Beeswax and carnauba wax modulate the crystallization behavior of palm kernel stearin

LWT - Food Science and Technology 115 (2019) 108446

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Beeswax and carnauba wax modulate the crystallization behavior of palm kernel stearin

T

Chunhuan Liu, Zhaojun Zheng, Zong Meng, Xiuhang Chai, Chen Cao, Yuanfa Liu* State Key Laboratory of Food Science and Technology, School of Food Science and Technology, National Engineering Research Center for Functional Food, National Engineering Laboratory for Cereal Fermentation Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People's Republic of China

ARTICLE INFO

ABSTRACT

Keywords: Beeswax Carnauba wax Palm kernel stearin Crystallization behavior X-ray diffraction

There is limited literature on the modulation of fat crystallization by natural waxes, which are underutilized and cost-effective food industry resources. In this study, the effects of two natural waxes (beeswax (BW) and carnauba wax (CW)) on the crystallization behavior of palm kernel stearin (PKS85) were systematically investigated by using differential scanning calorimetry (DSC), pulsed nuclear magnetic resonance (p-NMR), X-ray diffraction (XRD) and polarized light microscope (PLM). CW addition significantly promoted the isothermal crystallization process of PKS85, especially at 20 °C, while BW had no obvious effect on the crystallization rate. BW and CW addition changed the crystal growth mode at 20 °C. BW and CW could participate in the crystallization of TAGs in PKS85, induce the formation of a new hydrocarbon chain distances of 3.70 and 4.15 Å and decrease the lamellar distance (d(001)) and domain size (ξ) of the single crystallites, leading to a different morphology of fat crystals. Our findings indicated that BW and CW could modulate the fat crystal structure and possibly engineer the functional properties of fats and fat-structured materials, which also provided new application of natural waxes in the food industry.

1. Introduction Fat-containing food materials, such as chocolate, margarine and whipped cream, are an important component of our diet (Marangoni et al., 2012). The role that fat plays in structural properties (e.g., hardness and spreadability), eating properties (e.g., mouthfeel or meltability), physical stability (e.g., phase separation), and visual appearance of food products is due largely to its crystalline properties (Jahurul et al., 2014, 2019). These attributes depend on the microstructure and crystallization behavior of the fat crystal structure. For example, small and homogeneous crystals are expected in margarine and butter to achieve a smooth texture (Garbolino, Bartoccini, & Flöter, 2010). To obtain desired functionality, the use of additives is a common and practical process for fat modification by modulate the crystallization behavior and nano- and meso-scale structure of fats (Buscato et al., 2018; Martini, Carelli, & Lee, 2008). Hydrophobic emulsifiers are commonly used as additives to tune fat crystallization (Bayard, Leal-Calderon, & Cansell, 2017). Adding emulsifiers can the nucleation or growth step by either promoting or retarding fat crystallization depending on the concentration and the composition of the emulsifiers (Fontenele Domingues, Badan Ribeiro, *

Chiu, & Gonçalves, 2015). For example, monoglycerides from hydrogenated palm oil can be seeded and crystallized prior to TAGs to accelerate the nucleation process of palm oil. (Fredrick, Foubert, Sype, & Dewettinck, 2008). Phospholipids and diacylglycerols (DAGs) were shown to delay the onset time crystallization and slowed down AMF isothermal crystallization (Vanhoutte, Dewettinck, Foubert, Vanlerberghe, & Huyghebaert, 2002; Wright & Marangoni, 2002). However, most of them are industrial synthetic rather than natural. Natural waxes (e.g. carnauba wax and beeswax) are the lipids consisting of esters of long chain fatty acids and long chain alcohols (Moghtadaei, Soltanizadeh, & Goli, 2018). Martini et al. (2008) reported that waxes have the potential to modify the crystallization behavior of fats (e.g. anhydrous milk fat). Thereby, addition of waxes maybe a facile, low-cost, safe process of fat modification in food industry. Beeswax (BW) is an animal based wax which is has been approved and certified by FDA (FDA under regulation 21 CFR 184.1973) and recognized as E 901 (Emin & Mustafa, 2015). BW is derived from the genus Apis mellifera L of bees with the world output of approximately 60,000 metric tons (Fratini, Cilia, Turchi, & Felicioli, 2016). Currently, BW has found an increasingly wide utilization in food industry including stabilizer, glazing agent, texturizer in chewing gum,

