ORIGINAL PAPER

Effect of Temperature on the Crystalline Form and Fat CrystalNetwork of Two Model Palm Oil-Based Shortenings During Storage

Xia Zhang & Lin Li & He Xie & Zhili Liang & Jianyu Su &

Guoqin Liu & Bing Li

Received: 3 December 2012 /Accepted: 21 February 2013# Springer Science+Business Media New York 2013

Abstract Fat products experienced undesirable micro-structural property changes due to the temperatures dur-ing transportation and storage. In this study, the lipidcomposition and solid fat content (SFC) of two modelpalm oil-based shortenings, which are denoted as short-ening A (melting point of 35.1°C) and shortening B(melting point of 51.2°C), were evaluated, and theircrystallization behavior and polymorphism during stor-age at various temperatures (−10 °C to 30 °C) wasexamined by X-ray diffraction (XRD) and polarizedlight microscopy (PLM). The fractal dimension (Db)was calculated to qualify the change in the crystalnetwork during storage. The aggregation of high-melting triacylglycerols (TAGs) nanostructure and theformation of the most stable β polymorph were ob-served in shortening at higher temperatures (≥0 °C)during storage. At the same temperature, the intensityof β crystals in the two samples increased as the stor-age time increased, and this trend was obvious at hightemperatures (≥10 °C). In addition, the intensity of theβ crystals in the two samples gradually increased as thetemperature increased, and the size of the crystallineparticles became larger. The crystal size in shorteningA was larger than that in shortening B at high temper-atures (≥10 °C). The crystal network of shortening Bwas denser than that of shortening A. The Db valuereached a maximum at 10 °C and 30 °C for shorteningA and B, respectively. These findings have importantimplications on the storage stability and functional prop-erties of palm oil-based shortenings.

Keywords Palm oil-based shortening . Crystalline form .

Fat crystal network . Temperature . Storage

AbbreviationsPO Palm oilIV Iodine valueDSC Differential scanning calorimetryFAC Fatty acid compositionFAME Fatty acid methyl esterspNMR Pulsed Nuclear Magnetic resonanceTAG TriacylglycerolPLM Polarized light microscopeXRD X-ray diffractionDb Fractal dimensionS Saturated fatty acidU Unsaturated fatty acidSFC Solid fat contentPOP 1,3-Dipalmitoyl-2-oleoyl-glycerolPOO 1-Palmitoyl-2,3-dioleoyl-glycerolPPO 1,2-Dipalmitoyl-3-oleoyl-glycerolPOS 1-Palmitoyl-2-oleoyl-3-stearoyl-glycerolSOS 1,3-Distearoyl-2-oleoyl-sn-glycerol

Introduction

Shortenings are usually fats composed of oil (often with anemulsifier and other additives), which provides desirabletextural properties to a food product (Mattil 1964).Recently, per capita consumption of shortenings has in-creased around the world, and numerous types ofmanufactured shortenings have been introduced to the mar-ketplace. The characteristics of shortenings, such as spread-ability, hardness and mouth feel, are highly dependent onthe structure of the underlying fat crystal networks(Marangoni 2002). The fat crystal network is formed whenthe shortening is prepared. However, the fat crystal network

X. Zhang : L. Li :H. Xie : Z. Liang : J. Su :G. Liu : B. Li (*)College of Light Industry and Food Sciences, South ChinaUniversity of Technology, Guangzhou 510640, Chinae-mail: bli@scut.edu.cn

L. Li : J. Su :B. LiGuangdong Province Key Laboratory for Green Processing ofNatural Products and Product Safety, Guangzhou 510640, China

Food Bioprocess TechnolDOI 10.1007/s11947-013-1078-8

changes when the temperature fluctuates over a large rangeduring handling, storage and transportation (Jin et al. 2007).Palm oil (PO) is commonly used as one of the ingredients ofthe fat phase in shortening due to its easy availability,excellent physical characteristics, and price competitiveness(Aini and Miskandar 2007; Arifi et al. 2011; Saberi et al.2011; Sukumar et al. 2012). However, the use of PO-basedshortenings in food products has encountered serious struc-tural problems including the growth of granular crystals,which imparts heterogeneous fat crystals that impair theconsistency and plasticity of fat products (Meng et al.2010a). Due to the inevitable temperature fluctuations ex-perienced during transportation and storage, the fat crystalscan grow to a size of 30–200 μm in diameter, resulting in anobvious granular texture (Ishikawa et al. 1980). To improvethe quality of PO-based shortening and expand its applica-tion, it is very important to understand the mechanism oftemperature on the fat crystal network and the effect ofcomposition, polymorphic forms and structure on the short-enings during storage and transportation.

Temperature is one of the most important parameters inplastic oil production (Haighton 1976; Herrera and Hartel2000). In the tempering process, the temperature is wellcontrolled to produce fats with desirable properties, butduring transportation and storage most of the fat productsundergo undesirable changes caused by temperature fluctu-ations. The changes in the physical and textural properties ofplastic fats during storage have been investigated. Ishikawaet al. (1980); Watanabe et al. (1992) and Miura and Konishi(2001) observed the presence of granular crystals when thestorage temperature shifted between 5 °C and 20 °C every12 h and concluded that the slow crystallization rate of POmight lead to the β polymorph and the formation of granularcrystals was attributed to the most stable β1 polymorph of1,3-dipalmitoyl-2-oleoyl-glycerol (POP). Other studies haverevealed that the agglomeration of TAGs with high meltingpoints led to the formation of granular crystals (Jin et al.2007; Tanaka et al. 2007). Lee et al. (2008) found that thesamples stored at 5 °C (refrigerator temperature) were pri-marily composed of β′ form crystals together with a smallcontent of β form crystals, while those stored at 24 °C(room temperature) were only composed of β′ form crystals.The hardness of margarine samples stored at 15 °C for7 days increased upon storage, and the crystal habit changedfrom β′ to β despite having the same solid fat content (SFC)(Sabine and Claude 2004). Goli et al. (2009) prepared anexperimental table margarine enriched with conjugatedlinoleic acid (CLA) and stored it along with a commercialmargarine (as a control sample) at 5 °C and 15 °C for3 months. They found that the storage times and tempera-tures affected the SFC content of the products. The samplesstored at the lower temperature (5 °C) had more stability inβ′ crystal compared to those stored at 15 °C. The effects of

storage temperatures and times on the quality of the plasticoil have been studied, but there is limited research on theeffect of storage temperature and time on the crystalnetwork.

