Yingxi Gao, Jiayi Shi, Jie Wang, Fan Zhang, Shichao Tian, Zhiyong Zhou*, Zhongqi Ren*

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China

Keywords:

ABSTRACT

1. Introduction

Nervonic acid(C24:1 Δ15,cis-15-tetracosenoic acid,NA)is a ω-9 long-chain unsaturated monoethylenic fatty acid [1,2]. Nervonic acid is abundant in nerve and brain tissues and is an important part of biofilm. Nervonic acid is recognized by scientists from all over the world as the world’s first and only dual-effect magic substance that can repair and unblock damaged nerve pathways in the brain,nerve fibers, and promote nerve cell regeneration [3,4].

Nervonic acid can completely pass through the blood–brain barrier,directly act on nerve fibers to repair and dredge,regenerate damaged and shed protective sheaths, dissolve necrotic tissues that block channels, and induce self-growth and division of nerve fibers,which activates damaged,diseased and dormant nerve cells and reshapes neural networks to restore some or all the patient’s functions in language, memory, sensation, limbs, etc., to achieve a complete recovery from encephalopathy. Yamazaki et al. [5]found that the proportion of nervonic acid in serum lipids was associated with serum levels of plasmalogens and with metabolic syndrome, and probably reflected the peroxisomal dysfunction and enhancement of endoplasmic reticulum stress seen in common age-related diseases. Hu et al. [6] found that NA could alleviate the 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induc ed behavioral deficits dose-dependently, and NA significantly restored striatal dopamine, serotonin, and metabolites. Tanaka et al.[7]found that Lunaria oil rich in nervonic acid could also help treat Zellweger syndrome. After taking it for two weeks, the patient’s nervous system development, cholestasis symptoms and liver function were improved.Further research found that consuming vegetable oils rich in nervonic acids could repair damaged myelin sheaths, thereby treating demyelinating diseases [8]. Various special physiological functions of nervonic acid related to biofilms have received great attention in recent years [9–12].

Nervonic acid was first discovered in the brain nerve tissue of mammal. Japanese scholars extracted nervonic acid from shark oil in 1926 and confirmed it for the first time as a cis structure,thus, it is also called shark acid [13–15]. In the early days, the source of nervonic acid mainly depended on hunting sharks. Due to the upsurge of international protection of marine life and the prohibition of shark hunting, the source of nervonic acid caused discussions,and the extraction of nervonic acid from plants began to receive attention from all walks of life.

Research shows that a small amount of nervonic acid also exists in plants (Table S1, in Supplementary Material), including Lunaria annua (honesty), Acer truncatum Bunge (purpleblow maple),Tropaeolum speciosum(flame flower),Malania oleifera and Cannabis sativa (hemp), etc. [16–19]. Among them, Malania oleifera is the plant with the highest natural nervonic acid content, and the nervonic acid content is as high as 60%–70%. However, the inherent biological characteristics of Malania oleifera are not conducive to the development of the population, and is listed on the International Union for Conservation of Nature’s Red List of Threatened Species, and can’t be used as a source of nervonic acid. Lunaria annua and Tropaeolum speciosum contain 20%–40% nervonic acid,but the oil content of its plant seeds is too low.In addition,the lignocellulosic acids contained in Tropaeolum speciosum can cause the etiology of adrenoleukodystrophy and multiple sclerosis. Therefore, greater economic cost is required to separate lignocellulosic acid from nervonic acid [8].

The most noteworthy among all the plants currently containing nervonic acid is Acer truncatum Bunge. Acer truncatum Bunge seed kernel is the main material for the fat and protein with fat content as high as 47.88%, which facilitates the extraction of oils. Studies have shown that Acer truncatum Bunge oil is non-toxic and edible,which contains 4%–6% of nervonic acid [19,20]. Acer truncatum Bunge is a renewable energy tree species because its biomass can produce about 30 kg of fruit per tree after 20 years. The nervonic acid containing tree species represented by Acer truncatum Bunge have high oil content in seeds and rich resources, and have great potential to become the main resource for nervonic acid extraction[21].

The fatty acid composition of Acer truncatum Bunge oil is listed in Table S2.Acer truncatum Bunge oil contains more than ten types of fatty acids,of which erucic acid and nervonic acid are very similar in structure with only two carbon difference, which are difficult to separate by conventional methods. In addition, the saturated fatty acids like tetracosanoic acid and nervonic acid only have the difference in saturation,which further increases the difficulty of separation of nervonic acid. The wide variety of structurally similar fatty acids in Acer truncatum Bunge oil makes large-scale preparation of high-purity nervonic acid very difficult.

