Enzymatic Synthesis of Structured Monogalactosyldiacylglycerols

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Enzymatic Synthesis of Structured Monogalactosyldiacylglycerols Enriched in Pinolenic Acid Jiwon Kim, Min-Yu Chung, Hee-Don Choi, In-Wook Choi, and Byung Hee Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02599 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Journal of Agricultural and Food Chemistry

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Enzymatic Synthesis of Structured Monogalactosyldiacylglycerols Enriched in

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Pinolenic Acid

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Jiwon Kim,† Min-Yu Chung,‡ Hee-Don Choi,‡ In-Wook Choi,‡ and Byung Hee Kim*,§

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†

Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Korea

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‡

Korea Food Research Institute, Jeonbuk 55365, Korea

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§

Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Korea

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Short title: Enzymatic Synthesis of MGDGs Enriched in Pinolenic Acid

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* Corresponding author.

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Byung Hee Kim*

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Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Korea

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Tel.: + 82 220777241

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Fax: + 82 27109479

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E-mail address: [email protected]

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ABSTRACT: We enzymatically prepared structured monogalactosydiacylglycerols (MGDGs)

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enriched in pinolenic acid (PLA). PLA-enriched free fatty acids (FFAs) containing ~86 mol %

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PLA were produced from an FFA fraction obtained from pine nut oil (PLA content, ~13 mol %)

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by urea crystallization. Commercial MGDGs (5 mg) were acidolyzed with PLA-enriched FFAs

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using four commercial immobilized lipases as biocatalysts. The reaction was performed in

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acetone (4 mL) in a stirred-batch reactor. Lipozyme RM IM (immobilized Rhizomucor miehei

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lipase) was the most effective biocatalyst for the reaction. Structured MGDGs containing 42.1

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mol % PLA were obtained under optimal reaction conditions: temperature, 25 °C; substrate

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molar ratio, 1:30 (MGDGs:PLA-enriched FFAs); enzyme loading, 20 wt % of total substrates;

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and reaction time, 36 h. The structured MGDGs were separated from the reaction products at a

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purity of 96.6 wt % using silica column chromatography. The structured MGDGs could be

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possibly used as emulsifiers with appetite-suppression effects.

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KEYWORDS:

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monogalactosyldiacylglycerols, pinolenic acid, structured lipids

Acidolysis,

appetite

suppressants,

emulsifiers,

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Lipozyme

RM

IM,

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INTRODUCTION

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Monogalactosyldiacylglycerols (MGDGs) are glycoglycerolipids that contain one

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galactopyranosyl residue (predominantly in beta-form), which is linked to the sn-3 position of

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1,2-diacylglycerols.1 Naturally occurring MGDGs are mainly found in the thylakoid membrane

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within the chloroplasts of higher plants and occupy half of the chloroplast lipids, thereby

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indicating that MGDGs are the most abundant polar lipids on Earth.1,2 MGDGs are also found in

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other photosynthetic organisms, such as algae and fungi.3 Dietary MGDGs can help control body

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weight by inhibiting the activity of pancreatic lipase.4 Because dietary MGDGs are hydrolyzed

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by pancreatic lipase, they may compete with dietary triacylglycerols for hydrolysis of pancreatic

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lipase in the duodenum, thereby helping to delay digestion and absorption of triacylglycerols.4

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Undigested triacylglycerols in the ileum from delayed triacylglycerol digestion induce the

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secretion of gut satiety hormones, which slow gastric emptying and suppress appetite.5 MGDGs

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exhibit surface or interfacial active properties because they are amphiphilic molecules that

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contain a galactose moiety, which is a polar and uncharged head group.1 Therefore, MGDGs can

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act as non-ionic emulsifiers in food products. Several researchers have attempted to utilize

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MGDGs as emulsifiers to inhibit fat bloom in chocolate or to enhance the baking performance

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(e.g., loaf volume and crumb structure) of bread.6,7

D-

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Pinolenic acid (PLA; c5,c9,c12-18:3) is a ∆5-unsaturated polymethylene-interrupted fatty acid

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(FA). It is exclusively found in the oils of edible seeds (i.e., nuts) of pines, such as Korean pine

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nut (Pinus koraiensis; PLA content = ~14 mol %) or Siberian pine nut (P. sibirica; PLA content

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= ~19 mol %).8 Pine nut oil containing PLA or PLA itself is effective to reduce body weight

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because PLA provides an appetite-suppression effect that is derived from the increased secretion

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of gut satiety hormones, such as cholecystokinin-8 and glucagon-like peptide-1 in humans.8 3 ACS Paragon Plus Environment


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Structured lipids are lipids in which the composition and/or positional distribution of FAs have

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been modified from their natural biosynthetic form.9 Structured lipids can be synthesized

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chemically or enzymatically. Enzymatic synthesis of structured lipids using lipases as

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biocatalysts has several advantages over chemical methods because the enzymatic process

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reduces by-product formation, requires mild reaction conditions, and does not use deleterious

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chemicals.9 Furthermore, it is easy to recover the products and to reuse the lipases, which is

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environmentally friendly and economically feasible, when immobilized lipases are used.9 In the

