Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

An efficient method for the purification of arachidonic acid from fungal single-cell oil (ARASCO)

PUFA, such as arachidonic acid (AA), have several pharmaceutical applications. An efficient method was developed to obtain high-purity arachidonic acid (AA) from ARASCO, a single-cell oil from Martek (Columbia, MD). The method comprises three steps. In the first step, AA was enriched from saponified ARASCO oil by low-temperature solvent crystallization using a polar, aprotic solvent, which gave a FA fraction containing 75.7% AA with 97.3% yield. The second step involved enriching AA content via lipase-catalyzed selective esterification of FA with lauryl alcohol. When a mixture of 1 g FA/lauryl alcohol (2∶1 mol/mol), 50 mg Candida rugosa lipase, and 0.33 g water was incubated at 50°C for 24 h with stirring at 400 rpm, the AA content in the unesterified FA fraction was as much as 89.3%, with ca. 90% yield. Finally, a solvent extraction procedure, in which acetonitrile was the extracting solvent, was used to enrich AA from FA fraction dissolved in n-hexane. The best results were obtained when 2 g FA was dissolved in 80 mL hexane and extracted twice, each time with 20 mL acetonitrile at −20°C, by allowing 2 h storage. This step gave a FA fraction containing 95.3% AA with 81.2% yield. By using this three-step process the AA content in the saponified single-cell oil (ARASCO) was increased from 38.8 to 95.3% with a total yield of ca. 71%.     

Production of arachidonic acid by filamentous fungus, Mortierella alliacea strain YN-15

A filamentous fungus producing significant levels of arachidonic acid (AA, C20∶4n−6) was isolated from a freshwater pond sample and assigned to the species Mortierella alliacea. This strain, YN-15, accumulated AA mainly in the form of triglyceride in its mycelia. An optimized culture in 25 L of medium containing 12% glucose and 3% yeast extract yielded 46.1 g/L dry cell weight, 19.5 g/L total fatty acid, and 7.1 g/L AA by 7-d cultivation in a 50-L jar fermenter. Assimilation of soluble starch by YN-15 was notably enhanced by the addition of oleic acid, soybean oil, ammonium sulfate, or potassium phosphate to a starch-based medium. Using starch as a main carbon source in the pre-pilot scale cultivation improved the production of AA by up to 5.0 g/L. Mortierella alliacea strain YN-15 is therefore a promising fungal isolate for industrial production of AA and other polyunsaturated fatty acids.     

藻类花生四烯酸的提取工艺

花生四烯酸作为一种重要的保健、营养品,越来越受到人们的重视。从鱼油、真菌丝、微藻中都可以提取花生四烯酸,但这些工艺都有着明显的缺点。设计了一种从藻类提取花生四烯酸的新工艺过程。     

Enhancement of arachidonic acid production by Mortierella alpina 1S-4

The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O, and 0.05% MgCl₂·6H₂O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH₂PO₄ addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.     

Effect of mustard meal on the production of arachidonic acid by Mortierella elongata SC-208

A strain of Mortierella elongata SC-208 that was isolated from soil of a mustardseed extraction plant can produce arachidonic acid in significant amounts, and it was grown in three different media, one of which contained 0.5% deoiled mustard meal. The arachidonic acid content in the lipid part of dry mycelium (23.2 g/L) was as high as 33% w/w from the medium that contained mustard meal, and the overall yield of arachidonic acid was 0.49 g/L. The arachidonic acid contents in the phospholipid fraction and the triglyceride fraction were 39.5 and 30.2%, respectively.     

Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate

A fungal isolate Wuji-H4 with a dense-lobe rosette growth pattern on malt extract agar was identified as Mortierella alpina Peyronel. It was capable of producing 504 mg/L of arachidonic acid (AA) in the screening medium. Its AA content accounted for 42.4% of the total fatty acids. The AA yield was raised to 1,817 mg/L by a step-by-step approach, which uncovered that the preferred carbon source, nitrogen source, and temperature for fungal growth and lipid production were soluble starch, urea, and 24°C, respectively. Productivity was further optimized by exploiting the interactions between the constituents of the medium by the response surface method. A partial factorial design, followed by steepest ascent analysis, was carried out to locate the general vicinity of the optimal level of each nutrient. The response surface of AA production in this optimal region was then approximated with a full quadratic equation obtained from a three-factor/five-level central composite rotatable design. Maximum AA yield was predicted to occur in a medium that contained 99.7 g/L of soluble starch, 12.6 g/L of yeast extract, and 3.0 g/L of KH₂PO₄. Upon verification, the average experimental yield of AA (3,885 mg/L) was not significantly different from the predicted AA yield (3,940 mg/L), indicating that the response surface method had succeeded in exploiting the AA production potential of this new fungal isolate.     