Based on the fatty acid composition of Acer truncatum Bunge oil,researchers have conducted numerous studies on the isolation and purification of nervonic acid. Zhang et al. [22] further studied the separation of nervonic acid by molecular distillation, and sixstage molecular distillation enriched nervonic acid from 6 % to 78.35 %. However, since the mean free paths of C22 fatty acids and nervonic acid are very close to each other,multi-stage molecular distillation can’t completely separate C22 fatty acids either.Other commonly methods for separating unsaturated fatty acids like urea inclusion can only separate specific fractions of oleic and linoleic acids,etc.,from Acer truncatum Bunge oil.Studies have shown that the wide variety of fatty acids in Acer truncatum Bunge oil makes it impossible to extract high purity nervonic acid by a single method.

Since the purification of nervonic acid obtained by a single separation method is limited,researchers often apply a combination of processes to produce high-purity nervonic acid. Due to the presence of fatty acids in the oil such as erucic acid and lignocaine,which are similar in structure to nervonic acid, only chromatographic methods can completely remove them and obtain high purity nervonic acid. However, the disadvantages of column chromatography,such as small processing capacity,high cost and difficulty in industrial mass production, limit the industrialization of the preparation of high-purity nervonic acid. Liu et al. [23] used the preparative liquid chromatography instead of column chromatography to increase production of high-purity nervonic acid,and enriched nervonic acid in Acer truncatum Bunge oil from 5.05% to 95.8%. The preparative liquid chromatography also has the problems of expensive equipment and high production cost.Therefore, to efficiently prepare high-purity nervonic acid on a large scale, it is still necessary to find an alternative to chromatography.

Accordingly, in this study, a novel conventional combination process of molecular distillation-urea inclusion-solvent crystallization was proposed to enrich the nervonic acid in Acer truncatum Bunge oil. Among them, solvent crystallization was used to separate erucic acid and nervonic acid for the first time, which opened a new direction for the preparation of high-purity nervonic acid.In general, the objective of this work was to investigate the optimal conditions of extraction of high purity nervonic acid, including molecular distillation, urea inclusion and solvent crystallization,and explain the origin of the overall process route. This will facilitate the industrial preparation of high purity nervonic acid.

2. Materials and Methods

2.1. Materials and chemicals

Acer truncatum Bunge seeds (oil content of 46.09%) were purchased from Shaanxi Baofeng Garden Technology Engineering Co., Ltd (Baoji, China). Acer truncatum Bunge seeds were cracked and the kernels obtained were used for oil extraction. The kernels were dried in an oven at 70°C for 6 h to reach moisture content of 2.97%.

Ethanol absolute, propan-2-ol, n-propanol, methanol absolute and acetone were provided by Sinopharm Group Pharmaceutical Co.,Ltd(Beijing,China).Anhydrous sodium sulfate,n-butyl acetate,geranyl acetate, ethyl lactate and diethyl succinate were provided by McLean Technology Co.,Ltd(Shanghai,China).Petroleum ether,ethyl acetate and n-hexane were provided by Tianjin Damao Chemical Reagent Co., Ltd (Tianjin, China). Heptane, tetraethyl orthosilicate and pentanol were provided by Aladdin reagent Co.,Ltd (Shanghai, China). Potassium hydroxide was provided by J&K Scientific Ltd (Shanghai, China).

2.2. Free fatty acid preparation by saponification (SPOMFs)

SPOMFs(150 g)was mixed with 10 g KOH and 150 ml 95%ethanol, and the mixture was continuously stirred in an oil bath at 90°C for 5 h.After the completion of the reaction,20%hydrochloric acid was slowly added to adjust the pH to 2–3.Then 60–90 petroleum ether(25 ml)were added,and the mixture was transferred to a separating funnel to stand for separation. The petroleum ether layer was washed with water until neutral. Then, the extraction production was concentrated on a rotary evaporator at 80 °C to obtain SPOMFs.

2.3. Two-step molecular distillation processes

Molecular distillation was first used to remove short carbon chain fatty acids of C18–C20, including palmitic acid, stearic acid,oleic acid,linoleic acid and linolenic acid.The experimental molecular distillation apparatus used in this work is a self-made scraped membrane molecular distillation device (Beijing, China). The distance between the heating surface and the condensing surface,the evaporation area, the condensing area directly opposite to the evaporation surface, and the area of the complete inner condenser tube are 14 mm, 1.8 dm2, 0.56 dm2and 1.12 dm2, respectively. The condenser temperature was set at 25 °C, the feed temperature was kept at 40 °C, the feeding flow was around 40 g?h-1, the operation pressure was maintained at 0.12 Pa, rotor speed was set at 300 r?min-1, and the evaporation temperature was 130 °C. In the second step molecular distillation process, the feed rate was reduced to 35 g?h-1, and the other conditions remained the same.Finally,the composition of fatty acid was analyzed by gas chromatography (GC, 8890, Agilent, USA) and the yield of nervonic acid was calculated after two-step molecular distillation processes.

