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Enrichment of branched chain fatty acids from lanolin via urea complexation for infant formula use
LWT - Food Science and Technology 117 (2020) 108627
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Enrichment of branched chain fatty acids from lanolin via urea complexation for infant formula use
T
Xiaosan Wanga,b, Wang Xiaohana, Yang Chena, Wenhua Jina, Qingzhe Jina, Xingguo Wanga,∗ a Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China b Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), 11 Fucheng Road, Beijing, 100048, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Branched-chain fatty acids Lanolin Urea complexation Breast milk fat
Branched-chain fatty acids (BCFAs) play a crucial role in infants’ health. Currently, little information is available for BCFAs enrichment technologies. In this study, an improved method was developed for preparation of free fatty acids from lanolin. Afterwards, BCFAs were concentrated from the prepared free fatty acids by urea complexation. The effect of crystallization solvent, solvent/urea ratio, urea/fatty acid ratio, crystallization temperature and time was investigated. The results suggested that the urea/fatty acid ratio was a critical factor during the enrichment. Under optimal conditions (6:1 ratio of 95% aqueous ethanol/urea, 2:1 ratio of urea/fatty acid, 4 °C for 12 h), content and recovery of the BCFAs from lanolin were 94.69% and 40.02%, respectively. The major BCFAs in the concentrate were anteiso-C15:0, iso-C14:0 and anteiso-C17:0. Surprisingly, the concentrate had a similar BCFA composition and type with human milk fat. Thus, the BCFAs concentrate has the potential to be used in infant formulas.
1. Introduction Generally, branched-chain fatty acids (BCFAs) are saturated fatty acids with one or more methyl branches on the carbon chain. In humans, BCFAs are synthesized mainly by the skin, and they are also found in breast milk, with a content of up to 1.5 wt% (Gibson & Kneebone, 1981). Because BCFAs have a special branched-chain structure, their derivatives have special physical and chemical properties (Ran-Ressler, Bae, Lawrence, Wang, & Brenna, 2014; Shi et al., 2019). Additionally, BCFAs have unique physiological regulation functions (Wallace et al., 2018). Limited studies have shown that BCFAs have anticancer effects. In vivo and in vitro experiments have indicated that iso-C16:0 extracted from fermented soybean products can rapidly induce the programmed apoptosis of various human cancer cells and inhibit their growth (Yang et al., 2000). Both iso- and anteiso-BCFAs have anticancer effects that are even more significant than those of conjugated linoleic acid (Vlaeminck, Fievez, Cabrita, Fonseca, & Dewhurst, 2006). A recent experimental study indicated that BCFAs could reduce the prevalence of neonatal necrotizing enterocolitis (NEC) by more than 50% (Ran-Ressler et al., 2011). Thus, BCFAs are indispensable nutrients in infant formulas for infants fed with formulas. Although BCFAs occur widely in nature, e.g., in bacteria, ruminant milk, animal surface fats of cattle (Vlaeminck et al., 2006), human
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breast milk (Yan et al., 2017), and sheep wool (Jenkins, 1995), their contents in the materials are normally low and they are difficult to be extracted. At present, BCFAs are mainly obtained by chemical synthesis from a suitable starting material. Recently, the chemical synthesis of iso-C12:0–C19:0 from methyl undecylenate was carried out (Richardson & Williams, 2013). However, the method involved a series of complex chemical reactions, so its ability to provide naturally occurring foodgrade BCFA concentrate is debatable. Mudgal, Ran-Ressler, Liu, Brenna, and Rizvi (2016) reported that a two-step urea adduction process caused increase of BCFAs in butter from 2% to 11%, but the total recovery yield was less than 10%. Clearly, the BCFAs concentration was still low and could not meet market demands after two-step urea complexation. BCFAs are mostly enriched in skin lipid secretions, which account for more than 45% of the total fatty acids in lanolin and around 29% in human vernix caseosa. A recent study compared fatty acid composition of lanolin with vernix caseosa and found that lanolin included all the types of BCFAs (Ran-Ressler, Devapatla, Lawrence, & Brenna, 2008; Yao & Hammond, 2006). Currently, lanolin is an agricultural waste in China obtained from sheep wool and considered to be a potential natural source of BCFAs. It is a complex mixture of monoesters, diesters, and hydroxyl esters containing high-molecular-weight alcohols (aliphatic and steroidal alcohols) and approximately equal amounts of high-
Corresponding author. E-mail address: [email protected] (X. Wang).
https://doi.org/10.1016/j.lwt.2019.108627 Received 4 July 2019; Received in revised form 6 September 2019; Accepted 12 September 2019 Available online 13 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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molecular-weight acids (branched- or straight-chain fatty acids) (Church, Davie, James, Leong, & Tucker, 1994; Pelick & Shigley, 1967). Lanolin is difficult to be fully saponified and separated in the process of removing unsaponifiables due to emulsification and complicated composition in lanolin (Schlossman & Mccarthy, 1978). Therefore, it is considerably more difficult to obtain free fatty acids from lanolin than from net triglycerides. In this study, we aimed to obtain BCFAs resembling those from human milk fat. Therefore, a method for obtaining highly pure BCFAs was developed. First, we investigated the process of saponification, calcification, and ethanol washing for the preparation of lanolin fatty acids, and effectively solved the emulsification problem during saponification and washing. Subsequently, urea complexation was employed to enrich BCFAs from the lanolin fatty acids and several parameters, namely, the crystallization solvent, solvent/urea ratio, urea/ fatty acid ratio, crystallization temperature, and time were optimized. 2. Materials and methods 2.1. Materials Lanolin was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the solvents and reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). A standard mixture of fatty acid methyl esters (FAMEs) was purchased from Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China). BCFA standards including 12-methyltridecanoic acid (iso-14:0), 12-methyltetradecanoic acid (anteiso-15:0), 14-methylpentadecanoic acid (iso-16:0), and 14-methylhexadecanoic acid (anteiso-17:0) were purchased from Larodan Fine Chemicals (Malmö, Sweden). 2.2. Preparation of lanolin fatty acids Previous method was modified for the preparation of lanolin free fatty acids (Wang, Wang, & Wang, 2013). Current scheme is given in Fig. 1. (1) Saponification: 100 g of melted lanolin was added slowly to 400 mL of 80% ethanol (v/v) with 14 g of NaOH by refluxing at 70 °C for 4 h (2) Calcification: To achieve good separation of unsaponifiables, it was important to suitably adjust pH of the particular batch of lanolin with 1 mol/L HCl, so pH was selected to be between 8 and 9. Then, 200 mL of 7.0% calcium chloride (w/v) was added to the mixture, which was refluxed at 50 °C. After 2 h the precipitated calcium salts of fatty acids were collected by filtration. (3) Ethanol washing: The calcium salts were extracted with hot ethanol (100 mL) by refluxing at 70 °C for 2 h. After filtration, the purified calcium salts were obtained for acidification. The process was repeated four times to fully remove any impurities mainly consisting of long-chain alcohol. (4) Acidification: The purified calcium salts were mixed with 200 mL water and 100 mL hexane. Afterwards, 3 mol/L hydrochloric acid was added into the mixture to adjust pH to be 3. The organic phase containing free fatty acids was then washed with a 5% NaCl solution to remove excess hydrochloric acid, followed by drying over anhydrous sodium sulfate. Finally, the solvent was evaporated to obtain lanolin fatty acids.
Fig. 1. Flowsheet for preparation of free fatty acids from lanolin.
crystallized at different temperatures (−18, 0, 4, 10, and 20 °C) for different times (4, 8, 12, 16, 20, and 24 h). Five different solvents were investigated: absolute methanol, absolute ethanol, 95% ethanol, 90% ethanol, and 85% ethanol. The crystals (urea-fatty acids adducts, which are referred to as the urea complex (UC)) were separated from the liquid fraction (non-urea complex (NUC)) by filtration using a Bucher funnel. The NUC was washed with an equal volume of a 5% NaCl solution to remove urea; an equal volume of petroleum ether was subsequently added, and the mixture was stirred completely for an appropriate amount of time and then transferred to a separating funnel. The top petroleum ether layer, containing the liberated fatty acids, was dried using anhydrous sodium sulfate, and the solvent was removed at 65 °C using a vacuum rotary evaporator. Fatty acids were recovered from the UC using the same method.