Corresponding author. E-mail address: [email protected] (Y. Liu).

https://doi.org/10.1016/j.lwt.2019.108446 Received 10 April 2019; Received in revised form 15 July 2019; Accepted 27 July 2019 Available online 28 July 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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and carrier of food additives (Emin & Mustafa, 2015) and organogelator for oil structuring (Moghtadaei et al., 2018). Carnauba wax (CW) is a vegetable wax and obtained from the Copernicia prunifera tree found exclusively in Brazil, with the highest melting point of all vegetable waxes. CW is composed mainly of long chain aliphatic/aromatic esters (80%), with the remaining 20% comprised of fatty acids, fatty alcohols and hydrocarbons, which was granted the generally recognized as safe (GRAS) status and used widely in the food (e.g. coating fruits and vegetables) (Yi, Kim, Su, & Lee, 2017). Palm kernel stearin (PKS), a lauric acid-rich fat, is a stearin fraction obtained from palm kernel oil, which applied widely in food industry especially fat materials, such as margarine, cocoa butter substitutes, ice cream, and non-dairy whipping creams (Chai et al., 2018a,b). As a health ingredient, PKS consisting copious medium-chain triglycerides, possesses the characteristic of rapid and thorough hydrolysis. However, compared with hydrogenated oil, PKS has a certain gap in crystallization properties, which limited application in margarine and shortenings. To our knowledge, few study has been done to explore the influence of waxes on fat crystal structure. Therefore, this work attempts to collect more useful information on the effects of BW and CW on the crystal growth and nano- and meso-scale structure of fats, further favoring their popularization and utilization in the food industry.

For the crystallization kinetics, pNMR was used to measure the variation of SFC values as a function of time at different crystallization temperatures (4 and 20 °C). The completely melted samples were placed directly in a thermostatic waterbath at a preset crystallization temperature. SFC measurements were performed at appropriate time intervals. The data were fitted to the Avrami model by nonlinear regression (Smith, Cain, & Talbot, 2004).

SFCt =1 SFC

e

Kt n

(1)

Where SFCt describes the SFC at crystallization time t, SFC∞ is the limiting SFC when the time approaches infinity. K is the Avrami rate constant, depending primarily on crystallization temperature, and n is the Avrami exponent, a constant relating to the dimensionality of the transformation. Eq (1) can be rewritten as:

ln [ ln(1

Y )] = lnK + nlnt

(2)

Which allows the determination of the constants n and K from a plot of lnln (1/(1-Y)) vs ln(t). If the transformation follows the Avrami equation, this yields a straight line with gradient n and intercept lnK. K is directly related to the half-time of crystallization, t1/2, reflecting the magnitudes of the rate constants according to the following formula (Chai et al., 2018a,b):

2. Materials and methods

t1/2 =

2.1. Sample preparation

n

0.693 K

(3)

The SFC of the sample after isothermal crystallization at 4 °C and 20 °C for 24 h was determined using the following thermal treatment: The completely melted samples placed in pNMR tubes were kept at predetermined temperature for 24 h.

Palm kernel stearin (PKS85) was donated by Kerry Specialty Fats Co., Ltd. (Shanghai, China). Its fatty acid and triglyceride compositions were shown in Supplementary Table S1, measured following our group method (Liu et al., 2018). Beeswax (acid value: 5.55 mg KOH/g; iodine value: 98.2 gI2/100 g, melting point: 64.3 °C), Carnauba wax (acid value: 6.53 mg KOH/g; iodine value: 60.3 gI2/100 g, melting point: 82.5 °C) were provided by Likangweiye Technology Co., Ltd. (Beijing, China). BW and CW additives (2%, 4%, 6% and 8%, w/w) were mixed into completely melted PKS85, respectively. The mixtures were heated to 90 °C to erasure all crystal memory and stirred to obtain homogenous distribution. Then the samples were stored at 4 and 20 °C, which are commonly used in the industrial scale.