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lipase-catalyzed synthesis of structured lipids, acidolysis (the transfer of an acyl group between

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an acid and an ester) is one of the most effective means of incorporating targeted free FAs (FFAs)

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into lipids.10 In this study, we prepared structured MGDGs enriched in PLA using an

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immobilized lipase-catalyzed acidolysis of MGDGs with PLA-enriched FFAs obtained from pine

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nut oil. The structured MGDGs could be used as a new type of non-ionic emulsifier with

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appetite-suppression effects because they are expected to have the physicochemical and

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biological features of both MGDGs and PLA. Several recently published studies have attempted

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to synthesize structured lipids enriched in PLA, in the form of triacylglycerols11-13 or

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monoacylglycerols14 via lipase-catalyzed reaction, such as acidolysis11 or esterification.12-14

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However, no reports have examined the existence or preparation of MGDGs containing PLA.

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The aim of this study was to enzymatically prepare structured MGDGs enriched in PLA for

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potential use as emulsifiers with appetite-suppression effects. The structured MGDGs were

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synthesized from commercial MGDGs and PLA-enriched FFAs from pine nut oil by lipase-

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catalyzed acidolysis. The reaction was performed in acetone in a stirred-batch reactor. We

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evaluated the catalytic activity of four commercially available immobilized lipases. For a

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selected lipase, the effects of temperature, substrate molar ratio, and enzyme loading on the PLA 4 ACS Paragon Plus Environment

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content in the structured MGDGs were investigated; optimal reaction conditions to prepare

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MGDGs with a maximum PLA content were suggested by considering the economic feasibility

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of our reaction system. Finally, we suggested a purification procedure to separate the structured

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MGDGs from the reaction products.

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MATERIALS AND METHODS

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Materials. Pine nut oil (PLA content = 13.7 mol %) was obtained from Komega Co.

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(Eumseong, Korea). Commercial MGDGs (purity >99 wt %) were purchased from Avanti Polar

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Lipids, Inc. (Alabaster, AL). Immobilized lipases from Rhizomucor miehei (Lipozyme RM IM),

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Thermomyces lanuginosus (Lipozyme TL IM), and Candida antarctica (Novozym 435) were

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purchased from Novozymes A/S (Bagsværd, Denmark). Candida rugosa lipase (immobilized on

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Immobead 150) was purchased from Sigma Chemical Co. (St. Louis, MO). Urea (purity >99

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wt %) and dodecan-1-ol (purity >98 wt %) were purchased from Samchun Pure Chemical Co.

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(Pyeongtaek, Korea). Silica gel-coated glass plates for thin layer chromatography (TLC) were

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purchased from Merck (Darmstadt, Germany). The FA methyl ester (FAME) standards were

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obtained from Supelco (Bellefonte, PA). All other reagents were of analytical or high-

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performance liquid chromatography grade.

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Preparation of PLA-Enriched FFAs from Pine Nut Oil. PLA-enriched FFAs were obtained

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from pine nut oil by saponification followed by urea crystallization of the FFAs from the oil,

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according to the method described by Woo et al.12 with a slight modification. Pine nut oil (100 g)

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was saponified with 400 mL of 10 wt % NaOH in 75 vol% ethanol. Distilled water (200 mL) was

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added to the saponified mixture, and the resulting aqueous layer was acidified by adding 250 mL

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of 6 N HCl. n-Hexane (500 mL) and distilled water (300 mL) were added, and the aqueous layer 5 ACS Paragon Plus Environment


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was removed using a separatory funnel; the n-hexane layer containing the FFAs was washed five

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times with 100 mL of distilled water. The n-hexane layer was dried over anhydrous Na2SO4, and

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n-hexane was removed using a rotary vacuum evaporator at 40 °C. Then, the obtained FFAs (50

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g) and urea (200 g) were dissolved in 500 mL of methanol, and the solution was refluxed at

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250 °C until the urea dissolved completely. The solution was cooled to room temperature (23 °C)

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at a rate of ~2.5 °C/min and held at this temperature for 30 min. The solution was then held at

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4 °C for 16 h to crystallize the urea–FFA complex. The crystals that formed in the solution were

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removed by filtration through Whatman No. 1 filter paper in a Buchner funnel. Methanol was

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removed from the filtrate using a rotary vacuum evaporator at 40 °C, and the remaining filtrate

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was acidified with 300 mL of hot (~70 °C) 0.1 N HCl. The uncrystallized portion of the FFAs

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was recovered by extracting the acidified filtrate with 500 mL of n-hexane twice in a separatory

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funnel. The n-hexane layer was washed twice with 100 mL of water and dried over anhydrous

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Na2SO4. n-Hexane was removed using a rotary vacuum evaporator at 40 °C to obtain the PLA-

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enriched FFAs, which were then stored at −80 °C.