The structural hierarchy in shortening results from the anunderlying fat crystal network (Vreeker et al. 1992), whichis due to the interaction of polycrystalline fat particles thatprovide firmness or solid-like behavior to the plastic fats(Marangoni 2002; Narine and Marangoni 1999a; Wright etal. 2001; Tang and Marangoni 2006). The characteristics offat-structured materials such as spreadability, hardness andmouth feel, are highly dependent on the structure of theunderlying fat crystal networks. The plastic fats have beenreported to be fractal by Marangoni (2002), and the massfractal dimension was used to describe the crystal network.With increasing concern for the quality of oil products, it isimportant to determine the quality of the samples and howthe structure of the samples changes with time for samplesstored at a constant temperature. Therefore, clarifying thestabilization of PO-based plastic fats is helpful for fullyunderstanding the effects of temperature on their structureand properties.

The aim of this project is to characterize the effect oftemperature on the crystalline form and fat crystal networkof PO-based shortening during storage to provide pertinentinformation for optimization of spreadable products madefrom these systems. In this study, the lipid composition,thermal properties, polymorphism, crystal morphology, thestructural hierarchy of fat crystal networks and the crystalnetworks of two types of pure PO-based model shorteningsstored at different temperatures were systematicallyinvestigated.

Materials and Methods

Materials

Two kinds of commercially available shortenings (i.e.,shortening A and shortening B), which are the most widelyused in China, composed of pure PO with a certain amount(0.015%) of antioxidants such as butylhydroxyanisole andbutylated hydroxytoluene to prevent the changing of thecompositions caused by oxidation during storage, were gen-erously provided by Kerry Specialty Fats Ltd. (Guangzhou,China) as the model shortenings. Shortening A has a melt-ing point of 35.1°C with an iodine value (IV) of 8 and anacid value of 0.43 mg KOH/g, while shortening B exhibits amelting point of 51.2°C with an IV of 23 and an acid valueof 0.83 mg KOH/g. The shortening samples weremaintained at a temperature of 25±1°C while beingtransported from the factory to the laboratory where theywere immediately stored at different temperatures or

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analyzed. The shortening samples at 25±1°C were used asthe control samples.

All of the other reagents and solvents were of analyticalor chromatographic grade and were purchased fromSinopharm Chemical Reagent Co. Ltd. (Shanghai, China).Rounded aluminium pans with a diameter of 5 cm were usedto hold the samples.

Temperature Protocol

The shortening samples were cut into rectangular blocks thatwere 10×10×8 cm in length, width and height, respectively,and then placed on the aluminium pans and stored at −10 °C,0 °C, 10 °C, 20 °C or 30 °C for 1 week. The temperature wascontrolled by a DC-3006 low-temperature water bath circula-tor (Xinzhi, Ningbo, China). Three parallel experiments wereperformed. The sampling was carried out at a set time and thecharacteristics of the samples were analyzed by differentialscanning calorimetry (DSC), X-ray diffraction (XRD), pulsednuclear magnetic resonance (pNMR) and polarized lightmicroscopy (PLM).

Fatty Acid Composition

Fatty acid methyl esters (FAME) were prepared according tothe AOCS Official Method Ce 2-66 (2004) and were sub-sequently analyzed on a GC-14B gas chromatograph (GC)equipped with a fused silica capillary column (CP-Si188,100 m×0.25 mm×0.2 mm i.d.) with a flow rate of 1 mlN2/min using a flame ionization detector (Shimadzu, Tokyo,Japan). The temperature of both the injection port anddetector was maintained at 250 °C. Temperature programwas controlled as: initial temperature was 50 °C (1 min),which was heated to 190 °C at a rate of 8 °C/min and heldfor 2 min, and finally come to 220 °C with a rate of 3 °C/min.The fatty acid species were identified by comparing theretention times of the peaks with those of the respectiveFAME standards. The fatty acid content as mass percentwas quantified by the area of the corresponding peak relativeto the sum of the areas of all the peaks in the samples, usingcorrection factors experimentally determined, according toAOCS Official Method Ce 1-62 (1990). The fatty acidcompositions of the samples and the fatty acid content aregiven in Table 1. As shown in Table 1, the primary fatty

acids of the fat blends were palmitic acid (C16:0) and oleicacid (C18:1).

