Utilization of Amylose -Lipid Complexes as MolecularNanocapsules for Conjugated Linoleic Acid
Inbal Lalush, Hagit Bar, Imad Zakaria, Sigal Eichler, and Eyal Shimoni*
Department of Biotechnology and Food Engineering, TechnionsIsrael Institute of Technology,Haifa 32000, Israel
Received June 17, 2004; Revised Manuscript Received August 18, 2004
Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO)and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction(XRD), and complexes formed at 30, 60, and 90°C exhibit melting temperatures exceeding 88°C. Atomicforce microscopy (AFM) images showed distinct difference in complex organization, with complexes formedin water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superiorprotection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomachconditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release bypancreatin andR-amylase. Only moderate release was detected following hydrolysis by amyloglucosidaseandâ-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsulesfor protection and delivery of CLA.
Introduction
The interaction between amylose and lipids is oftencharacterized by amylose chains forming semicrystallineV-forms. The V-form is an amylose chain that forms a helixwith a large cavity in which various ligands can be situated,and the size of the ligand determines the number of glucosylresidues per turn (6, 7, or 8).1 It was suggested that thecrystalline state of amylose-fatty acid complex involves theV-amylose 6-fold single-chain left-handed helix, well-knownamong starch polymorphs.2 Accordingly, it is often assumedthat the fatty acid is a “stem” (planar zigzag) inside the helix,whose inner surface is hydrophobic because of the carbon-hydrogen of the 6-fold helix.3 The insoluble amylosecomplexes exist in two polymorphic forms, types I and II,each being characterized by the temperature at whichdissociation occurs: type I polymorphs have lower dissocia-tion temperatures.4,5 It is thought that V-helical complexsegments are interrupted by short sections of uncomplexedamylose that permit random orientation of the helicalsegments in the type I complexes, and folding into paralleland antiparallel arrays in the crystalline type II complexes.6,7
Most of the interest in amylose-lipid complexes focusedon their technological importance in starchy food systems,since it modifies the texture and structural stability of starchbased-products (e.g., reduction in stickiness, improvedfreeze-thaw stability, and retardation of retrogradation).5,6,8-10
Other researches studied amylose-lipid complexes in viewof their contribution to the bioavailability of starch, in termsof its enzymatic digestion.11-14 It was shown that the V-formscan be produced from mono- and diglycerides6,9,14-16 and
saturated fatty acids,5,8,17-19 as well as unsaturated fattyacids.1,5,8,9,20These studies showed that the complexes formedhave high melting temperatures, that the complexed fatty acidis efficiently protected from oxidation, and that the digest-ibility of starch is influenced by complex formation, whichdecreased the digestibility of starch.
In the present study, it is hypothesized that amylose-lipidcomplexes can be used as a delivery system for polyunsatu-rated fatty acids (PUFA). It is suggested that these complexeswill provide protection during processing and storage andwill release the PUFA in the intestine following enzymatichydrolysis of the amylose. The feasibility of this concept isexamined by use of conjugated linoleic acid (CLA) as amodel. CLA refers to a group of polyunsaturated fatty acidsthat exist as positional and stereoisomers of conjugateddienoic octadecadienoate (18:2). Numerous physiologicalproperties have been attributed to CLA including action asan antiadipogenic, antidiabetogenic, anticarcinogenic, andantiatherosclerotic agent. In addition, CLA has effects onbone formation and the immune system as well as fatty acidand lipid metabolism and gene expression in numeroustissues.21
The scope of this study was to develop amylose-CLAcomplexes, with optimal stability to oxidation and thermaltreatments, to dissolution in the stomach, and efficient releaseby mammalian amylases. The expected results, stableencapsulated PUFA, will enable the supplementation ofvarious staple foods with these important bioactive com-pounds.
Materials and Methods
Materials. Potato amylose (DP-900, according to manu-facturer), CLA (a mixture ofcis- andtrans-9,11 and -10,12-
octadecadienoic acids. Linoleic acid<1%), Amyloglucosi-dase (40 units/mg of protein, fromAspergillus niger),Pancreatin (amylase, 41 USP units; lipase, 4.9 USP units;protease, 29 USP units; from porcine pancreas),R-amylase(39.3 units/mg of protein, fromAspergillus oryzae), andâ-amylase (827 units/mg of protein, from sweet potato) wereall obtained from Sigma Chemical Co. (St. Louis, MO). Allother reagents were of analytical grade.
Preparation of Amylose-CLA Complexes. Complex-ation of amylose with CLA was carried out by two methods.Amylose-CLA complexes were first produced in DMSO/water solution.9 Amylose solution (25 mL containing 20 mgof amylose/mL) was prepared in dimethyl sulfoxide (DMSO)at 90 °C. Then the amylose solution was brought to thecrystallization temperatures: 90, 60, and 30°C. Amylose-CLA complexes were prepared by adding 50 mg of CLA tothe amylose solution; after the CLA had dissolved, water(475 mL, at the crystallization temperature) was added andthe mixture was incubated at the crystallization temperaturefor 15 min, with vigorous stirring to complete the complexformation. Then the suspension was cooled to 20°C, andthe amylose-CLA complexes were isolated by centrifugation(2000g, 15 min). The wet pellet of the complexes waswashed twice with ethanol/water mixture (50/50 w/w), toremove residues of uncomplexed CLA, and centrifuged asbefore. The complexes were then freeze-dried.