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Enzymatic Preparation of Structured MGDGs Enriched in PLA. To prepare structured

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MGDGs enriched in PLA, acidolysis was performed in acetone for commercial MGDGs with the

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PLA-enriched FFAs obtained from pine nut oil using Lipozyme RM IM as the biocatalyst

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(Figure 1). The reaction was performed in a flat-bottom glass vessel (15 cm × 5 cm internal

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diameter (i.d.)) equipped with a water jacket for temperature control. The vessel was maintained

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at the desired temperature (25, 30, 35, and 40 °C) using a water circulator (model RW-0252G,

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Jeio Tech, Seoul, Korea). The substrates contained 5 mg of MGDGs and 18.7, 37.3, 56.0, or 74.7

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mg of the PLA-enriched FFAs (corresponding to molar ratios of 1:10, 1:20, 1:30, and 1:40,

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respectively). After dissolving the substrates in 4 mL of acetone, the desired amounts (5, 10, 15, 6 ACS Paragon Plus Environment

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and 20 wt % of the total substrates) of Lipozyme RM IM were added, and the reaction mixture

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was agitated at 8.3 Hz using a magnetic stirrer during the reaction.

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Separation of Structured MGDGs from Reaction Products Using TLC. Samples (0.2 mL)

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of the reaction products were withdrawn from the reaction mixture at appropriate time intervals

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(i.e., 3, 6, 12, 24, 36, 48, and 60 h) during the reaction. The samples were dissolved in 2 mL of a

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mixture of chloroform and methanol (1:2, v/v) and filtered through a 0.45-µm hydrophilic

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polypropylene membrane filter with an anhydrous Na2SO4 layer on top to remove the enzyme.

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After completely drying the filtrates under a nitrogen flush, they were re-dissolved in 0.2 mL of

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chloroform and methanol (1:2, v/v), placed on a TLC plate, and then developed with

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chloroform/methanol/water/ethyl acetate/2-propanol (50:20:10:50:50, v/v/v/v/v). The plate was

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air-dried and sprayed with 0.2% 2,7-dichlorofluorescein in methanol, and the bands were

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visualized under ultraviolet light. The band corresponding to the MGDGs was scraped from the

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plates.

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Analysis of the FA Composition. We analyzed the total FA compositions of pine nut oil,

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FFAs of pine nut oil, PLA-enriched FFAs obtained from the FFAs of pine nut oil, commercial

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MGDGs, and structured MGDGs separated from reaction products following the method

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described by Kang et al.15 Lipid samples (20 mg) or MGDG bands scraped from the TLC plates

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were saponified with 3 mL of 0.5 N NaOH in methanol at 85 °C for 10 min and cooled to room

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temperature (23 °C). After methylating the saponifiable lipids with 3 mL of 14% BF3 in

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methanol at 85 °C for 10 min, the mixture was cooled to room temperature, 3 mL of isooctane

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and 5 mL of a saturated NaCl solution were added, and the mixture was vortexed. The upper

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isooctane layer containing the FAMEs was collected and passed through an anhydrous Na2SO4

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column. The FAMEs were analyzed by gas chromatography using an Agilent Technologies 7 ACS Paragon Plus Environment


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7890A gas chromatograph (Palo Alto, CA) equipped with a flame ionization detector and a fused

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silica column (SP-2560, 100 m × 0.25 mm i.d. × 0.2-µm film thickness, Supelco). A FAME

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sample (1 µL) was injected in split mode with a split

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20:1). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector and detector

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temperatures were maintained at 225 and 285 °C, respectively. The oven temperature was held at

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100 °C for 4 min, increased to 240 °C at a rate of 3 °C/min, and finally held at 240 °C for 17 min.

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The FAMEs were identified by comparing their retention times with those of the standards, and

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their contents (mol %) were calculated.

ratio of 200:1 (for MGDG analysis,

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Purification of Structured MGDGs from the Reaction Products Using Column

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Chromatography. Structured MGDGs enriched in PLA was purified from the reaction products

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obtained under optimal conditions using silica column chromatography. After packing a slurry of

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silica gel (5 g) and hexane/diethyl ether (60:40, v/v) into the glass column (30 mm × 3 mm i.d.),

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the products (40 mg) were loaded onto the silica gel bed. The column was then flushed with 250

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mL of hexane/diethyl ether (60:40, v/v) to remove FFAs, followed by 20 mL of acetone to elute

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the MGDGs. The eluted MGDG fractions were collected and dried under a nitrogen flush.

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Analysis of the Purity of Structured MGDGs Enriched in PLA. The purity of the

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structured MGDG fraction obtained from silica column chromatography was analyzed by liquid

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chromatography (LC) equipped with an evaporative light scattering detector (ELSD). The LC

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system (JASCO Corp., Tokyo, Japan) consisted of a pump (model PU-2089) and an autosampler

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(model AS-2057). The ELSD (model Alltech 3300, Associates Inc., Deerfield, IL) contained a

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drift tube that was maintained at 70 °C, and the flow of nitrogen was 2.5 L/min. The FFAs,

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MGDGs,

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LiChrospher® 100 Diol column (250 mm × 4 mm, 5-µm particle size; Merck, Darmstadt,

and

monogalactosylmonoacylglycerols

(MGMGs)


were

separated

using

a

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Germany). Optimal separation was achieved with a gradient elution using (A) n-hexane and (B)

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a mixture of n-hexane/2-propanol/1-butanol/water (60:30:7:3, v/v/v/v) at a flow rate of 1 mL/min.

