Characterization of enzymatically interesterified palm oil-based fats and its potential application as cocoa butter substitute

Journal Pre-proofs Characterization of enzymatically interesterified palm oil-based fats and its potential application as cocoa butter substitute Zhen Zhang, Jia Song, Wan Jun Lee, Xiaodong Xie, Yong Wang PII: DOI: Reference:

S0308-8146(20)30380-0 https://doi.org/10.1016/j.foodchem.2020.126518 FOCH 126518

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Food Chemistry

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6 September 2019 26 February 2020 27 February 2020

Please cite this article as: Zhang, Z., Song, J., Jun Lee, W., Xie, X., Wang, Y., Characterization of enzymatically interesterified palm oil-based fats and its potential application as cocoa butter substitute, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126518

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Characterization of enzymatically interesterified palm oil-based fats and its potential application as cocoa butter substitute To be revised to Food Chemistry

Zhen Zhanga, Jia Songa, Wan Jun Leea, Xiaodong Xiea, Yong Wanga* aJNU-UPM

International Joint Laboratory on Plant Oil Processing and Safety, Department of Food

Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, China. *Department of Food Science and Engineering, Jinan University, 601 Huangpu Ave West, Guangzhou 510632, China. E-mail: [email protected] (Dr. Yong Wang). Abstract Cocoa butter substitutes (CBS) used for chocolate preparation was produced using a mixture of palm kernel oil (PKO) and enzymatically interesterified fats. The interesterified fats consisted of palm olein (POL), fully hydrogenated palm oil (FHPO) and PKO that were catalyzed using Lipozyme TL IM at 65 °C in a solvent-free packed bed reactor. An interesterification degree of 97.10% was obtained using feed flow rate of 70 mL/min and the interesterified fats showed steep solid fat content (SFC) curve characteristics with low SFC at high temperature. In the binary system, PKO and the interesterified fats showed good compatibility at 5-10 °C, while eutectic effects were observed at 15-35°C. CBS produced from PKO and the interesterified fats in a mass ratio of 4:6 (CBS-46) and 3:7 (CBS-37) had crystals formed prominently in the β' form. Without the need of a tempering process, chocolate made using CBS-46 as the base oil exhibited the desired properties in terms of hardness and fracturability. Keywords ： enzymatic interesterification, cocoa butter substitutes, binary compatibility, chocolate texture 1. Introduction

Due to its unique flavor profile, cocoa butter (CB) has a wide range of applications as a conventional base material in the production of chocolates, whipping cream and confectionaries (Liu, Chang, & Liu, 2007). Moreover, the distinctive physicochemical properties provide CB with an exceptional mouthfeel and desirable processing characteristics. For instance, melting point of CB is close to the human body temperature and it has a narrow melting temperature profile whereby CB in the β crystal form (V) melts at 32-33°C while β crystal form (VI) melts at 35-36°C. Chocolate having crystals in V form is the most desirable as characteristics such as shiny, smooth texture, melt in the mouth and good snap properties are exhibited (Loisel, Keller, Lecq, Bourgaux, & Ollivon, 1998). CB also has a narrow plastic range, having high solid fat content (SFC) of more than 70% at 10°C and SFC of 0% at 37°C. This distinguishing characteristic allows the chocolate incorporated with CB to possess exclusive sensorial properties in which the chocolate exists as solid state when held in hands but melts rapidly in the mouth when consumed (Jahurul et al. 2013; Mohamed, 2012.). The application of CB from natural sources is limited by various factors such as the low CB yield as affected by climate and environment change, causing issues with supply shortages in manufacturing (Biswas, Cheow, Tan, Kanagaratnam, & Siow, 2017). Moreover, the chocolate tempering process which converts the unstable fat crystals into the stable β form, is largely affected by the CB triacylglycerol (TAG) structure. If improper tempering temperature is used, post-crystallization will occur, leading to the development of sandiness mouthfeel which affects the end product quality (Biswas, Cheow, Tan, Kanagaratnam, & Siow, 2017). Based on the aforementioned shortcomings of natural CB, cocoa butter alternatives has been introduced such as cocoa butter substitute (CBS), cocoa butter replacer (CBR) and cocoa butter equivalent (CBE). These three CB alternatives differ in terms of their (i) fatty acid profile whereby

