J Sci Food Agric 1998, 78, 491È497

Microstructural Features of Enzymatically TreatedOilseedsJorge Sineiro,1 Herminia Dom•�nguez,2 Ma Jose� Nu� n8 ez1* and Juan M Lema11 Departamento de Enxen8 eria Qu•�mica, Universidade de Santiago de Compostela, Avda das Ciencias s/n,15706 Santiago de Compostela, Spain2 Departamento de Enxen8 eria Qu•mica, Universidade de Vigo, Facultade de Ciencias de Ourense, AsLagoas, 32004 Ourense, Spain

(Received 14 July 1997 ; revised version received 23 February 1998 ; accepted 13 March 1998)

Abstract : Enzymatic hydrolytic treatments to enhance the oil extractability fromsoy and sunÑower seeds (low and high oil content, respectively) were performedat a range of experimental conditions (moisture content, particle size, incubationtime, pH and agitation). Light microscopy and scanning electron microscopyshowed the microstructural changes caused by the enzymatic attack. The extentof the cell wall degradation, closely related to the oil extractability, was observedto be dependent on the operational conditions under which the enzymatic treat-ment was carried out. Additionally, the e†ects of the enzymatic treatment on theseed structure were compared with those caused by thermal and mechanicaltreatments. 1998 Society of Chemical Industry.(

J Sci Food Agric 78, 491È497 (1998)

Key words : soybean ; sunÑower ; enzymatic treatment ; cell wall degradation ; oilextractability ; light microscopy ; scanning electron microscopy

INTRODUCTION

The beneÐcial e†ect of enzymatic treatment on theextraction yield of vegetable oils was observed in the1950s, when economic aspects did not allow its indus-trial application. In the 1970s, this subject againattracted attention and led to the implementation ofpilot and industrial processes (Montedoro and Pet-ruccioli 1973 ; Santos 1978 ; Cintra et al 1986 ; Alba et al1987 ; Sosulski and Sosulski 1990). The use of enzymesas processing aids in the extraction of oils from fruitsand seeds represented a new development for thisindustry because, under the mild conditions employed,the quality of the oil and protein is preserved (Caragay1983 ; Graille et al 1988 ; Dom•� nguez et al 1994).

To extract the vegetable oil, which is in intracellularvacuoles, cells must be ruptured and membranesdenatured. Mechanical and thermal conditioning causethe breakdown of the cellular structures but, on

* To whom correspondence should be addressed.Contract/grant sponsor : CICYTContract/grant number : BIO92-0568

occasion, can be inefficient, not allowing the completeextraction of the oil. An enzymatic treatment, whichalso damages the cell walls, improves the oil extraction,although a previous mechanical treatment is necessaryto facilitate the enzyme action (Marek et al 1990 ;Dom•� nguez et al 1994).

The vegetable cell wall is composed of a double mem-brane. The primary wall is an amorphous matrix ofpectins, hemicellulose and protein with cellulose micro-Ðbrils (Kisinger and Hock 1948). In the secondary wall,cellulose and hemicellulose prevail. For this reason, cel-lulase and hemicellulase are the most suitable enzymesto degrade the wall ; pectinases are also e†ective becausepectic substances are structural components of fruitsand vegetables and are largely responsible for coherenceand integrity of plant tissue. Enzyme mixtures andmulti-activity complexes are more efficient than puriÐedactivities (Fullbrook 1983 ; Bhatnagar and Johari 1987),although the resulting e†ect is not the sum of the indi-vidual enzymes (Du� sterho� ft et al 1993).

The process for the extraction of oil from seedsdepends on the type and structure of the seed, althoughextraction with organic solvents (hexane) constitutes,

4911998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

492 J Sineiro et al

alone or after pressing, the conventional process forboth low and high oil content seeds. The main draw-backs are related to economic, environmental and safetyaspects. The high temperatures reached at certain stagescan cause undesirable side-e†ects to the quality of theproducts, mainly proteins, limiting their use for foodpurposes. Furthermore, hexane is toxic and presentsoperational and environmental risks (Shoemaker 1981).Alternative extraction processes with biorenewable sol-vents have been devised ; among them, the most exten-sively studied is aqueous extraction, leading to highquality oil and a detoxiÐed proteinaceous product(Rhee et al 1972, Hagenmaier et al 1973 ; Hagenmaier1974 ; Lawhon et al 1981). Since water is not a speciÐcoil solvent, the extraction efficiency is low and theprocess must be aided by facilitating the oil evacuation.