TAG Composition Analysis

The sample was completely melted and then dissolved inchloroform to obtain a concentration of 0.5 mg/ml.Triacylglycerols (TAGs) were separated by reversed-phasehigh-performance liquid chromatography (HPLC) using aSymmetry C18 column (250×4.6 mm, particle size 5 μm)(Waters, Ireland) where the A phase was acetonitrile, the Bphase was n-hexane/isopropanol (4:5, v/v), and A/B=50:50was used as the eluent at a flow rate of 1.0 ml/min anddetected with an evaporative light scattering detector(ELSD). TAGs were identified by HPLC coupled to atmo-spheric pressure chemical ionization mass spectrometry(HPLC/APCI-MS) using the same HPLC conditions de-scribed above. A liquid chromatography-tandem mass spec-trometer (MS) (Bruker Daltonics Inc., USA) equipped withan APCI interface was run at an APCI source block andprobe temperature of respectively 100 °C and 400 °C and aMS multiplier voltage of 700 V. The measurement rangewas between m/z 200 and 1,200 (Jakab et al. 2002).Quantitative determination of individual TAG in the fatblends was performed with the HPLC results following theprocedures of Chen et al. (2007). Individual TAG contentwas calculated as percentage of the peak area of individualTAG relative to the total peak area of the total TAGs in thesamples. The structural designation for TAGs was based onreversed-phase HPLC-APCI-MS analysis of the observedTAGs corresponding to sodium adducts of the molecularions [M+Na]+ and one fatty acyl moiety cleaved ions[M-(R-COO)]+ ([DG]+).The TAG compositions, contentand the proposed TAG structures with the correspondingmass identification data are tabulated.

Solid Fat Content Determination

According to AOCS Official Method Cd 16b-93 (2004), theSFC of the samples was determined on a PC120 pulsednuclear magnetic resonance (pNMR) spectrometer (Bruker,Karlsruhe, Germany). The sample was placed in the NMRtube and successively melted at 70 °C for 30 min, temperedat 0 °C for 90 min, and then maintained at the 0.0 °C, 5.0 °C,

Table 1 Fatty acid compositions (%) of shortening A and B

C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Others

A 0.22±0.01A 1.21±0.02A 49.43±0.30A 5.43±0.09A 35.82±0.21A 6.48±0.04A 1.41±0.08A

B 0.10±0.01B 1.11±0.01A 57.61±0.04A 5.69±0.11A 27.54±0.44B 6.12±0.07B 1.83±0.23B

Different superscript capital letters denote significantly different groups (P<0.05), whereas the same letters denote similar groups (P>0.05) in thesame column

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10.0 °C, 21.1 °C, 26.7 °C, 33.3 °C, 40.0 °C and 45.0 °C,respectively, for 30 min prior to measurement. Triplicatemeasurements were performed. The SFC values were givenas means of triplicate and the final results plotted as percent-age unit (%). The plot of the SFC percentage of the samplesas a function of the temperatures during storage (0.0 °C,5.0 °C, 10.0 °C, 21.1 °C, 26.7 °C, 33.3 °C, 40.0 °C and45.0 °C) is performed.

Polarized Light Microscope (PLM)

The morphology of sample crystals was observed usingPLM (DMRX, Leica, Germany) with an attached CanonA640 digital camera (Canon, Tokyo, Japan). To observethe crystal network morphology of the shortening, the de-sired amount of sample (approximately 50 mg) was placedon a carrier glass slide pre-cooled to the desired tempera-tures. Then, a cover slip was placed parallel to the plane ofthe carrier slide and centred on the drop of sample to ensureuniformity and a desirable sample thickness. The photomi-crograph of the crystal was recorded at a 500× magnifica-tion. A number of images were acquired with eachrepresenting a typical field. The real-time PLM images ofthe shortenings, which were stored at −10 °C, 0 °C, 10 °C,20 °C or 30 °C for 0 h, 24 h, 120 h or 168 h are performed.

The images acquired were inverted, thresholded, andanalyzed using ImageJ 1.36b software that is available inthe public domain of the National Institutes of Health(National Institutes of Health, ImageJ 1.36b; http://rsb.info.nih.gov/ij). The fractal dimension was determinedusing a box counting algorithm, which was calculated as thenegative of the slope of the linear regression curve of thelog–log plot of the number of occupied boxes Nb versus theside length Lb, with the result denoted Db. The Db values ofthe recorded PLM images of the shortenings which werestored at −10 °C, 0 °C, 10 °C, 20 °C or 30 °C for 0, 24, 120or 168 h are recorded.

Crystal Polymorphism by XRD

The polymorphic forms of fat crystals in the blends weredetermined by D8 Advance XRD (Bruker, Germany), usingCu KR radiation with a Ni filter (k=1.54056 Å; voltage,40 kV; current, 40 mA; fixed 1.0, 1.0, and 0.1 mm diver-gence, antiscatter, and receiving slits, respectively). Sampleswere scanned from 1° to 30° (2θ scale) at a rate of 2.0°/minat ambient temperature. The intensity file was generated byremoving the background, smoothing, and correcting theraw data. The calculations were performed by MDI Jade5.0 software (Rigaku, Japan). The intensity of β′ was from3.8, 4.2 and 4.4 Å, and that of β was 3.7, 3.85 and 4.6 Å,and the other little peaks were ignored. The relative content(%) of the β′ and β forms was calculated from the intensity

(counts per second [CPS]) of the short spacings. XRDspectra of shortening samples stored at different tempera-tures (−10 °C, 0 °C, 10 °C, 20 °C and 30 °C) with differentstorage times (0, 24, 120 and 168 h) are reported with theshort spacings of the crystals. In addition, the relative con-tent (%) of the β′ and β forms at different temperatures(−10 °C, 0 °C, 10 °C, 20 °C and 30 °C) with differentstorage times (0, 24, 120 and 168 h) is listed.

Statistical Analysis

Triplicate analyses (n=3) were performed for each depen-dent variable (fatty acid composition, TAG composition,crystal polymorphism, SFC and crystal morphology).Analysis of variance (ANOVA) with Duncan’s multiple-range test was performed with the Statistical AnalysisSystem software (SAS, Cary, NC, USA). Differences wereconsidered to be significant at P<0.05. Values shown intables were the means of values from the triplicate analyses(standard deviations).