CBS normally contains lauric acid, CBR usually contains trans fatty acid by partial hydrogenation rather than lauric acid and CBE has fatty acid and TAG profile similar to CB; (ii) compatibility with CB (CBS: not compatible, CBR: partially compatible, and CBE: compatible); and (iii) CBS and CBR do not require tempering process in the end product while products from CBE require tempering (Biswas, Cheow, Tan, & Siow, 2016; Norazura, Nur Haqim, & Noor Lida, 2018). A CBE was produced using the esterified fat mixture consisted of palm oil, stearic acid and palmitic acid which was enzymatically esterified under the catalysis of Lipozyme TL IM at 60°C in a shaker reactor (Mohamed, 2012). Because the CBE product obtained had similar TAG composition as the natural CB, product tempering was required. Therefore, CBS can be a good alternative as tempering process of end product is not needed attributed to the variation in their fatty acid and TAG compositions and the crystallization characteristics comparing to that of natural CB (Kang, Jeon, Kim, & Kim, 2013; Torbica, Pajin, Omorjan, Lončarević, & Tomić, 2014; Sabariah, Ali, & Chong, 1998). In general, CBS produced from modified fats consisting of lauric acid is considered to be the only type of lipid that can be a complete substitute for the natural CB (Biswas, Cheow, Tan, Kanagaratnam, & Siow, 2017; Rossell, 1985; Zaidul, Norulaini, & Omar, 2007). Although interesterified palm-based mixture has been used to produce chocolate fats in several studies (Soekopitojo, 2009; Jin, Akoh, Jin, & Wang, 2018), the production of interesterified fats as a composition of CBS catalyzed by immobilized lipase using a pilot-scale PBR has not been reported. Up-scaling might cause alteration in the properties of the interesterified fats and therefore it is vital to characterize and determine the properties of the interesterified products and its application. This finding will serve as the basis for new operation technology for the enzymatic interesterification of confectionary fats and also to offer a theoretical foundation for subsequent industrialization studies

with potential applications in food specialty fats. In this study, interesterified fat mixture of palm olein (POL), fully hydrogenated palm oil (FHPO) and palm kernel oil (PKO) at mass ratio of 4:3:3 was used as a base oil of CBS for chocolate preparation. The effect of feed flow rate in a solvent-free packed bed reactor on the degree of interesterification (DI), SFC characteristics and physicochemical properties of modified fats were investigated. Characteristics and compatibility of the CBS for chocolate applications that were prepared using PKO and the enzymatically interesterified fat mixtures were also evaluated.

2. Materials and methods 2.1 Materials FHPO was provided by Masson Food Technology Co., Ltd. (Guangdong, China). POL and PKO were provided by Kerry specialty fats Co., Ltd. (Shanghai, China). Lipozyme TL IM (silica granulated Thermomyces lanuginose lipase, 250 IUN/g) was provided by Novozymes A/S (Bagsvaerd, Denmark). Commercial CB was purchased from local market (Guangdong, China). 2.2 Pilot scale enzymatic interesterification Enymatic interesterification was performed using raw materials consisting of POL:FHPO:PKO in mass ratio of 4:3:3. A pilot-scale test was carried out in a 5 kg (storage buffer, 1 batch) scale PBR (48 cm × 60 mm i.d.) packed with 783.0 g Lipozyme TL IM at fixed temperature of 65 °C (see supplementary Fig. 1). Selection of the interesterification parameters was based on the results from a preliminary work. The effect of different feed flow rates (60, 70, 80, 90, 100 mL/min) on the degree of interesterification (DI%) was investigated. 2.3 Analysis of the interesterification degree The TAG compositions were analyzed by using a reversed-phase high performance liquid

chromatography (RP-HPLC) system equipped with a Dikma C18 column (250 mm × 4.6 mm i.d., 5 μm) (Santa Clara, CA, U.S.A.) and an evaporative light-scattering detector (Alltech 2000ES, USA). Sample was prepared by dissolving 5 mg of interesterified fats in 5 mL of organic solvent (acetone: trichloromethane = 1:1 v/v). Sample volume of 10 μL was injected (duplicate) and the column temperature was set at 30 °C. The mobile phase consisting of acetone and acetonitrile was delivered in gradient mode whereby the initial concentration of acetone was linearly increased from 30% to 45% over 90 min at a flow rate of 0.8 mL/min before reversing to the initial concentration of acetonitrile. The peaks of TAGs were identified using TAG standards and equivalent carbon number (ECN). POO (1-palmitoyl-2,3-dioleylglycerol) was one of the main TAGs present in the raw material since POL and FHPO were rich in oleic and palmitic acid. Comparing the TAG profiles of samples before and after reaction, the change in the amount of POO ECN (48, Fig. S1) can be used to monitor the degree of reaction. The DI is defined as:

