Under optimum conditions, the highest ω-3PUFA enrichment ratio (92.98%) was obtained at an evaporator temperature of 110.4 °C, a feeding rate of 78.7 mL/h, a roller speed of 350 rpm, a feeding temperature of 80 °C, and an operating pressure 10 Pa.
1. INTRODUCTION Marine oils are considered as the major commercial source of omega-3 polyunsaturated fatty acids (ω-3PUFAs). However, fish oils are unattractive because of their content of substantial amounts of undesirable fatty acids and cholesterol.1 A few microalgae strains are known to contain high level of lipids, and they represent a great area of interest for research into sustainable sources for PUFA production.2,3 Schizochytrium limacinum, a heterotrophic marine fungus, contains large amount of PUFAs, such as docosahexenoic acid (DHA) and docosapentenoic acid (DPA), which have a unique effect on cardiovascular diseases.4,5 Refined Schizochytrium limacinum oil contains some saturated fatty acids, including tetradecanoic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0).6−9 It has been suggested that ω-3PUFA concentrates devoid of more saturated fatty acids are much better than marine oils themselves because they allow the daily intake of total lipids to be kept as low as possible. With growing public awareness of the nutritional benefits of consuming ω-3PUFA concentrates, the market for these products is expected to grow in the future.10,11 There are several methods for enriching ω-3PUFAs, such as winterization,12 urea complexation,13 molecular distillation,14 AgNO3 complexation,15 enzymatic purification,16 and supercritical CO2 extraction.17 Molecular distillation (or short-path distillation) is known for the distillation of thermally unstable materials and is the most economically feasible method of purification.18 This means that, under high vacuum conditions, the distance between the evaporation surface and the condensation surface is less than or equal to the mean free path; that is, the molecules which escape from the evaporation surface easily reach and are condensed on the surface before they collide with each other, without any resistance.19 The simplest and most efficient technique for obtaining ω3PUFA concentrates in the form of fatty acid ethyl esters (FAEEs) is molecular distillation.20 This is a well-established © 2013 American Chemical Society
technique for the elimination of saturated and monounsaturated fatty acids. Initially, the FAEEs are prepared from the triacylglycerol (TAG) of the oil by interesterification.21 The saturated and monounsaturated FAEEs can escape from the evaporation surface easily because of their short mean free path, and the long-chain PUFAs are enriched in the heavy phase. In this study, molecular distillation of Schizochytrium limacinum oil was carried out to concentrate the ω-3PUFAs of the oil. Factors such as evaporator temperature, feeding rate, feeding temperature, roller speed, and operating pressure were studied to optimize the conditions for obtaining the maximum concentration of ω-3PUFA.
2. MATERIALS AND METHODS 2.1. Materials. Refined, DHA-rich oil of Schizochytrium limacinum was obtained from local sources from Qingdao, China. Fatty acid ethyl esters were purchased from J&K Scientific. All other chemicals used in this study were of American Chemical Society quality or better. The model used in this work was a falling-film short-path evaporator (KDL1; UIC GmbH, Alzenau-Hörstein, Germany). The operating parameters are listed in Table 1. Table 1. Operating Parameters of Molecular Distillation operating parameter
technical specification
evaporation area feed quantity evaporating temperature operating pressure
1.8 dm2 0.1−0.4 kg/h maximum 250 °C as low as 0.001 mbar
Received: Revised: Accepted: Published: 3918
July 31, 2012 September 22, 2012 February 13, 2013 February 13, 2013 dx.doi.org/10.1021/ie3020044 | Ind. Eng. Chem. Res. 2013, 52, 3918−3925
Industrial & Engineering Chemistry Research
Article