Results and Discussion

TAG Composition

Table 2 shows the TAG composition of the samples obtainedfrom HPLC. Proposed TAG structures are listed with thecorresponding sodium adducts of the molecular ions [M+Na]+ and one fatty acyl moiety cleaved ions [M-(R-COO)]+

([DG]+). The HPLC chromatograms of the samples and themass spectra for individual TAGs are not shown to conservespace. The relative concentrations of the main TAGs weredifferent (P<0.05) when comparing shortening Awith short-ening B. The main TAGs in shortening A were POP and 1-palmitoyl-2, 3-dioleoyl-glycerol (POO), while those in short-ening B were PPP and POP/1, 2-dipalmitoyl-3-oleoyl-glycer-ol (PPO). TAGs have been classified into four types includingSSS, SUS, SUU and UUU (S — saturated fatty acid, U —unsaturated fatty acids) according to the saturation degree offatty acids. And the functionalities of margarine were closelyassociated with the TAG types (Wiedermann 1978). In short-ening, high melting point TAGs were the main source forcrystalline skeleton of the product, providing special structureand hardness to some extent. The high melting point TAGsinclined to agglomerate and form nuclei during crystallization.POP crystallized slowly and thus easily formed large non-uniform spherulites (Tanaka et al. 2007; Miura and Konishi2001). Shortening B contained a high amount of tripalmitoyl-glycerol (PPP), which was also likely to form nuclei andduring agglomerate crystallization, and POP, which easilyformed large non-uniform spherulites. Therefore, spherulitesmay be the most dominant structure in shortening B.

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SFC of Shortenings

SFC is a very important index used in plastic oil industry.Most functionalities of plastic oil can be determined fromSFC. SFC values of shortening A and B at different tem-peratures are shown in Fig. 1. It is clear that the SFC valuesof the two samples decrease with temperature increasing.Shortening A had higher SFC value than shortening B whenthe temperature was lower than 10 °C (P<0.05). In thetemperature range from 5 °C to 26.7 °C, the SFC value ofshortening A decreased much faster than that of shorteningB, and shortening Awas almost completely melted at 40 °C.This result was attributed to the fact that shortening Acontained more unsaturated fatty acids and low meltingpoint TAGs. As the temperature increased during the melt-ing process, the triunsaturated (UUU) TAGs usually meltedfirst, successively followed by monosaturated–diunsaturated(SU2) TAGs, disaturated–monounsaturated (S2U) TAGs,

and trisaturated (SSS) TAGs. When the temperature waslower than 10 °C, UUU TAGs in shortening B had relativelylower melting point than those in shortening A. ThereforeUUU TAGs in shortening B melted first resulting in thatSFC of shortening B was lower than that of shortening A.As the temperature increased, the relatively higher meltingpoint UUU, SUU, or SUS TAGs melted. Because of thehigher amount of SUU and SUS TAGs in shortening A, theSFC of shortening A decreased substantially.

Crystal Polymorphism

XRD spectra of shortening samples stored at different tem-peratures (−10 °C, 0 °C, 10 °C, 20 °C and 30 °C) withdifferent storage times (0, 24, 120 and 168 h) were detectedwith the short spacings of crystals (Figs. 2 and 3). Shortspacings refer to the cross-sectional packing of the hydro-carbon chains. The fat crystals mainly contain three types ofpolymorphs, such as α, β and β′. The polymorphs can bedetermined by the short spacing of the crystals. The shortspacing of α form appears near 4.15 Å, that of the β′ formappears close to 4.2 and 3.8 Å, and that of the β formappears at 4.6 Å (single strong spacing). In addition, thereare some intermediate polymorphs that are of interest.Pseudo-β′ is designated as a subform with a weak spacingat 4.4 Å (Kalnin et al. 2005). It has been reported that thetwo peaks with spacings close to 3.7 and 3.85 Å wereproduced by the subforms of β (Sabine and Véronique2007; Blaurock 1999). Levels of β′ and β crystals in themixtures have been estimated by the relative intensity of theshort spacings at 3.8, 4.2, and 4.6 Å (Lee et al. 2008). In thispaper, the relative content (%) of β′ and β form was calcu-lated from the intensity (in CPS) of the short spacings, inwhich the intensity of β′ form was 3.8, 4.2 and 4.4 Å, andthat of the β form was 3.7, 3.85 and 4.6 Å; the other littlepeaks were ignored. The results are shown in Table 3. The α

Table 2 TAG composition (%)in shortenings

Different superscript capital let-ters denote significantly differentgroups (P<0.05), whereas thesame letters denote similargroups (P>0.05) in the samerow

M myristic acid, P palmitic acid,S stearic acid, O oil acid, Llinoleic acid

TAG Shortening A Shortening B [M +Na]+ [DG]+ [DG]+ [DG]+

MPL/PLL 1.96±0.21A 2.14±0.15A 810/878 [MP]+ 520 [PL]+ 576 [LL]+ 600

OOL 1.57±0.10A 1.43±0.12A 905 [OO] + 604 [OL] + 602

MMP 8.48±0.06 A 1.49±0.22B 774 [MM]+494 [MP]+ 523

PLO 9.77±0.15A 8.05±0.12B 880 [PL]+ 576 [LO]+ 602 [PO]+ 578

PLP 6.47±0.02A 9.29±0.27B 854 [PP] + 552 [PL]+ 576

OOO 3.38±0.17A 1.41±0.17B 907 [OO] + 604

POO 21.60±0.36A 15.51±0.07B 882 [PO]+ 578 [OO] + 604

POP/PPO 37.97±0.23A 27.46±0.09B 856 [PP] + 552 [PO]+ 578

PPP 4.23±0.06A 20.71±0.24B 830 [PP] + 552

SOO 1.79±0.13A 1.32±0.06A 910 [SO] + 606 [OO] + 604

POS 8.24±0.22A 5.17±0.08B 884 [PO]+ 578 [SO] + 606 [PS] + 580

PPS 2.15±0.05A 6.02±0.34B 858 [PP] + 552 [PS] + 580

Fig. 1 SFC versus temperature profile for shortening A and B

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form is the least stable and has the lowest melting poly-morph, while the β form is the most stable and has thehighest melting point (Chong et al. 2007). The β′ form isintermediate in stability and melting point. In general, aconversion from one form to another (i.e., α to β′ and β′to β) occurs in the direction that favors the more stableforms. For margarines and shortenings, the β′ form is desir-able for providing good texture and good creaming proper-ties, while the one containing more β crystals produces adull and mottled product (Idris et al. 2007).