2.4. Polymorphism and crystalline domain size Fat polymorphism were analyzed by an X-ray diffractometer (XRD) (Bruker D2, Karlsruhe, Germany), The Cu-Ka radiation (k = 1.54056 Å) was set to 40 kV and 40 mA with the divergence slit, scatter slit, and receiving slit of 10, 1.0 and 0.1 mm, respectively. For the small-angle Xray diffraction analysis (SAXD), the samples were monitored at 2θ scale of 1-10° with a rate of 1°/min and a step size of 0.02, respectively. For the wide-angle X-ray diffraction analysis (WAXD), the samples were scanned from 10 to 35° (2θ scale) with a rate of 2.5°/min. The samples were heated to 90 °C for 30 min, after which they were kept at 4 and 20 °C for 24 h. The analyses were performed at ambient temperature. Data analysis were conducted using MDI Jade 6.0 software (Materials Data Inc., Livermore, USA). SAXD analysis was applied to evaluate the crystalline domain size (ξ) by the well-known Scherrer formula which is limited to nanoscale particles and it is not applicable to sizes larger than about 100 nm (Acevedo & Marangoni, 2010; Voda, den Adel, van Malssen, & van Duynhoven, 2017):

2.2. Differential scanning calorimetry Thermal properties of fats were analyzed using differential scanning calorimeter (DSC) (PerkinElmer DSC 8500, Massachusetts, USA). Approximately 5–8 mg melted samples were sealed in an aluminum pan, and an empty pan was used as a reference. Firstly, the sample was equilibrated at 90 °C for 5 min and then cooled at 5 °C/min to −20 °C to analyze crystal behavior. Secondly, the sample was held at −20 °C for 5 min, followed by heating to 90 °C at 5 °C/min to measure the melting behavior. Isothermal crystallization of fats was measured following by an indirect DSC method. Firstly, the sample was held at 90 °C for 5 min to erasure all crystal memory and then cooled at 30 °C/min to 4 °C or 20 °C, held at 4 °C or 20 °C for 10–20 min. All DSC tests were carried out in triplicate.

=

k FWHM cos

(4)

Where k is the shape factor with a value of 0.9 for crystallites of unknown shape and is the magnitude employed in this study. θ is the diffraction angle, FWHM is the full width at half of the maximum peak height in radians corresponding to the first small angle reflection reflecting the (001) plane, λ is the wavelength of the X-ray with the value of 1.54 Å for copper (Acevedo & Marangoni, 2010).

2.3. Solid fat content and crystallization kinetics Solid fat content (SFC) was measured by pulse-nuclear magnetic resonance (pNMR) (Niumag Electronic Technology Co., Ltd, Shang Hai, China). The instrument was firstly automatically calibrated using three standards (supplied by Niumag) containing 0, 30.0, and 70.0% solid. Then the melted fat samples (approximately 2.5 ± 0.2 g) were put into pNMR tubes, kept at 100 °C for 15 min to eliminate crystal memory.

2.5. Crystal morphology Fat crystals were visualized between crossed polarizers in a microscope (PLM) (DM2700P, Leica, Germany) installed with a video camera (DFC450, Leica Germany) according to previous method (Jahurul et al., 2

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2014). The sample was kept at 90 °C for 10 min and one drop (15 μL) molten sample was applied on a preheated carrier glass and covered with a preheated coverslip. Subsequently, sample slides were kept at 4 and 20 °C for 24 h before measured. The acquired images were then inverted, threshold, and analyzed by using ImageJ 1.36b software (USA) according to the method described by Meng et al. (2010).