2.2. Fatty Acid Components of the Initial Oil from the Microalgae Schizochytrium limacinum. 2.2.1. Methyl Esterification of Microalgae Oil.22 Microalgae oil (2−3 mg) and 2 mL of KOH/CH3OH solution (0.5 mol/L) were mixed in a glass tube and heated in a water bath at 60 °C for 30 min. Then, 2 mL of BF3/CH3OH solution (1:3, v/v) was added at 70 °C for 3−5 min. The oil drops disappeared, and the reaction mixture was cooled. The fatty acid methyl esters (FAMEs) were then extracted with 2 mL of hexane. After layer separation, the upper layer was removed and purged with nitrogen to a constant weight. The fatty acid methyl esters (FAMEs) were identified by gas chromatography (GC) analysis. 2.2.2. Gas Chromatography (GC) Analysis. A Shimadzu gas chromatograph (Shimadzu Scientific Instruments) was used for FAME analysis. The GC instrument was equipped with a flameionization detector (FID) and a CP-Sil88 capillary column (100 m × 0.25 μm × 0.2 μm). The temperature of the injection port and of the detector was 250 °C. The temperature program was as follows: maintain 120 °C for 2 min, increase to 175 °C at a rate of 8 °C/min and maintain 175 °C for 28 min, then increase to 215 °C at a rate of 3 °C/min, and maintain 215 °C for 30 min. The injection volume was 0.02 μL. The FAMEs in the sample were identified by comparing the retention times with those of standard fatty acid methyl esters (Sigma, St. Louis, MO). To quantify the fatty acids, first, the response factor of each fatty acid was determined by running the FAMEs and the internal standard (heptadecanoic acid methyl ester) through the gas chromatograph in equal amounts and comparing the peak area of the fatty acid to that of the internal standard. 2.3. Preparation of Fatty Acid Ethyl Esters from Triacylgycerol (TAG).21 Fatty acid ethyl esters (FAEEs) were obtained by interesterification. First, the water bath was heated to 70 °C, and then 40 mL of ethanol (ethanol absolute) was introduced into a 250 mL three-neck round-bottom flask. To the flask were then added 0.75 g of sodium hydroxide and 100 mL of microalgae oil. The reaction was started by mechanical stirring for 4 h. After the reaction had completed, the mixture was allowed to settle into two layers, and glycerol was removed by separatory funnel. The residue was washed with distilled water twice and then dried with anhydrous sodium sulfate. 2.4. Fatty Acid Composition Analysis of Fatty Acid Ethyl Esters. The FAEEs were analyzed by GC using the same instrument as for the FAME analysis. The temperature of the injection port was 250 °C, and that of the detector was 260 °C. The oven temperature was programmed to hold at 40 °C for 2 min, increase to 160 °C at a rate of 20 °C/min, then increase to 220 °C at a rate of 2 °C/min, and finally hold at 220 °C for 30 min. The FAEEs in the sample were identified by comparing the retention times with those of standard FAEEs (Sigma, St. Louis, MO). To quantify the fatty acids, first, the response factor of each fatty acid was determined by running the FAEEs and the internal standard (heptadecanoic acid ethyl ester) through the gas chromatograph in equal amounts and comparing the peak area of the fatty acid to that of the internal standard. The raw material and fractions obtained from molecular distillation were diluted with n-hexane and dried over anhydrous sodium sulfate before being analyzed. 2.5. Experimental Design and Data Analysis.23 In this study, single-factor experiments were first carried out to study the effects of evaporator temperature, feeding rate, feeding temperature, and roller speed on the contents of docosapentenoic acid (DPA) and docosahexenoic acid (DHA) in the final
product and the percentage recovery of the heavy phase. Feeding temperature, feeding rate, and roller speed, which had significant influences on the content of PUFA and the yield of the heavy phase, were screened out. Once the significant factors had been identified in the single-factor experiments, a central composite design was used to optimize their levels. The design matrix was a 23 full-factor design combined with six central points and six axial points at which one variable was set to an extreme level while other variables were set to their centralpoint levels (Table 4). The coded and real values of each parameter are listed in Table 3. Based on the experimental results reported in Table 4, a quadratic polynomial regression model was assumed to predict the individual response (Y) variables. The model proposed for each response Y was 3
Y=
3
3
∑ βi Xi + ∑ βiiXi
2
+
∑∑
i=1
i=1
βijXiXj
i
