Concentration of linoleic acid from cottonseed oil by starch complexation☆

Dan Liang,Yufeng Hu*,Weiting Ma,Zhengtang Zhao,Siqi Jiang,Yichuan Wang,Xianming Zhang

State Key Laboratory of Heavy Oil Processing,China University of Petroleum,Beijing 102249,China

Keywords:Starch Linoleic acid Starch complexation Starch-FA complex Cottonseed oil

ABSTRACT The extraction of linoleic acid from fatty acids(FA)of the cottonseed oil using starch-FA complexes was developed for the fi rst time.We showed that starch can form inclusion complexes of different strengths with FA and that the different strengths stem from the differences in chain length,degree of unsaturation,and position of double bonds of FA.The optimal separation conditions w ere determined as follow s:The inclusion temperature is 69°C,the inclusion time is 30 min,the starch/FA mass ratio is 10:1,and the ratio of the volume of methanolw ater solution and the mass of FA is 18:1.Compared to urea inclusion complexation,starch complexation has milder reaction temperature and shorter reaction time.Under these conditions,linoleic acid can be concentrated from 38.9%to 70.04%by one-off extraction.Moreover,the experimental results demonstrate the almost perfect reusability of starch.These results show that starch complexation isa promising method that can be used to obtain highly concentrated linoleic acid from cottonseed oil.

1.Introduction

The n-6 long-chain polyunsaturated fatty acids(PUFAs),especially linoleic acid(LA,18:2),are recognized as essential nutrients in the human diet[1].LA has long been know n for its role in reducing the risk of cardio-cerebrovascular disease,circulatory disorders and cancer.Therefore,LAhas a reputation of vascular scavenger.However,humans cannot biosynthesize LA,w hich means that healthful LA must be obtained from external sources.Because LA exists in vegetable oils,such as cottonseed oil,saf fl ow er seed oil,w alnut oil,etc.,ef fi cient methods are required to increase the concentration of LA.Up to now separation of LA from its analogs is still challenging due to the similarity of their molecular size,polarity and structure.

In recent years,separation methods tend to be diversi fi ed[2-4].Many methodshavebeen developed to produce LAconcentrate,including urea inclusion complexation[5-7],low temperature fractional crystallization[8],liquid-liquid extraction by aqueous silver nitrate solutions[9],solid phaseextractions[10],AgNO3-modi fi ed benzenesulfonic acid silica gel column separations[11],high performance liquid chromatography[12]and supercritical fl uid extraction[13].Among these methods,urea inclusion complexation is a favored process ow ing to its high separation capacity and simple manipulations.However,coal,petroleum and natural gas(non-renewable energy resource)are raw materials for urea production.The usage of urea inclusion complexation w ill eventually raise the consumption of non-renew able energy resource.Coupled w ith the grow ing aw areness of sustainable development,research on replacement of non-renew able materials w ith renewable but equally ef fi cient alternativesisdesirable[14].In addition,w ith the grow ing aw areness of green chemical principles[15],starch as a completely green,pollution-free material has an advantage.Previous studies in our lab have show n that cyclodextrin can be used in separation dueto itsremarkableproperty to accommodatemolecules in its cavity[16].Due to the high price of cyclodextrin,w e found that one of such alternatives is starch.Unbranchedα(1→4)glucan chains form helices w ith a hydrophobic interior,w hich can interact w ith a range of small non-polar molecules and the hydrophobic domains of amphiphilic molecules such as FA,monoglycerides and surfactants[17,18].The formation of a stable complex betw een FA that has only one carbon chain linked to the polar head and starch has been observed in previousstudies[19,20].Thestarch-FAcomplexeshavebeen studied by many scholars,w ho used the formed starch-FA complexes to improve the pasting properties(retarding gelatinization and retrogradation)of starchy food and to prepare novel starches(resistant or digestible)[19,21-24].How ever,up to now the starch-FA complexes have not been used to separate FA.

In the present study the starch-FA complexes w ere utilized to separate LA from cottonseed oil.The effects of inclusion temperature,inclusion time and the ratio of raw materials on the separation ef ficiency were investigated.The separation mechanism was discussed.

2.Materials and Methods

2.1.Chemicals

Starch w as obtained from a Hebeifood company.Cottonseed oil w as purchased from Jishui County Shunmin Pharmaceutical Flavor Oil Re fi nery.All reagents were of analytical reagent grade.Fatty methyl esters standards(GC)w ere purchased from Aladdin.