The hydrolytic enzymatic treatment can be incorpor-ated into conventional extraction or into aqueous pro-cessing with few changes in the basic schemes.SigniÐcant improvements in the oil extraction yieldsfrom aqueous extracted seeds can be achieved for tworeasons : the operational conditions (water content, pH,temperature and agitation) during mixing are compat-ible with the optimal values for the enzymatic action,and the yield achieved with water as extracting solventis low (Lanzani et al 1975 ; Fullbrook 1983 ; Marek et al1990 ; Badr and Sitohy 1992 ; Dom•� nguez et al 1995a,b).The application of the enzymatic treatment during con-ventional processing would either increase productivityor reduce operation time with minimal alterations tooperation. Since the enzymatic treatment requires theaddition of the enzyme in water and, once Ðnished, thematerial has to be dried before extraction, a compro-mise solution should be established between the wateradded to favour the enzymatic action and to avoidexcessive drying costs. Increased oil extraction yieldsand/or higher extraction rates have been reported forseveral seeds (Sosulski et al 1988 ; Sosulski and Sosulski1990 ; Dom•� nguez et al 1993, 1995c,d ; Smith et al 1993).

Microstructural information of foods and its relationto processing is fundamental to characterise, controland improve the properties of transformed foodmaterials (Aguilera and Stanley 1990). Studies on struc-ture and features of soybean that relate to mass transferduring solvent extraction have been reported (Schneider1978). The structural changes in the cell tissue of olivestreated with several enzyme activities have been studied(Montedoro and Petruccioli (1973) using transmissionelectron microscopy, observing the most drastic alter-ations when a mixture of cellulaseÈpapainÈpolygalacturonase was used. The e†ects of theapplication of the enzymatic treatment to canola seedsunder conditions of intermediate moisture have beenexamined through scanning electron microscopy bySosulski and Sosulski (1990), who reported a reductionin the cell wall thickness and a less organised structurethan for the control. Degradation of the cell wall was

also observed in other vegetable materials subjected toan enzymatic attack (Ghose et al 1969).

The present study focuses exclusively on the identiÐ-cation of structural modiÐcations caused by the applica-tion of an enzymatic treatment to oilseeds, with the aimof enhancing the oil extractability. Two oilseeds wereselected, based on their di†erent oil content and there-fore di†erent processing Ñowsheet. The enzymatic treat-ment was performed under a range of operationalconditions corresponding to those compatible with con-ventional and alternative extraction processes. ModiÐ-cations in the cytoplasmic network, cell wall structureand cell-to-cell junctions after enzymatic treatment werestudied, comparing the changes due to the e†ects of themechanical and thermal modiÐcations. Aspects relatedto the efficiency of the enzymatic action under di†erentoperation conditions are discussed.

MATERIALS AND METHODS

Seeds

Whole soybeans (Glycine max Amsoy N.2 cultivar) andcommercial soybean Ñakes were kindly supplied byCereol lbe� rica (La Corun8 a, Spain). SunÑower(Helianthus annuus) seeds were also used both as com-pletely dehulled seeds and as partially dehulled andÑaked seeds from an industrial extraction process usingpressure and solvents (Alco AAA, Maia, Portugal). Theseeds were stored at 4¡C until use.

Enzymes

Celluclast 1.5 L (Novo Nordisk A/S), a commercial cel-lulolytic enzyme, and Multifect (Finnish Sugars Co), amixed-activity enzyme mixture consisting of cellulase,hemicellulase and other degrading activities were usedfor soy and Celluclast 1.5 L, either alone or supplement-ed with Pectinex (Novo Nordisk A/S) was used for sun-Ñower. These enzymes were selected for enhancing theoil extractability as reported previously (Dom•� nguez etal 1993).

Mechanical treatment

The mechanical treatment consisted of grinding (toobtain a small particle size) or Ñaking (to cause distor-tion of the structure of the seeds). Samples were groundin a co†ee grinder, and Ñaked seeds were obtained fromindustrial processing.

Thermal treatment

Cross-sections of soy beans were subjected to thermaltreatment at 120¡C for 30 min. Samples were examined

Microstructural features of enzymatically treated oilseeds 493

by light microscopy (LM) and compared with a controlsample.

Enzymatic treatment

The enzymatic treatment was performed both duringthe aqueous extraction process and at an intermediatemoisture content in a conventional process.

Enzymatic treatment compatible with aqueous processesDehulled seeds were used either as ground seeds (usinga co†ee grinder and sieving to \0É2 mm) or as slices. Awater : seed ratio of 10 : 1 (w/w) was used at 50¡C for2 h. Concentrated HCl was added to maintain the pHat 4É5. Ground seeds were treated by stirring and slicesunder static conditions. Samples were examined by LMand compared with a control sample, incubated underthe same conditions without enzyme. The enzyme wasadded at 2 g per 100 g dry seeds.