As shown in Figs. 2 and 3, the main polymorphic form ofthe samples was quite different. For the shortening A controlsample that was not stored (stored for 0 h), there were threestrong peaks with spacing at 3.84, 4.20 and 4.34 Å, and a

weak peak at 4.60 Å. Ibrahim et al. (2006) reported that thespacing at 4.35 Å was produced by the crystals of β′ form.Rousseau et al. (2005) studied the polymorphic transition ofthe β′ to β in food fats and found that the spacing at 4.33 Åcorrespond to the β′ (β′-2) form. Therefore, the spacing at4.34 Å here was regarded as β′ form. Based on Fig. 2, themain form of the shortening A control sample was β′ form,which primarily consisted of the β′ form of the crystal(96.79±0.31%) with a small content of β form (3.21±0.16%) (Table 3 and Fig. 2). After shortening A was storedat the same storage temperature, the intensity of the peak at4.60 Å obviously increased as the storage time increased(Table 3 and Fig. 2), which indicated that the β formincreased with storage time. With an increase in time from

Fig. 2 X-ray diffraction spectraof shortening A stored at −10 °C,0 °C, 10 °C, 20 °C and 30 °Cafter 0, 24, 120 and 168 h

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24 to 168 h, the β form content increased from 3.20±0.64%to 5.76±0.68% at −10 °C, from 12.66±1.54% to 14.52±0.84% at 0 °C, from 85.22±2.61% to 86.96±2.18% at 10 °C,from 83.11±0.15% to 85.59±2.19% at 20 °C, and from86.21±0.15% to 87.74±0.15% at 30 °C (Table 3). For thesame storage duration, the intensity at 4.60 Å when thestorage temperature was ≥10 °C was markedly stronger thanthat when the storage temperature was <10 °C (P<0.05),which indicated that a higher storage temperature boostedthe formation of β form. Some new peaks with spacings at3.60, 3.74, 3.87, 4.03 and 4.45 Å appeared when the tem-perature was ≥10 °C, and the intensity of these new peaksincreased as the storage time increased. It has been reported

that the two peaks with spacings close to 3.7 and 3.85 Åwere produced by the subforms of β (Sabine and Véronique2007; Blaurock 1999). The peak at 4.03 Å corresponded tothe Bragg spacing of β′ form, and the peak at 3.60 Å wasdue to the Bragg spacing of the intermediate form betweenβ′ and β (Ibrahim et al. 2006). The peak with weak spacingat 4.45 Å corresponded to the pseudo-β′ form according tothe study by Kalnin et al. (2005). The appearance of thesenew peaks further indicated that there was a polymorphictransition from meta-stable β′ to stable β for the shorteningA samples. Therefore, the shortening A samples were verysensitive to high temperatures (10–30 °C), which was mostlikely due to an increased amount of unsaturated TAGs in

Fig. 3 X-ray diffraction spectraof shortening B stored at −10 °C,0 °C, 10 °C, 20 °C and 30 °Cafter 0, 24, 120 and 168 h

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the shortening A samples, such as POP, which was easilytransformed into the β polymorph (Tanaka et al. 2007).

For the shortening B control sample at room temperature,there was a distinct peak at 3.80 Å and a weak peak at4.20 Å (Fig. 3), which correspond to the β′ form, and therelative content of the β′ form was 96.79±0.13% (Table 3).In Fig. 3a–d, a strong peak at 3.80 Å, a weak peak at 4.20 Åand a weak peak at 4.60 Å which exhibited an intensity thatincreased as the storage time increasing, were observed.Table 3 shows that when the shortening B samples werestored at −10 °C, 0 °C, 10 °C and 20 °C, with a storage timethat ranged from 24 to 168 h, the relative content of the βform increased from 3.20±0.64% to 5.76±0.68% at −10 °C,from 9.92±1.07% to 30.11±1.67% at 0 °C, from 24.63±1.09% to 34.65±0.87% at 10 °C, and from 11.96±1.38% to26.63±0.15% at 20 °C. These results indicated that thedominant polymorph form in shortening B that was storedat ≤20 °C was β′ form. However, the relative content of βform increased as the storage time increased, which indicat-ed that the unstable β′ form was gradually transformed intothe stable β form during a long period of storage. In Table 3,the content of β form crystals at 20 °C was interestinglylower than that at 10 °C, which was most likely due to theTAGs such as 1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol(POL), POO, 1-oleoyl-2,3-distearoyl- glycerol (SOO)/1, 2-distearoyl-3-linoleoyl-glycerol (SSL) melt at 20 °C. As aresult, there was not enough space to allow the high-meltingTAGs (mainly β-tending property) to move and aggregate.At 30 °C, new smaller peaks with spacings at 3.60, 3.74,3.87, 4.03 and 4.40 Å were observed. According to the

studies by Blaurock (1999), the two peaks at 3.74 and3.87 Å corresponded to the β form, while the peaks at4.03 and 4.40 Å corresponded to the β′ form. In addition,the peaks at 3.60 Å was associated with the intermediateform between β′ and β. Figure 3e exhibits an evidentpolymorphic transition from β′ to β form. The relativecontent of β form in shortening B stored at 30 °C(Table 3) is higher than 80%, which is significantly higherthan that at other temperatures. Because many of the crystalsmelt at 30 °C, the β crystals of symmetric-type TAGs (i.e.,POP, 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol [POS], 1,3-distearoyl-2-oleoyl-sn-glycerol [SOS]) had the space tomove and aggregate to form larger crystals (Lawler andDimick 2002). In addition, the high temperature providedthe energy for the polymorph transition from meta-stable β′to stable β. Therefore, at 30 °C, β form was the dominantform observed for shortening B.