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The gradients (time, %B) were set as (0 min, 70), (5 min, 100), (20 min, 100), and (25 min, 70).

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The reaction products (1 mg) were dissolved in methanol (2 mL), and the solution was injected

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into the column for analysis. The FFAs, MGDGs, and MGMGs were identified by comparing

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their retention times with those of the standards, and their contents (wt %) were calculated.

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Statistical Analysis. All data are presented as the mean value with the standard deviation (i.e.,

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mean ± standard deviation). The difference in the lipid samples was determined using a Student’s

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t-test or one-way analysis of variance followed by Duncan’s multiple range test. The difference

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was considered significant if the P-value was < 0.05.

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RESULTS AND DISCUSSION

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FA Composition of the Substrates. Table 1 gives the FA compositions of the PLA-enriched

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FFAs and commercial MGDGs, which were used as the substrates in the acidolysis for preparing

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structured MGDGs enriched in PLA. Pine nut oil and the FFA fraction obtained from the oil,

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which was used as the starting materials to prepare PLA-enriched FFAs, were also given in the

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table. The PLA-enriched FFAs contained 85.5 mol % PLA. The major FAs of commercial

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MGDGs were n-3 polyunsaturated FAs, including α-linolenic (18:3n-3, 57.7 mol %) and

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c7,c10,c13-hexadecatrienoic acid (16:3n-3, 37.5 mol %), thereby occupying 95.2 mol % of the

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total FAs in the MGDGs.

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Evaluation of the Catalytic Activity of the Lipases. The catalytic activities of four

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commercial immobilized lipases were evaluated in the acidolysis of commercial MGDGs with

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the PLA-enriched FFAs obtained from pine nut oil to prepare structured MGDGs enriched in 9 ACS Paragon Plus Environment


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PLA. The reaction was performed in acetone because it was found that acetone was the most

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effective medium for the reaction (unpublished observations). Lipozyme RM IM and Lipozyme

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TL IM are sn-1,3-specific lipases, which show marked preferences for the acyl ester bonds at the

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sn-1 and sn-3 positions of the glycerol backbone. However, Novozym 435 and immobilized C.

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rugosa lipase on Immobead 150 are nonspecific lipases that do not show a distinct specificity

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with respect to the position of the acyl ester bond. Figure 2 shows the PLA content in the

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structured MGDGs that were separated from the reaction products by TLC as a function of the

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reaction time (3–60 h). For these trials, the temperature, substrate molar ratio, and enzyme

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loading were kept constant at 25 °C, 1:40 (MGDGs to PLA-enriched FFAs), and 20 wt % (based

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on total substrates), respectively. Lipozyme RM IM gave a significantly greater PLA content

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than any other lipases throughout the entire reaction and had a maximum PLA content of 42.3

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mol % at 36 h. For the trial with Lipozyme TL IM, a maximum PLA content of 33.5 wt % was

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obtained at 60 h. No PLA was found in the MGDGs present in the reaction products obtained

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using Novozym 435 and C. rugosa lipase after 60 h, thereby indicating that these two

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nonspecific lipases showed no catalytic activity in the reaction. These results suggest that

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Lipozyme RM IM has the greatest catalytic activity in the acidolysis of MGDGs with the PLA-

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enriched FFAs. Therefore, Lipozyme RM IM was selected as the most suitable biocatalyst for the

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preparation of structured MGDGs enriched in PLA.

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Effects of Reaction Conditions on the PLA Content in Structured MGDGs. We evaluated

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the effect of three reaction parameters—temperature, substrate molar ratio, and enzyme

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loading—on the Lipozyme RM IM-catalyzed acidolysis of commercial MGDGs with the PLA-

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enriched FFAs obtained from pine nut oil to prepare structured MGDGs enriched in PLA.

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Temperature. Temperature is a key parameter in an enzymatic reaction. In general, increasing 10 ACS Paragon Plus Environment

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the temperature increases the reaction rate by facilitating interactions between the enzymes and

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substrates.16 However, too high temperatures can decrease the reaction rate because of

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irreversible denaturation of the enzyme.10 The optimal temperature for Lipozyme RM IM lies

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between approximately 30–50 °C (information provided by the manufacturer). Figure 3 shows

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the effects of temperature on the PLA content in the structured MGDGs as a function of the

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reaction time (3–60 h). We examined the reaction at 25–40 °C by considering the boiling point

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(~56 °C) of acetone, which was used as the reaction medium, and the optimal temperature for

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Lipozyme RM IM. For these experiments, the substrate molar ratio and enzyme loading were

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maintained at 1:40 and 20 wt %, respectively. No significant difference was observed in the PLA

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content among all the experiments throughout the reaction period. Furthermore, the maximum

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PLA content (42.3 mol %) was achieved after 36 h at 25 °C; however, the PLA content was not

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significantly different from that in the other experiments—the maximum PLA contents were 42.1

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mol % (60 h) at 30 °C, 42.5 mol % (36 h) at 35 °C, and 42.8 mol % (36 h) at 40 °C. The

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temperature must be reduced to synthesize structured MGDGs enriched in PLA using the

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Lipozyme RM IM catalyst in an economically feasible manner. Therefore, 25 °C was considered

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as the optimal temperature for the acidolysis reaction.