different thermograms compared with pure PKS85. A super-highmelting peak which was clearly higher than that of pure PKS85, was appeared and shifted slightly to higher temperature with the increase of BW or CW addition. PKS85-BW and PKS85-CW were identified two-step crystallization. The first crystallizations started around 35–47 °C for PKS85-BW and 52–60 °C for PKS85-CW, respectively, and the subsequent crystallizations occurred at around 22 °C mainly due to PKS85. The temperature and shape of super-high-melting peaks were related to corresponding wax, as shown in Supplementary Fig. S1. There was no change in the PKS85 crystallization peak temperature location as the function of BW concentration. Interestingly, the major peak (peak1) of PKS85 gradually shifted toward higher temperatures with CW addition, suggesting some aliphatic chains of CW could form the initial crystals in combination with low-melting point TAGs of PKS85 (Cerdeira, Martini, Hartel, & Herrera, 2003; Chai, Meng, Jiang, Cao, Liang, Piatko, et al., 2018; Jana & Martini, 2016). Heated from −20 to 80 °C, pure PKS85 was observed two endothermic peaks at 31.5 and 40.5 °C. A new trend raised after adding BW or CW — the endothermic peak with the higher temperature (around 45 °C for BW and around 71 °C for CW) appeared, which probably caused by the melting of corresponding wax crystals. Further, the variations in the shape of the melting profile could be indicative of changes in polymorphism (Ishibashi, Hondoh, & Ueno, 2017). BW addition did not affect the endothermic peak temperature location of PKS85, just presented a new endothermic peak at higher temperature. However, the onset temperature of the low-melting peak of PKS85 (around 20 °C) shifted toward higher temperatures (around 25 °C) with the CW addition (denoted by the dotted lines). This is mainly caused by

2.6. Statistical analysis All the tests were employed at least in triplicate. The results of the analyses were reported as means ± standard deviations. Results of crystallization kinetic parameters were analyzed by oneway ANOVA (p < 0.05) with Dunnett's multiple comparisons test in statistical analysis system software of SPSS. 3. Results and discussion 3.1. Thermal properties Thermal profiles demonstrated phase transitions, melting and crystallization, proving details of complex interaction between lipid composition (P. D. Oliveira, Rodrigues, Bezerra, & Silva, 2017). DSC cooling and heating thermograms of PKS85, PKS85-BW and PKS85-CW are presented in Fig. 1. PKS85 exhibited two major exothermic peaks at 13.13 (peak 1) and 22.34 °C (peak 2) during cooling to −20 °C, representing the low-melting fraction and the high-melting fraction, respectively. Moreover, PKS85-BW and PKS85-CW were observed

Fig. 1. DSC crystallization (A) and melting (B) curves of PKS85, PKS85-BW and PKS85-CW obtained at 5 °C/min rate. 3

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Fig. 2. Solid fat content versus time profile during isothermal crystallization of PKS85, PKS85-BW and PKS85-CW at different crystallization temperatures of 4 (A) and 20 °C (B). Isothermal crystallization curves of PKS85, PKS85-BW and PKS85-CW at 4 (C) and 20 °C (D) by plotting the heat flow as function of the hold time obtained by DSC.

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the interactions between alkyl chain from CW and TAGs from PKS85. These results suggested that the addition of BW and CW maybe lead to the new polymorphic formation as described later.

