The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98/81/EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed earlier herein.

The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which it is introduced but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e., exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations where at least two of the combined sequences are foreign with respect to each other. The terms heterologous and exogenous specifically also apply to non-naturally occurring modified versions of otherwise endogenous nucleic acids or proteins.

The "specific activity" of an enzyme is herein understood to mean the amount of activity of a particular enzyme per amount of total host cell protein, usually expressed in units of enzyme activity per mg total host cell protein. In the context of the present invention, the specific activity of a particular enzyme may be increased or decreased as compared to the specific activity of that enzyme in an (otherwise identical) wild type host cell. Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.

A “mature polypeptide” is defined herein as a polypeptide in its final form and is obtained after translation of a mRNA into polypeptide and post-translational modifications of said polypeptide. Post-translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation and removal of leader sequences such as signal peptides, propeptides and/or prepropeptides by cleavage.

A phospholipid is also indicated as a glycerophospholipid. A phospholipid as used herein is an “intact” phospholipid and comprises a glycerol backbone comprising two fatty acids and a phosphoric acid. A phospholipid is also indicated as diacylglyceride comprising a phosphate group.

A lysophospholipid is a glycerol backbone comprising only one acyl (fatty acid) group, and a phosphate group. A lysophospholipid can be formed after removal of an acyl group from phospholipids by the action of a phospholipase A1 , and / or a phospholipase A2.

The wording triacylglyceride oil and triglyceride oil are used interchangeably herein. A triglyceride is an ester derived from glycerol and three fatty acids. A triacylglyceride oil can be an edible oil and I or an oil used as a biodiesel.

Percentages and parts-per-million (ppm) given herein are in weight-per-weight unless otherwise indicated.

Detailed description of the invention

The present inventors have surprisingly found that whereas conventional chemical refining of Brassica carinata oil is unable to sufficiently reduce phosphorus and metal levels to HVO reactor specifications, a simple treatment of crude B. carinata oil with a fungal phospholipase A1 is much more efficient in reducing phosphorus and metal levels in the oil. A further adsorption step then suffices to bring the enzyme-treated oil up to HVO reactor specifications.

In a first aspect, therefore, the invention relates to a process for reducing an amount of at least one of intact phospholipids and metals in a feedstock comprising oil from B. carinata. The process preferably comprises the step of incubating the feedstock with a polypeptide having phospholipase A1 activity, wherein the polypeptide comprises an amino acid sequence having at least 50%, more preferably at least 75%, identity to SEQ ID NO: 1 .

The processes described herein can accommodate a wide range of feedstocks comprising B. carinata oil. The B. carinata oil comprised in the feedstock can be classified as crude, degummed, and RBD (refined, bleached, and deodorized) grade, depending on level of pretreatment and residual phosphorus and metals content. In principle, feedstocks comprising any of these grades of B. carinata oil can be subjected to a process as described herein for further phosphorus and metals content. In one embodiment, a feedstock comprising B. carinata oil to be subjected to a process as described herein can be a mixture of B. carinata oil with other oils, e.g., other vegetable oils. In one embodiment, a feedstock comprising B. carinata oil to be subjected to a process as described herein comprises (in order of increasing preference) at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100% B. carinata oil, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100% crude B. carinata oil. Brassica carinata, commonly called Ethiopian mustard (known locally as gomenzer), is an amphidiploid formed through interspecific hybridization between the diploid parent species B. nigra L. and B. oleracea L. (Prakash and Hinata, 1980, “Taxonomy, cytogenetics and origin of crop Brassicas, a review”, Opera Botanica (Sweden), no. 55).

Crude vegetable oils such as crude B. carinata oil are obtained from pressing and/or solvent extraction methods and are a complex mixture of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, trace metals, and other minor compounds. In commonly known processes, the seeds are first pressed leading to a pressed oil fraction. The press cake can be further treated with a solvent to yield an extracted oil fraction and the two fractions combined are known as crude oil for B. carinata. Crude B. carinata oil, also referred to as non-degummed oil, is herein thus understood to refer to a pressed and/or extracted and non-degummed B. carinata oil.

A polypeptide having phospholipase A1 activity as used herein is a phospholipase A1 according to enzyme classification E.C. 3.1 .1 .32. A phospholipase A1 is an enzyme that cleaves a phospholipid at the SN1 position forming a lysophospholipid and a fatty acid. A phospholipase A1 as used herein may also cleave a lysophospholipid at the SN1 position forming a glycerophosphate and a fatty acid. The wording “phospholipase A1 ” and a “polypeptide having phospholipase A1 activity” is used interchangeably herein. A polypeptide having phospholipase A1 activity as disclosed herein preferably does not have phospholipase A2 activity.