As shown in Figs. 2 and 3 and Table 3, long periods ofstorage are harmful to the stabilization of shortening be-cause both shortening A and B exhibit an obvious transitionfrom β′ form to β form after 168 h of storage. Both short-ening A and B are sensitive to high temperature (i.e., 30 °C),and β form is the dominant polymorphic crystals at 30 °C.The lower the storage temperature resulted in better stabili-zation of β′ form. The content of β form crystals in short-ening A and B was lowest at −10 °C compared to that atother temperatures. However, Figs. 2 and 3 show that themain polymorphic form of shortening A and B was quitedifferent. The less stable polymorphs were found to convertto a more stable one. The speed of this transformation

Table 3 Content of polymor-phic forms (β and β′ Form) ofshortening A and B under thetemperature process

Different superscript capital let-ters in the same row and lower-case letters in the same columndenote significantly differentgroups (P<0.05)

Shortening A Shortening BContent (%) Content (%)

T (°C) Time (h) β β′ β β′

R.T. 0 3.22±0.16Aa 96.78±0.31Aa 3.21±0.03Aa 96.79+0.13Aa

−10 24 3.20±0.64Aa 96.80±1.46 Aa 6.93±0.15Bb 93.07±0.07Bb

120 3.59±0.21Aa 96.41±0.41Aa 6.52±0.83Bb 93.48±2.15Bb

168 5.76±0.68Aa 94.24±2.02 Aa 12.75±1.22Bc 87.25±2.35Bc

0 24 12.66±1.54Ab 87.34±2.67 Ab 9.92±1.07Bc 90.08±2.66Bc

120 10.90±0.89Ab 89.10±1.54 Ab 31.28±0.67Bd 68.72±1.45Bd

168 14.52±0.84Ab 85.48±1.99 Ab 30.11±1.67Bd 69.89±2.56Bd

10 24 85.22±2.61Ac 14.78±3.41 Ac 24.63±1.09Be 75.37±2.11Be

120 85.90±1.45Ac 14.10±2.79 Ac 27.74±0.17Be 72.26±0.89Be

168 86.96±2.18Ac 13.04±3.16 Ac 34.65±0.87Bd 65.35±1.98Bd

20 24 83.11±0.15Ad 16.89±0.28 Ad 11.96±1.38Bc 88.04±2.79Bc

120 84.16±2.41Ad 15.84±3.62 Ad 11.17±0.47Bc 88.83±0.83Bc

168 85.59±2.19Ac 14.41±2.44 Ac 26.63±0.15Be 73.37±1.12Be

30 24 86.21±1.35Ae 13.79±0.34 Ae 82.68±0.96Bf 17.32±0.45Bf

120 87.30±0.98Ae 12.30±0.32 Ae 83.68±1.24Bf 16.32±0.23Bf

168 87.74±0.21Ae 12.26±0.11 Ae 86.37±1.03Af 13.63±0.11Af

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depended on the fatty acid composition, and distribution andthe long or complicated fatty acid carbon chains resulted in alow transformation speed (Rousseau et al. 2005). When thetemperature was −10 °C and 0 °C, the relative content of βform in shortening B (6.52–31.28%) was higher than that ofshortening A (3.20–14.52%) (Table 3). As shown in Fig. 1,the SFC content of shortening A was higher than that ofshortening B when the temperature was <10 °C, whichindicated the crystals of shortening B had more space tomove to form β crystals compared to shortening A. Whenthe temperature ranged from 10 °C to 30 °C, the content ofβ form crystals of shortening A (85.22–87.74%) is muchhigher than that of shortening B (24.63–86.37%) (Table 3).The SFC value of shortening A decreased much faster thanthat of shortening B when the temperature was >10 °C,therefore shortening A had more space to move and gathertogether to form larger crystals compared to shortening B(Lawler and Dimick 2002). In addition, the high tempera-ture provided the energy for the polymorphic transition frommeta-stable β′ to stable β. Therefore, the content of β formcrystals in shortening A was much higher, which was inagreement well with the results reported by Shiota and hiscoworkers (2011) where the temperature provided the forcefor the transformation of β′ polymorphs to β ones.Moreover, the C48 and C54 type TAGs (such as PPP andtrioleoyl-glycerol [OOO]) easily form β polymorphs, whilethe C50 and C52 type TAGs (such as POP, POS) tend togenerate strong β′ polymorphs tendencies (Toro-Vazquez etal. 2000). Garbolino et al. (2005) also found that hightemperatures would make the polymorphs of POP trans-form. As shown in Figs. 2 and 3, β′ polymorphs were easilyformed in shortening A at lower temperatures, while β′ andβ polymorphs were observed for in shortening B. Thisdifference was due to POP being the main TAGs in short-ening A, while those in shortening B were PPP and POP(Table 2). As the temperature increased, it provided theenergy for the polymorphs of POP to transform. The resultssuggested that agglomeration of the high melting pointcrystals occurred with the temperature increasing, and concom-itantly the transformation into the most stable β polymorphtook place. The β polymorph is undesirable for shorteningquality; therefore, controlling storage time and storage temper-ature is significant for maintaining the quality of shortenings.