the Avrami model, SFC data of crystallization kinetic were fitted to the Avrami equitation (R2 > 0.99 in all cases) as shown in Table 1. The slight differences of Avrami constant (K) were observed in PKS85-BW crystallized at 4 °C, suggesting that BW addition slightly affect the crystallization rate of PKS85 at 4 °C. The K values of PKS85-CW at 4 °C were slightly increased. However, the value of Avrami constant (K) increased significantly and the value of crystallization half-time (t1/2) also decreased after adding CW crystallized at 20 °C (p < 0.05), suggesting CW could promote the fat crystallization. Concurrently, the value of Avrami constant (K) decreased significantly (p < 0.05) as the isothermal crystallization temperature increased from 4 to 20 °C in all samples. The K values obtained at 4 °C were significantly lower than the value at 20 °C, indicating the faster crystallization was existed at a higher degree of supercooling. The decreasing K values was a combined affection by the nucleation and/or growth rate of crystallization, suggesting the changes of crystal morphology, such as crystal type and size, spatial arrangement of crystals. The value of crystallization half-time (t1/2) also decreased with the decrease of crystallization temperature. These results also suggested that higher crystallization temperature could slow the crystallization rate, which was in agreement with the previous research of isothermal crystallization (Chai et al., 2018a,b; Meng et al., 2010). The Avrami exponent n reflects crystal growth geometry which indicated the information of crystal nucleation and growth mechanisms (Chai et al., 2018a,b). As shown in Table 1, the Avrami exponent n values of PKS85-BW and PKS85-CW were slightly lower than pure PKS85 crystallized at 4 °C, whereas at 20 °C, the n values of PKS85-BW and PKS85-CW obvious decreased, especially PKS85-CW. Besides, the n values for all samples decreased with decreasing temperature. The actual situation is complicated, although n should be an integer, fractional values are obtained in some analyses, even in cases where the Avrami equation is very accurately obeyed (Wright, Hartel, Narine, & Marangoni, 2000). For example, fractional values for n were consistently obtained from all samples crystallization curves, despite correlation coefficients of at least 0.99. The Avrami exponent does provide a phenomenological index of crystallization (Wright et al., 2000). Accordingly, the similarities in n between PKS85, PKS85-BW and PKS85CW at 4 °C pointed to the fact that BW and CW addition did not significantly change the growth mode. And the difference in the exponent n between PKS85, PKS85-CW and PKS85-BW at 20 °C suggested a change in crystal growth mechanism after adding BW and CW. Fig. 2C and D shows the heat flow versus time profile (another isothermal crystallization description obtained from DSC) for PKS85, PKS85-BW and PKS85-CW at isothermal crystallization temperature of 4 and 20 °C after fast cooling (30 °C/min). For all samples, two-step crystallization was observed. The two-step crystallization was related to the crystallization of different fractions, polymorphic transition with or without additional crystallization or a combination (Foubert, Dewettinck, Janssen, & Vanrolleghem, 2006). The heat flow can already be detected from the instant the temperature reaches pre-set temperature, which suggested that all samples started to crystallize during the preceded cooling. Effect of isothermal crystallization temperature on the crystallization mechanism and kinetics can be clearly observed, especially for the second crystallization step. As opposed to the samples crystallized at 4 °C, the heat flow returned to the baseline and some seconds later a clear crystallization peak representing the second crystallization step was visible. Meanwhile, the end time of the exothermic peak of all samples at 4 °C was similar at around 2.40 min (denoted by the dotted lines). BW addition did not significantly affect the location of exothermic peak at 20 °C. Interestingly, the exothermic peaks were clearly shifted toward lower time with CW addition at 20 °C, suggesting CW could promote PKS85 crystallization. This is possibly

3.2. Crystallization kinetics The SFC and crystallization kinetics are the key to describe the crystallization behavior of the fat (And & Mcgauley, 2002). As shown in Supplementary Fig. S2, the SFC values were around 89% and 70% after isothermal crystallization at 4 and 20 °C for 24 h, respectively. The SFC reduced by about 19% when the isothermal crystallization temperature increased from 4 to 20 °C. SFC values slightly increased after adding CW and showed no difference after adding BW. These results indicated that the isothermal crystallization temperature could change the equilibrium SFC of fat, adding CW could slightly increase the SFC, but adding BW could not affect. To further study the influence of BW and CW addition on dynamic process in fat crystallization, the isothermal crystallization kinetics of PKS85, PKS85-BW and PKS85-CW at 4 and 20 °C was studied (Fig. 2). As shown in Fig. 2A and B, all samples crystallized very rapidly at crystallization temperature (4 and 20 °C) with a clearly visible equilibrium value in SFC, showing hyperbolic patterns against time in all curves. Furthermore, comparison of the curves of two crystallization temperature (4 and 20 °C) clearly showed that the former has a steeper slope, which suggested that the fats at high super-cooling (4 °C) have faster crystallization rate and shorter times to achieve equilibrium SFC. Interestingly, PKS85-CW isothermal crystallization curves (the right of Fig. 2B) clearly showed a steeper slope than pure PKS85 at 20 °C, indicating that CW addition significantly promoted the crystallization process of PKS85. That is, PKS85-CW had a faster crystallization and a shorter time to get equilibrium SFC. BW addition showed no obvious effect on the crystallization as discussed later. The Avrami model is a useful method to characterize crystallization kinetics and demonstrate the nature of the crystallization process, especially nucleation and growth (And & Mcgauley, 2002; Singh, Bertoli, Rousset, & Marangoni, 2004). In order to acquire parameters of Table 1 Avrami exponents (n), Avrami constants (K), and half-times of crystallization (t1/2) for PKS85, PKS85-BW and PKS85-CW at different crystallization temperatures of 4 and 20 °C. Sample 4 °C PKS85 2 wt% BW 4 wt% BW 6 wt% BW 8 wt% BW 2 wt% CW 4 wt% CW 6 wt% CW 8 wt% CW 20 °C PKS85 2 wt% BW 4 wt% BW 6 wt% BW 8 wt% BW 2 wt% CW 4 wt% CW 6 wt% CW 8 wt% CW