2.4. One-step urea complexation process

First,3 g urea and 60 ml anhydrous ethanol were added in a 150 ml flask, and the mixture was stirred in an oil bath at 80 °C for 10 min. Then 5 g mixed fatty acids were added after the urea was completely dissolved. The reaction was refluxed for 20 min until the solution was clarified. Then the solution was taken out and cooled to room temperature, and encapsulated at –5 °C for 5 h.The urea inclusion crystals were separated by filtration, and the crystalline and filtrate fractions were obtained. The filtrate part was heated at 60 °C under reduced pressure rotary evaporation to remove impurities like ethanol, evaporated dry and dissolved with distilled water to remove urea. The upper organic phase was extracted with petroleum ether and separated with standing.The crude nervonic acid was obtained by rotary evaporation under reduced pressure at 60 °C to remove petroleum ether and water.

2.5. Five-step solvent crystallization processes

In general, SPOMFs of 0.5 g was mixed with a solvent at a certain ratio, and then the mixture was allowed to crystallize at a selected temperature. At the end of the crystallization, the liquid fraction was removed by low-temperature filtration to obtain the crystals which were rich in nervonic acid.

The optimization of several parameters including crystallization type of solvent, crystallization temperature, crystallization time and substrate ratio of SPOMFs to solvent was studied. Sixteen solvents including pentanol, propan-2-ol, n-propanol, ethanol absolute, n-butyl acetate, geranyl acetate, ethyl lactate, diethyl succinate,methanol absolute, acetone, petroleum ether, 90% ethanol, ethyl acetate, n-hexane, heptane and tetraethyl orthosilicate were used for screening.Finally,the first-stage solvent crystallization solvent was pentanol, the crystallization temperature was–9 °C, the crystallization time was 5.8 h and the substrate ratio of SPOMFs to solvent was 1:7.2.

As a lot of erucic acid still remained in the crystal,the second to fifth crystallization processes were conducted for further concentration of nervonic acid. The second to fifth crystallization processes were conducted by mixing 0.5 g nervonic acid product obtained from the previous crystallization process with 2 ml ethanol absolute at–18°C for 4 h.The resulting crystals were separated from the liquid by filtration. The fatty acid composition was analyzed by GC.

2.6. Gas chromatography analysis

The SPOMFs (0.01 g) was added into a 20 ml colorimetric tube with stopper and mixed with 5 ml 2% sulfuric acid ethanol solution. Then, the colorimetric tube was placed in a water bath at 60 °C, and allowed to react for 1 h. After reaction, the mixture was brought to room temperature and treated with 5 ml water and 5 ml n-hexane. The mixture was vigorously shaken. The organic phase was separated and fatty acid compositions of the SPOMFs were analyzed by GC.

The compositional analysis of fatty acid ethyl ester was performed using an Agilent GC equipped with a capillary column(DB-Fast FAME,30 m ×0.25 mm I.D., 0.25 μm film thickness,Agilent). The oven temperature program was as follows: 80 °C for 0.5 min, increased to 165 °C at 40 °C?min-1, held for 1 min, then increased to 230 °C at 4 °C?min-1, held for 4 min. 1 μl aliquot of FAMEs was injected into the column using a 100:1 split injection.The split temperature was set as 250 °C and the FID temperature was set as 260 °C.

3. Results and Discussion

3.1. Process route of separation and purification of nervonic acid

According to SPOMFs prepared from Acer truncatum Bunge oil(Table S2), it was segmented into C18–C20 short chain fatty acids containing palmitic acid, stearic acid, oleic acid, linoleic acid, cis-11-eicosenoic acid and linolenic acids occupying 72.92%of SPOMFs(63.04% C18 and 9.88% C20), C22–C24 long-chain saturated fatty acids containing docosanoic acid and tetracosanoic acid, C22–C24 long chain unsaturated fatty acids containing erucic acid and nervonic acid.