|𝐴3 ― 𝐴2| × 100% DI% = 1 ― |𝐴2 ― 𝐴1|

(

)

where A1 is the POO content in raw material; A2 is the theoretical POO content after the enzymatic interesterification; and A3 is the actual content of POO after the enzymatic interesterification (Li, Zhao, Xie, Zhang, & Wang, 2018). 2.4 Solid fat contents and thermodynamic behaviors The p-NMR tubes were filled with approximately 3 mL of melted samples and then placed in a water bath at 60 °C for 30 min. All the tubes were then transferred into a thermostatic water bath at 0 °C for 1 h. Following that, the tubes were kept at a particular temperature (the temperature range was 0– 45 °C, increased by 5 °C each time) for 30 min prior to measurement. The crystallization and melting behaviors were analyzed using a TA Q2000 DSC instrument (TA

Instruments, New Castle, USA). Interesterified samples of 8-12 mg were weighed into an aluminium pan and an empty pan was used as a reference. Samples were rapidly heated to 80 °C and was held for 10 min. A cycle of cooling and heating was subsequently performed. The aluminium pan was cooled to −50 °C and then heated to 80 °C at a rate of 10 °C/min. 2.5 Polymorphism and microstructure XRD patterns of samples were recorded on an X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany). The standard ceramic sealed tube using Cu as the X-ray source was operated at 30 mA and 40 kV. Scans from 1° to 35° were performed by using a step size of 0.3° and at a rate of 0.03 s/step at 25 °C. The microstructure was observed under polarized light using a DM2700P Leica microscope (Leica Microsystems, Wetzlar, Germany), which was equipped with a Charge-coupled Device camera. A drop of melted sample was added onto a microscopy slide, which was then covered with a glass slip. All the slides were chilled at -18 °C for 2 h, and then placed at 25 °C for 24 h. The slide was visualized under 200× magnification, and typical field images were acquired. 2.6 Analysis of texture A TA-XT Plus (Stable Microsystems, Surrey, UK) was used to test the hardness of each sample. Samples were melted and added into 30 mL flat-bottomed aluminium container. The containers were chilled at -18 °C for 2 h and conditioned at 25 °C for 24 h. A flat HDP/3PB probe was used for puncture experiments with the test speed of 1.0 mm/s, while pre-test speed and post-test speed were all set as 3.0 mm/s, and the penetration depth was 5 mm. The data were analyzed using the Texture exponent v.6.1.1.0 software. The peak force (g) is defined as hardness, and the depth (mm) of reactive force loss is defined as fracturability.

2.7 Compatibility test The compatibility of dualistic mixtures of interesterified fats with PKO was studied by calculating the ∆SFC. ∆SFC = practical SFC (P-SFC)  theoretical SFC (T-SFC), while T-SFC = SFCx X + SFCy Y which was calculated according to the SFC value of single oil. The X and Y were the percentages of total mass of x-oil and y-oil in the mixture, respectively. Oil blends with ∆SFC value close to zero has a high compatibility. When the ∆SFC value is above zero, blends are monotectic, otherwise eutectic (Zhang, Ma, Huang, & Wang, 2017; Jin, Zhang, Shan, Liu, & Wang, 2008). The suitable ratios of PKO and the interesterified fats were chosen for the fat formula of CBS according to the compatibility test. 2.8 Chocolate application The basic recipe of chocolate consisting of 46% sugar powder, 34% CBS, and 20% cocoa powder. All materials with CBS matrix were poured into a pastry blender and were blended at low speed until a homogenous mix was obtained. The mix was refined at 65 °C for 8 hours in a chocolate refiner. Two sets of chocolate were prepared; tempered and untampered. Tempering was done by heating the chocolate to 60 °C and was held for 30 min before reducing the temperature to 34 °C and finally to 38 °C. The liquid chocolate was then cooled at 10 °C in a mold for 30 min. Solid chocolates were then unmolded manually and were then subjected to different analyses. 2.9 Statistical analysis One-way ANOVA was performed using SPSS 16 statistical software (SPSS Inc., Chicago, IL). Differences were considered to be significant at p ≤ 0.05 according to Duncan’s Multiple Range Test.

