Practical Applications
According to our results, higher levels of fat bloom occurred in the chocolate sample without CBR (chocolate containing 100% CB, CBCH), compared with other chocolate samples. However, among the chocolates containing enzymatic interesterified sample (EIS) at various levels of 10%, 15% and 20%, the chocolate sample containing 10% of EI (EICH) showed lower fat bloom development during storage period (20 days). The results of polymorphic structure evaluation using X-ray diffractometer (XRD) showed new β-crystalline form with the XRD pattern close to that of β-VI and the m.p. appeared similar to β-V in CB. Also, XRD pattern of EIS showed β′-crystalline form in this sample. Presence of two crystalline forms (β and β′) in the EICH revealed that, adding 10% of the CBR prepared in the current study to chocolate formulation had probably no adverse effect on β-crystal formation in the chocolate sample.

Introduction
Fat bloom is a physical defect that during storage appears as dusty white spots or as a white film on the chocolate surface (Loisel et al., 1998; Sonwai and Rousseau, 2006). This defect is a major concern to chocolate industry because it makes the visual and textural quality of the product undesirable to consumer (Schenk and Peschar, 2004). Although fat bloom has been studied extensively for many years, the blooming mechanism is not clearly understood (Walter and Camillon, 2001; Smith et al., 2007). Nevertheless, it is now accepted that bloom occurs through polymorphic transition of CB (Guthrie et al., 2005; Smith et al., 2007). Polymorphism is defined as the ability of a substance to exist in more than one crystalline form in a crystal lattice (Oh and Swanson, 2006). This polymorphism, is a critical step in chocolate making, and has been studied extensively in the last century (Wille and Lutton, 1966; Chapman et al., 1971; Malssen et al., 1996; Schenk and Peschar, 2004). Several techniques such as differential scanning colorimetery, light microscopy, pulsed nuclear magnetic resonance, and X-ray diffractometer (XRD) have been used to determine polymorphic structures of lipids (Guthrie et al., 2005). Among these methods, XRD is a powerful tool, which provides direct structural information for identifying the types of crystals (Malssen et al., 1996; Langevelde et al., 2001; Schenk and Peschar, 2004; Guthrie et al., 2005). The technique shows at least five different crystalline forms with different characteristic patterns; γ, α, β′ and β (V, VI), in CB (Figure 1). In high quality chocolates, CB is crystallised in the β-V form. However, β-VI is the most stable polymorph and the transition of β-V to β-VI is one of the important theories in fat bloom formation (Wille and Lutton, 1966; Bricknell and Hartel, 1998; Graef et al., 2005; Khan and Rousseau, 2006). It is known that production parameters and storage conditions, including poor tempering, incorrect cooling methods, presence of soft fats in the centers of chocolates, warm storage conditions, and addition of fats as CBRs, can cause fat bloom formation in the chocolate formulation (Walter and Camillon, 2001; Lonchampt and Hartel, 2006). Due to CB’s unique physico-chemical properties, resulting from its symmetrical triacylglycerol species (TAGS, more than 85% of POP, POS and SOS), it is one of the most important ingredients in chocolate formulations (Foubert et al., 2003; Khan and Rousseau, 2006). As only a few countries cultivate cocoa beans, the price of CB is one of the highest among all commercial fats and oils. Therefore, industries are looking for CBRs (Zaidul et al., 2006; Liu et al., 2007). In recent years, enzymatic production of CBRs from lower value fats or oils is the subject of many investigations (Khumalo et al., 2002; Kurvinen et al., 2002; Osborn and Akoh, 2002; Abigor et al., 2003).

Figure 1 X-ray diffraction patterns of various cocoa butter phases (Schenk and Peschar, 2004)

 
Tea seed (Camellia sinensis), containing 30%~32% high quality oil, is known as a by-product in Iran. Therefore, in the first part of the study, tea seed oil was modified by enzymatic interesterification to produce a structured lipid that would be suitable for use as a CBR in the dark chocolate.

The aim of the second part of the study is to show the effect of CBR at various replacement levels of CB (10%, 15% and 20%) on crystalline forms and fat bloom formation in chocolate samples.

1 Results and Discussion
The results of determining TAGS of SF, EICH, and CB are shown in Table 1. According to the results obtained, among major TAGSs of CB (POS, SOS and POP), only SOS with the amount of 0.14% was found in SF. However, after enzymatic interesterification, not only the amount of SOS increased from 0.14% to 11.15% but also the percentages of 17.42% and 10.73% were observed for POS and POP, respectively. Furthermore, m.p. of EICH was comparable to that of the CB. Therefore, EICH was used in chocolate formulation as a CBR at the levels of 10%, 15% and 20% to study the fat bloom formation and polymorphic structure.