The second protocol to produce amylose-CLA complexeswas by KOH/HCl solution.5 Amylose solution (40 mL, 15mg/mL of 0.01 M KOH) and a solution of CLA (60 mL, 1mg/mL of 0.01 M KOH) preheated to 90°C were mixed atdifferent crystallization temperatures (90, 60, and 30°C);the mixture was neutralized with 10 mL of 0.1 M HCl. Thecomplex was then precipitated by adjusting the pH to∼4.7(by use of 2 M HCl). The mixture was then held at the presetcrystallization temperature for 24 h. All samples were thencentrifuged (2000g, 15 min), the supernatant was discarded,and the precipitate was washed twice with ethanol/watermixture (50/50 w/w) (to remove residues of uncomplexedCLA and to obtain a salt-free complexes) and centrifugedas before. The complexes were then freeze-dried.
Determination of CLA Content in the Complexes: (A)By Enzymatic Hydrolysis. The CLA content of the com-plexes was calculated on the basis of the free fatty acidreleased by full hydrolysis with pancreatic amylases. Fifteenmilligrams of the complex was incubated in 1 mL ofpancreatic amylases (140 units/mL enzymatic activity, pH) 6.9, 37°C) for 24 h. Then, the CLA was extracted byhexane, and the hexane was evaporated by a nitrogen flow.The CLA was quantified by use of a GC (Hewlett-PackardGCD system HP 5890; Avondale, PA) equipped with an HP-Innowax capillary column [30 m× 0.32 mm (i.d.) with 0.25µm film thickness; HP]. The temperature programming was120°C for 1 min, then ramped at 10°C/min to 250°C, andmaintained for 2 min. Inlet and detector temperatures were250°C. The nitrogen carrier gas flow rate was 2.4 mL/min,hydrogen flow to the detector was 25 mL/min, airflow was400 mL/min, and the flow of nitrogen makeup gas was 45mL/min. Peaks were identified by comparison with standardsfor CLA isomers from Sigma Chemical Co.
(B) By Acidic Hydrolysis. The determination of the CLAcontent was also done by methylation in a MeOH/H2SO4
(90/10 v/v) mixture.18 The sample (15 mg) was heated insealed tubes at 100°C for 30 min with 0.75 mL of MeOH/H2SO4 and 150µL of palmitic acid dissolved in toluene (1mg/mL). After cooling, 150µL of water was added to themixture. Then the methyl esters were extracted with 5 mLof hexane and quantified by GC, as described before. Thequantity of CLA present in complexes calculated from theratio of the peak area of CLA to that of an internal standard(palmitic acid, C16:0) of known content.
Characterization of the Complexes: (A) X-ray Dif-fraction. The formation of a V-type complex was verifiedby X-ray diffraction. XRD measurements were carried outby a Philips PW 3020 powder diffractometer equipped witha graphite crystal monochromator (Philips). The operatingconditions were Cu KR1 radiation (0.154 nm), voltage 40kV. and current 40 mA. Samples were scanned over the range5-35° 2θ in steps of 0.02° 2θ per 4 s, and the crystallinenature of the complex was determined by the position ofthe X-ray diffraction peaks.
(B) Differential Scanning Calorimetry. Thermal proper-ties of the complexes were examined on a Perkin-ElmerDSC-7 system (Perkin-Elmer Corp., Norwalk, CT). Samples(about 7 mg) were prepared and weighed in stainless steelpans. Distilled water was added to reach water contentsaround 75% before sealing. The DSC scan was preformedfrom 25 to 150°C with a 5°C/min ramping; the referencepan contained 20 mg of water. The transition temperaturesand enthalpies were calculated by use of the Pyris thermalanalysis system, version 3.72, of Perkin-Elmer LLC.
(C) Surface Characterization by Atomic Force Micros-copy. The surface of complexes samples was characterizedby scanning with a Nanonics NSOM/AFM 100 system(Nanonics Imaging Ltd., Jerusalem).22 For the AFM scans,mica was coated with various amylose-CLA complexessuspensions, followed by overnight oven dehydration (30°C).Scanning was performed in the tapping mode, by use of anuncoated Si AFM probe (MikroMasch, Madrid, Spain), witha tip curvature radius of less than 10 nm and force constantof 17.5 N/m. Data were analyzed by use of Quartz software(Cavendish Instrument, U.K.). Cross-section analysis wasused to estimate the dimensions of typical features.
Dissolution and Digestion Tests: (A) Stability Test ToSimulated Stomach Conditions.The release of CLA fromthe complex in stomach conditions was tested by incubatingamylose-CLA complexes in simulated stomach conditions,following the amount of CLA released. The complex (15mg) was incubated with 1 mL of HCl, pH) 2, for 2 h at 37°C under continuous stirring (15 rpm). The extent of CLArelease following the incubation under acidic conditions wasmeasured by extracting the reaction medium (complex andbuffer) with hexane (6 mL), followed by GC analysis (asdescribed before).