Microscopy and Image Quantitative Analysis

Real-time PLM observation of the shortenings which werestored at −10 °C, 0 °C, 10 °C, 20 °C or 30 °C for 0, 24, 120or 168 h was carried out (Figs. 4 and 5). Watanabe et al.(1992) found that the crystal size was important for thequality of the final products. Small crystals can form a morerigid product, and crystals that were 30–200 μm in sizecause a sandy mouth feel. In addition, when the size of the

aggregates was between 100 and 300 μm, a taste sandy wasproduced (Haighton 1976). Meanwhile, Piska et al. (2006)and Narine and Marangoni (1999b) observed that the size ofthe β′ crystal was 1–5 μm, and the size of the β crystal was20–100 μm. As shown in Fig. 4, the crystal size in shorten-ing A increased as the temperature increased, and the crys-talline network became more porous. The crystallinenetwork varied with time and temperature. When the tem-perature was −10 °C, the crystal was needle-like with asmall size (no more than 2 μm) corresponding to β′ crystal.The overall crystal network morphology did not changesignificantly, which was in agreement with the XRD results.When the temperature was 0 °C, the crystal size increased asthe needle-like crystals aggregated. However, the size ofmost of the crystals was between 5 and 20 μm, and thecrystals were still composed of the β′ type. Rousseau et al.(1998) and Meng et al. (2011) have observed the samephenomena when investigating the stability of the crystalli-zation of PO. As the time increased, the network graduallybecame denser and the β′ crystal was the main crystal. At10 °C, the crystal aggregated to form small spherulites withthe larger crystals being β crystals. In addition, β′ and βcrystals co-existed in the system, and the trend in crystallinenetwork morphological changes with time was not obvious.At 20 °C, the crystal size and the density further increasedresulting in β′ and β crystals, and the grain size graduallyincreased as the time increased. At 30 °C, the larger β-typespherulites (of approximately 150–250 μm) composed ofneedle-shaped crystals oriented radially from centre, whichincreased in size over time.

As the temperature increased for shortening B (shown inFig. 5), the crystal changed from a large number of irregularflocculent crystals to a large number of uniform spherulites.At −10 °C, there were a large number of irregular flocculentcrystals, which corresponded to β′ crystal, and the numberof flocculent crystals increased with time. At 0 °C, thecrystal was needle-like, and there was a small amount oflarge crystal aggregates. At 10 °C, the crystal size increased,the small crystals aggregated, and the network did notchange with time. At 20 and 30 °C, the larger spherulitesformed, while the network exhibited no significant variationover time. This aggregation process was most likely con-trolled by mass and heat transfer limitations.

Ribeiro et al. (2009) found that the network structure wasprimarily related to the main TAGs composition and crystaltransformation in the system, since the small crystalsappeared along with the SSS-type TAGs reducing. In addi-tion, Chu et al. (2002) noted that POP and PPP were βtrend-type TAGs. Therefore, when the temperature in-creased, the low melting-point TAGs (POL, POO,SOO/SSL) melt, resulting in the symmetrical TAGs (POP,POS, SOS) which still existed in the crystalline form mov-ing together to form nuclei. Subsequently, nuclei further

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grew to form β crystals. As shown in Table 2, the content ofPOP in shortening Awas higher than in shortening B. Whenthe temperature was higher (10–30 °C), these TAGs ofshortening A had enough space and energy to form βcrystals, resulting in larger crystals in shortening A

compared to those in shortening B at 10 °C, 20 °C and30 °C (Figs. 4 and 5). For the two shortening samples, thecrystal network of shortening B was denser than that ofshortening A (Figs. 4 and 5) because the melting point ofshortening Awas lower than that of B and the small crystals

Fig. 4 PLM image(×500) of shortening A stored at −10 °C, 0 °C, 10 °C, 20 °C and 30 °C after 0, 24, 120 and 168 h

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of shortening A melt at the same temperature. These resultssuggested that crystal size of shortening A was larger thanthat of shortening B at higher temperature (≥10 °C), whilethe crystal network of shortening B was denser than that ofshortening A. Our results are consistent with the conclusion

of Meng et al. (2010b). However, the crystal aggregates stilloccurred and would have a substantial impact on macro-scopic properties of the shortenings. Fats containing crystalsaggregated with compacted small crystals are desired be-cause these crystals could surround and stabilize air bubbles

Fig. 5 PLM image(×500)of shortening B stored at -10 °C, 0 °C, 10 °C, 20 °C and 30 °C after 0, 24, 120 and 168 h

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to yield a fine smooth product texture (Meng et al. 2010a). Ifthe crystal aggregates are too large, the quality of the prod-ucts would be damaged. All of the crystal aggregates inFigs. 4 and 5 appeared to be larger crystals except for thecrystals in shortening A at −10 °C. If the crystal network wastoo dense or loose, the macroscopic properties and processingcharacteristics of the shortening will deteriorate. Hence, forfurther study the influence of the temperature on the stabili-zation of the shortenings, it is necessary to conduct in-depthstudy of the crystalline network. Golding and Pelan (2008)noted that the crystalline network formed first followed by theformation of TAGs crystals. In addition, these crystalsintertwined to form spherulites, which aggregated to form abeam crystal that was interconnected in the liquid crystal oil toform three-dimensional network structure.

For a better understanding of the changes of fat crystalnetwork structure as a function of the various temperaturesduring storage, the fractal dimensions (Db) of fat crystalnetwork were calculated to quantify the spatial distributionof the crystals in the network. Box counting method wasused to analyze for each image in Figs. 4 and 5, the resultsare shown in Table 4.