Kinetics of methyl ester production from mixed crude palm oil by using acid-alkali catalyst

The production of biodiesel from high free fatty acid mixed crude palm oil using a two-stage process was investigated. The kinetics of the reactions was determined in a batch reactor at various reaction temperatures. It was found that the optimum conditions for reducing high free fatty acid (FFA) in MCPO (8-12 wt.%/wt oil) using esterification was a 10:1 molar ratio of methanol to FFA and using 10 wt.%/wt of sulfuric acid (based on FFA) as catalyst. The subsequent transesterification reaction to convert triglycerides to the methyl ester was found to be optimal using 6:1 molar ratio of methanol to the triglyceride (TG) in MCPO and using 0.6 wt.%/vol_TG sodium hydroxide as catalyst. Both reactions were carried out in a stirred batch reactor over a period of 20 min at 55, 60 and 65 °C. The concentration of compounds in each sample was analyzed by Thin Layer Chromatography/Flame Ionization Detector (TLC/FID), Karl Fischer, and titration techniques. The results were used for calculating the rate coefficients by using the curve-fitting tool of MATLAB. Optimal reaction rate coefficients for the forward and reverse esterification reactions of FFA were 1.340 and 0.682 l mol⁻¹ min⁻¹, respectively. The corresponding optimal transesterification, rate coefficients for the forward reactions of TG, diglyceride (DG), and monoglyceride (MG) of transesterification were 2.600, 1.186, and 2.303 l mol⁻¹ min⁻¹, and for the reverse reactions were 0.248, 0.227, and 0.022 l mol⁻¹ min⁻¹, respectively.     

Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S-4

The culture conditions for arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated by means of a morphological study with the aims of obtaining a high AA yield and scale-up. In a 50-L jar fermentor study, a medium containing 3.1% soy flour and 1.8% glucose with 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O and 0.05% MgCl₂·6H₂O was found to be optimum. The AA yield reached 9.8 g/L/7 d, and the major morphology was small pellets (1–2 mm). However, in the case of the only addition of KH₂PO₄, the major morphology was filaments. The apparent viscosity increased to 2240 cp, thereby requiring a high agitation speed to maintain adequate oxygen tension, which caused mycelial damage due to shear stress and therefore a decrease in the AA yield. When a medium with Na₂SO₄, CaCl₂, and MgCl₂ was used, the major morphology was large pellets (2–3 mm), and mass transfer limitation through the pellet wall caused a decrease in the AA yield. Based on these results, a scale-up study was carried out under the optimal conditions described above. An AA yield of 10.9 g/L/8 d was obtained in a 10-kL industrial fermentor, and the major morphology was small pellets.     

Preparation of malvalic and sterculic acid methyl esters from Bombax munguba and Sterculia foetida seed oils

A method has been developed for the preparation of highly pure malvalic (cis-8,9-methyleneheptadec-8-enoic) and sterculic (cis-9,10-methyleneoctadec-9-enoic) acid methyl esters starting from Bombax munguba and Sterculia foetida seed oils. The methyl esters of these oils were prepared by sodium methylate-catalyzed transmethylation followed by cooling (6°C) the hexane solution of crude methyl esters and separation of insoluble fatty acid methyl esters by centrifugation in the case of B. munguba and by column chromatography in the case of S. foetida. Subsequently, the saturated straight-chain fatty acid methyl esters were almost quantitatively removed by urea adduct formation. Finally, methyl malvalate and methyl sterculate were separated from the remaining unsaturated fatty acid methyl esters, in particular methyl oleate and methyl linoleate, by preparative high-performance liquid chromatography on C₁₈ reversed-phase using acetonitrile isocratically. Methyl malvalate and methyl sterculate were obtained with purities of 95–97 and 95–98%, respectively.     

从废弃油脂生物柴油中分离不饱和脂肪酸甲酯

以废弃油脂制生物柴油为原料,以95%醇为溶剂,采用尿素包合法提取不饱和脂肪酸甲酯,为生物柴油联产具有高附加值化工产品打下基础.重点考察了尿素用量、溶剂用量、包合时间和包合温度对不饱和脂肪酸甲酯分离效果的影响.结果表明,尿素包合法从生物柴油中分离不饱和脂肪酸甲酯的适宜工艺条件为:尿素,生物柴油质量比为1.4～1.7,溶剂/生物柴油质量比为4.6～6.0,包合温度为10℃,包合时间为18 h.在适宜条件下,不饱和脂肪酸甲酯含量可达93.5%,收率可达55.8%.     

FTIR estimation of free fatty acid content in crude oils extracted from damaged soybeans

A user-interactive computer-assisted Fourier transform infrared (FTIR) method has been developed for estimation of free fatty acids (FFA) in vegetable oil samples by deconvolution of the infrared (IR) absorbances corresponding to the triglyceride ester and FFA carbonyl bonds. Peak areas were used to determine FFA as a percentage of the total carbonyl areas in weighed standards of refined, bleached, deodorized soybean oil containing from 0 to 5% added oleic acid. These data for percent FFA by FTIR were compared to corresponding FFA data obtained by two titration methods-the AOCS Official Method Ca 5a-40 and the Official Method with a slight modification. Correlation coefficients were 0.999 for the Ca 5a-40, 0.999 for the modified and 0.989 for the FTIR methods. FFA in samples of crude soybean oils extracted from damaged beans (0.5 to 2.1% FFA) were measured by FTIR and compared to data obtained by titration of the same samples (correlation coefficient, 0.869). To whom correspondence should be addressed at National Center for Agricultural Utilization Research, Agriculture Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604. ¹The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