(B) Enzymatic Digestion Tests.The release of CLA byenzymatic digestion was measured by exposing the complexto mammalian pancreatic amylases, and well as pureR-amy-lase,â-amylase, and amyloglucosidase. Specifically, pan-creatin solution was prepared by dissolving 0.177 g of
122 Biomacromolecules, Vol. 6, No. 1, 2005 Lalush et al.
pancreatin in 20 mL of phosphate buffer (20 mM phosphate,pH 6.9, and 10 mM NaCl), followed by centrifugation (10min, 1500g), and the supernatant was used for the digestion.R-Amylase solution was prepared by dissolving 8.9 mg ofthe enzyme in 10 mL of phosphate buffer.â-Amylasesolution was prepared by dissolving 0.1 mL of the enzymein 5 mL of phosphate buffer. Amyloglucosidase solution wasprepared by dissolving 8.75 mg of the enzyme in 10 mL ofphosphate buffer. The activity values of all the other enzymes(exceptâ-amylase) were 35 units/mL, which is the minimalactivity in the intestine.23 The activity ofâ-amylase was muchhigher (700 units/mg) in order to obtain some degree ofhydrolysis and release. Amylose-CLA complexes (15 mg)were incubated with 1 mL of each of the above-mentionedenzyme solutions for 24 h at 37°C under continuous stirring(15 rpm).
The degree of enzymatic digestion was measured on a 0.2-mL portion of each sample by determination of the reducinghemiacetal groups.24 Hemiacetal groups determination wasperformed by adding of 100µL of 3,5-dinitrosalicylic acidto 100µL of each sample, incubating at 100°C for 5 min,cooling, and measuring the absorbance at 546 nm. Theconcentration of the reducing groups was determined ac-cording to a calibration curve of maltose or glucose,according to the enzyme. The extent of CLA releasefollowing the enzymatic digestion was measured by extract-ing the reaction medium (digested complex and buffer) withhexane, followed by GC analysis (as described before).
(C) Oxidation Stability Testing. The protection fromoxidation afforded to CLA by the complex was determinedby headspace-oxygen analysis.25 Serum sample bottles (2mL) containing 15 mg of complexes were incubated at 37°C for 70 h. The control was a free CLA with an equalcontent to the content of the complexes. Headspace-oxygencontent was measured with time (0, 4, 21, 28, 45, and 70 h),by injecting a 0.5 mL portion of headspace air from eachserum bottle into a GOW-MAC Instrument Co. GC series580, equipped with a thermal conductivity detector (TCD)and a CTRΙ column (6 ft× 1/4 in. outer and 6 ft× 1/8 in.inner ss, Alltech). Helium (99.99%) was used as a carriergas with a flow rate of 40 mL/min. The oven temperaturewas maintained at 40°C. The temperature of the injectorand detector was maintained at 80°C. The electronicresponse of oxygen in 0.5 mL of headspace air was recordedwith a Hitachi D-2500 chromatointegrator and converted tomoles of O2 consumed/mole of CLA in sample. Theheadspace oxygen (moles of O2/mole of CLA) was plottedagainst incubation time (hours) for the complexes created atthe three crystallization temperatures, by the two methodsof complexation.
(D) Data and Statistical Analysis.All experiments wereperformed in triplicate, and the results are expressed as theaverage. In the case of significant variability, the results arepresented also with their standard deviation. Statisticalanalysis was performed by JMP software.
Results
Complexation. The experimental approach in this studywas composed of three main aspects: complex formation
and characterization, evaluation of the oxidative stabilityafforded to CLA by complexation, and evaluation of complexdisintegration in simulated stomach conditions as well asenzymatic digestion. Complexation conditions, such ascrystallization temperature, water content, concentrationratios, and complexation time, affect the nature of theamylose-lipid V-complex. It is our hypothesis that bycontrolling these parameters one can tailor the physicalproperties of the complex, and thus eventually its biochemicalattributes. The purpose of this part of the study is to examinethe effect of the complexation temperature and the methodof creating the complexes on the inclusion efficiency. Wetherefore measured the weight yield of the two methods thatwere used for production of the complexes and the CLAcontent of the complexes. In addition, we characterized thephysical properties of the complexes by XRD, DSC, andAFM.
Percent Yield and CLA Content of the Complexes.Theweight yield of complexation was calculated on the basis ofthe quantity (milligrams) of complexes obtained at the endof the production process, as a percentage of the initialamounts (milligrams) of amylose and CLA used for thecomplexation (600 mg of amylose+ 60 mg of CLA usedfor complexation in KOH/HCl solution; 500 mg of amylose+ 50 mg of CLA used for complexation in DMSO/watersolution). The CLA content of the complexes was calculatedon the basis of the free fatty acid released by full hydrolysiswith pancreatic amylases (140 units/mL) for 24 h, extractionof the CLA by hexane, and quantification by GC. Peaks wereidentified by comparison with standards for CLA isomers.The results obtained were confirmed by the acidic hydrolysismethod. The results of the weight yield (percent) and theCLA content of complexes produced by the two methods ofcomplexation at three temperatures are presented in Table1.
Both methods used to create amylose-CLA complexesexhibited weight yield that does not exceed 60%. Complex-ation in water/DMSO solution exhibited higher weight yield(percent) than complexation by KOH/HCl solution (p <0.05). Complexes created in a water/DMSO solution exhib-ited an apparent increase in the weight yield as the crystal-lization temperature decreased. This trend was not detectedfor complexes created in a KOH/HCl solution. CLA contentin all complexes was up to 3.8%, with no significantdifferences between complexes created by the two methodsand the three temperatures.
