Enzymatic degumming

The first enzymatic degumming process to be used industrially was the EnzyMax® process that was launched in 1992; it used porcine phospholipase A2 (PLA2). Subsequently, various microbial phospholipases (PLases) with different specificities have been developed. They have the advantages of being kosher/halal and of having a non‐limited availability and lower cost. The first of these microbial enzymes were the phospholipases A1 (Lecitase® Novo and Ultra) and more recently, a phospholipase C (Purifine®) and a lipid acyl transferase (LysoMax®) with PLA2 acitivity have also become available in commercial quantities. These enzymes have different specificities. The Lecitases® and the LysoMax® enzymes catalyse the hydrolysis of all common phosphatides and differ in this respect from the Purifine® enzyme, which is specific for phosphatidyl choline and phosphatidyl ethanolamine. These phosphatides are hydrolysed to oil‐soluble diacylglycerol and water‐soluble phosphate esters. Since these diacylglycerols remain in the oil during refining, they contribute to the oil yield. That also holds for the sterol and stanol fatty esters formed as a consequence of the phosphatide hydrolysis catalysed by the LysoMax® enzyme. In addition, all enzymes cause less oil to be retained by the gums by decreasing the amount of gums and/or their oil retention, which also contributes to an improved oil yield. On the other hand and contrary to common belief, the enzymes are incapable of catalysing the hydrolysis of non‐hydratable phosphatides (alkaline earth salts of phosphatidic acid) under industrial conditions. Consequently, the industrial enzymatic degumming step has to be preceded by a chemical degumming step to arrive at a degummed oil with a sufficiently low residual phosphorus content that can be physically refined. Accordingly, it might well be preferable to limit the oil treatment to said chemical degumming and produce oil with a low residual phosphorus content and gums, and then treat the gums separately to recover their fatty matter, whereby this recovery can be enzymatic or chemical.     

Enzymatic Degumming of Crude Jatropha Oil: Evaluation of Impact Factors on the Removal of Phospholipids

Jatropha curcas seeds are rich in non‐edible oil, and this plant has received much interest in recent years, especially with respect to biodiesel production. Owing to the high content of phospholipids, crude jatropha oil has to be refined before further use. Conventional refining processes have several environmental and energetic shortcomings. Thus, the search for alternative degumming methods has become relevant. This study compares the enzymatic degumming of screw‐pressed crude jatropha oil with Lecitase Ultra (phospholipase A1) and LysoMax (phospholipase A2). Degumming with phospholipase A2 was less effective that degumming with phospholipase A1. Phospholipase A1 showed the highest reaction rate at 50 °C, 700 rpm stirring, 3 mL of water per 100 g of oil, and with 75 ppm of added phospholipase. To ensure optimum enzyme activity, the pH was adjusted to 5. The phosphorus content was reduced continuously for reaction times up to 3 h. The residual phosphorus content was found to be independent of its initial level. Laboratory experiments showed that enzymatic degumming of jatropha oil with phospholipase A1 at the adapted parameters enables the phosphorus content to be reduced to levels below 4 ppm.     

Fast synthesis of 1,3‐DAG by Lecitase® Ultra‐catalyzed esterification in solvent‐free system

Lecitase® Ultra, a phospholipase, was explored as an effective biocatalyst for direct esterification of glycerol with oleic acid to produce 1,3‐DAG. Experiments were carried out in batch mode, and optimal reaction conditions were evaluated. In comparison with several organic solvent mediums, the solvent‐free system was found to be more beneficial for this esterification reaction, which was further studied to investigate the reaction conditions including oleic acid/glycerol mole ratio, temperature, initial water content, enzyme load, and operating time. The results showed that Lecitase® Ultra catalyzed a fast synthesis of 1,3‐DAG by direct esterification in a solvent‐free medium. Under the optimal reaction conditions, a short reaction time 1.5 h was found to achieve the fatty acid esterification efficiency of 80.3 ± 1.2% and 1,3‐DAG content of 54.8 ± 1.6 wt% (lipid layer of reaction mixture mass). The reusability of Lecitase® Ultra was evaluated via recycling the excess glycerol layer in the reaction system. DAG in the upper lipid layer of reaction mixture was purified by molecular distillation and the 1,3‐DAG‐enriched oil with a purity of about 75 wt% was obtained. Practical applications: The new Lecitase® Ultra catalyzed process for production of 1,3‐DAG from glycerol and oleic acid described in this study provides several advantages over conventional methods including short reaction time, the absence of a solvents and a high product yield.     