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Substrate Molar Ratio. In the lipase-catalyzed acidolysis to prepare structured lipids, a

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relatively higher molar ratio of the targeted FFA to the lipids incorporates a higher amount of

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FFAs into the lipids.17 However, an excessive molar ratio can increase production costs for the

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structured lipids because of the increased amount of waste products (i.e., unreacted FFA).

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Therefore, for industrial applications, it would be preferable to prepare structured MGDGs

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enriched in PLA at a relatively lower substrate molar ratio to minimize costs. Figure 4 illustrates

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the effects of the substrate molar ratio on the PLA content in the structured MGDGs as a function 11 ACS Paragon Plus Environment


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of the reaction time (3–60 h). The investigated substrate molar ratio varied from 1:10 to 1:40

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(MGDGs to PLA-enriched FFAs). For these trials, the temperature and enzyme loading were

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held constant at 25 °C and 20 wt %, respectively. At a substrate molar ratio of 1:10, the PLA

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content in the MGDGs in the reaction products increased with longer reaction times but did not

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reach a plateau throughout the reaction. For this trial, the maximum PLA content was 32.9 mol %

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at 60 h. The PLA content obtained after 6 h in the trial with a substrate molar ratio of 1:20 was

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significantly greater than that of the trial with a substrate molar ratio of 1:10. However, the

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maximum PLA content (35.0 mol %) achieved at 60 h in the trial with a substrate molar ratio of

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1:20 was not significantly different from that of the trial with a substrate molar ratio of 1:10.

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When the substrate molar ratio increased from 1:20 to 1:30, a significant increase in the PLA

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content was observed after 24 h. The maximum PLA content (42.1 mol % at 36 h) in the trial

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with a substrate molar ratio of 1:30 was also significantly greater than that of the trial with a

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substrate molar ratio of 1:20. However, a further increase in the substrate molar ratio to 1:40 did

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not significantly increase the PLA content after 6 h over that obtained at a substrate molar ratio

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of 1:30, although a significant increase in the PLA content was observed during the initial phase

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(i.e., before 3 h) of the reaction. For this trial, the maximum PLA content (42.3 mol %) was

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achieved at 36 h and was not significantly different from that of the trial with a substrate molar

257

ratio of 1:30. Therefore, the most suitable molar ratio of commercial MGDGs to PLA-enriched

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FFAs for the acidolysis reaction was ≥1:30.

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Enzyme Loading. An increase in the enzyme loading increases the reaction rate.18 However,

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the enzyme loading must be reduced to attain an economically feasible reaction system. Figure 5

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shows the effects of enzyme loading on the PLA content in the structured MGDG as a function

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of the reaction time (3–60 h). The tested enzyme loading range was 5–20 wt %. For these trials, 12 ACS Paragon Plus Environment

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the temperature and substrate molar ratio were kept constant at 25 °C and 1:40, respectively. An

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increase in the enzyme loading generally increased the PLA content for reaction times ≤36 h.

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The reaction time required to achieve the maximum PLA content generally decreased with

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increased enzyme loading—the maximum PLA content was obtained at 60 h for 5 and 10 wt %,

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48 h for 15 wt %, and 36 h for 20 wt % enzyme loadings, although no significant difference was

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found in the maximum PLA content (41.9–42.5 mol %) in all trials. To enhance the economic

269

feasibility of the reaction system, it is important to reduce the reaction time and enzyme loading.

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Therefore, an enzyme loading of 15 or 20 wt % was more desirable than 5 or 10 wt %.

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Optimal Reaction Conditions. We attempted to establish optimal reaction conditions to

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maximize the PLA content in the structured MGDGs while enhancing the economic feasibility of

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the reaction system. From the experimental results in sections 3.1.1–3.3.3, four combinations of

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reaction factors were generated, and optimal reaction conditions were selected from these results

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(Figure 6). Combinations for the substrate molar ratio and enzyme loading include 1:30 and 15

276

wt %, 1:30 and 20 wt %, 1:40 and 15 wt %, and 1:40 and 20 wt %, respectively. The temperature

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was fixed at 25 °C. The PLA content obtained in the trials with 1:30 (substrate molar ratio) and

278

20 wt % (enzyme loading), 1:40 and 15 wt %, or 1:40 and 20 wt % were generally greater than

279

that of the trial with 1:30 and 15 wt % throughout the reaction time, whereas no significant

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difference was found in the PLA content in the former three conditions after 12 h. The maximal

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PLA contents (41.9–42.3 mol %) obtained under these three conditions were greater than that

282

(39.3 mol %) for the trial with a substrate molar ratio and enzyme loading of 1:30 and 15 wt %,

283

respectively, but the results were not significantly different from each other. However, the

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reaction time to achieve the maximum PLA content was shorter in the trials with 1:30 and 20 wt %

285

(36 h) or 1:40 and 20 wt % (36 h) than in the trials with 1:30 and 15 wt % (60 h) or 1:40 and 15 13 ACS Paragon Plus Environment


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wt % (48 h). Consequently, it was concluded that the optimal reaction conditions were at a

287

temperature of 25 °C, substrate molar ratio of 1:30 (MGDGs to PLA-enriched FFAs), enzyme

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loading of 20 wt % of the total substrates, and reaction time of 36 h. Structured MGDGs

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containing 42.1 mol % PLA were obtained under these conditions.