3. Results and discussion 3.1 Effect of feed flow rate on DI

The fatty acid compositions of all samples are shown in supplementary Table 1. Both FHPO and POL had high concentration of SFA (palmitic acid) whereas PKO had high concentration of medium chain fatty acid (lauric acid= 45.5 wt.%). Natural CB contained approximately equal concentration of palmitic acid (26.7 wt.%), stearic acid (36.0 wt.%) and oleic acid (33.1 wt.%). Supplementary Table 2 outlines the TAG composition of samples. Post interesterification, the content of PPP and PSS increased while POO, POP and lauric-based TAGs decreased which was also in accordance to the fatty acid composition results. It should be noted that CB had high concentration of SOS (31.2 wt.%), POS (40.4 wt.%) and low concentration of PPP, PSS and SSS comparing to that of interesterified fat. Using the concentration of POO as a basis, the effect of feed flow rate on the DI is shown in Fig. 1. DI above 80% for all samples were obtained under the range of feed flow rate (60 to 100 mL/min) investigated. As the feed flow rate increased, the DI decreased. At high feed flow rate, the contact and the residence time in between the enzyme and the substrate was short. Overall, as the residence time increased, the interesterification degree increased accordingly. Lower flow rate resulted in a higher DI (Yang et al., 2014). However, the increment in the DI was insignificant if the residence time was prolonged. Considering time cost and economic benefit for industrial application, the flow rate of 70 mL/min with a DI of 97.1% was optimal. Insert Fig. 1 3.2 Effect of feed flow rate on SFC profiles SFC is an important parameter used for the characterization of physical properties of food specialty fats and its products. The change in SFC under different temperature provides the different applications of the product. For instance, the SFC of shortenings affected its performance whereby at low temperature it must retain its plasticity while at room temperature it must maintain its shape without

oil leakage (Damodaran, Parkin, & Fennema, 2008; Lida, Sundram, & Siew, 2002). Also, SFC affects the crystallization characteristics of the fat products in terms of the crystal polymorphisms and the crystallization rates (Zhang, Ma, Huang, & Wang, 2017; Zhang, Shim, Ma, Huang, & Wang, 2018). Table 1 shows the effect of feed flow rates on the SFC at 0-45 °C of the interesterified samples. Especially at 70 mL/min, it was observed that the SFC trend significantly changed compared with the non-interesterified samples. It can be observed that the SFC increased at 5 °C (66.6% to 71.3%) and a notable decrease in the SFC at 45 °C (from 20.8% to 0%). Comparing to that of the interesterified fats, natural CB showed a steep SFC curve whereby highest SFC was obtained at temperature below 20 °C (75.4 % at 20 °C) and lowest SFC at temperature beyond 25 °C (1.6 % at 35 °C). This characteristic allowed the natural CB to exhibit an excellent melt-in-mouth property when used in chocolate. After interesterification, all interesterified samples showed steep SFC curves. At 0-20 °C, SFC of the starting mixture was lower than that of sample after enzymatic interesterification while at higher temperature range of 25-45 °C, the SFC of the interesterified samples were lower. Therefore, the change in the SFC profiles of modified samples met the requirements to be used as a base oil of CBS. Feed flow rate had significant effect on the SFC and this might be attributed by the high DI which proposed the different TAG compositions in between the samples (Lee, Akoh, & Lee, 2008). The variation in the SFC also implied that the interesterification changed the TAG ratios of different melting point (Zhao et al., 2014), which can be attributed to the acyl groups replacement via acyl exchange process between POL, PKO and FHPO. The ideal SFC profile for CBS is to have high SFC under low temperature for shape retention ability and low SFC at high temperature for melt-in-mouth properties. Hence, sample obtained using feed flow rate at 70 mL/min showed the best SFC properties as a potential CBS base oil besides of its