2.2.Hydrolysis of cottonseed oil and puri fi cation of FA

Cottonseed oil washydrolyzed at 80°Cusing potassium hydroxide to obtain FA,w hich w as used for extractions.The extraction procedures are brie fl y described as follows.First,3 g sodium hydroxide was dissolved in the mixed solvent[water(20 ml)+ethanol(40 ml)]in a 250 ml triangle bottle,and 20 g of cottonseed oil wasadded to thethus-obtained solution(sodium hydroxide+w ater+ethanol)to form the reaction mixture(cottonseed oil+sodium hydroxide+w ater+ethanol).This reaction mixturewasthen heated to 80°Cin an oilbath with the re fl ux condenser.After 0.5 h of reaction time,the reaction mixture w as transferred to 500 ml round-bottom fl ask.Excess ethanol in the soap was removed on a rotary evaporator at reduced pressureor by distillation at atmospheric pressure.200 ml of hot distilled w ater was added to aforementioned 500 ml round-bottom fl ask,along with the heating and stirring,to dissolve the soap.The solution of fatty acid salt became yellow upon the addition of a few drops of methyl orange indicator.Excess dilute sulfuric acid w as slow ly added to the reaction mixture,and the reaction mixture w as then separated into two obvious phases.After the reaction mixture w as cooled to room temperature,the ester and glycerol layersw ere separated in a separating funnel.FA w as dried over sodium sulfate anhydrous.A total of 18.46 g of FA per 20 g of cottonseed oil was obtained at the end of the puri fi cation process.

2.3.Preparation of starch-FAcomplex

The starch slurry wascooked in an oil bath at different temperatures with vigorousstirringfor 10 min.Thepasting temperatureof starch w as recorded around 70°C.The starch slurry w as heated at(40,55,65,70,75,and 80)°C,respectively.FA w as dissolved in methanol to form the solution[FA+methanol(35 w t%/mass percent)],w hich w as added to the sw elled starch dispersions.And the mixture(sw elled starch dispersions+FA+methanol)w as continuously heated at the determined temperature for an additional time w ith vigorous stirring to facilitate the interaction betw een starch and fatty acid.The suspension w as then cooled to room temperature,and the starch-FA complexes w ere isolated by negative-pressure fi ltration.The w et pellet of the complexes was w ashed tw ice with methanol to remove residues of uncomplexed FA.Excess methanol and water in the liquid phase were removed on a rotary evaporator at reduced pressure or by distillation at atmospheric pressure to obtain uncomplexed FA.

2.4.Determination of FAcomposition

Cottonseed oil contained triglycerides that w ere formed from various long chain FA and glycerin by ester bonds.Because FA analysis by GConly showed poor results,the triglycerides should be transformed to fatty acid methyl esters w ith a catalyst.For this reaction,an acid catalyst,such assulfuric acid,can be used to esterify the FAto methyl esters[25].Cottonseed oil(300 mg)w as w eighed into a clean round-bottom fl ask,and 3 ml fresh solution(methanol+concentrated sulfuric acid),of w hich the volume ratio w as 19:1,w as then added to the same round-bottom fl ask.The thus-obtained solution(cottonseed oil+methanol+concentrated sulfuric acid)w as heated at 100°Cfor 0.5 h.After being cooled to room temperature,1-hexane(5 ml)w as added to the above-mentioned solution and the mixture(1-hexane+cottonseed oil+methanol+concentrated sulfuric acid)w as separated into tw o phases.The superior phase that contained methyl linoleate w as transferred to another brow n bottle,dried over sodium sulfate anhydrous and centrifuged.

The fatty acid composition of cottonseed oil was analyzed quantitatively by the BeiFen Gas chromatograph instrument equipped w ith a fl ame ionization detector.An Agilent capillary column(HP-INNOWAX length:60 m,diameter:0.32 mm,fi lm thickness:0.25μm)was used.Analysisconditionswerethefollow ing.Theinjector and detector temperatureswereheld at 260 °Cand 280 °C,respectively.Theoven temperature w as held at 100 °Cfor 10 min,then increased to 230 °Cat 20 °C·min-1and held at 230°Cfor 10 min.The total run time was 31.5 min.Splitting ratio 1:20;sample size 0.2μl;carrier gas nitrogen of 0.4 MPa;reagent gases air of 0.4 MPa;hydrogen of 0.3 MPa.Under these conditions,quanti fi cation of the componentswascarried out with the help of an internal standard(butyl acetate).