Enzymatic treatment compatible with conventionalprocessesDuring incubation with enzymes and especially duringdrying, seeds become sticky and lose their shape, sosoybean seeds were cracked into grits and screened toselect the fraction size between 0É75 and 1 mm (16È22mesh). SunÑower was used both as whole kernels and asknife-cut half-kernels. To avoid the addition of salts, theenzymatic treatment was performed at pH values of 6É6for soybean and 6É8 for sunÑower. The enzyme wasadded in water, keeping 15È20% moisture for soybeanand 30% for sunÑower, at 1 g enzyme per 100 g dryseeds. The low water content for the enzymatic treat-ment of soybean was established to avoid shrinkingduring drying, after swelling during the enzymatic treat-ment if carried out at higher moisture conditions. Bothchanges are more marked in soybean tissue than in sun-Ñower, and shrinking hinders solvent drainage duringoil extraction which lowers the oil yields. The enzymatictreatment was carried out at 50¡C. Since the watercontent before extraction was severely restricted, thesamples were carefully dried in an air-oven at 60È70¡Cafter the enzymatic treatment and observed by scanningelectron microscopy (SEM) to investigate the inducedsuperÐcial changes. For LM, no drying step was per-formed. Seeds samples observed by either of the tech-niques were compared with a control sample, incubatedunder the same conditions without enzyme.

Microstructural examination

To get a better insight into the seed structure, and withthe aim of minimally altering the seeds during samplepreparation for microscopic examination, the samplesfrom di†erent treatments were viewed under the mostsuitable microscopic technique to facilitate the compari-son of mechanical, thermal and enzymatic modiÐcation.

LM was used to observe samples treated under aqueousconditions and treated at intermediate moisture con-tents without the necessity of drying after the enzymatictreatment. SEM was only used for studying the outersurface of samples treated with enzymes at intermediatemoisture content.

Preparation of samples for light microscopyThe sections were mounted on glass slides and washedwith distilled water until they presented a clear creamycolour. To di†erentiate the cell walls, the section werestained red by immersion for 1 min in Safranin solution(10 g safranin in 145 ml distilled water and 155 ml 95%ethanol ; this solution was diluted 1 : 1 with 50%ethanol before use). The cross-sections were rinsed withdistilled water and stained for 3 min with Alcian Bluesolution (0É1È0É5% in 3% acetic acid), rinsed again withdistilled water and with 96% ethanol until the redcolour disappeared, and Ðnally dehydrated in absoluteethanol. To stain protein, samples were immersed in0É5% Coomassie Brilliant Blue G-25 in 3% acetic acidand 50% methanol for 45 min and decolourized with10% acetic acid and 50% methanol for 45 min. Samplesprepared as described above were examined with aNikon microscope.

Preparation of samples for scanning electron microscopyThe samples were dried at 40¡C in an oven and storedover Dehydrated seed tissues mounted on stubsP2O5 .were subjected to metallic shadow casting in a PolaronE-5000 system to cover the material with a thick-350 Óness Au-Pd layer. Samples were observed in an ISI-SS60 SEM apparatus operating at an acceleratingvoltage of 30 kV.

RESULTS AND DISCUSSION

Cotyledonary structure

Figure 1 shows light micrographs of cotyledonary tissueof soybean and sunÑower stained by two methods.SafraninÈAlcian Blue (SAB) stained samples show thepolygonal elongated cells delimited by the cell wall,whereas the cytoplasmic content, vacuoles, proteinbodies and lipid bodies were clearer in Coomassie Bril-liant Blue (CBB) stained samples. In addition to thelipid content, oilseeds also have protein stored ingloboid bodies or aleurone grains ; lipids are stored insmaller spherosomes dispersed within the proteosomes.

Mechanical and thermal treatment

The mechanical and thermal processing steps prior tothe extraction of oil are known to cause drastic damageto the cell walls. Figure 2 shows light micrographs of

494 J Sineiro et al

Fig 1. Light micrographs of soybean and sunÑower cotyle-dons stained by two methods : (a) soybean SafraninÈAlcianBlue, (b) soybean Coomassie Brilliant Blue, (c) sunÑowerSafraninÈAlcian Blue, and (d) sunÑower kernel Coomassie

Brilliant Blue.

transverse sections of the soybean cell walls stained bySAB. The cell wall array as an intact compact skeletonof a matrix of polygonal cells can be observed in Fig 2a.In Fig 2b the mechanical treatment of this structureshows the rupture and dislocation of the cell wall array.The mechanical treatment of low oil content seeds, con-sisting of Ñaking between two rolling cylinders, causes adrastic demolition of the cellular structure. Figure 3apresents an SEM of soybean transverse cut, showing thedislocation in the di†erent layers of the cotyledon : theepidermal Ñat palisade cells of the outer surface, theelongated subepidermal pillar cells (also shown moredetailed in Fig 3c), and the inner cotyledon are dramat-ically damaged and destroyed. Figure 3bÈd showstransverse and longitudinal sections of a soybean Ñake.The complete rupture of the wall causes the release ofthe cytoplasmic material.