3.1.1. Separation and purification of nervonic acid by single method

Molecular distillation mainly separates substances by the difference in the mean free path of a particle of different substances.As can be seen from Table S3,when the distillation temperature is 130 °C and the system pressure is 0.12 Pa, the mean free paths of short chain fatty acids like palmitic acid and oleic acid in SPOMFs are greater than the distance between the condensing surface and the heating surface as 14 mm. However, the mean free paths of long carbon chain fatty acids like erucic acid and nervonic acid are less than 14 mm. Therefore, molecular distillation can quickly separate short carbon chain fatty acids from SPOMFs based on differences in the mean free path of fatty acids.For erucic acid,which has a similar structure to nervonic acid and a similar molecular mass,the molecular distillation conditions were set with a distillation temperature as 135°C and a system pressure as 0.11 Pa.Under these conditions, the mean free path of erucic acid is 14.10 mm,which is greater than the spacing of 14 mm. However, the mean free path of nervonic acid is 13.35 mm,which is only 0.65 mm different from the spacing of 14 mm.Thus,it is difficult to effectively separate nervonic acid from erucic acid by molecular distillation.

As shown in Table 1,the purity of nervonic acid was only 66.32%even after six-stage molecular distillation, and the remaining 28.34% of erucic acid was difficult to be separated from nervonic acid by molecular distillation.

Solvent crystallization is mainly used to separate and purify different fatty acids by the difference of their solubility in the solvent[24,25].Among SPOMFs,unsaturated fatty acids such as oleic acid,linoleic acid and cis-11-eicosenoic acid are soluble in the solvent and will remain in the solvent.Saturated fatty acids such as palmitic acid and stearic acid, which have lower solubility, can crystallize, precipitate out and separate from unsaturated fatty acids.

Table 1 Fatty acid composition after six-stage molecular distillation

Table 2 Crystalline fatty acid composition after three-stage solvent crystalline

As can be seen from Table 2,three-stage solvent crystallization reduced the content of short chain unsaturated fatty acids to about 1%,while erucic acid and nervonic acid were enriched after precipitation of saturated fatty acid due to the small solubility of long carbon chain,indicating that solvent crystallization can effectively separate short chain unsaturated fatty acids. As shown in Table 3,four-stage solvent crystallization could reduce the erucic acid content from 46.05% to 18.43%. It shows that solvent crystallization could separate erucic acid and nervonic acid by the difference of their solubility when they occupied the main components in the raw material of solvent crystallization, which forged a new direction for the separation of erucic acid and nervonic acid.

In summary, the separation and purification of nervonic acid from Acer truncatum Bunge oil by a single separation method,either molecular distillation or solvent crystallization, could only separate specific components and could not produce high-purity nervonic acid.Therefore,a combination process is needed to separate the components in sequential process.

3.1.2. Separation and purification of nervonic acid by combined process

Since the purification of nervonic acid by a single separation method could only separate a specific segment of the component,a large amount of fatty acid residues like erucic acid and tetracosanoic acid were still left, and high purity nervonic acid could not be obtained.Based on the separation results of a single method,a new separation method was introduced for separating residual fatty acids to form a combined process.

Molecular distillation could quickly separate short carbon chain fatty acids. However, for erucic acid, which has a similar structure to nervonic acid, it is difficult to separate efficiently by molecular distillation.In addition,solvent crystallization could be used based on the solubility difference of nervonic acid and erucic acid in the solvent. These two methods could be combined to form a joint three-stage molecular distillation and five-stage solvent crystallization processes.

As shown in Table 4,after three-stage molecular distillation,the content of short chain fatty acids like palmitic acid and oleic acid were reduced to less than 1%. Meanwhile, the content of nervonic acid increased to 41.31%, and the remaining major impurity was 50.19% content of erucic acid. Then the recombinant fraction obtained from the third stage molecular distillation was subjected to five stage solvent crystallization, the results of which showed that the erucic acid content was reduced to 16.59%,and the purity of nervonic acid increased to 74.45%. It is significantly that better effect of purification of nervonic acid could be obtained by the combined process compared with a single method. However, during solvent crystallization process, long carbon chain saturated fatty acids like docosanoic acid and tetracosanoic acid were enriched from 2.0% to 8.6%, which limited the separation and purification performances of high-purity nervonic acid, and required the addition of separation methods to the combined process to remove the enriched saturated fatty acids.

Since urea can form stable complexes with saturated fatty acids in the crystallization process,while unsaturated fatty acids are not easily encapsulated due to their complex spatial configuration,urea inclusion can effectively separate saturated and unsaturated fatty acids.In the combined process of three-stage molecular distillation and five-stage solvent crystallization, some saturated fatty acid impurities still existed. In addition, since the target product of nervonic acid is an unsaturated fatty acid, urea inclusion can be used to remove saturated fatty acid by adding urea package to the combined process.