2.3. Urea complexation
2.4. Fatty acid composition analysis
Urea complexation is a procedure primordially developed for polyunsaturated fatty acids enrichment (Wanasundara & Shahidi, 1999). This procedure was modified in this study to examine the effect of complexation variables on the enrichment of BCFAs. The BCFAs were separated from the fatty acid mixture of lanolin by carrying out urea complexation according to the scheme given in Fig. 2. Lanolin fatty acids were mixed with urea in a solvent and heated at 60 °C with stirring and refluxing until the mixture turned into a clear homogeneous solution. The refluxing time was 1 h. Then, the resulting solution was
Free fatty acids were converted to the corresponding methyl esters using 14% boron trifluoride in methanol (Wang, Zou, Miu, Jin, & Wang, 2019). Fatty acid composition was analyzed using a gas chromatograph system (7820A, Agilent, California, USA) equipped with a hydrogen flame ionization detector and capillary column (60 m × 0.25 mm × 0.25 μm, Trace TR-FAME, ThermoFisher, USA). Both the injector and detector temperatures were 250 °C. Nitrogen carrier gas was supplied at 25 mL/min, and the split ratio was 1:10. The pressure of the carrier gas was kept at 22.4 psi, and the flow rates of 2
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Fig. 2. Flowsheet for enrichment of BCFAs by urea complexation.
First, the melted lanolin should be added into NaOH alcohol solution drop by drop under stirring. The dropwise addition avoided incomplete reaction and increased the interaction between lanolin and NaOH. Second, pH should be adjusted to 8 to 9 before calcification because excess NaOH in the system will react with calcium chloride to form calcium hydroxide, which is not beneficial for the reaction between calcium chloride and fatty acid soaps for the formation of calcium salts of fatty acids. However, an acidic pH is also undesirable because of free fatty acids formation caused by the acidification of fatty acid soaps. Thus, the adjustment of pH with 1 mol/L HCl after saponification is very important. Subsequently, ethanol washing was conducted to remove any unsaponifiables. The lanolin contained 5.2% unsaponifiables and they must be removed before acidification of calcium salts of fatty acids because they can cause emulsification. Otherwise, the separation of free fatty acids from the unsaponifiables is particularly difficult. The impurities mainly included high molecular weight alcohol and hot ethanol was suitable for their removal by washing. After ethanol washing, the BCFAs was obtained by converting the calcium salts to free fatty acids. Surprisingly, overall free fatty acids yield was up to 82.3%, which was significantly higher than that (around 10%) obtained with a reported method (Wang et al., 2013). In short, BCFAs production is still challenging and little attention is paid for their preparation. Herein, we established an improved method for the preparation of BCFAs from lanolin. The current method is very effective and leads to a significantly higher yield compared to previous methods (Wang et al., 2013).
hydrogen and air were 30 and 400 mL/min, respectively. The oven temperature program was as follows: start at 60 °C for 3 min, increase to 175 °C at a rate of 5 °C/min, hold at 175 °C for 15 min, increase from 175 to 220 °C at a rate of 2 °C/min, and maintain at 220 °C for 10 min. The normal straight-chain FAMEs were identified by comparing the retention times of sample peaks with those of a mixture of FAME standards. The branched FAMEs were identified according to the chromatographic method reported in our laboratory previously (Jie et al., 2018). 2.5. Statistical analysis The software package Origin 8.0 (OriginLab, Northampton, MA) was used to calculate averages and standard deviations. The differences among the means were compared at P = 0.05 using Tukey's test. All the experiments were performed at least in duplicate. 3. Results and discussion 3.1. Preparation of lanolin fatty acids Even though free fatty acids preparation by saponification of a net triacylglycerol oil is easy to be conducted, the preparation of lanolin fatty acids using the existing method is very difficult due to emulsification. Currently, little information is available on methodologies of the preparation of lanolin fatty acids. When lanolin fatty acids were prepared based on the previously reported method containing saponification and acidification (Wang et al., 2013), free fatty acids yield only reached 10.6%. To obtain lanolin fatty acids in a high yield, we improved saponification method for fatty acid preparation. We found the following points were very crucial during the preparation of lanolin fatty acids.
3.2. Optimization of urea complexation conditions for BCFAs enrichment After the free fatty acids were prepared, we aimed to separate BCFAs from straight-chain fatty acids. A methyl group at the middle of the n-C13 carbon chain may effectively prevent urea complexation. In 3
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differences in terms of the recovery of BCFAs. Because methanol is highly toxic and provides low operational safety and a solvent containing a small amount of water is conducive to the dissolution of urea, 95% ethanol is selected as the urea complexation solvent for further study.
other words, any deviation from the straight-chain arrangement may weaken the stability of the adducted crystals, and hence, BCFAs are likely to be separated from straight-chain fatty acids through urea complexation. However, the application of this method to separate BCFAs from straight-chain fatty acids has been rarely reported. Such an application would require us to fully explore the effects of various urea complexation conditions on the purity and recovery of BCFAs. Previous study has shown that urea/fatty acid ratio and temperature have a significant effect on urea complexation (Mudgal et al., 2016). Additionally, solvent type and solvent/urea ratio affect dissolution of urea in solvent and thus influence adduction of urea with BCFA. Thus, in this study, we investigated the effect of solvent type, solvent/urea ratio, urea/fatty acid ratio, temperature and time on urea complexation.
3.2.2. Effect of solvent/urea ratio Afterwards, the effect of solvent/urea ratio was investigated. The results in Fig. 3B show that 95% ethanol/urea ratio had a significant effect on the recovery and content of BCFAs in the urea complexation process. Through multiple experiments, we found that when 95% ethanol/urea ratio was less than 3:1, it was quite difficult to dissolve urea. Therefore, the minimum 95% ethanol/urea ratio was selected to be 3:1. The BCFAs yield of the NUC improved significantly from 15.26% to 39.24% as the ratio increased from 3.0 to 6.0. However, when the 95% ethanol/urea ratio increased from 6.0 to 8.0, the BCFAs yield of the NUC basically remained the same and even slightly decreased sometimes. Bi, Ding, and Wang (2010) prepared low-melting-point biodiesel from corn oil using urea adduction and observed similar trends. This is attributed to the increased dissolution of BCFAs molecules as the amount of the solvent increases, which avoids BCFAs crystallization from the solution at a low temperature. Conversely, no significant differences were seen in BCFAs content in the ratio range of 3.0–6.0. However, when the ratio was higher than 6.0, BCFAs content significantly lowered, probably because of increased straight-chain fatty acids dissolution in the NUC. In a crystallization process, mass transfer and molecular motion occur in a solvent. In the filtering process carried out to avoid crystalline grain wrapping, an increase in the amount of 95% ethanol causes the increase of BCFAs yield in the NUC and causes the decrease in relative concentration and absolute amount of BCFAs in the UC. The amount of 95% ethanol can be increased further; however, if the amount of 95% ethanol is too high, the content of BCFAs in the NUC will decline slightly. In addition, the cost will increase because of the large consumption of the solvent. Therefore, the
3.2.1. Effect of crystallization solvent Solvent used may affect urea complexation as its effects on solubility of urea and free fatty acid (Kim & Liu, 1999). Herein, we examined the effects of five different solvents (absolute methanol, absolute ethanol, 95% ethanol, 90% ethanol, and 85% ethanol) and fixed the solvent/urea ratio, urea/fatty acid ratio, crystallization temperature, and crystallization time at 5:1, 2:1, 4 °C, and 12 h, respectively. Interestingly, the crystallization solvent had a significant impact on the enrichment of BCFAs (Fig. 3A). The results showed that under the same reaction conditions, when the ethanol concentration was lower than 95%, the content and recovery yield of BCFAs simultaneously reduced, which may be attributed to the fact that when the ethanol concentration was low, fatty acids solubility was low in the solvents. Thus, with the decrease of temperature, BCFAs crystallized out from the solution, leading to a decreased recovery and content in the liquid fraction containing BCFAs. Water content in the ethanol solution is expected to have a major effect on the partitioning of fatty acids, so the removal of saturated straight-chain fatty acids is strongly affected by the water content (Hayes, Bengtsson, Van Alstine, & Setterwall, 1998). Absolute methanol, 95% ethanol, and absolute ethanol have no significant
Fig. 3A. Effect of crystallization solvent on enrichment of BCFAs in NUC under following processing conditions: solvent/urea ratio of 5:1, urea/fatty acid ratio of 2:1, crystallization temperature of 4 °C, and crystallization time of 12 h. 4
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Fig. 3B. Effect of 95% ethanol/urea ratio on enrichment of BCFAs in NUC under following processing conditions: urea/fatty acid ratio of 2:1, crystallization temperature of 4 °C, and crystallization time of 12 h.
the number of carbon atoms exceeded 19, both the saturated straightchain and monomethyl-branched fatty acids could easily form inclusion compounds with urea, but the multi-branched fatty acids and polyunsaturated fatty acids, especially the latter, were more difficult to be adducted with urea because of the greater degree of carbon-chain bending. The monomethyl-branched fatty acids might also be removed from the NUC by crystallization at a low temperature as they have relatively high melting points and low solubility in the solvent. Ratnayake, Olsson, Matthews and Ackman (1988) reported that in urea complexation, fractionation mainly depends on the degree of fatty acid unsaturation but the chain lengths also influence the fractionation efficiency. Hayes et al. (1998) studied the changes in the fatty acid composition in LEAR oil by urea complexation and found that the content of C20:0 in filtrates (NUC) was less than 0.1% after urea complexation. It was noteworthy that the maximum enrichment in BCFAs was obtained at a urea/fatty acid ratio of 3:1. However, when the ratio was higher than 2:1, BCFAs content was not significantly increased in the range of 2:1 to 3:1 and the overall yield dropped significantly. Therefore, in view of the consumption of urea per unit weight of the fatty acids, further studies were conducted using the urea/fatty acid ratio of 2:1.