Optimization of degumming process for soybean oil by phospholipase B

BACKGROUND: Enzymatic degumming, the ‘EnzyMax® process’, in which a phospholipase (type A₁, A₂ or B) was used to convert nonhydratable phospholipids into their hydratable forms. Compared with conventional methods, enzymatic degumming offers a safe biological route and eco‐friendly solution to industrial processes. To date, only phospholipases A₁ and A₂ have been used for enzymatic oil‐degumming. In this study, phospholipase B from Pseudomonas fluorescens BIT‐18 was applied for the first time in soybean oil degumming. RESULTS: Three major factors (temperature, pH and PLB dosage) were screened out through Plackett–Burman design. Then, response surface modeling combined with central composite design and regression analysis were employed for optimization of the final degumming process. The optimum conditions for the minimum residual phosphorus content in the oil were achieved at 40 °C, pH 4.7 and with PLB dosage of 500 U kg^?1. Under optimal conditions, the residual phosphorus content decreased to 4.9 mg kg^?1, which was comparable with predicted response values. CONCLUSION: These results suggested that Plackett‐Burman design combined with response surface modeling were proved effective in determining the optimum soybean oil degumming conditions. The results also revealed that phospholipase B from Pseudomonas fluorescens BIT‐18 was a good candidate for degumming various vegetable oils. Copyright © 2011 Society of Chemical Industry     

Optimization of enzymatic degumming process for rapeseed oil

An enzymatic process optimization and a largescale plant trial for rapeseed oil degumming were carried out by a novel microbial lipase. Response surface methodology was used to obtain the desired data in the process optimization. Enzyme dosage, temperature, and pH were important determining factors affecting oil degumming. The optimal set of variables was an enzyme dosage of 39.6 mg/kg, a temperature of 48.3°C, and a pH of 4.9. The phosphorus content could be reduced to 3.1 mg/kg at the optimal levels of the tested factors. An enzymatic degumming plant trial was performed on a 400 tons/d oil production line. pH was found to play an important role in degumming performance. When the pH was 4.6–5.1, the corresponding phosphorus content of degummed rapeseed oil could be reduced to less than 10 mg/kg, which met the demands of the physical refining process.     

Enzymatic oil‐degumming by a novel microbial phospholipase

Phospholipase A‐mediated oil‐degumming is a well‐established process step (Enzy‐Max^®) in physical refining of vegetable oils (rape seed, soy bean, sunflower seed). A screening programme for microbial phospholipases of type A has been carried out. The target has been to develop a stable and robust phospholipase with optimal oil‐degumming performance in the pH‐range 4—5 and in the temperature range 30— 70 °C. One phospholipase of type A1 from Fusarium oxysporum, given the trade name Lecitase^® Novo, has been studied in detail. Some of the characteristics of this novel microbial phospholipase in the oil‐degumming application are: pH optimum ∼5, temperature optimum 40—45 °C. In laboratory tests the new phospholipase Lecitase^® Novo has proven to be superior to porcine pancreatic Lecitase^® 10L and other phospholipases with respect to oil‐degumming performance, and it has proven to be suited for degumming of different oil qualities ranging from water‐degummed to crude oil. A further advantage is that the new phospholipase acts at very low water content, which will make the problematic sludge recycling in the EnzyMax^® process superfluous. As demonstrated by an HPLC study, phospholipase‐mediated degumming is a unique process quite distinct from the well‐known acid degumming variations, since the phospholipids (both hydratable and non‐hydratable) present in the oil are hydrolysed to the corresponding lyso‐phospholipids, which migrate to the aqueous phase under the conditions employed. Lecitase^® Novo was introduced successfully for degumming of rapeseed oil at Cereol (Mannheim, Germany) mid 2000.     