X-ray Diffraction. The formation of a V-type complexwas verified by X-ray diffraction (XRD), and the results of
Table 1. Weight Yield and CLA Content of Complexes Made byWater/DMSO and KOH/HCl Methods, at 90, 60, and 30 °C
the analysis are presented in Figure 1. We examinedcomplexes created by both complexation methods at the threetemperatures. For all complexes, X-ray diffraction patternswere typical of the Vh form of amylose18 with the three mainreflections corresponding to the Bragg angles 2θ ) 7.4°,12.9°, and 19.8°. Complexes created in KOH/HCl solutionat the three temperatures had another reflection at 2θ ) 28.2°,with complexes created at 60 and 30°C having an additionalreflection at 2θ ) 32°. The peaks of the diffractogramsobtained from complexes made in KOH/HCl solution werenarrower than those obtained from complexes created bywater/DMSO solution. This may indicate that these com-plexes are composed of larger crystals. The three tempera-tures of crystallization did not show significant influence onthe diffractograms obtained for complexes created in water/DMSO solution. However, in complexes created with KOH/HCl solution, narrower peaks were obtained with the increasein crystallization temperature, possibly due to larger crystals.
Differential Scanning Calorimetry. The influence ofcomplexation method and crystallization temperature on themelting temperature and enthalpies of amylose-CLA com-plexes was evaluated by DSC. The results of the DSCanalyses are reported in Table 2. DSC thermograms con-tained one endotherm. The melting point of all the complexes
was higher than 88°C, and the enthalpies ranged from 7 to17.4 J/gr dry matter. Complexes created in water/DMSOsolution at 90°C showed the highest enthalpy and meltingtemperature. In complexes made in water/DMSO solution,there was a mild increase in melting temperature and enthalpyof melting with the increase in complexation temperature.For complexes created in KOH/HCl solution, the highestmelting point and enthalpy were measured in complexesproduced at 60°C. However, there was no statisticallysignificant influence of the complexation method or tem-perature on the melting temperatures and enthalpies of thedifferent amylose-CLA complexes.
Atomic Force Microscopy.Complexes were scanned byAFM in order to correlate their structure with their physicalproperties. The complexes created in water/DMSO and KOH/HCl solutions at different crystallization temperatures werecharacterized by AFM in the tapping mode, and the imagesof complexes created by the two complexation methods at90 °C, at 5µm × 5 µm scan, are shown in Figures 2 and 3.
The complexes created in water/DMSO solution at 90°C(Figure 2) revealed globular structures of heterogeneousnature with an averagez-range (n ) 15) of 71.6( 59 nmand diameter of 152( 39 nm. Larger structures are seen,but closer inspection indicates that these are clusters of the
Figure 1. V-type X-ray diffractogram of amylose (DP-900)-CLAcomplexes created by a water/DMSO solution (A) and in KOH/HClsolution (B) at 90, 60, and 30 °C.
Table 2. Melting Temperatures and Enthalpies of DSCEndotherms of Amylose-CLA Complexes Created by the TwoComplexation Methods at 90, 60, and 30 °C
a Data presented as the average ( standard deviation of threereplicates.
Figure 2. AFM images: phase (A) and amplitude (B) of amylose-CLA complexes created in water/DMSO solution at 90 °C. Scale bar) 500 nm (bottom). Scan range ) 5 µm × 5 µm.
124 Biomacromolecules, Vol. 6, No. 1, 2005 Lalush et al.
previously described spheres. Complexes created by KOH/HCl solution at 90°C (Figure 3) exhibited mainly rodlikestructures of heterogeneous nature with an averagez-range(n ) 15) of 37.8( 18 nm, width of 43-160 nm, and lengthranging from 0.35 to 3.2µm. Globular structures withaverage diameter of 60( 8 nm were also present. Bothcomplexes exhibit high similarity between the phase image(A) and the amplitude image (B). Preliminary results ofcomplexes created by KOH/HCl solution at 60 and 30°Cseem to exhibit a decrease in thez-range as the temperaturedecreases (results not shown).
Stability of CLA Complex to Oxidation. It is ourhypothesis that CLA complexation with amylose protects itfrom oxidation and that this protection depends on thephysical properties of the complex. This part of the studywas designed to measure the extent of protection againstoxidation afforded to CLA by its inclusion in the amylosecomplex. The results are presented in Figure 4. In all testedsamples, CLA oxidation was lower in complex with amylose.The protective effect of the complexes against CLA oxidationwas higher for complexes created in water/DMSO solutionthan that for complexes created by KOH/HCl solution. Thisdifference was more pronounced for complexes created at
90 and 30°C. Complexes created by KOH/HCl solution at60°C showed the best protective effect among the complexescreated by the same method, whereas for complexes madeby water/DMSO solution, 60°C was the temperature thatsupplied the lower protection among complexes made bythis method.
The headspace-oxygen consumption of the control sample(CLA alone) increased from 0.07 to 0.78 mol of O2/mol ofCLA, whereas for complexes created in water/DMSOsolution, the headspace-oxygen consumption increased from0.01 to 0.15 mol of O2/mol of CLA, with the exception ofcomplexes created at 60°C, which increased from 0.01 to0.35 mol of O2/mol of CLA. For complexes created in KOH/HCl solution it increased from 0.01 up to 0.7 mol of O2/molof CLA, with the exception of complexes created at 60°C(increased from 0.01 to 0.27 mol of O2/mol of CLA).
Complex Stability in Simulated Stomach Conditions.It is suggested that the complexes are stable in the stomachand that this stability is affected by their physical properties.
Figure 3. AFM images: phase (A) and amplitude (B) of amylose-CLA complexes created by KOH/HCl solution at 90 °C. Scale bar )500 nm (bottom). Scan range ) 5 µm × 5 µm.