The higherDb values indicate a fuller network. The micro-structural factors, such as the crystal shape, size and crystalsize region, have a significant influence on Db (Chen et al.2007). In general, higher fractal dimensions were observed innetworks that were more ordered, whereas networks thatarose from a more disordered nucleation and growth processresulted in lower fractal dimensions (Lee et al. 2008). Table 4shows that the temperature increase results in theDb values ofsample A first decreasing, then increasing and decreasing last.Db reached a maximum at 10 °C and a minimum at 30 °C,which suggested that the re-crystallization at different tem-peratures changed the spatial distribution of crystals. Thecrystal network was more ordered and fuller at 10 °C, whichis consistent with the change in the polymorphic forms. Theminimum value appeared at 30 °C, and the network appearedloose comparing to that observed for networks at other tem-peratures. These results may be due to the larger crystal size

and shape. Dietler et al. (1986) and Marangoni (2002) notedthat the larger particles or needle-like particles effectivelyincreased the Db value for networks. Tang and Marangoni(2006) found that the Db value increased with the crystal sizeand crystal area when the system had a low SFC value. Inaddition, the Db value often increased with the crystal arearather than the crystal size when the system had a high SFCvalue. When the temperature was 30 °C, the SFC value waslow, the crystal size and area was large. Therefore, the Db

value was low. At 10 °C, the SFC value was relatively high,which was higher than that at −10 °C or 0 °C and lower thanthat at 20 °C or 30 °C. The lower Db values corresponded tolarger microstructures at lower SFC, whereas samples with alarger number of smaller clusters at higher SFC were charac-terized by higher Db (Awad et al. 2004). Hence, the Db valuereached a maximum at 10 °C and a minimum at 30 °C. Forsample B, the Db value reached a maximum at 30 °C, asshown in Table 4. The differences in Db were not obviousfor the different temperatures. Db is also sensitive to thedegree of fill within a network; therefore, higher Db valuesindicate an increase in space-filling mass. The results inTable 4 suggested more filled network with higher Db. Thevalue of Db for shortening B was greater than that of short-ening A, indicating that shortening B was more populatedthan shortening A at the same temperature. These results wereconsistent with the PLM images of the two shortening sam-ples (Figs. 4 and 5). At 30 °C, the difference inDb for the twosamples was most significant (P<0.05) because the crystalsize of shortening Awas very larger and the crystal network ofshortening B was fully filled at this temperature. This resultindicated that when the crystal size became large enough, thelarge crystal size and the filling degree of crystal network hadthe same effect on Db. For the samples stored at differenttemperatures, the higher Db with more space-filling mass ofshortening B appeared at 30 °C. However, the main crystal ofthe shortening A at 30 °C was β form. Therefore, a higher Db

does not mean a fine and smooth product texture because thetexture of products is affected by the crystal form and the fatcrystal network.

Table 4 Fractal dimension (Db) of shortening A and B at different temperature after stored for a week

shortening Time, T −10 °C 0 °C 10 °C 20 °C 30 °C

A 0 h 1.9195±0.0040Aa 1.9195±0.0040Aa 1.9195±0.0040Aa 1.9195±0.0040Aa 1.9195±0.0040Aa

24 h 1.9307±0.0130Ab 1.9104±0.0198Aa 1.9581±0.0069Bb 1.9157±0.0139Aa 1.8805±0.002 Cb

120 h 1.9247±0.0183Ab 1.9187±0.0103Aa 1.9591±0.0077Bb 1.9134±0.0018Aa 1.8108±0.0756Cc

168 h 1.9174±0.0024Aa 1.9156±0.0182Aa 1.9574±0.0034Bb 1.8748±0.0262Cb 1.7723±0.0855Dd

B 0 h 1.9121±0.0038Aa 1.9121±0.0038Aa 1.9121±0.0038Aa 1.9121±0.0038Aa 1.9121±0.0038Aa

24 h 1.9142±0.0081Aa 1.9097±0.0469Aa 1.8802±0.0135Bb 1.8899±0.0103Bb 1.8822±0.0193Bb

120 h 1.9012±0.0058Ac 1.8437±0.0047Bb 1.9163±0.0297Aa 1.9077±0.0070Aa 1.9279±0.0044Ce

168 h 1.9091±0.0050Ac 1.9037±0.0183Aa 1.9233±0.0094Ba 1.8895±0.0093Cb 1.9226±0.0684Be

Different superscript capital letters in the same row and lowercase letters in the same column denote significantly different groups (P<0.05)

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Conclusion

In conclusion, the transformation into the most stable β poly-morph took place as the driving force provided by an increase inthe temperature. At the same temperature,the intensity ofβcrystal in the two sampleswas gradually increased asthe storagetime increased, andthis trend was most apparentat high temper-atures (≥10 °C).As the temperature increased, the intensity ofβcrystal in the two sampleswas gradually increased, and thecrystallineparticlessize became larger.In addition,the crystalsof shortening A changed from needle-like crystals with smallsize to lager β-type spherulites, when the crystals of shorteningB changed from a large number of irregular flocculent crystals toa large number of uniform spherulites. These large crystalsexceeded the sensory threshold and impaired the consistencyand plasticity of fat products. For the lower-melting shorteningA, the increased temperature loosened the crystal network,which could not stabilize air bubbles and compromised thesensory and functional properties of the finished products.

Acknowledgments This work is supported by the State Key Pro-gram of National Natural Science of China (No. 31130042 and No.20976061), the National Key Technology R&D Program (No.2012BAD37B01), NCET-10-0395 and the Fundamental ResearchFunds for the Central Universities, SCUT (No. 2011ZZ0084).

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