Immobilization of Phospholipase A<Subscript>1</Subscript> and its Application in Soybean Oil Degumming

Phospholipase A₁ (PLA₁), or Lecitase^® Ultra, was immobilized on three different supports, calcium alginate (CA), calcium alginate-chitosan (CAC), and calcium alginate-gelatin (CAG), and crosslinked with glutaraldehyde. The results indicated that PLA₁–CA retained 56.2% of the enzyme’s initial activity, whereas PLA₁–CAC and PLA₁–CAG retained 65.5 and 60.2%, respectively. Compared with free PLA₁, the optimal pH of immobilized PLA₁ shifted to the basic side by 0.5–1.0 pH units and the pH/activity profile range was considerably broadened. Similarly, the temperature-optima of PLA₁–CAC and PLA₁–CAG increased from 50 to 60 °C, and their thermal stability increased with relative activities of more than 90% that covered a wider temperature range spanning 50–65 °C. In a batch oil degumming process, the final residual phosphorus content was reduced to less than 10 mg/kg with free PLA₁, PLA₁–CAC and PLA₁–CA in less than 5, 6 and 8 h respectively while PLA₁–CAG was only able to reduce it to 15 mg/kg in 10 h. When the PLA₁–CAC was applied in a plant degumming trial, the final residual phosphorus content was reduced to 9.7 mg/kg with 99.1% recovery of soybean oil. The recoveries of immobilized PLA₁–CAC and activity of PLA₁ were 80.2 and 78.2% respectively. Therefore, it was concluded that PLA₁–CAC was the best immobilized enzyme complex for the continuous hydrolysis of phospholipids in crude vegetable oils.     

Enzymatic degumming

The oldest enzymatic degumming process (the Lurgi EnzyMax® process) was launched in 1992. It used porcine phospholipase A₂, which has the disadvantages of limited availability and not being kosher/halal. To overcome these disadvantages, various microbial enzymes have been developed; they have different specificities and therefore offer different advantages. Phospholipase C for instance has the advantage that it leads to the formation of diacylglycerols that remain in the oil being degummed. This constitutes a significant yield improvement which also results from the formation of lysophospholipids that retain less oil than their precursors. In the laboratory, a fine dispersion of the aqueous enzyme solution in the oil can be maintained so that the phospholipase enzymes can be made to interact with non‐hydratable phosphatides (NHP) in the oil phase and catalyse their hydrolysis. On an industrial scale, dispersions coalesce before the enzymatic NHP hydrolysis is complete. Accordingly, enzymatic degumming processes that claim NHP‐removal and a low residual phosphorus content in the enzymatically degummed oil are invariably preceded by an acid treatment in which a degumming acid (citric acid) is finely dispersed into the oil to be degummed and made to react with the NHP present in the oil before the enzyme is added. This enzyme then only interacts with the phospholipids present in the water phase. This raises the question whether the yield increase resulting from the use of enzymes should be realised by treating the oil to be degummed or the gums that have already been isolated from the oil during a degumming treatment. Lack of experimental evidence prevents a firm answer to this question but the arguments in favour of treating the gums look more impressive than what can be said in favour of treating the oil. In short: Enzymes do not degum the oil but can be used to de‐oil the gums.     

磷脂酶用于大豆油脱胶的研究

植物油的酶法脱胶是一种新的大豆油脱胶方法。利用新型微生物磷脂酶Lecitase Ultra进行大豆油脱胶的研究,探讨了若干操作参数对大豆油脱胶效果的影响,确定了该酶较优的反应条件:反应时间200 min,加酶量25 mg/kg,pH值4.8,温度46~48℃,大豆油含磷量能降到4.7 mg/kg。结果表明,Lecitase Ultra应用于植物油脱胶效果好且稳定,是一种更适宜于工业化应用的酶种。     

Insight into the Enzymatic Degumming Process of Soybean Oil

An enzymatic degumming trial of soybean oil was carried out at a capacity of 400 tons/day by applying microbial phospholipase A₁ from Thermomyces lanuginosus/Fusarium oxysporum. When the pH was kept in the range of 4.8–5.1, less than 10 mg/kg of phosphorous content of The oil was obtained. The gum and oil were easily separated after centrifugation and the oil loss was minimal under the process conditions. Through analysis of phospholipids compounds in the gum by Electrospray Ionization-Mass Spectrometer and phosphorous content, it could be seen that both glycerophospholipids and lysophospholipids existed with contents of 45.7 and 54.3%, respectively. The performance of enzymatic degumming was found to be related to the production of glycerophospholipids.     