Figure 4. Headspace-oxygen analysis of complexes created at 90°C (A), 60 °C (B), and 30 °C (C) in water/DMSO solution (b) and byKOH/HCl solution (9), with comparison to free CLA (2).
Therefore, in this part of the study the protection affordedby complexation to CLA from release in the stomach wastested by incubating amylose-CLA complexes in simulatedstomach conditions (HCl, pH) 1.5, 2 h at 37°C), andfollowing the amount of CLA released. The results arepresented in Figure 5. Complexes created in water/DMSOsolution exhibited significantly better stability in simulatedstomach conditions than complexes created by KOH/HClsolution (p < 0.05). Apparently, the two methods ofcomplexation affect inversely the amount of CLA releasedin simulated stomach conditions from the complexes: forcomplexes created in water/DMSO solution, as the crystal-lization temperature rose, the smaller was the amount of CLAreleased; whereas for complexes made by KOH/HCl solution,as the temperature rose, the higher was the amount released.However, there were no statistically significant differencesbetween the crystallization temperatures for the two methodsof complexation (p < 0.05).
Release of CLA by Enzymatic Digestion.A majorattribute of the amylose-CLA complex as a delivery systemis its ability to release CLA due to enzymatic digestion inthe intestine. Therefore, the aim of this part of the study wasto evaluate the effect of specific parameters in the formationof the amylose-CLA complex on its degradation by amy-lases and the release of CLA. The susceptibility of thecomplex to enzyme hydrolysis was examined by use ofglucoamylase,R-amylase,â-amylase, and pancreatin. Thevalues of the amylolytic activity used were typical of theminimal activity reported in the intestine, 35 units/mL.23 Thedegree of enzymatic digestion was measured on a portionof 0.2 mL of each sample by determination of the reducinghemiacetal groups.24 CLA release was measured by extrac-tion of the reaction medium (complex and buffer) withhexane followed by GC analysis. The extent of hydrolysis(percent) and CLA release (percent) of amylose-CLAcomplexes created in water/DMSO solution and by KOH/HCl solution (at 90, 60, and 30°C) are presented in Figures6 and 7, respectively.
The enzymatic hydrolysis of complexes created by bothcomplexation methods increased in the following order:pancreatin> R-amylase> amyloglucosidase> â-amylase.Complete hydrolysis (100%) was obtained by pancreatin,whereasR-amylase hydrolyzed up to 87% of the complexes.Amyloglucosidase exhibited low rate of hydrolysis (up to
36%), and theâ-amylase hardly hydrolyzed the complexes(up to 8.5%), despite extremely high enzyme activity (700units/mL). For pancreatin, amyloglucosidase, andâ-amylase,there was insignificant difference in the hydrolysis betweencomplexes prepared by the two methods being used. Theexception wasR-amylase, with a higher extent of hydrolysisof complexes created by KOH/HCl solution at 60°C.
To test the release of CLA due to the enzymatic degrada-tion, we first incubated a control experiment including thecomplexes with no enzyme. Here, only mild release of CLAwas observed; for complexes made in water/DMSO solutionit ranged from 3% to 6%, and for complexes created byKOH/HCl solution, from 7% to 11%. When incubated inthe presence of amylases, among complexes created by KOH/HCl solution, the maximum release due to amylolytic activitywas obtained with pancreatin in complexes produced at 90°C (p < 0.05). Unlike pancreatin,R-amylase caused higherhydrolysis of complexes produced at 60 and 30°C. Therelease of CLA by pancreatin and amyloglucosidase in-creased with the increase in crystallization temperature (p< 0.05), and the maximal release by both enzymes wasobtained in complexes produced at 90°C. The crystallizationtemperature had no significant effect on the release of CLAfrom these complexes whenR-amylase was used.
The release after the action of all enzymes was similarfor complexes made in water/DMSO solution at the threecrystallization temperatures. When the activity of different
Figure 5. Amount of CLA released in simulated stomach conditions((HCl, pH ) 1.5, 2 h at 37 °C) from amylose-CLA complexes, createdin water/DMSO solution (black bars) and by KOH/HCl solution (whitebars), at 90, 60, and 30 °C.
Figure 6. Extent of hydrolysis (%) (A), and CLA release (%) (B) ofamylose-CLA complexes created in water/DMSO solution at 90 °C(gray bars), 60 °C (white bars), and 30 °C (black bars). Hydrolysiswas performed by pancreatin (pan), amyloglucosidase (gluco),R-amylase (R), and â-amylase (â) at concentrations of 35 units/mL.The control contained no enzyme.
126 Biomacromolecules, Vol. 6, No. 1, 2005 Lalush et al.
enzymes on complexes created by different complexationmethods was compared, a significantly different release wasobtained for complexes created by KOH/HCl solution at 90°C by use of amyloglucosidase. This difference occurred inspite of the fact that no significant difference in hydrolysiswas obtained for this enzyme. Furthermore, for complexescreates by the two methods at 60 and 30°C, no significantdifferences in the release of CLA were obtained as a resultof hydrolysis by amyloglucosidase. It should be noted,however, that regardless of the system tested, there was acorrelation between the release and extent of hydrolysis.
Discussion
Percent Yield and CLA Content of the Complexes.Amylose-CLA complex formation is strongly influencedby the type of lipids involved.5 It is known that amylosecan form complexes with free fatty acids (FA). Variousstudies obtained reasonably efficient complexing with un-saturated free FA and monoglycerides.5,7,9,26-28 However,early reports claim that cis-unsaturated FA complex poorlywith amylose, giving low yields and enthalpies of dissoci-ation.9,28-31 This has been attributed to inefficient complexingby such FAs, which is depicted as nonlinear or kinked dueto the cis double bond. The results of this study show thatamylose can also complex with the unsaturated CLA.