95% ethanol/urea ratio of 6:1 (v/w) was the best choice for further study.
3.2.3. Effect of urea/fatty acid ratio Several studies on urea complexation have shown that different urea/fatty acid ratio can lead to corresponding changes in fatty acid composition in the NUC and UC (Kim & Liu, 1999; Wu, Ding, Wang, & Xu, 2008). Thus, the ratio influences the content and recovery of BCFAs in the NUC. These studies have also shown that the optimal urea/fatty acid ratio for fatty acid enrichment changes with the change in the oil source. Therefore, it was necessary to explore the optimal urea/fatty acid ratio for enriching BCFAs from lanolin. In combination with the previous urea complexation conditions, we chose five urea/fatty acid ratios (1:1, 1.5:1, 2:1, 2.5:1, and 3:1) to determine the optimal urea/ fatty acid ratio. As expected, the urea/fatty acid ratio had a remarkable effect on the content and recovery yield of BCFAs in the NUC (Fig. 3C). When the amount of urea was low, some saturated straight-chain fatty acids in the system could not be adducted, resulting in a lower content and higher recovery of BCFAs in the NUC. With an increase in the urea amount, the excessive urea in the system formed inclusion compounds with BCFAs, leading to an increase in the loss of BCFAs and a decrease in the recovery yield of BCFAs. For a urea/fatty acid ratio of 2:1, the BCFAs concentration was 94.24%, which was an increase of approximately 46% over the corresponding value for lanolin, and the recovery was up to 39.46%. Through the detection and analysis of the fatty acid composition in the NUC, we observed that the maximum enrichment was obtained by anteiso-C15:0, followed by iso-C14:0, iso-C16:0, and anteiso-C17:0, and the total content of the four BCFAs was more than 65%. The increase in the BCFA percentage composition can be attributed to a significant decrease in the saturated straight-chain fatty acids. After urea inclusion, the content of cinnamic acid, palmitic acid, and stearic acid decreased from 5.79%, 6.95%, and 4.06%–1.86%, 0.66%, and 0.13%, respectively. Moreover, no fatty acids with the number of carbon atoms greater than 19 were detected in the NUC. It was speculated that when
3.2.4. Effect of crystallization temperature To better explore the effect of the crystallization temperature on urea complexation, we selected the commonly used value of 4 °C, 20 °C, and other values (−18, 0, and 10 °C) reported in the literature (Iverson & Sheppard, 1986; Liu, Zhang, Hong, & Ji, 2006; Zheng, Dai, & Shen, 2018). Fig. 3D shows that as the crystallization temperature increased, the recovery of BCFAs increased in the range of −18 to 4 °C and decreased subsequently. The content of BCFAs decreased with increase of temperature. The maximum content and minimum recovery yield of BCFAs were obtained when the crystallization temperature was −18 °C. In the process of urea complexation with seal blubber oil, low crystallization temperatures resulted in high purities but led to a significant reduction in the overall recovery yield of omega-3 fatty acids 5
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Fig. 3C. Effect of urea/fatty acid ratio on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, crystallization temperature of 4 °C, and crystallization time of 12 h.
but the recovery of BCFAs was low. Compared to the content of BCFAs at the crystallization temperature of 0 °C, the BCFAs content at 4 °C showed no obvious change, but the recovery increased significantly from 24.11% to 40.02%. According to the results, 4 °C was chosen as
(Ratnayake, Olsson, Matthews, & Ackman, 1988). The crystallization process was accompanied by exothermal activities, and low temperatures were favorable for the formation of urea adducts, so under the low-temperature conditions, the content of BCFAs in the NUC was high
Fig. 3D. Effect of crystallization temperature on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, urea/fatty acid ratio of 2:1, and crystallization time of 12 h. 6
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Fig. 3E. Effect of crystallization time on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, urea/fatty acid ratio of 2:1, and crystallization temperature of 4 °C.
acids and all fatty acids with number of carbon atoms above 19 were separated from the liquid fraction. Previous study indicated that human milk was mainly composed of six BCFAs, namely iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, isoC17:0, and anteiso-C17:0, whose contents in the BCFAs concentrate were more than 75% (Yan et al., 2017). BCFAs in human milk play a very important role for infants’ health. Neonatal NEC is the most dangerous disease for infants. Currently, there is no good therapy for such a disease. Previous studies have demonstrated that the incidence of NEC is significantly reduced in infants fed with human milk compared to those fed with infant formulas (Lucas & Cole, 1990; Mcguire & Anthony, 2003). Further study found that BCFAs could reduce the incidence of NEC (Ran-Ressler et al., 2011). Thus, it is necessary to add BCFAs to formulas to provide health benefits for infants, especially preterm and low birth weight infants. However, commercially infant formulas do not intend to add any BCFAs probably due to difficult preparation from a native resource. Some ruminant milks also contains some BCFAs. However, BCFAs composition and type from the milks totally differ from human milk. At present, BCFAs preparation from a native source has received much little attention. Surprisingly, we found that the major BCFAs in the NUC were anteiso-C15:0 and anteiso-C17:0, which were also high in human milk fat. Composition and type of BCFA of the concentrate resembled that of human milk fat. Therefore, the enriched BCFAs product has potential application in infant formulas for improved quality.