high DI of 97.1 %. Enzymatic interesterified sample from 70 mL/min was selected for the subsequent analyses. Insert Table 1a 3.3 Thermodynamic properties DSC is commonly used to evaluate oil thermodynamic properties. The DSC curves of the interesterified samples under different flow rates are shown in Fig. 2. Simultaneously, the main transition peak temperature as well as the crystallization onset temperature (Ton) and melting offset temperature (Toff) are summarized in Table 1b. From Fig. 2 and Table 1b, single crystallization peak was observed for both the starting mixture and interesterified samples with the later exhibiting lower crystallization onset temperature. This was attributed to the presence of FHPO in the starting mixture which existed in the form of tri-saturated TAG that had high crystallization temperature. On the contrary, natural CB had low onset and offset melting temperature which is in consistent with its SFC profile. After interesterification, the concentration of the tri-saturated TAG decreased which lead to the lower crystallization onset temperature, especially at 70 mL/min, the onset temperature reduced from 52.9 °C to 45.1 °C. This observation was consistent to the change in the SFC of samples. There were insignificant differences in between the samples obtained at different esterification feed flow rate. This was due to the similar chemical compositions of the interesterified products and the crystallization peak temperatures were mainly dependent on the chemical compositions rather than on the crystalline state (Lida, Sundram, & Siew, 2002). Fat mixture showed two endothermic peaks after modification with large span in between the two melting peaks. On the other hand, interesterified samples exhibited two small endothermic peaks with each peak closer to the other with melting temperature in the range of 22-50 °C.

Feed flow rate showed insignificant effect on the melting characteristic. Among the feed flow rates that were investigated, sample obtained from the flow rate at 70 mL/min had the lowest offset melting temperature at 45.1±0.1 °C. Together with the results obtained from the SFC analysis, the sample obtained from 70 mL/min was selected as the interesterified fats in CBS base oil for the following work. Insert Fig. 2 and insert Table 1b 3.4 Analysis of the binary mixture compatibility In order to prepare the CBS with a desired SFC profile and to provide the end product with a good mouthfeel, PKO was chosen as the CBS base oil which is also commonly used in chocolate and confectionary fat products. Fig. 3a shows the iso-solid phase diagram of the mixture of PKO and interesterified fats. The solid line signifies the SFC at various temperatures based on different ratios. At 5 °C, the SFC of the binary mixture system showed an increasing value when higher amount of enzymatically interesterified fats was added. Maximum SFC of 80% was recorded with the addition of 30-40% of interesterified fats. The SFC was at its minimum value when 80% of interesterified fats was added. Along with the increase in the amount of interesterified fats added, the SFC increased. This observation showed that at 5 oC, the SFC was greatly influenced by the mixture composition and it was suggested that the compatibility in the binary system varied (Liu, Wu, & Yang, 2010). Good compatibility in the binary system was observed with the addition of 0-80% of interesterified fats at 10 °C. The iso-solid curve with SFC of 73% at 10 °C appeared to be slightly concave with the addition of 80% interesterified fats. It was suggested that under these conditions, the eutectic effect occurred

which can be used as one of the indexes for evaluating oil compatibility. At 15 °C, a steeper curve was observed with the addition of 20-80% interesterified fats. This indicated that the eutectic effect increased along with temperature. Above 45 °C, when less than 90% of interesterified fats was added, SFC with zero value was recorded which showed that there was no plasticity in the product. In order to determine the suitable base oil for the chocolate production, the compatibility of the components in the binary mixture need to be further evaluated using oil compatibility test (ΔSFC) as a basis. Insert Fig. 3a and insert Fig. 3b The ΔSFC-T curve of the mixture of PKO and enzymatically interesterified fats mixed in different proportions (mass ratios) is shown in Fig.3b. Generally, the compatibility of different fat components is divided into four categories. (i) System which is highly compatible when in any ratio, the different components in the system exhibits a continuous solid state. (ii) A partially compatible system exists when eutectic effect occurs and the system has low melting point. (iii) A partially compatible system arises when components are mixed in a certain proportion and develops a single component system in crystallized state. (iv) Incompatible system whereby the components in the mixture co-existed in their respective crystalline state (Lida & Ali, 1998; Buning & Bartsch, 1989). The compatibility of oil blends refers to the degree of mutual consistency between different oils. The compatibility among various stock oils directly influences the processing operations, qualities and shelf lives of the specialty fats. The specialty fat products made by processing incompatible oil blends tend to have sand streaking, oil leaking, lowered caseation valence and etc (Lida & Ali, 1998). From Fig. 3b, the ΔSFC variation in the binary system was complex. At 5 °C, each mixed system exhibited a crystallized state which