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Purification of the Structured MGDGs from the Reaction Products. Structured MGDGs

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enriched in PLA were purified from the reaction products obtained under the optimal conditions

292

established in this study using silica column chromatography. Figure 7 compares the LC-ELSD

293

chromatograms of the reaction products and purified products obtained from the reaction

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products. Before the purification, the reaction products contained 0.9 wt % MGDGs, 98.8 wt %

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FFAs, and 0.3% MGMGs. Using silica column chromatography, the FFAs were almost

296

completely removed during the first elution using hexane/diethyl ether (60:40, v/v). Then the

297

residual FFAs, MGDGs, and MGMGs were eluted in order from the silica column using acetone

298

as the second eluent. The final purified products obtained after silica column chromatography

299

contained 96.6 wt % MGDGs and 3.4 wt % FFAs.

300

FA Composition of the Structured MGDGs. Table 1 compares the FA composition of the

301

structured MGDGs that were purified by silica column chromatography and that of the

302

commercial MGDGs. The structured MGDGs contained 42.1 mol % PLA, 31.1 mol % 16:3n-3,

303

8.0 mol % palmitic acid (16:0), 7.5 mol % 18:3n-3, 7.3 mol % linoleic acid (18:2n-6), 2.6 mol %

304

stearic acid (18:0), and 1.4 mol % oleic acid (18:1n-9). The contents of the two major FAs (i.e.,

305

18:3n-3 and 16:3n-3) of commercial MGDGs were significantly reduced in the structured

306

MGDGs; in particular, the 18:3n-3 content was more drastically reduced compared to the 16:3n-

307

3 content. These results suggest that 18:3n-3 was more predominant at the sn-1 position of

308

commercial MGDGs than 16:3n-3. However, further investigations into the positional 14 ACS Paragon Plus Environment

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309

distribution of FAs in the commercial MGDGs and structured MGDGs are required to reveal the

310

cause of this phenomenon.

311

In conclusion, the enzymatic reaction system used for acidolysis of commercial MGDGs with

312

PLA-enriched FFAs obtained from pine nut oil to prepare structured MGDGs enriched in PLA

313

used Lipozyme RM IM as the biocatalyst and acetone as the medium. It was demonstrated that

314

the structured MGDGs containing ~42 mol % PLA could be obtained under optimal reaction

315

conditions, which were established in this study. The structured MGDGs were successfully

316

purified at a high purity of ~97 wt % using silica column chromatography with a two-step elution.

317

The resulting structured MGDGs enriched in PLA would be suitable as emulsifiers with appetite-

318

suppression effects. However, for these applications, this study contains some limitations,

319

including the use of commercial MGDGs as the substrates and the use of very small quantities of

320

the substrates. Therefore, further investigations into the development of new MGDG sources and

321

scaling up the reaction system would be required for their possible applications.

322 323

ABBREVIATIONS USED

324

ELSD, evaporative light scattering detector; FA, fatty acid; FAME, fatty acid methyl ester; FFA,

325

free

326

monogalactosylmonoacylglycerol; PLA, pinolenic acid; TLC, thin layer chromatography

fatty

acid;

MGDG,

monogalactosydiacylglycerol;

MGMG,

327 328

Funding

329

This research was supported by Main Research Program (E0124200-05) of the Korea Food

330

Research Institute (KFRI) funded by the Ministry of Science, ICT & Future Planning and was

331

also supported by the Sookmyung Women’s University Research Grants (1-1703-2047). 15 ACS Paragon Plus Environment


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332 333

Notes

334

The authors declare no competing financial interest.

335 336 337 338 339 340

REFERENCES (1) Gounaris, K.; Barber, J. Monogalactosyldiacylglycerol: The most abundant polar lipid in nature. Trends Biochem. Sci. 1983, 8, 378–381. (2) Dörmann, P.; Benning, C. Galactolipids rule in seed plants. Trends Plant Sci. 2002, 7, 112– 118.

341

(3) Nakamura, Y.; Shimojima, M.; Ohta, H.; Shimojima, K. Biosynthesis and function of

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monogalactosyldiacylglycerol (MGDG), the signature lipid of chloroplasts. In The Chloroplast.

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Advances in Photosynthesis and Respiration (vol. 31), 1st ed.; Rebeiz, C. A., Benning, C.,

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Bohnert, H., Daniell, H. J., Hoober, J. K., Lichtenthaler, H. K., Portis, A. R., Tripathy, B. C.

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Eds.; Springer: Dordrecht, The Netherlands, 2010; pp 185–202.

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(4) Banskota, A. H.; Steevensz, A. J.; Stefanova, R.; Sperker, S.; Melanson, R.; Osborne, J. A.;

347

O’Leary,

S.

J.

B.;

Melanson,

J.

E.

Pancreatic

lipase

inhibitory

activity

of

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monogalactosyldiacylglycerols isolated from the freshwater microalga Chlorella sorokiniana. J.