A Comparative Study of Phospholipase A1 and Phospholipase C on Soybean Oil Degumming

The degumming of crude soybean oil with phospholipase A₁ (PLA₁) and phospholipase C (PLC) was studied, and optimal conditions were obtained for each enzyme. During degumming with PLA₁, more fatty acid was found in the oil than would be expected by hydrolysis of only the terminal fatty acid chains, and glycerophosphophorylcholine and glycerophosphoethanolamine were detected in the gums. These observations indicate that acyl‐migration of phospholipid fatty acids occurred during PLA₁ degumming. In addition, results showed that PLA₁ degumming was capable of reducing the phosphorus content in the oil to levels acceptable for physical refining (<10 mg/kg). During degumming with PLC, an increase of 1,2‐diacylglycerol was found, as most phosphatidylcholine and phosphatidylethanolamine were hydrolyzed by this enzyme. Treatment with either enzyme slightly decreased the oxidative stability of the oil and most metals were separated with the gums fraction.     

Effect of Different Deacidification Methods on Phytonutrients Retention in Deacidified Fractionated Palm Oil

Crude red palm oil of 11.4 % free fatty acid content was dry fractionated to obtain liquid crude red palm olein which was deacidified using enzyme (lipase from Rhizomucor miehei), solvent (ethanol), and chemical (aqueous sodium hydroxide), and its effect on physicochemical characteristics and phytonutrients retention was evaluated. Enzymatic deacidification showed 100 % product yield and no neutral lipid loss, whereas yields of 78 and 62 % and neutral lipid loss of 12 and 30 % were observed for solvent and chemical deacidification, respectively. Variation in viscosity (25.3–37.2 cSt at 40 °C), slip melting point (15–36 °C), monoacylglycerols (1.7–3.3 %), and diacylglycerols (5.8–27.9 %) were also observed. Carotenoid content was slightly reduced by enzymatic (535 mg/kg), solvent (556 mg/kg), and chemical (526 mg/kg) deacidification. Retention of phytonutrients such as phytosterols (1,235 mg/kg), total tocopherols (965 mg/kg), squalene (301 mg/kg), coenzyme Q₁₀ (25.9 mg/kg), and total phenolics (3 mg/kg) was highest following enzymatic deacidification. The IC₅₀ value of the enzymatic deacidified sample (21.8 mg/ml) indicates more radical scavenging activity than in samples obtained using solvent (42.0 mg/ml) and chemical (28.8 mg/ml) methods.     

Optimization of the Degumming Process for Aqueous-Extracted Wild Almond Oil

Wild almond (Amygdalus scoparia) oil is rich in oleic acid and, considering both nutritional and stability points of view, it can be utilized for future food applications. In the current study, acid degumming was investigated based on a method by response surface methodology using four degumming parameters, namely the amount of phosphoric acid (0.0–0.2%, w/w), the amount of water (1.0–5.0%, w/w), degumming temperature (30–70 °C), and degumming time (10–50 min). Optimum conditions for the minimum phosphorus level in the oil were found to be 0.15% phosphoric acid, 3.0% water, 40 °C degumming temperature, and 28 min degumming time, resulting in an almost complete removal of phosphorus. The final degummed wild almond oil had less than 1 mg kg⁻¹ phosphorus (reduced from an original value of 206 mg kg⁻¹). The experimental value of phosphorus reduction at optimum conditions agreed well with that predicted by the model. Peroxide value, anisidine value, iron, copper, and lead contents, phytosterols, unsaponifiable matter, and color of the oil decreased significantly during the degumming process; however, the fatty acid composition did not change. Also, degumming did not significantly impact the free fatty acid level, refractive index, density, iodine value, and the saponification value of the oil. However, tocopherols and the oxidative stability of the oil increased during degumming. Crude wild almond oil contained a trace level of amygdalin, which was completely eliminated during the degumming process.     