was favorable for the formation of a stable and dense TAG network structure (Calliauw et al., 2005). At 5-10 °C, each mixture showed good compatibility and this was probably attributed to the development of β' crystals in the mixture of PKO and interesterified fats (Kadivar, De Clercq, Danthine, & Dewettinck, 2016). At 15-45 °C, the ΔSFC value of the binary system mixture showed different degrees of eutectic effects in the samples. It has been reported that the negative value of ΔSFC was closely related to the eutectic effect, and the large negative value of ΔSFC indicated the occurrence of a strong eutectic effect (Calliauw et al., 2005). Therefore, the eutectic effect was most prominent at 25 °C for all the binary mixture with an exception for 80% and 90% of interesterified fats addition. Herngvist found that in a system consisted of a large amount of fatty acids with various chain lengths, the TAG components will be crystallized differently on their own and the system will be of higher melting point (Herngvist, 1988). In this work, the main fatty acid composition in the binary mixture consisted of lauric acid from PKO while the interesterified fats consisted of palmitic, stearic and oleic acids. The significant difference between the fatty acid chain lengths of these two components caused the crystallization process to take place on their own. Nevertheless, the ΔSFC value does not always reflect the eutectic effect in this work. At 40 °C, in the mixture with less than 50% addition of interesterified fats showed SFC with zero value, indicating the absence of crystallization. This observation was also reported by Saberi, Lai, and Miskandar (2012). In addition, PKO showed melting characteristics at 25 C. As a result, the phenomenon of eutectic crystal weakened and the compatibility increased. However, when the temperature exceeded 25 C, the systems were highly compatible. Even though the addition of higher amount of PKO was able to provide a positive boost in the mouthfeel by reducing the melting point, a higher proportion of PKO will not be able to provide the mixture with sufficient solid fats below 35 C. Hence, taking into

consideration of the mixtures’ melting point and SFC profiles, binary system consisted of PKO and enzymatically interesterified fats in a mass ratio of 40:60 (CBS-46, 46.6 C) and 30:70 (CBS-37, 46.5 C) were applied as the CBS for the chocolate preparation. 3.5 Crystal polymorphism From Fig. 4, CBS-37 and CBS-46 showed main diffraction peaks at 4.2 Å and 3.8 Å. This indicated that the crystals existed mainly in the β' form and there was less crystals in β form which was consistent with our previous findings on the effect of interesterification on crystal polymorphisms (Zhang, Ma, Huang, & Wang, 2017; Zhang, Shim, Ma, Huang, & Wang, 2018). Formation of β′ crystals in CBS37 and CBS-46 was probably attributed to the presence of fatty acids with various chain lengths and the structure was unattached due to the folding of fatty acid methyl tail, leading to the formation of β′ crystals (Larsson, 1994). Meanwhile, the small β′ crystals formed dense crystal network structure which contributed to the smooth appearance of the end product. Fig. 4 shows the crystal morphology of the two CBS samples. Both CBS-46 and CBS-37 showed finer crystals with approximately 2.6 μm crystal diameters. Combined with the SFC results, it can be suggested that the higher ratio of interesterified fats contributed to the high solid state of the CBS with an increased crystallization rate (Zhang, Ma, Huang, & Wang, 2017). Thereby, the binary system crystal size was smaller and the crystal network formed was denser and dominant in β′ crystals. Insert Fig. 4 3.6 Effect of tempering on the chocolate hardness and fracturability When producing food products containing plastic fats, it is important to be aware of the fact that the physicochemical and sensory properties of the end products will be influenced by the physical state of the fats (Damodaran, Parkin, & Fennema, 2008). In general, tempering process is not required if stable

crystal in CBS can be obtained through direct cooling. CBS containing crystals in unstable form needs to be heated and cooled to obtain a stable β crystal to prevent chocolate blooming caused by crystal conversion. CBE has similar fatty acids and TAG composition as CB and hence, the symmetrical TAG can only achieve stable β crystals after being tempered. Crystals are essential for end products’ consistency and the microstructure is easily affected by processing conditions and had a significant effect on oils and fats macroscopic physical properties compared with SFC and polymorphism (Narine & Marangoni, 1999a; Narine & Marangoni, 1999b). Under different processing conditions, texture may change inevitably (Narine & Marangoni, 1999c). The lauric-acid based CBS greatly simplifies the production process because it can naturally stabilize into β' crystal without the need of tempering process. The results from the previous crystallization characteristics demonstrated that CBS-37 and CBS-46, which were prepared from a mixture of interesterified fats and PKO, exhibited the dominance of β' crystals. In order to verify the application characteristics of CBS-37 and CBS-46, they were incorporated into the chocolate formulation. The hardness and brittleness of the produced chocolate were compared to investigate the effect of the different CBS and the effect of tempering process on the textural properties of the chocolate. From Fig. 5, all chocolate samples had a similar fracturability value of approximately 39 mm. Although CBS-46 contained higher content of PKO, its brittleness in chocolate products was not inferior to that of CBS-37. The tempering process had insignificant effect on the chocolate brittleness. Chocolate prepared with CBS-37 had higher hardness value than that of CBS-46 and this was due to the higher concentration of interesterified fats in the CBS-37 with high SFC. The hardness value of CBS-37 increased after tempering while the hardness of CBS-46 did not change significantly. The XRD results of chocolate products before and after tempering were analyzed (data not shown).