 

Table 1 Tags (percent) of SF and EIS in comparison with CB

 
The WI values, as bloom formation index, are plotted versus days of cycling in Figure 2. This result shows that the SFC of EICH, in all temperature conditions, was lower than that of the CB (Table 2). This is probably due to higher content of triolein (OOO) and some other TAGSs (PLP, OOO, PLS and SOO) in the sample compared with those of the CB (Table 1). Therefore, increasing the level of EIS from 10% to 20% in chocolate formulation enhances the rates of fat bloom formation (Figure 2). Several studies also have shown that an increase in liquid fat content (decrease in SFC) and the presence of TAGSs incompatible with those of the CB, increasing the fat bloom formation markedly on the chocolate surface (Ali et al., 2001; Langevelde et al., 2001; Guthrie et al., 2005; Lonchampt and Hartel, 2006; Smith et al., 2007). Yet, in the case of the CB chocolate, bloom development was higher than EI chocolate samples. This is perhaps due to the more complex crystalline forms (polymorphic structures) provided by modified oil (EIS) in the chocolate sample (Osborn and Akoh, 2002). This result is similar to that of Sonwai and Rousseau (2006), who reported that addition of cocoa butter equivalent to chocolate formulation delays bloom development by 1~2 weeks. Our finding is also in agreement with Osborn and Akoh (2002). They studied the effect of enzymatically modified beef tallow, as cocoa butter substitute, on fat bloom rate in dark chocolate and have found that the fat bloom rates were lower in chocolate prepared with modified beef tallow compared with chocolate prepared with CB.

Figure 2 Bloom development on the surface of dark chocolate samples containing cocoa butter and cocoa butter blending with 10, 15 and 20 percent of enzymatically modified sample

Table 2 Solidfat content of cocoa butter and enzymatically interesterified sample

 
The polymorphism patterns of CB, EICH, CB and EI chocolate samples are shown in Table 3. According to the results obtained, a single strong spacing was found in CB at 4.59 Å, which indicates the formation of β-polymorph. In the literature, two different β-polymorphs (V and VI) have been reported with different m.p. and XRD patterns. Since fat bloom formation is linked to transformation of CB from β-V to β-VI, determining the structures of these crystalline forms is very important.

Table 3 Short spacing values [Å] of polymorphic forms of cocoa butter

Some studies have demonstrated that depending on the CB origin, β-V and β-VI show different XRD patterns (Langevelde et al., 2001). A comparison of the XRD patterns of CB from various origins with our sample (Figure 3) reveals that XRD characteristics of our sample is closer to that of CB originated from Bahaia. This is while the iodine value (34.90) is closer to that of the CB obtained from Malaysia (33.9). Our CB samples also shows XRD patterns which look much like that of β-VI (Figure 1), while its melting point (31.43℃) was closer to β-V (30.8~33.8℃), as reported by Chapman et al. (1971) and Wille and Lutton (2001). Malssen et al. (1996) also observed that in some CB samples originated from twelve different countries, XRD patterns of β-phase are closer to those of β-VI but their m.p. resembled that of β-V. They concluded that phase V and VI should be regarded as two sub phases of β. In comparison, we concluded that there is probably one new crystalline form in CB with the behavioral characteristics between β-V and β-VI. 

Figure 3 X-ray diffraction patterns of cocoa butters from different origins (A) (Langevelde et al., 2001), CB sample (malasian CB) was used in this study (B)

 
According to the results shown in Table 3, a very strong spacing at 4.13 Å and a medium spacing at 3.74 Å were found in EIS, which indicate the formation of β′-polymorph in this sample. Moreover, according to some literatures, many vegetable oils or fats, which are used as CBRs, crystallize in the β′ form due to increase of the OOO and other undesirable TAGS percentages (Ali et al., 2001; Osborn and Akoh, 2002; Graef et al., 2005). As shown in Table 1, in the current study, the EIS contained the PLP, OOO, PLS and SOO (3.19%, 14.82%, 6.30% and 4.81%, respectively), which are higher than those of the CB (1.66%, 0.62%, 2.21% and 1.86%, respectively), and can induce β′-polymorph formation in this sample.

The results illustrated in Table 3 also show that CB chocolate contains one crystal polymorph (β structure with very strong and strong short spacing at 4.59 Å and 3.70 Å, respectively), while CB and EI chocolate contains two polymorph structures (β and β′ forms). Consequently, blending 10% of EIS has probably no adverse effect on β-crystal formation in the chocolate product. This result is in accordance with that reported by Osborn and Akoh (2002), who observed a mixture of β and β′ crystal forms in chocolate prepared with enzymatically interesterified beef tallow as cocoa butter substitute.

2 Materials and Methods
Tea seeds (Lahijan variety) were harvested from Iranian farms in Lahijan (in the North of Iran). The oil was extracted by the solvent (Hexane) method after grinding the seeds. Lypozime TL IM (1, 3-specific lipase), a silica granulated Thermomyces lanuginosus lipase, and Malaysian CB were donated by Novozymes A/S (Bagsvaerd, Denmark) and Minoo Chocolate Factory (Tehran, Iran), respectively. TAG standards were purchased from Larodan Fine Chemicals AB (Malmo, Sweden).