Fig 2. Light micrographs of transversal cuts of soybean coty-ledons : (a) compact packaging of cells, and (b) e†ect of the

mechanical treatment.

Fig 3. Scanning electron micrographs of soybean (a) externalsurface, (b) transversal section of Ñaked seed, (c) and (d) longi-

tudinal section of Ñaked soybean.

During processing, oilseeds are thermally conditioned(60È70¡C) before Ñaking to impart increased pliability.Moist heat treatment of soybean not only improves thequality and reÐnability of the oil but also enhances thepercolation rate during extraction and lowers solventretention. The structural changes during this treatmentare presented in Fig 4. Figure 4a shows a section ofsoybean cotyledon, and Fig 4b shows the same sectionafter treatment. Heating leads to physical and chemicalalterations, a†ecting the structure of cell walls by twoopposite e†ects : loss of water, observed as shrinkage,and retraction of the protein matrix (Vardar-Sukan et al1993), epidermal cell swelling and proteosome dis-tention. This process has also been reported to enhancethe release of b-glucan, the amount depending on thecooking method, duration and extent of cell wall dis-ruption (Yiu 1993). The most remarkable e†ect of thethermal treatment is the swelling of the cytoplasm, evenwhen the rigidity of the cell walls (that appear thinner)maintains their shape. Protein bodies became more dis-tended, although neither cellÈcell separations nor cyto-plasmic network disruption occurred in the epidermisor in the mid-region of parenchymal tissue. Since thesee†ects are time-dependent, more time is probablyrequired for these modiÐcations to be apparent.

Fig 4. Light micrographs of soybean (a) transversal sectionand (b) e†ect of the thermal conditioning.

Microstructural features of enzymatically treated oilseeds 495

Enzymatic treatment

The enzymatic treatment was integrated in a schemecompatible with the processing, either conventional oralternative for oil extraction. Therefore, a prior mecha-nical treatment, more or less severe, is usually per-formed, and the rate and extent of enzymatic hydrolysisof cellulose is signiÐcantly a†ected by both the structur-al features of substrate and the operational conditions.

Enzymatic treatment compatible with the aqueous processFigure 5 shows light micrographs of aqueously pro-cessed soybean seeds. Figure 5a presents the morphol-ogy of a control sample, and Fig 5bÈd show the e†ect ofthe enzymatic treatment. The control sample reÑects therupture caused by the Ðne grinding, performed prior tothe mixing and necessary for extraction of oil with hotwater. The enzyme-treated samples have, in general, asmaller particle size than the control, with a degradedsurface as a result of the enzymatic action. Groundseeds submitted to shearing action su†er additionalfractures across the cell wall. However, it is worthnoting that the prevailing e†ect in control samples isthat caused by the mechanical rupture, whereas, inenzymatically treated samples, it is the cell separationcaused by the degradation of the middle lamellae. Thin-ning and degradation of the cell wall, which maybecome dissolved, liberates the cellular material. There-fore, cell tissues gradually lost cellular and subcellularorganisation as the walls and cytoplasm became dis-rupted.

Analogous e†ects are observed in the series of lightmicrographs shown in Fig 6, before and after enzymatictreatment of soybean and sunÑower cotyledon understatic conditions. Figure 6a shows untreated soybeantissue and Fig 6b after enzymatic treatment. Figure 6cshows untreated sunÑower tissue, where the protein

Fig 5. Light micrographs of soybean cotyledons stained withSAB (a) untreated and (b), (c) and (d) enzymatically treated

during aqueous processing.

Fig 6. Light micrographs of seed cross sections (a) untreatedsoybean, (b) enzymatically treated soybean at high watercontent under static conditions, (c) untreated sunÑower and(d) enzymatically treated sunÑower at high water content

under static conditions.

bodies are particularly visible and di†erentiated. Figure6d shows enzymatically treated sunÑower sections. As ageneral trend, the examination of the treated cross-sections revealed the progressive degradation of the cellwall polysaccharides, but, opposite to the treatmentunder stirred conditions, the original structure is main-tained after the treatment.