K (min-n)

n

R2

t1/2 (min)

2.220 2.532 2.373 2.612 2.580 2.234 2.413 2.326 2.045

± ± ± ± ± ± ± ± ±

0.071bc 0.096 ab 0.082bc 0.097a 0.104a 0.091bc 0.095abc 0.100cd 0.077d

0.224 0.214 0.236 0.225 0.242 0.283 0.343 0.374 0.419

± ± ± ± ± ± ± ± ±

0.012a 0.013b 0.013b 0.013c 0.015c 0.018d 0.017d 0.019d 0.018e

1.663 1.591 1.575 1.538 1.503 1.493 1.338 1.304 1.279

± ± ± ± ± ± ± ± ±

0.049a 0.038 ab 0.117 ab 0.004bc 0.012bc 0.060c 0.012d 0.048d 0.021d

0.998 0.997 0.998 0.997 0.997 0.996 0.997 0.996 0.997

2.992 2.579 2.532 2.405 2.412 1.818 1.797 1.813 1.546

± ± ± ± ± ± ± ± ±

0.028a 0.124b 0.149b 0.137b 0.133b 0.116c 0.100c 0.111c 0.078c

0.011 0.039 0.031 0.032 0.031 0.137 0.173 0.154 0.258

± ± ± ± ± ± ± ± ±

0.002d 0.007d 0.006d 0.006d 0.006d 0.019c 0.020b 0.019bc 0.021a

4.024 3.535 3.411 3.592 3.626 2.439 2.334 2.292 1.895

± ± ± ± ± ± ± ± ±

0.104a 0.058b 0.110b 0.019b 0.124b 0.133c 0.025c 0.058d 0.256d

0.990 0.990 0.990 0.991 0.992 0.991 0.990 0.990 0.991

Different lowercase letters in a column indicate that the means are significantly different at P < 0.05.

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Fig. 3. XRD spectra for short spacings (A) of PKS85-BW at 4 (A-1) and 20 °C (A-2) and PKS85-CW at 4 (A-3) and 20 °C (A-4), and the schematic illustration of subcell packing (B) of PKS85-BW and PKS85-CW. XRD spectra for long spacings (C) of PKS85-BW at 4 (C-1) and 20 °C (C-2) and PKS85-CW at 4 (C-3) and 20 °C (C-4) and schematic illustration lamellar structures (D) of PKS85-BW and PKS85-CW. Inset: magnification of long spacings.

caused by the co-crystallization between PKS85 and CW compositions. These results were consistent with the trend of the results of the Avrami constant (K) and crystallization half-time (t1/2).