the crystallization temperature for further research. 3.2.5. Effect of crystallization time Six different time periods (4, 8, 12, 16, 20, and 24) were selected to examine the effect of the urea complexation time on BCFAs enrichment. The formation of urea adducts requires a certain period of time. In this study, a minimum crystallization time of 4 h was selected because many previous studies on urea complexation with milk fat and other fats used a crystallization time of at least 4 h (Crexi, Monte, Monte, & Pinto, 2011). A high BCFAs content and recovery were obtained in the NUC for a solvent/urea ratio, urea/fatty acid ratio, and crystallization temperature of 6:1, 2:1, and 4 °C, respectively. We can clearly observed that the crystallization time had little impact on the concentration of BCFAs (Fig. 3E). With an increase in the crystallization time, overall change in the content of BCFAs was not obvious, whereas the recovery gradually increased first and then kept at a constant level. The total BCFAs content and recovery in the NUC were 94.69% and 40.02% for a crystallization time of 12 h. Our study showed that the crystallization time had no significant influence on the content of BCFAs but had a significant influence on its recovery. Taking both of the content and recovery of BCFAs into account, the crystallization time of 12 h was the optimum choice. 3.3. Comparison of fatty acid composition of lanolin, urea complexation product and human milk fat
4. Conclusion
The fatty acid composition of lanolin, NUC and UC after the urea complexation under optimal conditions are shown in Table 1. We could find that an enriched fraction of BCFAs in the NUC was obtained. The BCFA percentage in the NUC was up to 94.69%, which was considerably higher than that in lanolin (64.35%). In the UC, BCFAs content was the lowest (47.44%) compared to NUC and raw material. After urea complexation, the most abundant BCFA in the NUC was anteisoC15:0 (34.09%), followed by iso-C14:0 (14.64%), anteiso-C17:0 (12.33%) and iso-C16:0 (9.52%). Most of saturated straight-chain fatty
The addition of BCFAs to infant formulas is crucial for the prevention of neonatal NEC. In this study, we established an improved method to enrich BCFAs from lanolin. Under the optimal conditions (6:1 ratio of 95% ethanol/urea, 2:1 ratio of urea/fatty acid, 4 °C, 12 h), the total content of BCFAs can be increased up to 94.69% with a yield of 40.02%. The concentrate had a very similar BCFAs composition and type with human milk fat. The prepared BCFAs product can be used as human 7
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References
Table 1 The fatty acid composition (%) of lanolin, NUC and UC after the urea complexation under optimal conditions. Fatty acid
Lanolin
UC
NUC
BCFA in breast milk fat a
iso-C10:0 C10:0 anteiso-C11:0 C11:0 iso-C12:0 C12:0 iso-C13:0 anteiso-C13:0 C13:0 iso-C14:0 C14:0 iso-C15:0 anteiso-C15:0 C15:0 iso-C16:0 C16:0 iso-C17:0 anteiso-C17:0 C17:0 iso-C18:0 C18:1 C18:0 iso-C19:0 anteiso-C19:0 C19:0 iso-C20:0 C20:0 anteiso-C21:0 C21:0 iso-C22:0 C22:0 iso-C23:0 anteiso-C23:0 C23:0 iso-C24:0 C24:0 anteiso-C25:0 C25:0 iso-C26:0 C26:0 anteiso-C27:0 iso-C28:0 C28:0 anteiso-C29:0 Total BCFAs
0.09 ± 0.02 0.31 ± 0.03 0.36 ± 0.03 0.07 ± 0.01 0.76 ± 0.08 0.71 ± 0.12 0.10 ± 0.01 1.16 ± 0.18 0.30 ± 0.03 3.23 ± 0.21 5.79 ± 0.27 1.43 ± 0.09 5.66 ± 0.16 1.69 ± 0.07 6.66 ± 0.24 6.95 ± 0.31 0.63 ± 0.02 5.81 ± 0.26 0.68 ± 0.08 5.79 ± 0.22 0.85 ± 0.17 4.06 ± 0.19 0.47 ± 0.04 5.08 ± 0.31 0.29 ± 0.02 5.44 ± 0.29 1.43 ± 0.08 4.33 ± 0.31 0.35 ± 0.06 2.65 ± 0.20 1.68 ± 0.12 0.23 ± 0.03 2.73 ± 0.15 0.91 ± 0.09 2.74 ± 0.30 3.09 ± 0.28 4.04 ± 0.30 0.62 ± 0.08 2.37 ± 0.18 2.75 ± 0.21 2.35 ± 0.12 0.72 ± 0.10 0.65 ± 0.04 0.71 ± 0.08 64.35 ± 1.84
0.12 ± 0.05 0.11 ± 0.02 0.09 ± 0.07 0.12 ± 0.03 0.26 ± 0.10 1.97 ± 0.14 tr 0.28 ± 0.08 0.86 ± 0.16 0.45 ± 0.07 10.59 ± 1.21 0.43 ± 0.14 1.36 ± 0.26 3.27 ± 0.24 1.46 ± 0.35 12.32 ± 1.24 0.08 ± 0.02 1.48 ± 0.53 1.18 ± 0.05 6.37 ± 1.42 0.67 ± 0.14 8.42 ± 1.25 0.67 ± 0.07 5.54 ± 0.21 0.41 ± 0.04 5.26 ± 0.28 1.63 ± 0.12 4.42 ± 0.22 0.52 ± 0.07 2.76 ± 0.11 1.85 ± 0.25 0.27 ± 0.06 2.95 ± 0.24 1.03 ± 0.17 2.86 ± 0.12 3.32 ± 0.31 3.85 ± 0.26 0.86 ± 0.19 2.42 ± 0.21 2.86 ± 0.17 2.44 ± 0.15 0.76 ± 0.08 0.57 ± 0.10 0.87 ± 0.14 47.44 ± 2.31
0.55 ± 0.04 0.75 ± 0.08 2.84 ± 0.12 tr 3.93 ± 0.28 0.65 ± 0.11 0.52 ± 0.07 7.01 ± 1.02 tr 14.64 ± 1.32 1.14 ± 0.34 2.51 ± 0.08 34.09 ± 2.13 0.40 ± 0.08 9.52 ± 0.17 0.91 ± 0.14 2.17 ± 0.15 12.33 ± 1.56 tr 1.02 ± 0.21 0.84 ± 0.17 0.42 ± 0.12 tr 2.74 tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr 94.69 ± 1.21
tr – tr – tr – 0.01 tr – 0.04 – 0.07 0.19 – 0.07 – 0.09 0.15 – 0.01 – – tr tr – tr – tr – tr – tr tr – tr – tr – tr – tr tr – tr
Bi, Y., Ding, D., & Wang, D. (2010). Low-melting-point biodiesel derived from corn oil via urea complexation. Bioresource Technology, 101, 1220–1226. Church, J. S., Davie, A. S., James, D. W., Leong, W. H., & Tucker, D. J. (1994). Determination of wool wax in raw wool by Raman spectroscopy. Journal of the American Oil Chemists Society, 71, 1163–1167. Crexi, V. T., Monte, M. L., Monte, M. L., & Pinto, L. A. A. (2011). Polyunsaturated fatty acid concentrates of carp oil: Chemical hydrolysis and urea complexation. Journal of the American Oil Chemists Society, 89, 329–334. Gibson, R. A., & Kneebone, G. M. (1981). Fatty acid composition of human colostrum and mature breast milk. The American Journal of Clinical Nutrition, 34, 252–257. Hayes, D. G., Bengtsson, Y. C., Van Alstine, J. M., & Setterwall, F. (1998). Urea complexation for the rapid, ecologically responsible fractionation of fatty acids from seed oil. Journal of the American Oil Chemists Society, 75, 1403–1409. Iverson, J. L., & Sheppard, A. J. (1986). Determination of fatty acids in butter fat using temperature-programmed gas chromatography of the butyl esters. Food Chemistry, 21, 223–234. Jenkins, T. (1995). Butylsoyamide protects soybean oil from ruminal biohydrogenation: Effects of butylsoyamide on plasma fatty acids and nutrient digestion in sheep. Journal of Animal Science, 73, 818–823. Jie, L., Qi, C., Sun, J., Yu, R., Wang, X., Korma, S. A., et al. (2018). The impact of lactation and gestational age on the composition of branched-chain fatty acids in human breast milk. Food & Function, 9, 1747–1754. Kim, Y. J., & Liu, R. (1999). Selective increase in conjugated linoleic acid in milk fat by crystallization. Journal of Food Science, 64, 792–795. Liu, S., Zhang, C., Hong, P., & Ji, H. (2006). Concentration of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of tuna oil by urea complexation: Optimization of process parameters. Journal of Food Engineering, 73, 203–209. Lucas, A., & Cole, T. J. (1990). Breast milk and neonatal necrotising enterocolitis. Lancet, 336, 1519–1542. Mcguire, W., & Anthony, M. Y. (2003). Donor human milk versus formula for preventing necrotising enterocolitis in preterm infants: Systematic review. ADC-Fetal and Neonatal Edition, 88, 11–14. Mudgal, S., Ran-Ressler, R. R., Liu, L., Brenna, J. T., & Rizvi, S. S. (2016). Branched chain fatty acids concentrate prepared from butter oil via urea adduction. European Journal of Lipid Science and Technology, 118, 669–674. Pelick, N., & Shigley, J. (1967). The fatty acids of degras. J. Am. Oil Chem. Soc. 44, 121–125 1967. Ran-Ressler, R. R., Bae, S., Lawrence, P., Wang, D. H., & Brenna, J. T. (2014). Branched-chain fatty acid content of foods and estimated intake in the USA. British Journal of Nutrition, 112, 565–572. Ran-Ressler, R. R., Devapatla, S., Lawrence, P., & Brenna, J. T. (2008). Branched chain fatty acids are constituents of the normal healthy newborn gastrointestinal tract. Pediatric Research, 64, 605–609. Ran-Ressler, R. R., Khailova, L., Arganbright, K. M., Adkins-Rieck, C. K., Jouni, Z. E., Koren, O., et al. (2011). Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model. PLoS One, 6, 29032–29041. Ratnayake, W., Olsson, B., Matthews, D., & Ackman, R. (1998). Preparation of omega‐3 PUFA concentrates from fish oils via urea complexation. Lipid/Fett, 90, 381–386. Richardson, M. B., & Williams, S. J. (2013). A practical synthesis of long-chain iso-fatty acids (iso-C12–C19) and related natural products. Beilstein Journal of Organic Chemistry, 9, 1807. Schlossman, M. L., & Mccarthy, J. P. (1978). Lanolin and its derivatives. Journal of the American Oil Chemists’ Society, 55, 447–450. Shi, J., Zhan, Y., Zhou, M., He, M., Wang, Q., Li, X., et al. (2019). High-level production of short branched-chain fatty acids from waste materials by genetically modified Bacillus licheniformis. Bioresource Technology, 271, 325–331. Vlaeminck, B., Fievez, V., Cabrita, A., Fonseca, A., & Dewhurst, R. (2006). Factors affecting oddand branched-chain fatty acids in milk: A review. Animal Feed Science and Technology, 131, 389–417. Vlaeminck, B., Fievez, V., Tamminga, S., Dewhurst, R., Van Vuuren, A., De Brabander, D., et al. (2006). Milk odd-and branched-chain fatty acids in relation to the rumen fermentation pattern. Journal of Dairy Science, 89, 3954–3964. Wallace, M., Green, C. R., Roberts, L. S., Lee, Y. M., McCarville, J. L., Sanchez-Gurmaches, J., et al. (2018). Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues. Nature Chemical Biology, 14, 1021–1031. Wanasundara, U. N., & Shahidi, F. (1999). Concentration of omega 3-polyunsaturated fatty acids of seal blubber oil by urea complexation: Optimization of reaction conditions. Food Chemistry, 65, 41–49. Wang, X., Wang, X., & Wang, T. (2013). Enrichment of arachidonic acid for the enzymatic synthesis of arachidonoyl ethanolamide. Journal of the American Oil Chemists Society, 90, 1031–1039. Wang, X., Zou, S., Miu, Z., Jin, Q., & Wang, X. (2019). Enzymatic preparation of structured triacylglycerols with arachidonic and palmitic acids at the sn-2 position for infant formula use. Food Chemistry, 283, 331–337. Wu, M., Ding, H., Wang, S., & Xu, S. (2008). Optimizing conditions for the purification of linoleic acid from sunflower oil by urea complex fractionation. J. Am. Oil Chem. Soc. 85, 677–684. Yang, Z., Liu, S., Chen, X., Chen, H., Huang, M., & Zheng, J. (2000). Induction of apoptotic cell death and in vivo growth inhibition of human cancer cells by a saturated branched-chain fatty acid, 13-methyltetradecanoic acid. Cancer Research, 60, 505–509. Yan, Y., Wang, Z., Wang, X., Wang, Y., Xiang, J., Kothapalli, K. S., et al. (2017). Branched chain fatty acids positional distribution in human milk fat and common human food fats and uptake in human intestinal cells. Journal of Functional Foods, 29, 172–177. Yao, L., & Hammond, E. G. (2006). Isolation and melting properties of branched-chain esters from lanolin. Journal of the American Oil Chemists Society, 83, 547–552. Zheng, Z., Dai, Z., & Shen, Q. (2018). Enrichment of polyunsaturated fatty acids from seal oil through urea adduction and the fatty acids change rules during the process. Journal of Food Processing and Preservation, 42, e13593.
± 0.00
± 0.00 ± 0.01 ± 0.00 ± 0.00 ± 0.00 ± 0.01 ± 0.00
tr means < 0.05. — indicates that the fatty acid is not BCFA. NUC: non-urea complex. UC: urea complex.
milk fat substitutes for infant formulas. The improved method was very effective in enriching BCFAs. The successful preparation of high-valueadded BCFAs is beneficial for the cost reduction and sustainable utilization of lanolin.
Declaration of interest statement The authors have declared no conflict of interest.
Acknowledgements This study was financially supported by “National Natural Science Foundation of China (Grant No.: 31701559 and Grant No.: 31972035)” and “The Natural Science Foundation of Jiangsu Province (Grant No: BK20150137)”.
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Enrichment of branched chain fatty acids from lanolin via urea complexation for infant formula use
T
Xiaosan Wanga,b, Wang Xiaohana, Yang Chena, Wenhua Jina, Qingzhe Jina, Xingguo Wanga,∗ a Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China b Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), 11 Fucheng Road, Beijing, 100048, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Branched-chain fatty acids Lanolin Urea complexation Breast milk fat
Branched-chain fatty acids (BCFAs) play a crucial role in infants’ health. Currently, little information is available for BCFAs enrichment technologies. In this study, an improved method was developed for preparation of free fatty acids from lanolin. Afterwards, BCFAs were concentrated from the prepared free fatty acids by urea complexation. The effect of crystallization solvent, solvent/urea ratio, urea/fatty acid ratio, crystallization temperature and time was investigated. The results suggested that the urea/fatty acid ratio was a critical factor during the enrichment. Under optimal conditions (6:1 ratio of 95% aqueous ethanol/urea, 2:1 ratio of urea/fatty acid, 4 °C for 12 h), content and recovery of the BCFAs from lanolin were 94.69% and 40.02%, respectively. The major BCFAs in the concentrate were anteiso-C15:0, iso-C14:0 and anteiso-C17:0. Surprisingly, the concentrate had a similar BCFA composition and type with human milk fat. Thus, the BCFAs concentrate has the potential to be used in infant formulas.
1. Introduction Generally, branched-chain fatty acids (BCFAs) are saturated fatty acids with one or more methyl branches on the carbon chain. In humans, BCFAs are synthesized mainly by the skin, and they are also found in breast milk, with a content of up to 1.5 wt% (Gibson & Kneebone, 1981). Because BCFAs have a special branched-chain structure, their derivatives have special physical and chemical properties (Ran-Ressler, Bae, Lawrence, Wang, & Brenna, 2014; Shi et al., 2019). Additionally, BCFAs have unique physiological regulation functions (Wallace et al., 2018). Limited studies have shown that BCFAs have anticancer effects. In vivo and in vitro experiments have indicated that iso-C16:0 extracted from fermented soybean products can rapidly induce the programmed apoptosis of various human cancer cells and inhibit their growth (Yang et al., 2000). Both iso- and anteiso-BCFAs have anticancer effects that are even more significant than those of conjugated linoleic acid (Vlaeminck, Fievez, Cabrita, Fonseca, & Dewhurst, 2006). A recent experimental study indicated that BCFAs could reduce the prevalence of neonatal necrotizing enterocolitis (NEC) by more than 50% (Ran-Ressler et al., 2011). Thus, BCFAs are indispensable nutrients in infant formulas for infants fed with formulas. Although BCFAs occur widely in nature, e.g., in bacteria, ruminant milk, animal surface fats of cattle (Vlaeminck et al., 2006), human
∗
breast milk (Yan et al., 2017), and sheep wool (Jenkins, 1995), their contents in the materials are normally low and they are difficult to be extracted. At present, BCFAs are mainly obtained by chemical synthesis from a suitable starting material. Recently, the chemical synthesis of iso-C12:0–C19:0 from methyl undecylenate was carried out (Richardson & Williams, 2013). However, the method involved a series of complex chemical reactions, so its ability to provide naturally occurring foodgrade BCFA concentrate is debatable. Mudgal, Ran-Ressler, Liu, Brenna, and Rizvi (2016) reported that a two-step urea adduction process caused increase of BCFAs in butter from 2% to 11%, but the total recovery yield was less than 10%. Clearly, the BCFAs concentration was still low and could not meet market demands after two-step urea complexation. BCFAs are mostly enriched in skin lipid secretions, which account for more than 45% of the total fatty acids in lanolin and around 29% in human vernix caseosa. A recent study compared fatty acid composition of lanolin with vernix caseosa and found that lanolin included all the types of BCFAs (Ran-Ressler, Devapatla, Lawrence, & Brenna, 2008; Yao & Hammond, 2006). Currently, lanolin is an agricultural waste in China obtained from sheep wool and considered to be a potential natural source of BCFAs. It is a complex mixture of monoesters, diesters, and hydroxyl esters containing high-molecular-weight alcohols (aliphatic and steroidal alcohols) and approximately equal amounts of high-
Corresponding author. E-mail address: [email protected] (X. Wang).