The e†ect of separation of single cells was observedassociated with both wall and cytoplasmic disruption.The intense breakdown of the middle lamella, togetherwith the degradation of the cell wall, causing the disper-sion of the cytoplasmic material, is favoured by the useof combined enzyme activities (Dominguez et al 1993).Montedoro and Petruccioli (1973), Bathnagar andJohari (1987), Sosulski et al (1988), Badr and Sitohy(1992) and Du� sterho� ft et al (1993) also reported that thebeneÐcial e†ect of mixtures of cellulase and pectinaseprovoked a complete demolition of the cotyledonarystructures and subsequently the release of the oil boundto pectin molecules, whereas pure activities alone didnot show either the tissue degrading or the oil extrac-tion enhancing e†ect.

Enzymatic treatment compatible with the conventionalprocessWith the aim of observing more clearly the e†ect of theenzymatic action on the cell walls, instead of usingÑaked samples, the soybean seeds samples subjected toenzymatic treatment used in this work were cut in gritsand sunÑower kernels cut in halves.

As can be seen in Fig 7, no di†erences were observedin the sections of untreated (a) and treated (b) soybean

496 J Sineiro et al

Fig 7. Scanning electron micrographs of soybean sections (a)untreated and (b) enzymatically treated under low moisture

conditions.

grits. However, SEMs of samples allows visualisation ofthe di†erences in the outer surface of the cell wall, asshown in Fig 8a for untreated soybean surface and inFig 8b for enzymatically treated samples at intermediatemoisture conditions, which is seen more clearly athigher magniÐcation in Fig 8c. SEM revealed that themost striking feature is the individualisation of thesingle cells after the enzymatic treatment, similar to thee†ect observed during the aqueous treatment. Closerexamination of Fig 8c revealed that this external attackwas the only visible e†ect of the enzymatic treatment,probably due to the reduced moisture and lack of agita-tion. The degradation e†ect was less severe than thatobserved when the enzymatic treatment was performedat high moisture content.

The e†ects of the enzymatic attack on the cell walls ofsunÑower kernels, treated at intermediate moisture, canbe observed in the LMs of Fig 9. Located degradedareas might result from the enzymatic attack inrestricted zones due to the inability of the enzyme to

Fig 8. Scanning electron micrographs of soybean external cellwalls (a) untreated, (b) enzymatically treated at intermediatemoisture and (c) enzymatically treated at intermediate mois-

ture observed at higher magniÐcation.

Fig 9. Light micrographs of sunÑower kernel (a) untreated, (b)and (c) enzymatically treated at intermediate moisture.

di†use in the low water content medium under staticconditions.

Figure 10 shows SEMs of the surface of sunÑowerkernels, untreated (a) and enzymatically treated at inter-mediate moisture conditions (b and c). Enzymatic treat-ment resulted in some noticeable changes in the seedmicrostructure. Whereas the untreated sample hadsmoother cell walls and quite spherical oil accumula-tions, treated samples had an irregular surface lessstructured than the controls and di†erently shapedaggregates of oil. This result is in agreement with thestudy of Sosulski and Sosulski (1990), who noticed thedegradation of the structure and also observed a thin-ning e†ect in the walls resulting from the enzymaticattack of the variety of enzymatic activities present inthe mixture used.

Fig 10. Scanning electron micrograph of the outer surface ofsunÑower kernels (a) untreated, (b) and (c) enzymatically

treated at intermediate moisture.

Microstructural features of enzymatically treated oilseeds 497

The degree of attack of the cell walls was lower whenthe treatment was performed at intermediate moistureconditions than in aqueous processing. In addition tothe water content of the medium, which not onlyfavours the action of hydrolases but also the di†usion ofthe enzymes and products of hydrolysis (many acting asenzyme inhibitors), the agitation conditions had astrong inÑuence. During the enzymatic treatment atintermediate moisture, static conditions were main-tained, whereas in aqueous treatment mechanical dis-ruption is caused by the mechanical shear stress,dependent on the impeller geometry and stirring rate,and is responsible for an additional rupture e†ect on theattacked tissues.

Although a qualitative relationship was establishedbetween cell wall degradation and oil extractability(Marek et al 1990 ; Dominguez et al 1993, 1995b), theoptimum operational conditions during the enzymatictreatment are not necessarily those leading to the moredramatic rupture of the tissues, but those selected toincorporate the enzymatic treatment in the selectedextraction process.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the CICYT for eco-nomical support, (Project BIO92-0568). Thanks are alsodue to the Department of Anatomy, Faculty of Medi-cine, University of Santiago, for taking SEM micro-graphs.

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