Therefore, two new combination processes were proposed. The first one was that solvent crystallization was first used to enrichdocosanoic acid and tetracosanoic acid to a certain concentration after molecular distillation, then they could be removed by urea inclusion, and finally erucic acid was removed by solvent crystallization. The second one was that docosanoic acid and tetracosanoic acid were directly removed by urea inclusion at low concentration after molecular distillation, and finally erucic acid was remove by solvent crystallization to obtain high purity nervonic acid. As can be seen from Table 5, during the first combination process, docosanoic acid and tetracosanoic acid could not be completely removed using urea encapsulation at high concentration,resulting in continued enrichment in subsequent solvent crys-tallization. As can be seen from Table 6, during the second combination process, behenic acid could be removed better at low concentration by urea encapsulation, and behenic acid remained at very low concentration after urea encapsulation and did not continue to be enriched in subsequent low-temperature crystallization.

Table 5 Fatty acid composition obtained after two-stage molecular distillation, three-stage solvent crystallization, one-stage urea inclusion and two-stage solvent crystallization

Table 6 Fatty acid composition obtained after two-stage molecular distillation,one-stage urea inclusion and five-stage solvent crystallization

3.1.3.Purification of nervonic acid from Acer truncatum Bunge oil by a combined process of molecular distillation,urea inclusion and solvent crystallization

Separation and purification of high purity nervonic acid from Acer truncatum Bunge oil using the integrated process of molecular distillation, urea inclusion and solvent crystallization was established (Fig. 1). Among the combined process, the short chain fatty acids and long chain saturated fatty acids can be separated by molecular distillation and urea inclusion, while the residual short chain fatty acids and erucic acid can be finally separated by solvent crystallization to obtain high purity nervonic acid.

3.2. Optimization of molecular distillation parameters

3.2.1.Effect of evaporation temperature on enrichment performance of nervonic acid

Fig. 1. Combination process of molecular distillation, urea inclusion and solvent crystallization for purification of nervonic acid from Acer truncatum Bunge oil.

Molecular distillation can be used for the separation of homologs and mixtures with different molecular weights and partial vapor pressures. In general, molecular distillation is conducted under vacuum condition,which decreases the evaporation temperature and the residence time[26,27].The chain length and number of double bonds of a fatty acid affect its boiling point,and the distillation order follows the sequence palmitoleic acid > linoleic acid > stearic acid > oleic acid > linolenic acid > cis-11-eicosenoic acid>erucic acid>nervonic acid.When the temperature is higher than 129°C and the pressure is 0.12 Pa,cis-11-eicosenoicacid,erucic acid and nervonic acid are expected to remain in the residue while palmitoleic acid,linoleic acid,stearic acid and oleic acid will be collected in the distillate.

In the case of fixed system pressure, temperature is the main factor affecting the average molecular free range (Table S4). As shown in Fig. 2(a), with the gradual increase of temperature, the average molecular free range of fatty acids increase, and the content of short carbon chain fatty acids like oleic acid, linoleic acid and stearic acid, whose average molecular free range is greater than the gap distance between the condensation surfaces of the heating surface, gradually decrease in the fatty acid mixture. Long chain fatty acids like nervonic acid and erucic acid are always enriched in the recombinant fraction due to their small average molecular free range, and the content of nervonic acid gradually increases. After the temperature exceeds 130 °C, the average molecular free range of short carbon chain fatty acids like oleic acid and linoleic acid is much larger than the molecular distillation gap distance,which entrains away part of the nervonic acid during the separation process,resulting in a rapid decrease in the content and yield of nervonic acid.Accordingly,130°C was selected as the optimal temperature for molecular distillation.

3.2.2. Effect of feed rate on enrichment performance of nervonic acid

In the process of molecular distillation, the feed rate of raw materials determines the stay time of the raw materials on the inner wall of the distillation device, which has a great influence on whether the separation is enough. When the feed rate is low,raw materials stay on the inner wall of the distillation device for too long, which leads to the increase of heating time and the proportion of nervonic acid distilled with the light components,resulting in decrease of the purity and yield of nervonic acid.As shown in Fig.2(b),when the feed rate is 30 g?h-1,the heating time of the raw materials is suitable,leading to good separation effect. As the feed rate continues to increase,the heating time of the raw materials is shortened, resulting in no separation of the light components.Therefore, the content of the light components in the heavy components increases, and the content of nervonic acid decreases accordingly. However, when the feed rate increases, the nervonic acid is entrained by great decrease of light components, and the yield of nervonic acid increases rapidly. Accordingly, 40 g?h-1was selected as the optimal feed rate for molecular distillation.