3.Results and Discussion

3.1.Species and content of FAin cottonseed oil

Thefatty acid composition of cottonseed oil wasobtained by GC-MS,and three main species(LA,oleic acid,and palmitic acid)of FAwere detected.The contentsof FAw ere obtained by GC.The concentration of LA in the concentrate was 38.9%.Other FAs in the concentrate w ere oleic acid and palmitic acid,of w hich the concentrations w ere 43.2%and 16.8%,respectively.

3.2.Optimum separation conditions

The complexation conditions such as inclusion temperature,inclusion time,and concentration ratiosaffected the purity of LAin enriched FA.The physical propertiesof thecomplex can betailored by optimizing the complexation conditions.

3.2.1.Effect of inclusion temperature

Fig.1.The in fl uence of inclusion temperature on the concentration of LA.Inclusion time w as 30 min,and the starch/FA mass ratio(m/m)w as 8:1.The ratio of the volume of methanol-water solution and the mass of FA(v/m)was 20:1.

The concentration of LA of cottonseed oil in extraction phase w as collected for a mixture of 38.9%LA,43.2%oleic acid,and 16.8%palmitic acid in a feed.Six groups of experiments w ere conducted to determine the in fl uence of inclusion temperature on the concentration of LA.The resultsareshow n in Fig.1.It can be seen that theconcentration of LAincreased as inclusion temperature increased from 40 °Cto 69 °C.However,as inclusion temperature exceeded 69°C,further increase in inclusion temperature decreased the concentration of LA.By comparison,oleic acid and palmitic acid showed an oppositetendency.Theseresults indicated that the rise in inclusion temperature favored the formation of starch-FA complexes in the temperature range 40-69°C and w hen the temperature exceeded 69°C(gelatinization temperature),as the temperature further rose,some of the chemical bonds in the starch molecule became unstable,w hich was conducive to the breakage of these bonds.How ever,the process for the intramolecular bonds to disintegrate completely did not fi nish at a single temperature point.It is evident that,when gelatinization took place under the above-mentioned conditions,the gelatinization process initiated at low er temperatures and continued until the granules w ere completely disrupted at higher temperatures[26].The maximum LAconcentration in the concentrate w as 65.5%at 69 °C.Therefore,69 °Cw as chosen as the optimum inclusion temperature to enhance LA content through crystallization.We also determined the optimum reaction temperature for the enrichment of linoleic acid by the urea inclusion method.The result w as-5°C.Therefore,the starch complexation has a milder reaction temperature than the urea inclusion complexation.

3.2.2.Effect of inclusion time

On the basis of the aforementioned results,69°Cw as chosen as the optimum inclusion temperature to investigate thein fl uenceof inclusion time on the extraction of LA.The results are show n in Fig.2.As can be seen from this fi gure,increasing inclusion time in the time range 5-30 min enhanced theconcentration of LAapparently.The LApercentage in the concentrate remained almost a constant in the 30-75 min time range.In other words,the reactions between starch and FA completed w ithin 30 min.Therefore,the complex can be formed easily and rapidly under simple conditions.Such a short reaction time con fi rmed that there existed suf fi cient interaction forces between starch and FA.Note that reaction timeisa key element to evaluate the ef fi ciency of the technique.The optimum reaction time and the optimum reaction temperature determined in the present study for the enrichment of LA by the ureainclusion method were3 h and-5°C.Compared to theureainclusion complexation,the starch complexation has shorter reaction time and milder reaction temperature.

Fig.2.The in fl uence of inclusion time on the concentration of LA.Inclusion temperature w as 69°C,and the starch/FA mass ratio(m/m)was 8:1.The ratio of the volume of methanol-water solution and the mass of FA(v/m)was 20:1.

3.2.3.Effect of ratio of raw materials

In order to acquire a deeper insight on the selectivity of starch for LA from FA,the effects of the ratios of raw materials including starch/FA mass ratio(m/m)and the ratio of the volume of methanol-w ater solution and the mass of FA(v/m)w ere investigated.The results are show n in Figs.3 and 4.