349

Appl. Phycol. 2016, 28, 169–175.

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(5) Chu, B. S.; Rich, G. T.; Ridout, M. J.; Faulks, R. M.; Wickham, M. S.; Wilde, P. J. (2009).

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Modulating pancreatic lipase activity with galactolipids: effects of emulsion interfacial

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composition. Langmuir 2009, 25, 9352–9360.

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(6) Nakae, T.; Kometani, T.; Nishimura, T.; Takii, H.; Okada, S. Preparation of

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glyceroglycolipids from pumpkin and their effects on polymorphic transformation of cocoa 16 ACS Paragon Plus Environment

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butter. Food Sci. Technol. Res. 2000, 6, 263–268. (7) Selmair, P. L.; Koehler, P. Role of glycolipids in breadmaking. Lipid Technol. 2010, 22, 7– 10. (8) Xie, K. Y.; Miles, E. A.; Calder, P. C. A review of the potential health benefits of pine nut oil and its characteristic fatty acid pinolenic acid. J. Funct. Food. 2016, 23, 464–473. (9) Kim, B. H.; Akoh, C. C. Recent research trends on the enzymatic synthesis of structured lipids. J. Food Sci. 2015, 80, C1713–C1724.

362

(10) Willis, W. M.; Marangoni, A. G. Enzymatic interesterification. In Akoh, C. C., Min, D. B.

363

Eds., Food Lipids–Chemistry, Nutrition, and Biotechnology, 3rd ed.; CRC Press: Boca Raton, FL,

364

2008; pp 807–840.

365

(11) Choi, J. H.; Kim, B. H.; Hong, S. I.; Kim, Y.; Kim, I. H. Synthesis of structured lipids

366

containing pinolenic acid at the sn-2 position via lipase-catalyzed acidolysis. J. Am. Oil Chem.

367

Soc. 2012, 89, 1449–1454.

368

(12) Woo, H.; Kim, J.; Kim, I. H.; Choi, H. D.; Choi, I. W.; Kim, B. H. Substrate selectivity of

369

Novozym 435 in the esterification of glycerol with an equimolar mixture of linoleic, conjugated

370

linoleic, and pinolenic acids. Eur. J. Lipid Sci. Technol. 2016, 118, 928–937.

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(13) Chung, M. Y.; Woo, H.; Kim, J.; Kong, D.; Choi, H. D.; Choi, I. W.; Kim, I. H.; Noh, S.

372

K.; Kim, B. H. Pinolenic acid in structured triacylglycerols exhibits superior intestinal lymphatic

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absorption as compared to pinolenic acid in natural pine nut oil. J. Agric. Food Chem. 2017, 65,

374

1543–1549.

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(14) Pyo, Y. G.; Hong, S. I.; Kim, Y.; Kim, B. H.; Kim, I. H. Synthesis of monoacylglycerol

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containing pinolenic acid via stepwise esterification using a cold active lipase. Biotechnol. Prog. 17 ACS Paragon Plus Environment


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2012, 28, 1218–1224.

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(15) Kang, K. K.; Kim, S.; Kim, I. H.; Lee, C.; Kim, B. H. Selective enrichment of symmetric

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monounsaturated triacylglycerols from palm stearin by double solvent fractionation. LWT–Food

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Sci. Technol. 2013, 51, 242–252.

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(16) Garcia, H. S.; Keough, K. J.; Arcos, J. A.; Hill, C. G. Continuous interesterification of

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butteroil and conjugated linoleic acid in a tubular reactor packed with an immobilized lipase.

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Biotechnol. Tech. 1999, 13, 369–373.

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(17) Kim, B. H.; Akoh, C. C. Modeling of lipase-catalyzed acidolysis of sesame oil and

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caprylic acid by response surface methodology: Optimization of reaction conditions by

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considering both acyl incorporation and migration. J. Agric. Food Chem. 2005, 53, 8033–8037.

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(18) Blasi, F.; Maurelli, S.; Cossignani, L.; D’Arco, G.; Simonetti, M. S.; Damiani, P. Study of

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some experimental parameters in the synthesis of triacylglycerols with CLA isomers and

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structural analysis. J. Am. Oil Chem. Soc. 2009, 86, 531–537.

390


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391


Figure captions

392 393

Figure 1. Preparation of structured MGDGs enriched in PLA by Lipozyme RM IM-catalyzed

394

acidolysis of commercial MGDGs with PLA-enriched FFAs obtained from pine nut oil.

395 396

Figure 2. PLA content in the structured MGDGs present in the reaction products obtained by

397

acidolysis of commercial MGDGs with PLA-enriched FFAs mediated by various commercial

398

immobilized lipases. The reaction was performed at 25 °C, substrate molar ratio of 1:40

399

(MGDGs to PLA-enriched FFAs), enzyme loading of 20 wt % (based on total substrates), and

400

agitation speed of 8.3 Hz in acetone in a stirred-batch reactor. All trials were conducted in

401

duplicate. Means indicated by the same letter (a–c) are not significantly different (P > 0.05).