Although, given the chemical structure of CLA, it is difficultto think of a formation of a helical inclusion, it is possiblethat only part of the aliphatic segment, adjacent to theconjugated bonds of the CLA, is inside the helical amyloseand the rest of the CLA molecule is outside the helix. Thissuggestion is in agreement with the structure of amylose-FA complexes suggested by computer modeling.17 It wasshown that the cis-trans part of the FA could be outsidethe helix. Moreover, complexation with other bulky mol-ecules such as naphthol was demonstrated,6,15which supportsthe possibility of inclusion of the CLA inside the helix. Inaddition, the XRD results also support the formation of aninclusion complex with CLA, especially for complexescreated in water/DMSO solution. As for complexes createdby KOH/HCl solution, the additional two peaks observed inthe XRD results may imply the presence of an additionalform of amylose-CLA complexes, as will be discussed later.
Another major factor governing the formation of com-plexes is the solubility/dispersability of the FA in thecomplexation medium, and this in turn depends on variablessuch as temperature and FA molecular weight.8,28,32 Thecomplexation in DMSO/water solution occurs in pureDMSO, and the water added is used to precipitate thecomplexes formed. Since DMSO is an organic solvent, thesolubility of the FA is very high; hence the FA moleculesare available to create inclusion complexes with amylose,and only a V-type is formed, as seen by the XRD results.For the complexation by KOH/HCl solution, the FA isdissolved in aqueous solution of KOH before mixing withthe amylose solution. But, although the alkaline improvesthe solubility of FA by ionization, the solubility/dispersabilityof the CLA in water is less than in organic solvent. Therefore,less FA is available to form helical inclusion complexes, asis also supported by the XRD analysis.
The weight yield of the complexes obtained in the differentconditions ranged from 52% to 60%, with CLA content of2%-3.5% (Table 1). Previously reported yields of amylosecomplexes were 60%-83% with FA content of about 4%(complexes created with a mixture of mainly 18:1, 18:2, and18:3).20 Others obtained complexes with FA content of4.9%-8.3% depending on the crystallization temperature.5
Apparently, the yield and CLA content obtained for thedifferent complexes were lower than reported for otherunsaturated free FAs. This may arise from the structure ofCLA. Computer modeling of amylose-FA complex dem-onstrated that the “all-trans” segment for the FA, which isenergetically favored (global minimum), fit into the helix.19
Free rotation about C-C bonds adjacent to CdC bondsallows the unsaturated FA to adopt a quasi-linear conforma-tion around the double bond, which would reduce the sterichindrance for complexation.5 Unlike other unsaturated FA,the conjugated double bonds of CLA are not separated byC-C bonds; hence CLA cannot adopt a fully linearconformation and the molecule is bent by the conjugatedCdC bonds. It is therefore possible that, due to the bend ofCLA molecules, a greater steric hindrance for complexationis created, thus resulting in lower yield and content comparedto other unsaturated FA.
Figure 7. Extent of hydrolysis (%) (A) and CLA release (%) (B) ofamylose-CLA complexes created by KOH/HCl solution at 90 °C (graybars), 60 °C (white bars), and 30 °C (black bars). Abbreviations areas described for Figure 6.
Complexes created in water/DMSO solution exhibited anincrease in the yield as the crystallization temperaturedecreased. This is in agreement with effective complexationof unsaturated monoglycerides and FAs in ambient temper-atures.5,9 In contrast, complexes created by KOH/HCl solu-tion did not show any significant difference between thecrystallization temperatures.
Characterization of the Complexes.Amylose-FA com-plexes exist in two polymorphic forms, namely, types I andII. The type I forms are considered to be amorphous whilethe semicrystalline type II forms exhibit X-ray diffractionpatterns characteristic of the 6-fold single helices in crys-tallites.1,6 At low temperatures (Tc), formation of complex Iis the favored process, whereas form II is favored at highTc.6,18 In this study all complexes created by the twocomplexation methods, at the three crystallization temper-atures, exhibited a characteristic diffraction patterns of theV-complex, meaning that at all crystallization temperatures(90, 60, and 30°C) form II complexes were produced, whichis in contradiction with other observations.5,6 Complexescreated by KOH/HCl solution exhibited narrower peaks thanthose obtained from complexes created in water/DMSOsolution, suggesting rather larger crystallites, but accordingto the AFM characterization, the morphology of the com-plexes created by the two methods was different (rods vsglobular); hence no comparison of crystallite sizes can bedone from the AFM images.