Fig.3.The in fl uence of the starch/FA mass ratio(m/m)on the concentration of LA.Inclusion temperature was 69°C,and inclusion time was 30 min.The ratio of the volume of methanol-water solution and the mass of FA(v/m)was 20:1.m c:the mass of corn starch.m FA:the mass of FA.

Fig.4.Thein fl uence of the ratio of the volumeof methanol-water solution and the massof FA(v/m)on the concentration of LA.Inclusion temperature w as 69°C,and inclusion time was 30 min.The starch/FAmass ratio(m/m)was 10:1.v m:the volume of the methanolwater solution.m FA:the mass of FA.

Fig.3 shows the effect of starch/FA mass ratio(m/m)on the enrichment of LA.Increasing the m/m value in the m/m range 6:1-10:1 apparently increased the concentration of LA and apparently decreased the oleic acid concentration.As the m/m value w as greater than 10:1,the concentrationsof LAand oleic acid becamenearly constant.The concentration of palmitic acid remained almost alw ays a constant in the m/m range of 6:1-12:1.Therefore,the optimum value of m/m was 10:1.

Fig.4 show s the effect of the ratio of the volume of methanol-water solution and the mass of FA(v/m)on the concentration of LA.The concentration of LA increased w ith increasing the v/m value in the v/m range 15:1-18:1.Further increase in the v/m value apparently decreased the LAconcentration.The maximal concentration of LAwasobtained at v/m=18:1.By comparison,the content of oleic acid showed opposite tendency.The concentration of palmitic acid remained almost a constant in the v/m range of 15:1-30:1.Therefore,the optimum value of v/m w as 18:1.Note that,because palmitic acid is a saturated fatty acid,the complexation reaction of palmitic acid w ith the starch molecule w as easy to be completed at the initial condition.Therefore,the change of the starch dosage did not affect the content of palmitic acid in the later stage.As a consequence,the concentration of palmitic acid remained almost alw ays a constant both in the m/m range of 6:1-12:1 and in the v/m range of 15:1-30:1.3.3.Separation mechanism

The main ingredient of starch isa covalent polymer,w hich isformed by the alpha-D-glucose bond w ith alpha-1,4-D-glycosides or alpha-1,6-D-glycosides,and the molecular formula is(C6H10O5)n.Starch is a mixture of two polymers,i.e.,amylose which is predominantly a linear glucose polymer and amylopectin w hich is a branched glucose polymer.The amylose is constituted by double helix structure,whose inner surface is hydrophobic because of the carbon-hydrogen of the 6-fold helix[27,28].The carbon-hydrogen group is inside the helix structure and the hydroxyl is outside the helix.Therefore,the interior of the helix has the lipophilic and hydrophobic properties.The main chain of amylopectin isconstituted by the helix w ith the hydrophobic inner surface,and thispart isidentical to amylose.There areplenty of side chains attached to the main chain w ith different spacing distances.The side chainsof the amylopectin can form ‘fascicle’through hydrogen bonding interaction,and thechainsin ‘fascicle’are parallel to each other,and the branch points are located inside the fascicle,and the external branched side chains form a double helix crystal structure.

The presence of a suitable ligand induces a compact helical conformation of starch.This results in a helix w ith a hydrophobic cavity w hich provides binding sites w ith a high af fi nity for the apolar(part of the)ligand[29].Intramolecular forces,such as Van der Waals forces and hydrogen bonding,occur betw een the turns along the helix,and stabilize a single chain helix[30].Intermolecular forces,on the other hand,stabilize the interaction between starch and its ligand.The starch helix is hydrophilic to the outside and hydrophobic inside itscavity,favoring the formation of hydrophobic interactions[31].It isgenerally accepted that,for most starch-FA complexes,the hydrocarbon chains of FA are inside the helix cavity and the carboxyl groups are outside the helix due to steric and electrostatic repulsions[32].