shown in Fig. S3). Adding waxes induced a new hydrocarbon chain distance of 3.70 and 4.15 Å as illustrated in Fig. 3B. Small angle diffraction (SAXD) data provides an information related to lamellar crystalline structures, with d-spacings appearing at 1:1/2:1/ 3 corresponding to the main reflection and higher order reflections from the (001) crystallographic plane (Wang et al., 2018). As shown in Fig. 3C, SAXD peaks of PKS85 stored at 4 and 20 °C were observed at 36.1 and 37.1 Å (d(001)), respectively, indicating a double chain length (2 L) stacking of TAGs (Chai et al., 2018a,b). After adding BW and CW, the SAXS peak at 36.1 and 37.1 Å gradually shifted to a lower value of 34.7 Å, indicating a closer lamellar distance (d(001)) in PKS85-BW and PKS85-CW. Due to the complexity of the composition in BW and CW, there is currently no clear compositional analysis (molecular level). The crystallization zone is concerned rather than amorphous zone of waxes for this research. Therefore, based on the previous studies of BW (Basson & Reynhardt, 2000a; Fratini et al., 2016) and CW (Basson & Reynhardt, 2000b; Bayer et al., 2011), the illustration of crystallization zone was established (as shown in Supplementary Fig. S4). Adding BW and CW could influence the lamellar packing during the isothermal

3.3. Crystal polymorphism and crystalline domain size Crystal polymorphism and crystalline domain size are essential foundation for our understanding the nature of fat crystallization (Chai et al., 2018a,b). In Fig. 3, the short spacing patterns (2θ ≥ 15°) of PKS85 stored at 4 and 20 °C showed similar WAXD peaks at 3.80, 4.05, 4.20 and 4.40 Å, characteristic of the β′ polymorphism (D'Souza, Deman, & Deman, 1990), which is an orthorhombic perpendicular (O⊥) subcell structure (G. M. D. Oliveira, Ribeiro, Santos, Cardoso, & Kieckbusch, 2015). The lateral hydrocarbon chain distances of the PKS85 β′ form were primarily 3.80 and 4.20 Å accompanied by 4.05 and 4.40 Å, respectively (Fig. 3A). With the addition of waxes, the WAXD additional peaks at 3.70 and 4.15 Å were displayed, which corresponded to the β′ form of wax crystals (Wang, Miyazaki, & Marangoni, 2018). This is related to the polymorphism of pure wax (as

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sensitive to fat globules partial aggregation for aerated structure food such as ice cream and whipping topping (Boode & Walstra, 1993). Compared with PKS85, the crystals of PKS85-CW had various differences in morphology. Large feather-like layer crystals were clearly observed for PKS85-CW. The number and size of large crystals in the view increased with the increase of CW addition and the size of the large crystals decreased when the temperature increased from 4 to 20 °C. These results suggested that BW and CW could participate in the seeding of the fat crystals, affect the growth of the fat crystal clusters, leading to the different crystal microstructures. The fat crystal structures modulated by BW and CW exhibited the variants of morphology due to the different compositions of BW and CW. Isothermal crystallization temperature also affected the fat crystallization network processing by modifying the process of nucleation and growth of the crystals. These variations in microstructure related to the regulation obtained in Avrami analysis. The fractal dimension (Df) might be useful as a quantitative, which may evaluate the spatial distribution of the crystals in the crystal network (Chai et al., 2018a,b). As shown in Fig. 5, no significant differences observed in the Df between PKS85 crystallized at 4 °C (Df = 1.964 ± 0.024) and PKS85-BW (Df = 1.952 ± 0.031–1.967 ± 0.018) crystallized at the 4 and 20 °C. BW addition can change the appearance of PKS85 crystallized at 20 °C to at 4 °C. However, the Df values of PKS85-CW ranging from 1.909 ± 0.015 to 1.957 ± 0.021 were significantly lower than PKS85 at 4 °C, especially with 6 and 8 wt% CW addition, which indicated less ordered structures were formed. Microscopy of PKS85-CW indicated a larger crystal aggregated to form the larger clusters at both 4 and 20 °C. These also suggested that BW and CW addition could affect the cluster-cluster interactions of fat crystal structure. Additionally, the Df values of pure PKS85 decreased significantly when the temperature increased from 4 to 20 °C, suggesting pure PKS85 nucleation proceeded very rapidly and resulted in a fine pattern of crystal structures at a high degree of supercooling at 4 °C. But regulations of PKS85-BW and PKS85-CW is different: the values of Df observed at 4 °C were lower than those at 20 °C. Upon cooling, the long straight carbon chain molecules of BW and CW firstly formed the stable molecular aggregation stacking in pairs side by side as crystal, and then TAG molecules aggregation stacking. Interestingly, aliphatic chains in CW were combined with the lowmelting point TAGs (as shown in the results of DSC, Fig. 1). Furthermore, BW and CW could participate in the crystal matrix which in turn resulted in the decrease of lamellar distance (d(001)) and domain size (ξ) of the single crystallites and alter the morphology of crystals by joining the growth sites. Subsequently, the single crystallites further aggregated, grew, and formed crystal structure, forming various structure of fats (Chai et al., 2018a,b).