氯代甲氧基脂肪酸甲酯增塑性能的研究

本文以不同比例的氯代甲氧基脂肪酸甲酯替代电缆料配方中的增塑剂DOP,考察了电缆料试片的最大力、抗张强度、断裂伸长率及邵氏硬度等指标。试验表明,当DOP为增塑剂总量40%,氯代甲氧基脂肪酸甲酯为增塑剂总量60%时,电缆料材料的各项性能指标均好于100%DOP的电缆料材料,其最大力为375N、抗拉强度为23.78MPa、断裂伸长率为324.42%、邵氏硬度75.86度。当DOP为增塑剂总量20%,氯代甲氧基脂肪酸甲酯为增塑剂总量80%时,电缆料材料的各项指标性能与100%DOP的电缆料材料性能相当。     

Transesterification processes for vegetable oils: A simple control method of methyl ester content

One of the main problems in the study or industrial application of transesterification processes for vegetable oils is how to measure the methyl ester content. In this work, a quick analytical method was developed for assessing the methyl ester content of purified “fuel grade” transesterification products by applying a simple correlation with viscosity. The correlation was tested on a wide range of samples with various methyl ester contents; the results were in agreement with the values measured by gas-chromatographic analysis. In a defined range of weight fractions the correlation allows for the determination of the methyl ester content of purified transesterification products by a single viscosity measurement. This method is especially suitable for process control purposes as it determines the methyl ester content quickly and simply.     

桐酸甲酯中甘油水洗去除及脱水精制工艺

为制备高纯桐酸甲酯,研究了桐酸甲酯中甘油水洗去除及脱水精制工艺。以桐酸甲酯的含水率及甘油含量为分析指标,研究了精制工艺中的水洗比、脱水真空度、脱水温度及脱水时间等参数的影响。研究表明,桐酸甲酯精制最优工艺条件是:水洗比为3、真空度为80 kPa、温度为80℃、脱水时间为3 h。该条件下精制的桐酸甲酯的含水率为0.122 6%、甘油含量为0.070 2%。     

双液相萃取棉籽油制备脂肪酸甲酯

以双液相萃取技术处理棉籽,在得到脱毒棉粕的同时得到含有高质量毛油的非极性相。以非极性相作为与甲醇进行酯交换反应的原料,得到脂肪酸甲酯和甘油。考察了非极性相溶剂石油醚与棉籽油的比例对酯交换转化率和洗涤粗产品用水量的影响,确定了石油醚与棉籽油的最佳质量比为3,在此条件下,洗涤用水量可降低一半。考察了醇油比、催化剂用量、反应温度、反应时间等参数对转化率的影响。应用正交实验的方法找出酯交换反应的适宜条件为：醇油比6：1,催化剂用量1.1％,反应温度60℃,反应时间120min。在此反应条件下,产物中脂肪酸甲酯的含量可达97.4％。     

强酸性阳离子树脂催化棕榈油副产物合成脂肪酸甲酯

以强酸性阳离子交换树脂为催化剂,经过自制的固定床反应器,使棕榈油脱臭馏出物(PODD)中的脂肪酸与甲醇起酯化反应,合成脂肪酸甲酯。结果表明,用固定床可从PODD连续制备脂肪酸甲酯,酯化反应的最佳条件为:n(甲醇)/n(PODD)=17.3;反应温度在甲醇正常沸点以下时,温度越高,转化率越大;转化率随催化反应时间增大而增大,但增大速度逐渐趋缓。当在常压下,64℃反应56 min时,游离脂肪酸的转化率可达87%左右。     

Synthesis of fatty acid esters from acid oils using lipase B from Candida antarctica

Esterification of corn and sunflower acid oils with straight‐ and branched‐chain alcohols were conducted using lipase B from Candida antarctica (Novozym 435) in n‐hexane. Sunflower acid oil consisted of 55.6% free fatty acids and 24.7% triacylglycerols, while the free fatty acids and triacylglycerols contents of corn acid oil were 75.3% and 8.6%, respectively. After 1.5 h of methanolysis of sunflower acid oil, the highest fatty acid methyl ester content (63.6%) was obtained at 40 °C and the total fatty acid/methanol molar ratio was 1/1, using 15% enzyme based on acid oil weight. The conversion of both acid oils with straight‐ and branched‐chain alcohols was not significantly affected by the chain length of the alcohols. However, the lowest fatty acid methyl ester content (50%) was obtained in the reaction of corn acid oil with methanol. Sunflower acid oil was converted to fatty acid esters using primer alcohols such as n‐propanol, i‐ and n‐butanol, n‐amylalcohols, n‐octanol, and a mixture of amylalcohol isomers, resulting in a fatty acid ester content of about 70% at 40 °C.