Scale-up of canola oil degumming

The chemical degumming of canola oil was optimized using citric acid and maleic anhydride as degumming agents. These chemicals were selected from a group of 54 degumming agents, reported previously. The effect of temperature, chemical addition level, water addition level and contact times was investigated. Best results were obtained at 40 C, using 10 min contact with the chemical, followed by the addition of 2% water and agitation for 20 min. Chemical degumming reduced the residual phosphorus level from 1049 mg/kg to 50 mg/kg using either maleic anhydride or citric acid. Refining tests gave excellent deodorized or hydrogenated products. The optimized reaction conditions were applied to 330 kg test batches of oil in the P.O.S. Pilot Plant. Results were identical to those obtained in the laboratory, indicating that the process may be scaled up readily for industrial application.     

Effect of water quality on degumming and stability of soybean oil

Solvent-extracted crude soybean oil was degummed with deionized distilled water containing various amounts of CaCO₃−MgCO₃ FeCl₂, and NaCl. The total phosphorus content remaining in the degummed oil was determined and the peroxide value of the degummed oil held at 98–101 C was measured daily for 10 days. The results were compared statistically with those from oil degummed with deionized distilled water as a control. It was found that 250 mg/L of CaCO₃−MgCO₃ significantly reduced the efficiency of the degumming process. FeCl₂ at concentrations of 150 and 250 μg/L and NaCl at 300 mg/L resulted in the removal of more phosphorus than the control at the 5% level of significance. Generally, the stability of the degummed oils decreased as the salt concentrations increased. The rate of oxidation was greater for oils degummed in the presence of FeCl₂ than of NaCl and CaCO₃−MgCO₃ under the same conditions.     

Preparation of diacylglycerol‐enriched palm olein by phospholipase A1‐catalyzed partial hydrolysis

Partial hydrolysis of palm olein catalyzed by phospholipase A₁ (Lecitase Ultra) in a solvent‐free system was carried out to produce diacylglycerol (DAG)‐enriched palm olein (DEPO). Four reaction parameters, namely, reaction time (2–10 h), water content (20–60 wt‐% of the oil mass), enzyme load (10–50 U/g of the oil mass), and reaction temperature (30–60 °C), were investigated. The optimal conditions for partial hydrolysis of palm olein catalyzed by Lecitase Ultra were obtained by an orthogonal experiment as follows: 45 °C reaction temperature, 44 wt‐% water content, 8 h reaction time, and an enzyme load of 34 U/g. The upper oil layer of the reaction mixture with an acid value of 54.26 ± 0.86 mg KOH/g was first molecularly distilled at 150 °C to yield a DEPO with 35.51 wt‐% of DAG. The DEPO was distilled again at 250 °C to obtain a DAG oil with 74.52 wt‐% of DAG. The composition of the acylglycerols of palm olein and the DEPO were analyzed and identified by high‐performance liquid chromatography (HPLC) and HPLC/electrospray ionization/mass spectrometry. The released fatty acids from the partial hydrolysis of palm olein catalyzed by phospholipase A₁ showed a higher saturated fatty acid content than that of the raw material.     

Rapeseed oil methyl esters with low phosphorus content

The phosphorus content in rapeseed oil methyl esters (RMEs) used as an alternative diesel engine fuel is an important indicator of the fuel quality because it affects the function of automobile catalytic converters. In this study, preparation procedures of RMEs with a phosphorus content of 0.1—1 mg kg^—1 are described. Oil with a phosphorus content below 20 mg kg^—1 can be obtained by cold pressing of rapeseeds. After few simple refining steps, RMEs prepared from this oil contain less than 1 mg kg^—1 of phosphorus. Even enzymatic degumming of a rapeseed oil prepared by pressing and extraction with a high phosphorus content of about 600 mg kg^—1 results in a phosphorus content in final RMEs lower than 1 mg kg^—1. The use of short-path wiped-film evaporators for distilling raw RMEs prepared even from untreated rapeseed oil leads to a phosphorus level in the distilled RME lower than 0.1 mg kg^—1.     