It should be noted that as the crystallization temperatureincreased, complexes created by KOH/HCl solution exhibitnarrower peaks, suggesting the formation of larger crystal-lites, as seems to be supported by the dimensions of thecomplexes as analyzed by cross-section analysis of the AFMsoftware (results not presented). For complexes created inwater/DMSO solution, the crystallization temperature seemedto have no influence on the peak width, which is in contrastto the AFM results (decrease in height of complexes as thecrystallization temperature decreased; results not presented).This may be explained if the crystallite size increase occursalong the macromolecular chain axis, which may not bevisible on the width of major diffraction lines observed.33
Two additional diffraction peaks are seen in samplesprepared by KOH/HCl complexation. Other studies thatobserved additional peaks to the peaks characteristic to theV-form17,32 suggested that these peaks reflect the presenceof pure crystalline FA, which can also be demonstrated byappearance of another endotherm in the DSC spectrum thatcorresponds to the melting of the FA itself. These studiesused FA (C12-C18) with melting temperatures higher than44.2°C.8 These FAs are solid at room temperature, whereasin the present research we used CLA, which has a very lowmelting temperature and thus is liquid at room temperature.Therefore, the additional peaks seen for complexes createdby KOH/HCl complexation are unlikely to stem from purecrystalline FA. An alternative possible explanation could bethe presence of another type of amylose-FA complexes, asseen by Fourier transform infrared (FTIR) analysis of starch-hydrocarbon complexes.34 The FTIR analysis revealed thatcomplexing hydrocarbons expelled part of the amorphouscontent of starch to form internal empty domains. In such
domains, hydrocarbons were held with involvement of localvan der Waals and dispersion forces ofD-glucose units ratherthan by formation of helical complexes with amylose.
Form II is believed to have a lamellarlike organization ofamylose complexes; that is, the polysaccharide chains areso folded as to have their chain axes perpendicular to thesurface of the lamella.6,15,35 This molecular organizationseems to be similar to the lamellar stacks shown byamylose-palmitic acid complexes in TEM; their thicknesswas 4.6 nm, which corresponds to the total length of twopalmitic acid molecules.10 The AFM images of the currentstudy cannot support the lamellarlike organization, mainlysince much higher magnification is needed. Their observa-tion, however, can be supported by the AFM image ofamylose-polyether inclusion complexes at a magnificationof 100 nm × 100 nm, revealing structures separated bydistances of 3-3.5 nm.36 The structures (both rods andglobular) presented in the AFM images (Figures 2 and 3)exhibit a diameter of about 100 nm, which is supported byAFM images (scan 3µm × 3 µm) of amylose complexescreated with carbon nanotubes,37 that exhibit a width of about75 nm.
The melting temperature (Tm) of all the complexes washigher than 88°C (Table 2). It is known that complexes ofform II give high melting temperatures and enthalpies.Linoleic acid (LA)-amylose complexes, type II, haveTm
of about 100°C.5 Since the position of the double bonds ofthe unsaturated FA influences the melting point of thecomplexes, the lowerTm of complexes created with CLAcan be attributed to the conjugated double bonds of CLA.No significant influence of the crystallization temperatureon theTm was observed, which is in contrast to the increaseof Tm with the increase of crystallization temperature.6 Themelting enthalpies of the complexes ranged from 7 to 17.4J/g (Table 2), which is in contrast to the higher enthalpiesreported for type II complexes but yet in agreement withthe influence of the structure of CLA compared to LA(maximum enthalpy of 24 J/g at all crystallization temper-atures).5 An interesting outcome of the measuredTm is thatamylose-CLA complexes may be used to protect CLAduring thermal processing such as pasteurization.
Stability of the Complexed CLA against Oxidation.Theprotection against oxidation afforded to CLA by its inclusionin an amylose complex (Figure 3) demonstrates the potentialof the complexes, especially those created in water/DMSOsolution, to efficiently protect CLA from oxidation. Theseresults are in agreement with other studies.20,38Interestingly,the extent of CLA oxidation correlates well with theirenthalpy of melting (Figure 8). As the enthalpy increased,the extent of oxidation decreased. Since the enthalpy reflectsthe complexes’ degree of crystallinity, it may be suggestedthat the protection against CLA oxidation depends on thedegree of crystallinity.
It seems that complexes created in water/DMSO solutionexhibited higher crystallinity than complexes created byKOH/HCl solution. Hence, it is in agreement with the factthat complexes created in water/DMSO solution exhibitedbetter protective ability from oxidation. Also, it is possiblethat the differences between the morphology of the com-
128 Biomacromolecules, Vol. 6, No. 1, 2005 Lalush et al.
plexes (rods vs globular) affect the extent of oxidation dueto the surface exposed to oxidation, which is greater for rods(complexes created by KOH/HCl solution). Another possibleexplanation could stem from better molecular arrangement,as well as more compact structure of the complexes createdin water/DMSO solution.
Stability of Amylose-CLA Complexes in SimulatedStomach Conditions.The complexation method appears tohave a significant effect on the protection afforded bycomplexation to CLA from release in stomach conditions.Complexes created in water/DMSO solution exhibited higherstability to stomach conditions than complexes created byKOH/HCl solution (Figure 4). One explanation could be thepresence of another type of amylose-CLA complex forcomplexes created by KOH/HCl solution (suggested by theXRD), which is probably less stable to pH changes. Anotherexplanation could be related to possible conformationaldifferences between complexes created in the differentmethods; complexation in DMSO depends on solvation,meaning that the complex formation is driven mainly by thedissolution of amylose and CLA in the organic solvent,whereas complexation by KOH solution is restricted by sterichindrance due to charged FA, and hydrostatic forces createdby the water molecules (hydration), which may result infewer interactions between amylose and CLA and hence lessstable CLA inside the helix. Therefore, the conformation ofthese complexes can be affected more easily by pH changesthan complexes created in DMSO. In general, the hypothesisthat the stability of the complexes is affected by their physicalproperties was approved. Thus, smart control on CLA releasecan be enabled, and the CLA release probably will not occurin stomach conditions.