3.2.3. Effect of wiper rolling speed on enrichment performance of nervonic acid

The rotation speed of the wiper affects the thickness and uniformity of the film formed on the inner surface of the heater, and is also one of the factors affecting the separation performance. With the speed of the film scraper increases,the centrifugal force on the rotor of the film scraper becomes larger, the scraper fits more closely with the wall,the liquid film formed becomes thinner,and the renovation of the liquid film is accelerated, which is conducive to heat transfer.As shown in Fig.2(c),with the increase of the scraper speed,the distillation efficiency increased,and the content of short chain fatty acids like oleic acid and linoleic acid in the recombinant fraction decreased, while the content of long chain fatty acids like nervonic acid and erucic acid increased significantly.The film scraper speed exceeding 300 r?min-1will make the rotor head wave aggregation serious, which not only leads to the increase of heat transfer coefficient fluctuation, but also generates liquid splash and reduces molecular efficiency. Therefore, 300 r?min-1was selected as the optimal scraper speed for primary molecular distillation.

3.2.4. Effect of feed temperature on enrichment performance of nervonic acid

The feed temperature determines the viscosity of the raw material, directly affecting the fluidity of the raw material in the separation device, making it as one of the factors affecting the separation effect. If the viscosity of the raw material is too large and the evaporation area used for preheating the raw material in the distillation unit is large, the effective evaporation area and the purity of the nervonic acid in the product are low. As shown in Fig.2(d),with the increase of the feed temperature,the viscosity of the raw material decreases and the fluidity increases obviously,resulting in decreasing the evaporation area for preheating raw materials and increasing the effective evaporation area. The light components are sufficiently distilled,leading to increasing the purity of nervonic acid in the product, but the yield of nervonic acid decreases.Accordingly,40°C was selected as the optimal feed temperature for molecular distillation.

3.2.5. Effect of two-stage molecular distillation on enrichment performance of nervonic acid

In summary, the optimal parameters of one-stage molecular distillation consisted of evaporation temperature as 130 °C, the feed rate as 40 g?h-1, the wiper rolling speed as 300 r?min-1, and the feed temperature as 40 °C. After one-stage molecular distillation process,the content of C18–C20 short carbon chain fatty acids was reduced from 65.09% to 29.41%, demonstrating great effect of the molecular distillation. However, the content of short carbon fatty acids as 29.41%is still high,and a second-stage molecular distillation process is needed. The conditions of the second-stage molecular distillation process were slightly adjusted based on the results of the first-stage molecular distillation process. After the first-stage molecular distillation process,the content of low carbon fatty acids has been greatly reduced.By reducing the feed rate,the entrainment of nervonic acid by light components was reduced,thereby improving the yield and content of nervonic acid.The optimal parameters of second-stage molecular distillation process consisted of evaporation temperature as 130 °C, the feed rate as 35 g?h-1,the wiper rolling speed as 300 r?min-1,and the feed temperature as 40 °C. As shown in Table 7, after two-stage molecular distillation processes,the content of short-carbon chain fatty acids was reduced from 29.41% to 13.02%, the content of oleic acid was reduced to 5.21%, the content of linoleic acid was reduced to 6.37%, and the short-chain fatty acids were basically removed.The remaining main components were long carbon chain fatty acids, including unsaturated fatty acids cis-11-eicosenoicacid and erucic acid,saturated fatty acids docosanoic acid and tetracosanoic acid.

3.3. Optimization of urea inclusion parameters

Urea inclusion can separate fatty acids according to differences in fatty acid carbon chain length and saturation.The ease of formation and stability of urea inclusion complexes increases with theincrease of chain length. The straight chain saturated fatty acids have small molecular diameter and spatial structure, and are easy to enter the hexagonal channel of urea crystals to form relatively stable inclusion complexes. However, due to the presence of double bonds, unsaturated fatty acids have an enlarged spatial structure, making it difficult to form stable inclusion compounds.Therefore, the urea inclusion ability follows the sequence: tetracosanoic acid > docosanoic acid > nervonic acid > erucic acid. By controlling the addition amount of urea,docosanoic acid and tetracosanoic acid can be included, and nervonic acid and erucic acid can remain in solution. According to the calculation of urea inclusion, the theoretically required amount of urea to remove docosanoic acid and tetracosanoic acid was 0.19 g?g-1SPOMFs.

As shown in Fig.3(a),with the increase of urea dosage,the contents of docosanoic acid and tetracosanoic acid decreased. When the dosage of urea was higher than 0.6,the contents of docosanoic acid and tetracosanoic acid remained basically constant. The content of docosanoic acid was 0.21%,and the content of tetracosanoic acid was 0.07%. Due to the extremely low content of docosanoic acid, continues increasing the dosage of urea make urea preferentially included nervonic acid,resulting in a gradual decrease in the yield of nervonic acid.