Tempered CBS-37 chocolate product showed transition of β' crystal into the co-existence of β'+β crystals and the texture was altered. On the other hand, tempering process had little effect on the crystallization tendency of the CBS-46 chocolate product and the presence of β' crystal was still dominant. This also explains on the reasons for the hardness of CBS-46 chocolate product to remain unchanged after the tempering process. The sensorial properties of chocolate are significantly influenced by its fracturability. It is hard to form and mold the cholate when the fracturability value is too low while high fracturability value affects the textural properties. Based on the above consideration, CBS-46 was able to provide a better fracturability value for the chocolate without excessive hardness compared to that of CBS-37. Combined with its steeper SFC characteristics and desired crystallization characteristics which does not require the tempering process, CBS-46 was suggested to be a better substitute for the natural CB for chocolate making.

Insert Fig. 5

4. Conclusions In this work, a CBS was produced from enzymatic interesterification using a fat mixture consisting of POL:FHPO:PKO with a mass ratio of 4:3:3. During the interesterification process, the highest interesterification degree of 97.1% was obtained using a feed flow rate of 70 mL/min. Chocolate made using the CBS from the combination of PKO and enzymatically modified fat with a ratio of 4:6 had better characteristics in terms of the textural properties. Tempering process for the chocolate was not required as the crystals existed dominantly in β' form. Findings from this study strongly indicate the

high utility of enzymatic PBR systems for industrial bioprocessing and make PBR a promising alternative industrial approach to the conventional production of confectionary fats. Acknowledgements The financial support from National Natural Science Foundation of China under grant 31671781, China Postdoctoral Science Foundation under grant 2019M663388, the National Key Research and Development Program of China under grant 2017YFD0400200, the Bureau of Science and Information of Guangzhou under grant 201803020032, the Program for Guangdong YangFan Introducing Innovative and Enterpreneurial Teams (Grant 2016YT03H132) are gratefully acknowledged. Notes The authors declare no competing financial interest.

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Figure captions Fig. 1 Effect of feed flow rate on the degree of interesterification (DI) during reaction. The means ± standard deviation with different letters denote significant difference at p ≤ 0.05 (n=3). Fig. 2 The crystallization and melting curves for the enzymatic interesterified products obtained from different feed flow rate.a (aA: starting mixture (POL:FHPO:PKO 4:3:3, w/w/w); enzymatic interesterified samples obtained from different feed flow rates at B: 60 mL/min; C: 70 mL/min; D: 80 mL/min; E: 90 mL/min and F: 100 mL/min.) Fig. 3a Iso-solid diagrams consisting of PKO and enzymatic interesterified fats mixed in different mass ratios. 3b ΔSFC-T curves of PKO and enzymatically interesterified fats mixed in different mass proportions. Fig. 4 Crystal microstructures and polymorphic forms of the CBS-46 (A) and CBS-37 (B). Size bar = 100 mm. Fig. 5 Effects of tempering process on the hardness and fracturability of CBS-46 and CBS-37 based chocolates. a (a CBS-46, CBS-based chocolate (PKO:interesterified fats = 4:6, w/w); CBS-46 T, CBSbased chocolate after tempering process (PKO:interesterified fats = 4:6, w/w); CBS-37, CBS-based chocolate (PKO:interesterified fats = 3:7, w/w); CBS-37 T, CBS-based chocolate after tempering process (PKO:interesterified fats = 3:7, w/w))

Figure 1 100

a

a b

DI %

95

c

90

d

85

80 60

70

80

Feed flow rate (mL/min)

Figure 2

90

100

Figure 3

a

b

Figure 4

Figure 5

Table 1 (a) Effect of feed flow rates on the solid fat content profiles. ab Temperature (°C)