Fig. 4. Domain size (ξ) calculated from XRD data of PKS85, PKS85-BW and PKS85-CW at 4 (A) and 20 °C (B).

crystallization, possibly related to linear fatty acid carbon chain length (Ishibashi et al., 2017) of BW and CW as illustrated in Fig. 3D. We further calculated the crystalline domain size (ξ) following the well-known Scherrer equation. As shown in Fig. 4, the tendency of domain size (ξ) was consistent with the lamellar thickness (d(001)): PKS85 had a slightly bigger domain size (ξ) than PKS85-BW and PKS85CW. That is, BW and CW could participate in the onset of crystallization of TAGs molecules, causing the changes of lateral hydrocarbon chain distances and lamellar packing structures of PKS85, which would further influence the microstructure of fats. In order to clarify this hypothesis, polarized light micrographs (PLM) was used to monitor the morphology and distribution of the clusters.

4. Conclusion The addition of BW and CW can modify the crystallization behavior of PKS85. The crystallization and melting process were significantly affected by adding BW and CW. CW addition significantly promoted the isothermal crystallization process of PKS85, showing a faster crystallization and a shorter time to get equilibrium SFC, especially at 20 °C. BW addition had no obvious effect on the crystallization rate. BW and CW addition did not significantly change the growth mode at 4 °C, but changed at 20 °C. In summary, BW and CW could participate in the crystallization of TAGs, induce the formation of a new hydrocarbon chain distances of 3.70 and 4.15 Å on the original basis and decrease of lamellar distance (d(001)) and domain size (ξ) of the single crystallites, leading to a different morphology of fat crystals. These results indicated

3.4. Microscopy and fractal dimension Same as hypothesis, BW and CW had an obvious effect on the fat crystal structure. PKS85 displayed smaller and more numerous needlelike appearance at 4 °C and spherical shape formed by layer crystal along with granular crystal at 20 °C, respectively (Supplementary Fig. S5). As shown in Fig. 5, PKS85-BW showed smaller needlelike appearance with a little layer crystal at both 4 °C and 20 °C, similar to the morphology of PKS85 crystallized at 4 °C and obviously different from the morphology of PKS85 crystallized at 20 °C. The fat crystals with BW addition tended to be finer and uniform needle crystals, which were

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Fig. 5. Polarized light micrographs of PKS85-BW (A) and PKS85-CW (B) crystallized at 4 and 20 °C for 24 h with different wax concentration (2, 4, 6 and 8 wt%). Df –fractal dimension.

that BW and CW could structure lipids and modify their functional properties, further possibly engineer the functional properties of fats and fat-structured materials for various applications in the food industry.

Abbreviations BW beeswax CW carnauba wax DSC differential scanning calorimetry p-NMR pulsed nuclear magnetic resonance XRD X-ray diffraction PLM polarizing microscope TAGs triglycerides DAGs diacylglycerols SFC solid fat content PKS palm kernel stearin PKS85 a model of palm kernel stearin PKS85-BW mixtures of PKS85 and BW PKS85-CW mixtures of PKS85 and CW n avrami exponents K avrami constants t1/2 half-times of crystallization SAXD small-angle X-ray diffraction analysis WAXD wide-angle X-ray diffraction analysis Df fractal dimension d(001) lamellar distance ξ domain size.

Note No competing financial interest was declared by the authors. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This research was supported by the Taishan industry leading talents innovation project in Shandong Province (LJNY2015007), National Key Research and Development Program of China (2016YFD0401404), the National Natural Science Foundation of China (31471678, 31772008), and also supported by General Projects of China Postdoctoral Science Foundation (2018M632235) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1813).

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Appendix A. Supplementary data

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