Enzymatic Digestion and Release of CLA.Complexformation is thought to decrease the digestibility of starchand modulate the glycemic response to ingested carbohy-drates.13 The in vitro digestibility of amylose complexesdepends on enzyme concentration, incubation time, natureof lipid, and conformational hindrance to enzymatic attack.Most studies investigated the digestibility of amylose-lipidcomplexes by amylolytic enzymes at concentrations muchlower than in physiological conditions, where the amylolyticactivity values typical to the intestine are 35 units/mL(minimal) and 120 units/mL (average).23 These studiesshowed that complexes containing cis-, mono-, or diunsat-
urated monoglycerides were significantly less stable towardenzymatic breakdown than complexes containing saturatedmonoglyceride9 and that amylose-oleic acid complexes werehydrolyzed to a greater extent than amylose-palmitic acidcomplexes.12 The results of the present study show thatamylose-CLA complexes can be fully digested in physi-ological conditions, because of the nature of CLA. This canalso be supported by the in vivo study, which showedpractically complete absorption within 120 min of amylose-lipid complexes that had reached the small intestine,12 andalso by the fully degraded complexes under prolongeddigestion time and high enzyme levels.14
In this study enzyme hydrolysis of the complexes wasperformed by the exoamylases glucoamylase andâ-amylase(not found in mammals) and by the endoamylasesR-amylaseand pancreatin (amylolytic activity related toR-amylase).The hydrolysis of complexes from both complexationmethods increased in the following order: pancreatin>R-amylase> amyloglucosidase> â-amylase. ForR-amy-lases, no significant differences were obtained in the hy-drolysis of complexes created by the two methods (p < 0.05),despite differences observed by the AFM and X-ray diffrac-tion. This is in agreement with similar degradation kineticsdespite the apparent differences in thermal stability andsupermolecular organization.6 Significantly low hydrolysiswas obtained by the exoamylases glucoamylase andâ-amy-lase, indicating better digestibility by endoamylases.
While hydrolysis by endoamylases occurs randomly alongthe amylose chain, exoamylases begin their action from thenonreducing terminus. It is possible that a steric hindrance,caused by the structure of the complexes, hampers the actionof exoamylases, resulting in low hydrolysis. Interestingly,the rate and extent of hydrolysis of the complexes areinversely related to the degree of organization: complexeswith greater crystallinity were more resistant to enzymaticdegradation.14 The results of the present study found nosignificant differences (p < 0.05) in the crystallinity of thedifferent complexes (DSC) and hence no differences inhydrolysis of complexes created by the two methods.
It should be noted that some spontaneous CLA releasefrom the complexes was detected. This release is not likelyto occur from uncomplexed CLA, since the complexes werewashed twice by a water/ethanol mixture to remove accessof CLA. The location of FA molecules within the crystallattice between the amylose helices is not possible, since thesmall cavities present between helices cannot accommodatea FA molecule.18 The phosphate buffer has an effect ofstabilization on the complexes;39 hence it is also not likelythat the spontaneous release is due to the buffer. A possibleexplanation for the spontaneous release could be the stabi-lization of the CLA inside the helix. The bend in the CLAmolecule created by the conjugated double bonds mightprevent the formation of some of the interactions betweenthe amylose and the CLA, resulting in a less stabilized CLAinside the helix.
The results of the present work support the hypothesis thathydrolytic activity will release the CLA from the complexes.It is evident that the hydrolysis and the release of CLA area function of the specific digesting enzyme. Despite the fact
Figure 8. Correlation between the maximal extent of oxidation (molesof O2/moles of CLA), obtained after 70 h at 37 °C, of differentcomplexes and their enthalpies of melting.
that full hydrolysis was not necessarily followed by acomplete CLA release from the complexes, the release ofCLA was proportional to the hydrolysis. Overall, the CLArelease due to amylolytic activity of pancreatin indicates thatthe CLA-amylose complex system can serve as a vehiclefor delivery of CLA to the intestine.
Conclusions
The results of the presented research show that amylosecan complex with CLA. The complexes formed providestability to oxidation and thermal treatments, such aspasteurization. Control of CLA release is enabled, and theCLA release does not occur in simulated stomach conditions;rather, it is driven by amylolytic activity of pancreatin, whichindicates that the location of release in the digestive tractwill probably be in the intestine. Complexes created in water/DMSO solution at 90 or 30°C provided the maximal stabilityto oxidation and thermal treatments, dissolution in simulatedstomach conditions, and efficient release by mammalianamylases. Overall, the results indicate that the amylose-lipid complex system could serve as a vehicle for deliveryof polyunsaturated fatty acids (PUFA) to the intestine. Hence,potential use of amylose-lipid complexes can be supple-mentation of various staple foods with PUFA.
Acknowledgment. The research was supported in partby the Israel Science Foundation.
References and Notes
(1) Snape, C. E.; Morrison, W. R.; Maroto-Valer, M. M.; Karkalas, J.;Pethrick, R. A.Carbohydr. Polym.1998, 36, 225-237.
(25) Kim, S. J.; Park, G. B.; Kang, C. B.; Park, S. D.; Jung, M. Y.; Kim,J. O.; Ha, Y. L.J. Agric. Food Chem2000, 48, 3922-3929.
(26) Morrison, W. R. In New Approaches to Research on CerealCarbohydrates; Progress in Biotechnology; Hill, R. D., Munck, L.,Eds.; Elsevier: Amsterdam, 1985; Vol. 1, pp 61-70.
