花生四烯酸(arachidonic acid,ARA)可直接参与婴幼儿认知和视觉功能的构建,以中长链甘油三酯(medium- and long-chain triglycerides,MLCT)为载体制备ARA型的结构脂质可提升婴幼儿对ARA的吸收效率。该研究以表面活性剂生物印迹脂肪酶提升酶活力,并催化裂殖壶菌微生物油与中链甘油三酯(medium-chain triglycerides,MCT)的酯交换反应合成ARA型MLCT。通过单因素试验和正交试验确定生物印迹脂肪酶的最佳制备条件如下:以Lipozyme 435为催化剂,吐温-20为生物印迹模板,表面活性剂质量浓度为50 mg/L,缓冲液pH值为7.0,这使得印迹后脂肪酶的初始活力比印迹前提高了1.36倍。MLCT合成的最佳条件为脂肪酶添加量的质量分数为6%、MCT与微生物油摩尔比1.5∶1、温度70 ℃、反应时间6 h,产物组成的质量分数为13.12% MCT、66.80% MLCT和20.08%长链甘油三酯(long-chain triglycerides,LCT)。其中,位于sn-1,3位的MCFA的摩尔分数为58.13%,位于sn-2位的ARA摩尔分数为15.61%。生物印迹脂肪酶重复使用7次后MLCT含量仍保持61.05%。该研究合成ARA型MLCT,提高了ARA生物利用率,在功能食品和营养保健品中具有潜在应用价值。
Arachidonic acid (ARA) plays a key role in infant cognitive and visual development.Enhancing ARA absorption efficiency can be achieved by structuring lipids using medium- and long-chain triglycerides (MLCT) as carriers.This study aimed to synthesize ARA-enriched MLCT through enzymatic interesterification of Schizochytrium sp.microbial oil with medium-chain triglycerides (MCT), using surfactant-imprinted lipase to improve catalytic activity.The optimal preparation conditions of bio-imprinted lipase were determined through single-factor experiments and orthogonal experiments as follows:Lipozyme 435 served as the catalyst, Tween 20 as the imprinting template, surfactant concentration at 50 mg/L, and buffer pH at 7.0.These conditions increased the lipase’s initial activity by 1.36-fold.For MLCT synthesis, the best reaction conditions included 6% (mass fraction) lipase addition amount, a 1.5∶1 MCT-to-microbial oil molar ratio, 70 ℃, and 6 h.The mass fraction of the final product contained 13.12% MCT, 66.80% MLCT, and 20.08% long-chain triglycerides (LCT).Notably, the molar fraction of MCFA at the sn-1,3 position was 58.13%, and the molar fraction of ARA at the sn-2 position was 15.61%.After BIL was reused for 7 times, the MLCT content still remained at 61.05%.The synthesized ARA-enriched MLCT demonstrates improved bioavailability, offering potential applications in functional foods and nutritional supplements for infants.
[1] GARG P, PEJAVER R K, SUKHIJA M, et al.Role of DHA, ARA, & phospholipids in brain development:An Indian perspective[J].Clinical Epidemiology and Global Health, 2017, 5(4):155-162.
[2] BOURLIEU C, MÉNARD O, BOUZERZOUR K, et al.Specificity of infant digestive conditions:Some clues for developing relevant in vitro models[J].Critical Reviews in Food Science and Nutrition, 2014, 54(11):1427-1457.
[3] YUHAS R, PRAMUK K, LIEN E L.Human milk fatty acid composition from nine countries varies most in DHA[J].Lipids, 2006, 41(9):851-858.
[4] ABRAHAMSE E, MINEKUS M, VAN AKEN G A, et al.Development of the digestive system:Experimental challenges and approaches of infant lipid digestion[J].Food Digestion, 2012, 3(1):63-77.
[5] LEE Y Y, TANG T K, CHAN E S, et al.Medium chain triglyceride and medium-and long chain triglyceride:Metabolism, production, health impacts and its applications - a review[J].Critical Reviews in Food Science and Nutrition, 2022, 62(15):4169-4185.
[6] AOYAMA T, NOSAKA N, KASAI M.Research on the nutritional characteristics of medium-chain fatty acids[J].The Journal of Medical Investigation, 2007, 54(3-4):385-388.
[7] YUAN T L, WEI W, ZHANG X H, et al.Medium- and long-chain triacylglycerols composition in preterm and full-term human milk across different lactation stages[J].LWT, 2021, 142:110907.