https://doi.org/10.1016/j.lwt.2019.108627 Received 4 July 2019; Received in revised form 6 September 2019; Accepted 12 September 2019 Available online 13 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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molecular-weight acids (branched- or straight-chain fatty acids) (Church, Davie, James, Leong, & Tucker, 1994; Pelick & Shigley, 1967). Lanolin is difficult to be fully saponified and separated in the process of removing unsaponifiables due to emulsification and complicated composition in lanolin (Schlossman & Mccarthy, 1978). Therefore, it is considerably more difficult to obtain free fatty acids from lanolin than from net triglycerides. In this study, we aimed to obtain BCFAs resembling those from human milk fat. Therefore, a method for obtaining highly pure BCFAs was developed. First, we investigated the process of saponification, calcification, and ethanol washing for the preparation of lanolin fatty acids, and effectively solved the emulsification problem during saponification and washing. Subsequently, urea complexation was employed to enrich BCFAs from the lanolin fatty acids and several parameters, namely, the crystallization solvent, solvent/urea ratio, urea/ fatty acid ratio, crystallization temperature, and time were optimized. 2. Materials and methods 2.1. Materials Lanolin was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the solvents and reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). A standard mixture of fatty acid methyl esters (FAMEs) was purchased from Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China). BCFA standards including 12-methyltridecanoic acid (iso-14:0), 12-methyltetradecanoic acid (anteiso-15:0), 14-methylpentadecanoic acid (iso-16:0), and 14-methylhexadecanoic acid (anteiso-17:0) were purchased from Larodan Fine Chemicals (Malmö, Sweden). 2.2. Preparation of lanolin fatty acids Previous method was modified for the preparation of lanolin free fatty acids (Wang, Wang, & Wang, 2013). Current scheme is given in Fig. 1. (1) Saponification: 100 g of melted lanolin was added slowly to 400 mL of 80% ethanol (v/v) with 14 g of NaOH by refluxing at 70 °C for 4 h (2) Calcification: To achieve good separation of unsaponifiables, it was important to suitably adjust pH of the particular batch of lanolin with 1 mol/L HCl, so pH was selected to be between 8 and 9. Then, 200 mL of 7.0% calcium chloride (w/v) was added to the mixture, which was refluxed at 50 °C. After 2 h the precipitated calcium salts of fatty acids were collected by filtration. (3) Ethanol washing: The calcium salts were extracted with hot ethanol (100 mL) by refluxing at 70 °C for 2 h. After filtration, the purified calcium salts were obtained for acidification. The process was repeated four times to fully remove any impurities mainly consisting of long-chain alcohol. (4) Acidification: The purified calcium salts were mixed with 200 mL water and 100 mL hexane. Afterwards, 3 mol/L hydrochloric acid was added into the mixture to adjust pH to be 3. The organic phase containing free fatty acids was then washed with a 5% NaCl solution to remove excess hydrochloric acid, followed by drying over anhydrous sodium sulfate. Finally, the solvent was evaporated to obtain lanolin fatty acids.
Fig. 1. Flowsheet for preparation of free fatty acids from lanolin.
crystallized at different temperatures (−18, 0, 4, 10, and 20 °C) for different times (4, 8, 12, 16, 20, and 24 h). Five different solvents were investigated: absolute methanol, absolute ethanol, 95% ethanol, 90% ethanol, and 85% ethanol. The crystals (urea-fatty acids adducts, which are referred to as the urea complex (UC)) were separated from the liquid fraction (non-urea complex (NUC)) by filtration using a Bucher funnel. The NUC was washed with an equal volume of a 5% NaCl solution to remove urea; an equal volume of petroleum ether was subsequently added, and the mixture was stirred completely for an appropriate amount of time and then transferred to a separating funnel. The top petroleum ether layer, containing the liberated fatty acids, was dried using anhydrous sodium sulfate, and the solvent was removed at 65 °C using a vacuum rotary evaporator. Fatty acids were recovered from the UC using the same method.
2.3. Urea complexation
2.4. Fatty acid composition analysis
Urea complexation is a procedure primordially developed for polyunsaturated fatty acids enrichment (Wanasundara & Shahidi, 1999). This procedure was modified in this study to examine the effect of complexation variables on the enrichment of BCFAs. The BCFAs were separated from the fatty acid mixture of lanolin by carrying out urea complexation according to the scheme given in Fig. 2. Lanolin fatty acids were mixed with urea in a solvent and heated at 60 °C with stirring and refluxing until the mixture turned into a clear homogeneous solution. The refluxing time was 1 h. Then, the resulting solution was
Free fatty acids were converted to the corresponding methyl esters using 14% boron trifluoride in methanol (Wang, Zou, Miu, Jin, & Wang, 2019). Fatty acid composition was analyzed using a gas chromatograph system (7820A, Agilent, California, USA) equipped with a hydrogen flame ionization detector and capillary column (60 m × 0.25 mm × 0.25 μm, Trace TR-FAME, ThermoFisher, USA). Both the injector and detector temperatures were 250 °C. Nitrogen carrier gas was supplied at 25 mL/min, and the split ratio was 1:10. The pressure of the carrier gas was kept at 22.4 psi, and the flow rates of 2
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Fig. 2. Flowsheet for enrichment of BCFAs by urea complexation.
First, the melted lanolin should be added into NaOH alcohol solution drop by drop under stirring. The dropwise addition avoided incomplete reaction and increased the interaction between lanolin and NaOH. Second, pH should be adjusted to 8 to 9 before calcification because excess NaOH in the system will react with calcium chloride to form calcium hydroxide, which is not beneficial for the reaction between calcium chloride and fatty acid soaps for the formation of calcium salts of fatty acids. However, an acidic pH is also undesirable because of free fatty acids formation caused by the acidification of fatty acid soaps. Thus, the adjustment of pH with 1 mol/L HCl after saponification is very important. Subsequently, ethanol washing was conducted to remove any unsaponifiables. The lanolin contained 5.2% unsaponifiables and they must be removed before acidification of calcium salts of fatty acids because they can cause emulsification. Otherwise, the separation of free fatty acids from the unsaponifiables is particularly difficult. The impurities mainly included high molecular weight alcohol and hot ethanol was suitable for their removal by washing. After ethanol washing, the BCFAs was obtained by converting the calcium salts to free fatty acids. Surprisingly, overall free fatty acids yield was up to 82.3%, which was significantly higher than that (around 10%) obtained with a reported method (Wang et al., 2013). In short, BCFAs production is still challenging and little attention is paid for their preparation. Herein, we established an improved method for the preparation of BCFAs from lanolin. The current method is very effective and leads to a significantly higher yield compared to previous methods (Wang et al., 2013).
hydrogen and air were 30 and 400 mL/min, respectively. The oven temperature program was as follows: start at 60 °C for 3 min, increase to 175 °C at a rate of 5 °C/min, hold at 175 °C for 15 min, increase from 175 to 220 °C at a rate of 2 °C/min, and maintain at 220 °C for 10 min. The normal straight-chain FAMEs were identified by comparing the retention times of sample peaks with those of a mixture of FAME standards. The branched FAMEs were identified according to the chromatographic method reported in our laboratory previously (Jie et al., 2018). 2.5. Statistical analysis The software package Origin 8.0 (OriginLab, Northampton, MA) was used to calculate averages and standard deviations. The differences among the means were compared at P = 0.05 using Tukey's test. All the experiments were performed at least in duplicate. 3. Results and discussion 3.1. Preparation of lanolin fatty acids Even though free fatty acids preparation by saponification of a net triacylglycerol oil is easy to be conducted, the preparation of lanolin fatty acids using the existing method is very difficult due to emulsification. Currently, little information is available on methodologies of the preparation of lanolin fatty acids. When lanolin fatty acids were prepared based on the previously reported method containing saponification and acidification (Wang et al., 2013), free fatty acids yield only reached 10.6%. To obtain lanolin fatty acids in a high yield, we improved saponification method for fatty acid preparation. We found the following points were very crucial during the preparation of lanolin fatty acids.
3.2. Optimization of urea complexation conditions for BCFAs enrichment After the free fatty acids were prepared, we aimed to separate BCFAs from straight-chain fatty acids. A methyl group at the middle of the n-C13 carbon chain may effectively prevent urea complexation. In 3
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differences in terms of the recovery of BCFAs. Because methanol is highly toxic and provides low operational safety and a solvent containing a small amount of water is conducive to the dissolution of urea, 95% ethanol is selected as the urea complexation solvent for further study.
other words, any deviation from the straight-chain arrangement may weaken the stability of the adducted crystals, and hence, BCFAs are likely to be separated from straight-chain fatty acids through urea complexation. However, the application of this method to separate BCFAs from straight-chain fatty acids has been rarely reported. Such an application would require us to fully explore the effects of various urea complexation conditions on the purity and recovery of BCFAs. Previous study has shown that urea/fatty acid ratio and temperature have a significant effect on urea complexation (Mudgal et al., 2016). Additionally, solvent type and solvent/urea ratio affect dissolution of urea in solvent and thus influence adduction of urea with BCFA. Thus, in this study, we investigated the effect of solvent type, solvent/urea ratio, urea/fatty acid ratio, temperature and time on urea complexation.