A

B

C

D

E

F

CB

0

67.0 ± 0.7 a

70.0 ± 0.6 bc 71.4 ± 0.5 d 70.3 ± 0.6 cd 69.3 ± 0.7 bc 69.0± 0.9 b

94.8± 0.9 e

5

66.6 ± 0.4 a

69.7 ± 0.3 bc 71.3 ± 0.4 d

70.0 ± 0.7 c 69.6 ± 0.6 bc 69.0 ± 0.6 b

94.0± 0.8 e

10

63.0 ± 0.4 a

67.8 ± 0.5 b

68.9 ± 0.4 c

67.4 ± 0.9 b 67.0 ± 0.5 b 67.0 ± 0.4 b

93.0± 0.9 e

15

58.6 ± 0.7 a

63.0 ± 0.8 b

64.3 ± 0.7 c

62.7 ± 0.7 b 62.1 ± 0.6 b 62.3 ± 0.7 b

89.3± 0.7 e

20

50.9 ± 0.5 a

55.0 ± 0.9 cd 55.9 ± 0.8 d 54.2 ± 0.5 bc 53.8 ± 0.3 b 54.0 ± 0.5 bc 75.4± 0.9 e

25

42.8 ± 0.3 c

42.3 ± 0.5 c

40.7 ± 0.4 b 40.5 ± 0.9 b 40.9 ± 0.4 b

30.2± 0.4 a

30

37.3 ± 0.5 e

31.4 ± 0.4 d 31.0 ± 0.5 d 29.7 ± 0.6 bc 29.5 ± 0.9 b 30.7 ± 0.6 cd

5.1± 0.1 a

35

32.1 ± 0.5 d

20.1 ± 0.4 c

18.1 ± 0.5 b 17.8 ± 0.4 b 18.2 ± 0.6 b

19.4 ± 0.7 c

1.6± 0.1 a

40

27.3 ± 0.5 e

11.7 ± 0.7 d

8.2 ± 0.5 b

9.9 ± 0.4 c

9.8 ± 0.7 c

11.3 ± 0.4 d

0a

45

20.8 ± 0.7 d

3.6 ± 0.6 c

0 ± 0.1 a

2.1 ± 0.2 b

2.4 ± 0.1 b

4.2 ± 0.3 c

0a

42.4 ± 0.9 c

(b) Comparison of DSC-measured onset, offset and transition temperatures for the main crystallization and melting curves of the samples at different feed flow rate. ab

Toff (°C)

A

B

C

D

E

F

CB

52.9 ±

47.9 ±

45.1 ±

46.9 ±

47.1 ±

47.5 ±

31.9±

0.1f

0.1e

0.1b

0.1c

0.2c

0.1d

0.2a

54.5 ±

45.7 ±

43.1 ±

43.6 ±

44.2 ±

44.8 ±

21.3±

0.2g

0.1f

0.1b

0.1c

0.2d

0.2e

0.1a

36.7±

29.7 ±

26.6 ±

27.1 ±

27.0 ±

27.5 ±

16.6±

0.1f

0.1e

0.0b

0.1c

0.1c

0.2d

0.1a

32.2 ±

26.6 ±

25.9 ±

26.3 ±

26.5 ±

26.6 ±

11.6±

0.1e

0.2d

0.1b

0.1c

0.1cd

0.1d

0.1a

Melting Tp (°C)

Ton (°C) Crystallization Tp (°C) a A:

starting mixture (POL:FHPO:PKO 4:3:3, w/w/w); enzymatic interesterified samples obtained from different feed

flow rates at B: 60 mL/min; C: 70 mL/min; D: 80 mL/min; E: 90 mL/min; F: 100 mL/min; CB: cocoa butter. b The

means ± standard deviation with different letters denote significant difference at p ≤ 0.05 (n=3).

Credit author statement Author contributions Zhen Zhang: Conceptualization, Methodology, Software, Investigation, Writing - Original draft preparation. Jia Song: Validation, Formal analysis, Visualization, Software. Wan Jun Lee: Validation, Formal analysis, Writing - Review & Editing. Xiaodong Xie: Resources, Visualization, Data Curation. Yong Wang: Resources, Writing - Review & Editing, Supervision.

1. Enzymatic interesterification of palm oil-based matrix showed steep SFC profiles. 2. Process feed flow rate influenced the fats’ SFC and crystallization behaviors. 3. Binary mixture of PKO and interesterified fats was dominant in β′ crystal. 4. This mixture was used as CBS and was incorporated into chocolate formulation. 5. The chocolate showed consistent texture before and after tempering process.