[8] AKRAM F, FATIMA T, UL HAQ I.Auto-induction, biochemical characterization and application of a novel thermo-alkaline and detergent-stable lipase (S9 peptidase domain) from Thermotoga petrophila as cleaning additive and degrading oil/fat wastes[J].Bioorganic Chemistry, 2024, 151:107658.
[9] ZOU X Q, SU H, ZHANG F C, et al.Bioimprinted lipase-catalyzed synthesis of medium- and long-chain structured lipids rich in docosahexaenoic acid for infant formula[J].Food Chemistry, 2023, 424:136450.
[10] CHENG C, JIANG T Y, WU Y L, et al.Elucidation of lid open and orientation of lipase activated in interfacial activation by amphiphilic environment[J].International Journal of Biological Macromolecules, 2018, 119:1211-1217.
[11] ZHU Y T, FENG Y H, WANG J W, et al.Selective esterification design of lipases for TAG synthesis based on the unique structure of curved DHA[J].ACS Food Science & Technology, 2024, 4(7):1722-1730.
[12] BILAL M, ZHAO Y P, NOREEN S, et al.Modifying bio-catalytic properties of enzymes for efficient biocatalysis:A review from immobilization strategies viewpoint[J].Biocatalysis and Biotransformation, 2019, 37(3):159-182.
[13] DE S BRANDÃO L M, BARBOSA M S, SOUZA R L, et al.Lipase activation by molecular bioimprinting:The role of interactions between fatty acids and enzyme active site[J].Biotechnology Progress, 2021, 37(1):e3064.
[14] FISHMAN A, COGAN U.Bio-imprinting of lipases with fatty acids[J].Journal of Molecular Catalysis B:Enzymatic, 2003, 22(3-4):193-202.
[15] OTZEN D E.Protein unfolding in detergents:Effect of micelle structure, ionic strength, pH, and temperature[J].Biophysical Journal, 2002, 83(4):2219-2230.
[16] OTZEN D E, OLIVEBERG M.Burst-phase expansion of native protein prior to global unfolding in SDS1[J].Journal of Molecular Biology, 2002, 315(5):1231-1240.
[17] ZOU X Q, HUANG J H, JIN Q Z, et al.Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid:Combination of enzymatic and physical methods[J].Journal of Agricultural and Food Chemistry, 2012, 60(37):9415-9423.
[18] XU X, BALCHEN S, HØY C E, et al.Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor[J].Journal of the American Oil Chemists’ Society, 1998, 75(11):1573-1579.
[19] AMENAGHAWON A N, ESHIEMOGIE S A, EVBARUNEGBE N I, et al.Surfactant-facilitated metabolic induction enhances lipase production from an optimally formulated waste-derived substrate mix using Aspergillus niger:A case of machine learning modeling and metaheuristic optimization[J].Bioresource Technology Reports, 2024, 28:101993.
[20] CARRASCO-LÓPEZ C, GODOY C, DE LAS RIVAS B, et al.Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements[J].Journal of Biological Chemistry, 2009, 284(7):4365-4372.
[21] ROCHA S, RUFINO A T, FREITAS M, et al.Methodologies for assessing pancreatic lipase catalytic activity:A review[J].Critical Reviews in Analytical Chemistry, 2024, 54(8):3038-3065.
[22] YANG T K, XU X B, HE C, et al.Lipase-catalyzed modification of lard to produce human milk fat substitutes[J].Food Chemistry, 2003, 80(4):473-481.
[23] XIE W L, ZHANG C.Propylsulfonic and arenesulfonic functionalized SBA-15 silica as an efficient and reusable catalyst for the acidolysis of soybean oil with medium-chain fatty acids[J].Food Chemistry, 2016, 211:74-82.
[24] ZOU X Q, KHAN I, WANG Y X, et al.Preparation of medium- and long-chain triacylglycerols rich in n-3 polyunsaturated fatty acids by bio-imprinted lipase-catalyzed interesterification[J].Food Chemistry, 2024, 455:139907.
[25] HE Y J, QIU C Y, GUO Z, et al.Production of new human milk fat substitutes by enzymatic acidolysis of microalgae oils from Nannochloropsis oculata and Isochrysis galbana[J].Bioresource Technology, 2017, 238:129-138.