In one embodiment, the polypeptide having phospholipase A1 activity reduces at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, or at least 95% % of the amount of intact phospholipids originally present in a crude B. carinata oil when the polypeptide is incubated with the crude B. carinata oil in an amount of 2500 PLAU / kg oil in the presence of 300 - 500 ppm citric acid, at a temperature of 60°C for 3 hours, for example as tested and using assays as described in the Examples herein. “PLAU” refers to phospholipase A1 units as defined and determined in WO 2019/215078. Phosphorous components such as phospholipids, lysophospholipids and phosphate esters can be determined using³¹P-NMR and/or HPLC, as are known in the art, for instance as disclosed in the Examples herein.

In one embodiment, the polypeptide having phospholipase A1 activity reduces at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, or at least 95% % of the amount of metals, preferably at least one of calcium, magnesium and iron, originally present in a crude B. carinata oil when the polypeptide is incubated with the crude B. carinata oil in an amount of 0.6 mg active protein / kg oil in the presence of 300 - 500 ppm citric acid, at a temperature of 60°C for 3 hours, for example as tested and using assays as described in the Examples herein. Metals content can be determined using Inductively Coupled Plasma (ICP) spectroscopic methods such as ICP-AES (atomic emission spectroscopy) and ICP-OES (optical emission spectroscopy), such as AOCS, as are known in the art, for instance as disclosed in the Examples herein. Reducing the amount of metals is herein understood as a reduction of the total metals. Such metals can include, but are not limited to, As, Ca, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Pb, Sr, Zn, or a combination of any two or more thereof. For example, in any embodiment herein, the total metals can include Ca, Fe, K, Mg, and Na, or can at least include Ca, Fe and Mg.

In one embodiment, the polypeptide having phospholipase A1 activity is a phospholipase A1 derived from a fungus, preferably from a filamentous fungus, more preferably from a filamentous fungus of the genus Aspergillus, most preferably from a filamentous fungus of the subgenus Circumdati, section Nigri. A suitable polypeptide having phospholipase A1 activity can thus be phospholipase A1 derived from a filamentous fungus of the genus Aspergillus selected from the group consisting of A. niger, A. tubingensis, A. foetidus, A. carbonarius, A. awamori, A. ficuum, A. saitoi, A. phoenicis, A. welwitschiae, A. piperis, A. vadensis, A. eucalypticola, A. luchuensis, A. neoniger, A. costaricaensis, A. brasiliensis, A. ibericus, A. sclerotiicarbonarius, A. sclerotioniger, A. ellipticus, A. homomorphus, A. heteromorphus, A. pseudoheteromorphus, A. japonicus, A. rambellii, A. iacticoffeatus, A. ochraceoroseus, A. aculeatus, and A. ellipticus.

In one embodiment, the polypeptide having phospholipase A1 activity is a polypeptide comprising an amino acid sequence having (in order of increasing preference) at least 55, 60, 65, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 1 . In a preferred embodiment, the polypeptide having phospholipase A1 activity is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 . The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of the mature phospholipase A1 from A. niger CBS 513.88, that comprises amino acids 30 - 298 of the pre-pro-phospholipase A1 amino acid sequence of Genbank accession no. XM_001393495.2. It is understood herein that the polypeptide having phospholipase A1 may lack a few amino acids from the N- and/or C-terminus as compared to a polypeptide having an above-defined percentage of sequence identity to SEQ ID NO: 1 , due to exoproteolytic activity during the manufacture of the enzyme.

In one embodiment, the polypeptide having phospholipase A1 activity is a naturally occurring polypeptide. In one embodiment, the polypeptide having phospholipase A1 activity is a variant or modified polypeptide, having one or more amino acid substitutions, insertions and/or deletions as compared to a corresponding naturally occurring polypeptide.

Means and methods for producing a polypeptide having phospholipase A1 activity as described herein are generally known in the art and are e.g., described in WO 2019/215078.

Incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity in a process as disclosed herein comprises converting phospholipids in the oil into lysophospholipids, and free fatty acids. Incubating the feedstock with a polypeptide having phospholipase A1 activity in a process as disclosed herein may further comprise converting phospholipids and/or lysophospholipids in the feedstock into lysophospholipids and I or glycerophosphates, and free fatty acids. In one embodiment, incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity in a process as disclosed herein is performed at a pH value of from 2 to 8, for instance from 3 to 7, for instance from 4 to 6.

In one embodiment, incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity in a process as disclosed herein is performed in the presence of an acid. Accordingly, a process for reducing an amount of at least one of intact phospholipids and metals in a feedstock comprising B. carinata oil as disclosed herein comprises adding an acid such that the amount of acid in the feedstock is from 100 to 1000 ppm of acid, such as from 200 to 900 ppm of acid, for instance from 300 to 800 ppm of acid, for instance from 400 to 600 ppm of acid. A suitable acid used in a process as disclosed herein is an acid selected from the group consisting citric acid, phosphoric acid, acetic acid, tartaric acid, succinic acid, and mixtures thereof.