Enzymatic activity of Chromobacterium viscosum lipase in an AOT/Tween 85 mixed reverse micellar system

To enhance the Chromobacterium viscosum lipase (glycerol‐ester hydrolase; EC 3.1.1.3) activity for the reaction of water‐insoluble substrates, the AOT/isooctane reverse micellar interface was modified by co‐adsorption of a non‐ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non‐ionic surfactant and olive oil as a water‐insoluble substrate. An appreciable increase of lipase activity was observed and at higher W_o values (where W_o = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell‐shaped profile. The kinetic model for the lipase‐catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems. © 1999 Society of Chemical Industry     

Emulsifier and antioxidant properties of by‐products obtained by enzymatic degumming of soybean oil

The enzymes used in degumming, called phospholipases, specifically act on phospholipids without degrading the oil itself. Degumming using a phospholipase C enzyme allows to meet all market specifications while it increases the oil yield. The aim of this study was to evaluate antioxidant and emulsifier properties of the recovered gum (RG) obtained by enzymatic oil degumming process of crude soybean oil subjected to modifications as deoiling (RG deoiled) or ethanol fractionation (RG soluble and insoluble). RG soluble allowed obtaining more stable oil‐in‐water emulsions (30:70 w/w) in comparison with those by‐products assayed at different concentrations (0.1–1.0%). Also, deoiled soybean lecithin (DSL) and RG deoiled had a similar behavior in relation to the kinetic destabilization (% backscattering profiles), despite the different degumming processes used to obtain these samples. The study on induction times (Metrohm Rancimat) showed a significant antioxidant effect (p<0.05) against a refined sunflower oil associated with all the by‐products analyzed. However, RG soluble and DSL showed a strong effect on the oil stability at high concentrations (1000–2000 ppm). These results showed that the deoiled recovered gum and its derivates obtained by ethanol fractionation are a potential alternative for industrial application as additive. Practical applications: The economic benefits of enzymatic degumming process have also been quantified by several oilseed processors. This process allows obtaining a by‐product with a high concentration of different phospholipids. This study intends to increase the commercial value of this recovered gum contributing to the food industry with useful information about their functional properties.     

Extraction of lipids from Yarrowia Lipolytica

BACKGROUND: Microorganisms have often been considered for the production of oils and fats as an alternative to agricultural and animal resources. Extraction experiments were performed using a strain of the yeast Yarrowia lipolytica (Y. lipolytica), a high‐lipid‐content yeast. Three different methods were tested: Soxhlet extraction, accelerated solvent extraction (ASE) and supercritical carbon dioxide (SCCO₂) extraction using ethanol as a co‐solvent. Also, high pressure solubility measurements in the systems ‘CO₂ + yeast oil’ and ‘CO₂ + ethanol + yeast oil’ were carried out. RESULTS: The solubility experiments determined that, at the conditions of the supercritical extractor (40 °C and 20 MPa), a maximum concentration of 10 mg of yeast oil per g of solvent can be expected in pure CO₂. 10% w/w of ethanol in the solvent mixture increased this value to almost 15 mg of yeast oil per g of solvent. Different pretreatments were necessary to obtain satisfactory yields in the extraction experiments. The Soxhlet and the ASE method were not able to complete the lipid extraction. The ‘SCCO₂ + ethanol’ extraction curves revealed the influence of the different pretreatments on the extraction mechanism. CONCLUSION: Evaluating the effectiveness of a given pretreatment, ASE reduced the amount of material and solvent used compared with Soxhlet. In all three cases, the best total extraction performance was obtained for the ethanol‐macerated yeast (EtM). Addition of ethanol to the solvent mixture enhanced the oil solubility. Oil can be extracted from Y. lipolytica in two different steps: a non‐selective ethanol extraction followed by TAG‐selective SCCO₂ purification. © 2012 Society of Chemical Industry