3.2.2. Effect of solvent/urea ratio Afterwards, the effect of solvent/urea ratio was investigated. The results in Fig. 3B show that 95% ethanol/urea ratio had a significant effect on the recovery and content of BCFAs in the urea complexation process. Through multiple experiments, we found that when 95% ethanol/urea ratio was less than 3:1, it was quite difficult to dissolve urea. Therefore, the minimum 95% ethanol/urea ratio was selected to be 3:1. The BCFAs yield of the NUC improved significantly from 15.26% to 39.24% as the ratio increased from 3.0 to 6.0. However, when the 95% ethanol/urea ratio increased from 6.0 to 8.0, the BCFAs yield of the NUC basically remained the same and even slightly decreased sometimes. Bi, Ding, and Wang (2010) prepared low-melting-point biodiesel from corn oil using urea adduction and observed similar trends. This is attributed to the increased dissolution of BCFAs molecules as the amount of the solvent increases, which avoids BCFAs crystallization from the solution at a low temperature. Conversely, no significant differences were seen in BCFAs content in the ratio range of 3.0–6.0. However, when the ratio was higher than 6.0, BCFAs content significantly lowered, probably because of increased straight-chain fatty acids dissolution in the NUC. In a crystallization process, mass transfer and molecular motion occur in a solvent. In the filtering process carried out to avoid crystalline grain wrapping, an increase in the amount of 95% ethanol causes the increase of BCFAs yield in the NUC and causes the decrease in relative concentration and absolute amount of BCFAs in the UC. The amount of 95% ethanol can be increased further; however, if the amount of 95% ethanol is too high, the content of BCFAs in the NUC will decline slightly. In addition, the cost will increase because of the large consumption of the solvent. Therefore, the
3.2.1. Effect of crystallization solvent Solvent used may affect urea complexation as its effects on solubility of urea and free fatty acid (Kim & Liu, 1999). Herein, we examined the effects of five different solvents (absolute methanol, absolute ethanol, 95% ethanol, 90% ethanol, and 85% ethanol) and fixed the solvent/urea ratio, urea/fatty acid ratio, crystallization temperature, and crystallization time at 5:1, 2:1, 4 °C, and 12 h, respectively. Interestingly, the crystallization solvent had a significant impact on the enrichment of BCFAs (Fig. 3A). The results showed that under the same reaction conditions, when the ethanol concentration was lower than 95%, the content and recovery yield of BCFAs simultaneously reduced, which may be attributed to the fact that when the ethanol concentration was low, fatty acids solubility was low in the solvents. Thus, with the decrease of temperature, BCFAs crystallized out from the solution, leading to a decreased recovery and content in the liquid fraction containing BCFAs. Water content in the ethanol solution is expected to have a major effect on the partitioning of fatty acids, so the removal of saturated straight-chain fatty acids is strongly affected by the water content (Hayes, Bengtsson, Van Alstine, & Setterwall, 1998). Absolute methanol, 95% ethanol, and absolute ethanol have no significant
Fig. 3A. Effect of crystallization solvent on enrichment of BCFAs in NUC under following processing conditions: solvent/urea ratio of 5:1, urea/fatty acid ratio of 2:1, crystallization temperature of 4 °C, and crystallization time of 12 h. 4
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Fig. 3B. Effect of 95% ethanol/urea ratio on enrichment of BCFAs in NUC under following processing conditions: urea/fatty acid ratio of 2:1, crystallization temperature of 4 °C, and crystallization time of 12 h.
the number of carbon atoms exceeded 19, both the saturated straightchain and monomethyl-branched fatty acids could easily form inclusion compounds with urea, but the multi-branched fatty acids and polyunsaturated fatty acids, especially the latter, were more difficult to be adducted with urea because of the greater degree of carbon-chain bending. The monomethyl-branched fatty acids might also be removed from the NUC by crystallization at a low temperature as they have relatively high melting points and low solubility in the solvent. Ratnayake, Olsson, Matthews and Ackman (1988) reported that in urea complexation, fractionation mainly depends on the degree of fatty acid unsaturation but the chain lengths also influence the fractionation efficiency. Hayes et al. (1998) studied the changes in the fatty acid composition in LEAR oil by urea complexation and found that the content of C20:0 in filtrates (NUC) was less than 0.1% after urea complexation. It was noteworthy that the maximum enrichment in BCFAs was obtained at a urea/fatty acid ratio of 3:1. However, when the ratio was higher than 2:1, BCFAs content was not significantly increased in the range of 2:1 to 3:1 and the overall yield dropped significantly. Therefore, in view of the consumption of urea per unit weight of the fatty acids, further studies were conducted using the urea/fatty acid ratio of 2:1.
95% ethanol/urea ratio of 6:1 (v/w) was the best choice for further study.
3.2.3. Effect of urea/fatty acid ratio Several studies on urea complexation have shown that different urea/fatty acid ratio can lead to corresponding changes in fatty acid composition in the NUC and UC (Kim & Liu, 1999; Wu, Ding, Wang, & Xu, 2008). Thus, the ratio influences the content and recovery of BCFAs in the NUC. These studies have also shown that the optimal urea/fatty acid ratio for fatty acid enrichment changes with the change in the oil source. Therefore, it was necessary to explore the optimal urea/fatty acid ratio for enriching BCFAs from lanolin. In combination with the previous urea complexation conditions, we chose five urea/fatty acid ratios (1:1, 1.5:1, 2:1, 2.5:1, and 3:1) to determine the optimal urea/ fatty acid ratio. As expected, the urea/fatty acid ratio had a remarkable effect on the content and recovery yield of BCFAs in the NUC (Fig. 3C). When the amount of urea was low, some saturated straight-chain fatty acids in the system could not be adducted, resulting in a lower content and higher recovery of BCFAs in the NUC. With an increase in the urea amount, the excessive urea in the system formed inclusion compounds with BCFAs, leading to an increase in the loss of BCFAs and a decrease in the recovery yield of BCFAs. For a urea/fatty acid ratio of 2:1, the BCFAs concentration was 94.24%, which was an increase of approximately 46% over the corresponding value for lanolin, and the recovery was up to 39.46%. Through the detection and analysis of the fatty acid composition in the NUC, we observed that the maximum enrichment was obtained by anteiso-C15:0, followed by iso-C14:0, iso-C16:0, and anteiso-C17:0, and the total content of the four BCFAs was more than 65%. The increase in the BCFA percentage composition can be attributed to a significant decrease in the saturated straight-chain fatty acids. After urea inclusion, the content of cinnamic acid, palmitic acid, and stearic acid decreased from 5.79%, 6.95%, and 4.06%–1.86%, 0.66%, and 0.13%, respectively. Moreover, no fatty acids with the number of carbon atoms greater than 19 were detected in the NUC. It was speculated that when
3.2.4. Effect of crystallization temperature To better explore the effect of the crystallization temperature on urea complexation, we selected the commonly used value of 4 °C, 20 °C, and other values (−18, 0, and 10 °C) reported in the literature (Iverson & Sheppard, 1986; Liu, Zhang, Hong, & Ji, 2006; Zheng, Dai, & Shen, 2018). Fig. 3D shows that as the crystallization temperature increased, the recovery of BCFAs increased in the range of −18 to 4 °C and decreased subsequently. The content of BCFAs decreased with increase of temperature. The maximum content and minimum recovery yield of BCFAs were obtained when the crystallization temperature was −18 °C. In the process of urea complexation with seal blubber oil, low crystallization temperatures resulted in high purities but led to a significant reduction in the overall recovery yield of omega-3 fatty acids 5
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Fig. 3C. Effect of urea/fatty acid ratio on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, crystallization temperature of 4 °C, and crystallization time of 12 h.
but the recovery of BCFAs was low. Compared to the content of BCFAs at the crystallization temperature of 0 °C, the BCFAs content at 4 °C showed no obvious change, but the recovery increased significantly from 24.11% to 40.02%. According to the results, 4 °C was chosen as
(Ratnayake, Olsson, Matthews, & Ackman, 1988). The crystallization process was accompanied by exothermal activities, and low temperatures were favorable for the formation of urea adducts, so under the low-temperature conditions, the content of BCFAs in the NUC was high
Fig. 3D. Effect of crystallization temperature on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, urea/fatty acid ratio of 2:1, and crystallization time of 12 h. 6
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Fig. 3E. Effect of crystallization time on enrichment of BCFAs in NUC under following processing conditions: 95% ethanol/urea ratio of 6:1, urea/fatty acid ratio of 2:1, and crystallization temperature of 4 °C.