Tea seed oil was hydrogenated in a laboratory reactor (Zero Max, USA) at 180℃ temperature, 400 rpm mixing rate, and 0.4% Ni catalyst. Solid fraction (SF) of the oil was prepared by dry fractionation in a test chamber (Binder, Germany) at −20℃ at 20~30 rpm mixing rate for 4 h and centrifugation at 10000 g for 10 min at −20℃. Oil blend was then prepared by blending hydrogenated tea seed oil and SF at 30:70 (wt/wt) ratio. Enzymatic interesterification of the oil blend was carried out in screw-capped sealed glass vials with a magnetic stirrer (700 rpm) at 60℃ for 8 h with 10% enzyme dosage by weigh of the oil. Interesterified sample was immediately filtered through Whatman filter paper No. 4 to remove fine enzyme particles. The sample was then stored at −24℃ for further analysis (Zhang et al., 2001; Abigor et al., 2003).

TAGS of samples were determined by reversed-phase high-performance liquid chromatography (Younglin, Acme 9000, South Korea) equipped with a refractive index (RI) detector and a LiChrosphere RP C-18 column (25 cm length × 4 mm i.d, 4 µm particle size, Spain). The mobile phase was acetone: acetonitrile (60:40, v/v) at a flow rate of 0.6 mL/min. The column and detector temperatures were set at 35℃ and 40℃, respectively. The samples were prepared at 5% concentration with chloroform as the solvent and 10 μL of aliquots was injected into the column. Individual peaks were identified by comparing the retention times with those of TAG standards (AOCS, 1997). Each sample was analyzed three times and the data was reported as average of percentage areas.

For preparation of dark chocolate samples, 50% sieved sugar (mesh 100), 35% CB (CB was replaced at the rates of 10%, 15% and 20% of EICH), 14.5% cocoa powder, and 0.5% soy lecithin were carefully blended to form a homogeneous paste. Then, chocolate samples were prepared according to Briggs and Wang (2004), and Bricknell and Hartel (1998). Briefly, all ingredients were mixed completely at 200 rpm at 60℃ for 20 min in a 100 mL glass container. Lecithin was added and the mass was mixed for another 20 min at 60℃. A stainless steel stirrer connected to the speed controller was used to mix all ingredients. For adjusting the temperatures, glass container and stirrer are located in a laboratory oven (Memmert, Germany). The mass was then tempered at the next step by cooling to 27~28℃ within 20 min to produce seed crystals. The mass was then maintained at the temperature for several minutes to develop crystals, and then heated again to 30~31℃ to melt unstable crystals (Bricknell and Hartel, 1998, Briggs and Wang, 2004). The chocolate was poured into plastic molds, cooled down to 10℃, and maintained at the temperature for 24 h (Osborn and Akoh, 2002). The chocolate samples were later stored in refrigerator conditions for further analysis.

Bloom was determined in triplicate using an accelerated bloom test with continuous temperature cycling between 30℃, 8 h and 20℃, 16 h for 20 days (every other day) (Ali et al., 2001). Values L*, a* and b* were measured using a Hunter-Lab colorimeter (Hunter Associated Laboratory, Inc., Reston, Virginia, USA) and converted to whiteness index (WI) values according to the following equation (Sonwai and Rousseau, 2006):

Sqr{(100−L^*)² + a^*2 + b^*2}

An X-ray diffractometer (Philips, X’ pert, The Netherlands) equipped with a CuKα (λ=1.54 Å) radiation source was employed to determine the polymorphic forms of the samples. The instrument setting was 40 kV with filament currents of 40 mA. The angle scanned was considered from 15º to 30º (2Ó©) with a step size of 0.02º. Identification of polymorphic forms in the samples was performed based on the short spacing values reported in literature (Ali et al., 2001; Osborn and Akoh, 2002; Zhang et al., 2004).

Solid fat content (SFC) was determined using a Bruker minispec NMR (Karlsruhe, Germany). The samples were placed into glass test tubes of 10 mm diameter and tempered at 60℃ for 5 min, followed by cooling at 0℃ for 60 min prior to analysis. SFC was measured in triplicate at 5℃, 10℃, 15℃, 20℃, 30℃, 35℃ and 40℃.

3 Conclussion
In the current study, production of CBR from tea seed oil and application of this modified oil in chocolate formulation was studied. According to the results obtained, we demonstrated that enzymatically modified tea seed oil at the level of 10% of CB does not negatively influence β-crystal formation. Also, it decreases the rate of bloom development in chocolate sample treated in this way. Therefore, applying this product may be useful as a partial CBR in chocolate and related confectionary applications and industries.

Acknowledgments
The authors thank the financial support of Tarbiat Modares University Research Council and Center of Excellency for Recycling and Losses of Strategic Agricultural Products.

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