In this study,the main FA of the cottonseed oil including palmitate acid,oleic acid and LA have been investigated.Their structures are shown in Fig.5.The differencesbetw een these FAare chain length,adegree of unsaturation,and the position of double bonds.These distinctions impact the interactions betw een starch and the aforementioned FAand are bene fi cial for the separation of FA.First,the longer hydrocarbon chain allow smore hydrophobic interactionsw ith the interior of the helix due to the low er hydrophilicity of longer carbon chains,and the resulting stronger preference to reside w ithin the hydrophobic helix cavity[33].Second,it w as further show n that FA w ith a cis double bond in the chain show ed limiting areas different from those of FA w ith a saturated chain[34].The addition of a double bond to an alkyl

Fig.5.Molecular structures of palmitate,oleic,and LA.chain limitsthe fl exibility of the chain in this region.The rise of cis double bond numbers result in chainsthat kink and bend at the position of the cis double bond[34]and it thusseemsmore probable that lesslinear ligandsrequire a larger helix cavity.Third,thehydrocarbon chainsof FA are inside the helix cavity and the carboxyl groups are outside the helix due to steric and electrostatic repulsions.Moreover,the addition of a doublebond to an alkyl chain limits fl exibility of thechain in thisregion.Therefore,the inclusion process betw een starch and FAis more dif fi cult to complete w hen the double bond is closer to the non-polar end.

Palmitate acid and oleic acid have different hydrocarbon chain lengthsand degreesof unsaturation,and oleic acid haslonger hydrocarbon chain length and a higher degree of unsaturation.Oleic acid has stronger preference to reside w ithin the hydrophobic helix cavity because of the longer hydrocarbon chain length compared with palmitate acid and hasagreater steric hindrancesincethedoublebond limits fl exibility of the chain in this region compared to palmitate acid[35].Obviously,there arecontradictionsto thesetwo parts.Oleic acid and LAhave different degrees of unsaturation and positions of double bonds.It seems more probable that less linear ligands require a larger helix cavity.LA requires a larger helix cavity,because it has more double bonds than oleic acid and the location of the double bond of LA is closer to the non-polar end than that of oleic acid.The following results showed that the physical(or the thermal)stability of the complexes follow ed the order palmitate acid＞oleic acid＞LA,which indicated that a higher degree of unsaturation and a closer location of the double bond to the non-polar end of the chain decreased the physical stability of the complexes.

The fact that the complex has a higher melting temperature re fl ects that the helical length of the complex is longer.That the complex has a higher melting temperature indicates that the complex has a greater physical stability.By measuring the temperature at which the inclusion compound disintegrated during the process of gradual heating,the physical(or the thermal)stability of different inclusion complexes w as obtained.The order thus-determined for the physical stability of the complexes w as palmitate acid＞oleic acid＞LA,w hich agreed w ell with the previous results[36].

Both amylose and amylopectin have the helix w ith hydrophobic inner surface.How ever,the interaction between starch and FA is often characterized by amylose chains forming semi-crystalline V-forms.The V-form is an amylose chain that forms a helix w ith a large cavity in w hich variousligandscan besituated,and thesizeof theligand determinesthe number of glucosyl residues per turn(6,7,or 8).The effect or assistanceof theamylopectin to the inclusion experiment israrely mentioned.For thisreason,the experiments are performed using pure amylose or amylopectin asraw material.The thusobtained concentration of LAis68.38%and 40.59%,respectively.These resultsindicated that there is little complex formation betw een amylopectin and FA,probably because the short chain branches are not long enough to form a complex w ith FA.Furthermore,complex formation is prevented by steric hindrance between the branches of amylopectin.3.4.Reusability of starch

It iscritically important to seek reusablesubstances.Therefore,three experiments w ere carried out to con fi rm that starch can be recycled.The solid containing the starch-FA complexes and unpacked starch w as obtained from the aforementioned inclusion experiment.FA was extracted from the solid w ith the use of n-hexane.The rest of the solid w as dried in the oven,and was then weighed.The results are shown in Fig.6.The nearly horizontal plot of Fig.6 revealed apronounced reusability of starch.More speci fi cally,the properties of starch stayed the same except mass.The initial mass of starch w as 10.03 g.It decreased to 8.12 g at the end of the 4th extraction process.Such loss of extractant arose from the transfer between the glassvessels.Therefore,the results of Fig.6 demonstrated the almost perfect reusability of the starch.

Fig.6.Reusability of starch.Inclusion temperature w as 69°C,inclusion time w as 30 min,the starch/FA mass ratio(m/m)w as 10:1,and the ratio of the volume of methanolw ater solution and the mass of FA(v/m)w as 18:1.

4.Conclusions

Starch has been successfully used for selective extraction of FAfrom cottonseed oil.Experimental resultsshow ed a relatively high selectivity and a high complexation constant betw een starch and FA.Moreover,the temperature of the inclusion process w as mild,the reaction time w as short,and the reaction w as reversible,w hich makes starch have good reusability.