3.4. Optimization of solvent crystallization parameters

3.4.1. Screening of solvent

After two-stage molecular distillation and one-stage urea inclusion separation, it can be seen from Table 8 that the remaining components are mainly 21.35% short carbon chain fatty acids and 45.78% erucic acid. Short carbon chain fatty acids and nervonic acids have a large difference in carbon number and solubility.However, the carbon number of erucic acid is close to that of nervonic acid,and the solubility gap is small.Short carbon-chain fatty acids and erucic acid can be separated by solvent crystallization with different solvents. Therefore, the solvent was first screened with the purity and yield of nervonic acid as the main evaluation criterion and the results are shown in Fig. 3(b).

It can be seen from Fig. 3(b) that the increase amount of nervonic acid by more than 13% was mainly obtained by alcohols and esters. Other commonly used solvents such as acetone, nhexane and petroleum ether had little effect on increasing the content of nervonic acid. Among these two types of solvents with excellent effects, alcohol solvents showed better increasing effect.As shown in Fig.3(c),with the carbon number of the alcohol gradually increased,the polarity of the solvent decreased,and the crystal purity of nervonic acid increased,demonstrating that the weak polar alcohol solvent was beneficial to the first-stage solvent crystallization. Finally, pentanol with the highest purification performance was selected as the solvent for the first-stage solvent crystallization.

Fig. 3. Influences of urea/SPOMFs mass ratio (a), solvent type (b) and alcohol solvent type (c) on enrichment behavior of nervonic acid.

Table 8 Fatty acid composition obtained by urea complexation

The main goal of the one-stage solvent crystallization process was to remove the residual short chain unsaturated fatty acids.Three factors, crystallization temperature, crystallization time and SPOMFs/pentanol ratio,which affect the separation and purification performances of primary low-temperature crystallization,were investigated.

As shown in Fig.4(a),contrary to the results of most literatures,with the gradual increase of crystallization temperature,the purity of nervonic acid gradually increased, but the purity of erucic acid gradually decreased[28,29].The reason may be that nervonic acid quickly reached saturation solubility and then precipitated rapidly at low temperature,but some erucic acid was entrained in this process,resulting in lower purity of nervonic acid.As the temperature gradually increased,the saturated solubilities of nervonic acid and erucic acid gradually increased.However, the increase rate of nervonic acid might be much higher than that of erucic acid,resulting in a larger difference between the solubility of nervonic acid and erucic acid, which is conducive to the separation of erucic acid and nervonic acid. Therefore, –9 °C was selected as the optimal crystallization temperature.

However,the precipitation rate of nervonic acid was slow at–9°C. As shown in Fig. 4(b), with the increase of crystallization time,nervonic acid gradually precipitated and the content increased.The content of nervonic acid in the crystals reached the peak at 6 h.With the prolongation of crystallization time,other fatty acids with a slightly higher solubility than nervonic acid, such as docosanoic acid and cis-11-eicosenoicacid, were precipitated in large quantities,resulting in gradual decreasing in the content of nervonic acid in the crystal.

Fig. 4. Effects of crystallization temperature (a), crystallization time (b) and SPOMFs/pentanol ratio (c) on enrichment behavior of nervonic acid by one-stage solvent crystallization.

As shown in Fig. 4(c), as the dosage of solvent increased, the concentration of the solution decreased. When the temperature was lowered at a certain speed, the primary nucleation phenomenon was not easy to occur, and the crystal growth rate was slow. Meanwhile, the viscosity of the solution was small, leading to low impurities adsorbed on the surface of the crystal,which was easy to be eluted. In this paper, with the increase of the dosage of pentanol, the crystallization rate of nervonic acid decreased, and the content of highly soluble substance like oleic acid and linoleic acid adsorbed on the crystallization decreased too.When the ratio of SPOMFs to solvent was 1:7,the nervonic acid content reached its maximum value. Continue to increase the dosage of solvent, the solution concentration was too low, which is not conducive to the precipitation of nervonic acid crystallization, resulting in a decrease in nervonic acid content.

3.4.3. Optimization of one-stage solvent crystallization factors using response surface methodology

The one-stage solvent crystallization factors on nervonic acid enrichment were optimized by response surface methodology(RSM) and the analytical factors and levels of RSM are listed in Table S5. A second-order polynomial equation was established to estimate the relationship between the nervonic acid content and variables. The model could be expressed as Eq. (1):

The results of the analysis of the models are listed in Tables S6 and S7. The determination coefficient (R2= 0.9996) was obtained by variance analysis (ANOVA) of the quadratic regression model,demonstrating a very high degree of precision and a good deal of reliability of the experimental values. The model was found to be adequate for prediction within the range of experimental variables.The linear terms(X2, and X3), and all the quadratic terms(X12, X22,and X32) were of highly significant with very small P-values(P < 0.0001). However, the linear term X1was not so significant with a higher P-value(P=0.6569).The reason was that in a smaller temperature range, the temperature had little change in the solubility difference between nervonic acid and erucic acid. Therefore,temperature showed little effect on increasing nervonic acid content.