acids and all fatty acids with number of carbon atoms above 19 were separated from the liquid fraction. Previous study indicated that human milk was mainly composed of six BCFAs, namely iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, isoC17:0, and anteiso-C17:0, whose contents in the BCFAs concentrate were more than 75% (Yan et al., 2017). BCFAs in human milk play a very important role for infants’ health. Neonatal NEC is the most dangerous disease for infants. Currently, there is no good therapy for such a disease. Previous studies have demonstrated that the incidence of NEC is significantly reduced in infants fed with human milk compared to those fed with infant formulas (Lucas & Cole, 1990; Mcguire & Anthony, 2003). Further study found that BCFAs could reduce the incidence of NEC (Ran-Ressler et al., 2011). Thus, it is necessary to add BCFAs to formulas to provide health benefits for infants, especially preterm and low birth weight infants. However, commercially infant formulas do not intend to add any BCFAs probably due to difficult preparation from a native resource. Some ruminant milks also contains some BCFAs. However, BCFAs composition and type from the milks totally differ from human milk. At present, BCFAs preparation from a native source has received much little attention. Surprisingly, we found that the major BCFAs in the NUC were anteiso-C15:0 and anteiso-C17:0, which were also high in human milk fat. Composition and type of BCFA of the concentrate resembled that of human milk fat. Therefore, the enriched BCFAs product has potential application in infant formulas for improved quality.
the crystallization temperature for further research. 3.2.5. Effect of crystallization time Six different time periods (4, 8, 12, 16, 20, and 24) were selected to examine the effect of the urea complexation time on BCFAs enrichment. The formation of urea adducts requires a certain period of time. In this study, a minimum crystallization time of 4 h was selected because many previous studies on urea complexation with milk fat and other fats used a crystallization time of at least 4 h (Crexi, Monte, Monte, & Pinto, 2011). A high BCFAs content and recovery were obtained in the NUC for a solvent/urea ratio, urea/fatty acid ratio, and crystallization temperature of 6:1, 2:1, and 4 °C, respectively. We can clearly observed that the crystallization time had little impact on the concentration of BCFAs (Fig. 3E). With an increase in the crystallization time, overall change in the content of BCFAs was not obvious, whereas the recovery gradually increased first and then kept at a constant level. The total BCFAs content and recovery in the NUC were 94.69% and 40.02% for a crystallization time of 12 h. Our study showed that the crystallization time had no significant influence on the content of BCFAs but had a significant influence on its recovery. Taking both of the content and recovery of BCFAs into account, the crystallization time of 12 h was the optimum choice. 3.3. Comparison of fatty acid composition of lanolin, urea complexation product and human milk fat
4. Conclusion
The fatty acid composition of lanolin, NUC and UC after the urea complexation under optimal conditions are shown in Table 1. We could find that an enriched fraction of BCFAs in the NUC was obtained. The BCFA percentage in the NUC was up to 94.69%, which was considerably higher than that in lanolin (64.35%). In the UC, BCFAs content was the lowest (47.44%) compared to NUC and raw material. After urea complexation, the most abundant BCFA in the NUC was anteisoC15:0 (34.09%), followed by iso-C14:0 (14.64%), anteiso-C17:0 (12.33%) and iso-C16:0 (9.52%). Most of saturated straight-chain fatty
The addition of BCFAs to infant formulas is crucial for the prevention of neonatal NEC. In this study, we established an improved method to enrich BCFAs from lanolin. Under the optimal conditions (6:1 ratio of 95% ethanol/urea, 2:1 ratio of urea/fatty acid, 4 °C, 12 h), the total content of BCFAs can be increased up to 94.69% with a yield of 40.02%. The concentrate had a very similar BCFAs composition and type with human milk fat. The prepared BCFAs product can be used as human 7
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References
Table 1 The fatty acid composition (%) of lanolin, NUC and UC after the urea complexation under optimal conditions. Fatty acid
Lanolin
UC
NUC
BCFA in breast milk fat a
iso-C10:0 C10:0 anteiso-C11:0 C11:0 iso-C12:0 C12:0 iso-C13:0 anteiso-C13:0 C13:0 iso-C14:0 C14:0 iso-C15:0 anteiso-C15:0 C15:0 iso-C16:0 C16:0 iso-C17:0 anteiso-C17:0 C17:0 iso-C18:0 C18:1 C18:0 iso-C19:0 anteiso-C19:0 C19:0 iso-C20:0 C20:0 anteiso-C21:0 C21:0 iso-C22:0 C22:0 iso-C23:0 anteiso-C23:0 C23:0 iso-C24:0 C24:0 anteiso-C25:0 C25:0 iso-C26:0 C26:0 anteiso-C27:0 iso-C28:0 C28:0 anteiso-C29:0 Total BCFAs
0.09 ± 0.02 0.31 ± 0.03 0.36 ± 0.03 0.07 ± 0.01 0.76 ± 0.08 0.71 ± 0.12 0.10 ± 0.01 1.16 ± 0.18 0.30 ± 0.03 3.23 ± 0.21 5.79 ± 0.27 1.43 ± 0.09 5.66 ± 0.16 1.69 ± 0.07 6.66 ± 0.24 6.95 ± 0.31 0.63 ± 0.02 5.81 ± 0.26 0.68 ± 0.08 5.79 ± 0.22 0.85 ± 0.17 4.06 ± 0.19 0.47 ± 0.04 5.08 ± 0.31 0.29 ± 0.02 5.44 ± 0.29 1.43 ± 0.08 4.33 ± 0.31 0.35 ± 0.06 2.65 ± 0.20 1.68 ± 0.12 0.23 ± 0.03 2.73 ± 0.15 0.91 ± 0.09 2.74 ± 0.30 3.09 ± 0.28 4.04 ± 0.30 0.62 ± 0.08 2.37 ± 0.18 2.75 ± 0.21 2.35 ± 0.12 0.72 ± 0.10 0.65 ± 0.04 0.71 ± 0.08 64.35 ± 1.84
0.12 ± 0.05 0.11 ± 0.02 0.09 ± 0.07 0.12 ± 0.03 0.26 ± 0.10 1.97 ± 0.14 tr 0.28 ± 0.08 0.86 ± 0.16 0.45 ± 0.07 10.59 ± 1.21 0.43 ± 0.14 1.36 ± 0.26 3.27 ± 0.24 1.46 ± 0.35 12.32 ± 1.24 0.08 ± 0.02 1.48 ± 0.53 1.18 ± 0.05 6.37 ± 1.42 0.67 ± 0.14 8.42 ± 1.25 0.67 ± 0.07 5.54 ± 0.21 0.41 ± 0.04 5.26 ± 0.28 1.63 ± 0.12 4.42 ± 0.22 0.52 ± 0.07 2.76 ± 0.11 1.85 ± 0.25 0.27 ± 0.06 2.95 ± 0.24 1.03 ± 0.17 2.86 ± 0.12 3.32 ± 0.31 3.85 ± 0.26 0.86 ± 0.19 2.42 ± 0.21 2.86 ± 0.17 2.44 ± 0.15 0.76 ± 0.08 0.57 ± 0.10 0.87 ± 0.14 47.44 ± 2.31
0.55 ± 0.04 0.75 ± 0.08 2.84 ± 0.12 tr 3.93 ± 0.28 0.65 ± 0.11 0.52 ± 0.07 7.01 ± 1.02 tr 14.64 ± 1.32 1.14 ± 0.34 2.51 ± 0.08 34.09 ± 2.13 0.40 ± 0.08 9.52 ± 0.17 0.91 ± 0.14 2.17 ± 0.15 12.33 ± 1.56 tr 1.02 ± 0.21 0.84 ± 0.17 0.42 ± 0.12 tr 2.74 tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr 94.69 ± 1.21
tr – tr – tr – 0.01 tr – 0.04 – 0.07 0.19 – 0.07 – 0.09 0.15 – 0.01 – – tr tr – tr – tr – tr – tr tr – tr – tr – tr – tr tr – tr
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± 0.00
± 0.00 ± 0.01 ± 0.00 ± 0.00 ± 0.00 ± 0.01 ± 0.00
tr means < 0.05. — indicates that the fatty acid is not BCFA. NUC: non-urea complex. UC: urea complex.
milk fat substitutes for infant formulas. The improved method was very effective in enriching BCFAs. The successful preparation of high-valueadded BCFAs is beneficial for the cost reduction and sustainable utilization of lanolin.
Declaration of interest statement The authors have declared no conflict of interest.
Acknowledgements This study was financially supported by “National Natural Science Foundation of China (Grant No.: 31701559 and Grant No.: 31972035)” and “The Natural Science Foundation of Jiangsu Province (Grant No: BK20150137)”.
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