402 403

Figure 3. Effects of temperature on the PLA content in the structured MGDGs present in the

404

reaction products obtained by Lipozyme RM IM-catalyzed acidolysis as a function of the

405

reaction time. The reaction was performed at substrate molar ratio of 1:40 (MGDGs to PLA-

406

enriched FFAs), enzyme loading of 20 wt % (based on total substrates), and agitation speed of

407

8.3 Hz in acetone in a stirred-batch reactor. All trials were conducted in duplicate. Means

408

indicated by "n.s." (not significant) are not significantly different (P > 0.05).

409 410

Figure 4. Effects of substrate molar ratio (MGDGs to PLA-enriched FFAs) on the PLA content

411

in the structured MGDGs present in the reaction products obtained by Lipozyme RM IM-

412

catalyzed acidolysis as a function of the reaction time. The reaction was performed at 25 °C,

413

enzyme loading of 20 wt % (based on total substrates), and agitation speed of 8.3 Hz in acetone 19 ACS Paragon Plus Environment


Page 20 of 29

414

in a stirred-batch reactor. All trials were conducted in duplicate. Means indicated by the same

415

letter (a–c) are not significantly different (P > 0.05).

416 417

Figure 5. Effects of enzyme loading (based on total substrates) on the PLA content in the

418

structured MGDGs present in the reaction products obtained by Lipozyme RM IM-catalyzed

419

acidolysis as a function of the reaction time. The reaction was performed at 25 °C, substrate

420

molar ratio of 1:40 (MGDGs to PLA-enriched FFAs), and agitation speed of 8.3 Hz in acetone in

421

a stirred-batch reactor. All trials were conducted in duplicate. Means indicated by the same letter

422

(a–d) are not significantly different (P > 0.05).

423 424

Figure 6. PLA content in the structured MGDGs in the reaction products obtained by Lipozyme

425

RM IM-catalyzed acidolysis for four different combinations of the substrate molar ratio

426

(MGDGs to PLA-enriched FFAs) and enzyme loading (based on total substrates). The reaction

427

was performed at 25 °C and agitation speed of 8.3 Hz in acetone in a stirred-batch reactor. All

428

trials were conducted in duplicate. Means indicated by the same letter (a–c) are not significantly

429

different (P > 0.05).

430 431

Figure

432

chromatograms of (A) reaction products obtained by Lipozyme RM IM-catalyzed acidolysis of

433

commercial MGDGs with PLA-enriched FFAs under the optimal conditions, and (B) purified

434

products obtained from the reaction products using silica column chromatography.

7.

Liquid

chromatography-evaporative

light


scattering

detector

(LC-ELSD)

Page 21 of 29


Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



Page 28 of 29

Table 1. Total FA Composition of PLA-enriched FFAs, Commercial MGDGs, and Structured MGDGs Prepared by Lipozyme RM IM-Catalyzed Acidolysis of Commercial MGDGs with PLA-Enriched FFAs (mol %) FA

pine nut oil

FFA fraction obtained from pine nut oil

PLA-enriched FFAs

commercial MGDGs

structured MGDGs enriched in PLA

16:0

5.3 ± 0.0

4.5 ± 0.0

n.d.a

1.4 ± 0.0

8.0 ± 1.0*

16:1n-7

0.1 ± 0.0

n.d.

n.d.

n.d.

n.d.

16:3n-3

n.d.

n.d.

n.d.

37.5 ± 0.1

31.1 ± 0.8*

18:0

2.1 ± 0.0

2.3 ± 0.0

n.d.

0.1 ± 0.0

2.6 ± 0.1*

18:1n-9

25.7 ± 0.1

27.1 ± 0.1

0.2 ± 0.0

0.4 ± 0.1

1.4 ± 0.1*

18:1n-7

0.5 ± 0.0

0.5 ± 0.0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.1 ± 0.0

n.d.

c5,c9-18:2

2.2 ± 0.0

2.3 ± 0.0

n.d.

n.d.

n.d.

18:2n-6

46.0 ± 0.1

45.3 ± 0.2

4.6 ± 0.0

2.6 ± 0.2

7.3 ± 0.6*

c5,c9,c12-18:3 (PLA)

13.7 ± 0.0

13.0 ± 0.1

85.5 ± 0.2

n.d.

42.1 ± 0.5

18:3n-3

0.6 ± 0.0

0.2 ± 0.0

n.d.

57.7 ± 0.0

7.5 ± 0.3*

18:3t

n.d.

n.d.

n.d.

0.2 ± 0.0

n.d.

20:0

0.3 ± 0.0

0.5 ± 0.0

0.9 ± 1.2

n.d.

n.d.

20:1

1.1 ± 0.0

1.5 ± 0.1

n.d.

n.d.

n.d.

20:2

0.6 ± 0.0

0.8 ± 0.0

n.d.

n.d.

n.d.

c5,c11,c14-20:3

1.1 ± 0.0

1.3 ± 0.0

1.7 ± 0.0

n.d.

n.d.

Unidentified

0.7 ± 0.1

0.7 ± 0.0

7.0 ± 1.4

n.d.

n.d.

18:1t

All values represent mean ± standard deviation (n = 2). a Not detected. * Significantly different from commercial MGDGs, P < 0.05.


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Enzymatic Synthesis of Structured Monogalactosyldiacylglycerols

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