As shown in Fig. S1(a) and (c), the content of nervonic acid increased with the increase of time and solvent ratio within a certain range. Since the increase in the dosage of solvent led to a decrease in the concentration of the solution,a decrease in the rate of crystal nucleation, and an increase in the time required for the precipitation of nervonic acid. However, with the gradual increase of crystallization time,other components like erucic acid gradually precipitated,resulting in a decrease in the content of nervonic acid.The interaction between crystallization time and temperature wasalso obvious. As the temperature gradually increased, the time required for the precipitation of nervonic acid gradually increased.

Table 9 Fatty acid composition obtained by one-stage solvent crystallization

Based on the mathematical predicted model, the optimal experimental conditions consisted of crystallization temperature as –9 °C, crystallization time as 6 h and SPOMFs/pentanol ratio as 7.2. Nervonic acid content of 47.32% (Table 9) was obtained under these conditions,which is a good fit for the value forecasted(47.37%) by the regression model. Therefore, the nervonic acid enrichment conditions of the one-stage solvent crystallization obtained by RSM were reliable and practical.

3.5. Multistage solvent crystallization

Since a large amount of erucic acid was still contained in the crystal after the first stage solvent crystallization, multistage solvent crystallization was carried out to remove the erucic acid and further increase the purity of nervonic acid. The purpose of multistage solvent crystallization is to separate erucic acid and nervonic acid. According to the results of solvent screening, anhydrous ethanol was selected as the solvent for subsequent solvent crystallization. The experimental conditions of the second to fifth stage solvent crystallization consisted of crystallization temperature as –18 °C, crystallization time as 5 h and SPOMFs/pentanol ratio as 5. As shown in Table 10, during the subsequent fourstage solvent crystallization, each stage could remove about 10%of erucic acid. In addition, fatty acids other than erucic acid were basically removed, and the final nervonic acid purity reached up to 96.53%.

It can be seen from Table 11 that high-purity nervonic acid could not be obtained from Acer truncatum Bunge oil by a single method at present. In order to obtain high-purity nervonic acid,column chromatography or high performance liquid chromatography should be used.However,these methods have a series of problems such as low efficiency, low yield, large use of toxic and harmful organic solvents, and difficulty in large-scale production.The solvent crystallization used in this paper has the advantages of low energy consumption, simple operation, green environmental protection,and can efficiently separate erucic acid and nervonic acid to prepare high-purity nervonic acid.Therefore,the combined process of molecular distillation, urea complexation and solvent crystallization proposed in this paper provides a novel method and scientific basis for the industrial production of high-purity nervonic acid.

4. Conclusions

In this study, the combination process of two-stage molecular distillation, one-stage urea complexation and five-stage solvent crystallization was proposed for extraction of nervonic acid from Acer truncatum Bunge oil and the final purity of nervonic acid was up to 96.53%. Molecular distillation was proved to be a good method for the rapid removal of short carbon chain fatty acids,and suitable for the initial accumulation of nervonic acid content.The nervonic acid content rapidly increased from 6.08% to 33.26%, and short carbon chain fatty acids decreased from 65.09%to 13.02% after two-stage molecular distillation. The long carbon chain saturated fatty acids like tetracosanoic acid and docosanoic acid could be effectively removed by urea inclusion, which solved the problem of accumulation of saturated fatty acid during solvent crystallization.Solvent crystallization was used for the first time toseparate erucic acid and nervonic acid with very similar structures.Pentanol and anhydrous ethanol as solvents could efficiently separate short carbon-chain fatty acids and erucic acid, respectively.Compared with traditional separation methods,solvent crystallization showed the advantages of simple operation, short process route,low energy consumption,large processing capacity,and easy industrialization. Separation of erucic acid by solvent crystallization created a new direction for large-scale and efficient production of high purity nervonic acid.

Table 10 Fatty acid composition obtained by a combination of two-stage molecular distillation, one-stage urea complexation and five-stage solvent crystallization from Acer truncatum Bunge oil

Table 11 Comparison of separation performances of nervonic acid in this work and reported in literatures

Data Availability

Data will be made available on request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(22125802 and 22078010),Beijing Natural Science Foundation (2222017) and Big Science Project from BUCT(XK180301). The authors gratefully acknowledge these grants.

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2022.10.007.