Usually, water is present in a process as disclosed herein. A process as disclosed herein may further comprise adding water to the oil, for instance an amount of water is added such that an amount of 0.5 to 5 wt%, such as an amount of 1 to 4 wt%, such as an amount of 2 to 3 wt% of water is present in the oil in a process as disclosed herein.

In one embodiment, incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity in a process as disclosed herein, is performed at a temperature in the range of 40 °C to 75 °C, preferably a temperature in the range of 50 °C to 70 °C, more preferably as a temperature in the range of 55 °C to 65 °C and most preferably as a temperature in the range of 57 °C to 63 °C.

A suitable period for incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity in a process as disclosed herein, is from 0.5 to 10 hrs, for instance from 1 to 8 hrs, such as from 2 to 6 hrs, or from 2.5 to 4 hours.

In one embodiment of a process as disclosed herein, the feedstock comprising B. carinata oil is incubated with a polypeptide having phospholipase A1 activity as described herein, as sole lipolytic enzyme. Thus, when the feedstock comprising B. carinata oil is incubated with a polypeptide having phospholipase A1 activity as described herein, no further lipases and/or (lyso)phospholipases (A2, B, C or D) need be present.

In one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity as disclosed herein, comprises a further step of separating at least one of the phosphorous-containing components and metal-containing components, from the processed feedstock as obtained after incubation of the feedstock with the polypeptide. In one embodiment, the step of separating at least one of the phosphorous-containing components and metal-containing components, from the processed feedstock is performed by centrifugation. The centrifuging can include use of a disc-stack centrifuge, a decanter centrifuge, and/or a 3-phase centrifuge. Other methods, systems, and apparatus for separating and/or centrifuging the processed feedstock can be included, such as methods, systems, and apparatus such as settling tanks and are known to the skilled person.

In one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity as disclosed herein, further comprises an adsorption step. Thus in one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity as disclosed herein, comprises a step wherein the processed feedstock as obtained after incubation of the feedstock with the polypeptide, optionally further processed by separating at least one of the phosphorous-containing components and metalcontaining components, is combined with an adsorption medium to generate a slurry, and wherein the adsorbed processed feedstock is subsequently separated from the adsorption medium.

Suitable adsorption media (also referred to herein as “sorbent media”) can include, but are not limited to, silica (e.g., silica hydrogels, silica hydrogel particles), diatomaceous earth, activated carbon, bleaching earths (also referred to as bleaching clays), perlite, cellulosic media, bauxite, silica aluminates, natural fibers, natural flakes, synthetic fibers, or a combination of any two or more thereof. In a preferred embodiment, the adsorption medium is a bleaching earth.

Means and methods for performing an adsorption step on oil feedstocks are described in more detail in e.g., US 11 ,459,523.

In one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity, as disclosed herein, is a process for at least one of degumming and refining the feedstock. Crude vegetable oils such as B. carinata oil contain lecithins, phospholipids, and metals, which are generally called, because of their appearance, mucilaginous gums or simply “gum”. A process of elimination of these lecithins, phospholipids, and metals, i.e. gums is herein understood as “degumming”. It is further understood that a process of refining a vegetable oil, such as a feedstock comprising B. carinata oil, at least includes degumming of the vegetable oil and can further include one or more of adsorption, bleaching, deodorizing, water washing, FFA stripping, glycerolysis, and alkalinity reduction.

In one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity, as disclosed herein, can thus further comprise one or more of bleaching, deodorizing, water washing, FFA stripping, glycerolysis, and alkalinity reduction.

In one embodiment, a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity as disclosed herein, the feedstock comprises crude B. carinata oil, or wherein preferably the feedstock is crude B. carinata oil.

In a second aspect, there is provided a degummed feedstock comprising B. carinata oil. In one embodiment, the degummed feedstock is characterized by at least one of: a) a phosphorus content of less than 10, 5, 2 or 1 ppm, preferably as determined by³¹P NMR; and, b) a total metals content of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method, wherein preferably the degummed feedstock is a refined feedstock.

In one embodiment, there is provided a degummed B. carinata oil, wherein the oil is characterized by at least one of: a) a phosphorus content of less than 10, 5, 2 or 1 ppm, preferably as determined by³¹P NMR; and, b) a total metals content of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method, wherein preferably the degummed oil is a refined oil. In one embodiment, there is a provided a degummed feedstock, wherein the feedstock is characterized by at least one of: i) a content of calcium of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method; ii) a content of iron of less than 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method; and, iii) a content of magnesium of less than 5, 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method, wherein preferably the degummed feedstock is a refined feedstock.

In one embodiment, there is provided a degummed B. carinata oil, wherein the oil is characterized by at least one of: i) a content of calcium of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method; ii) a content of iron of less than 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method; and, iii) a content of magnesium of less than 5, 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method, wherein preferably the degummed oil is a refined oil.

In one embodiment, there is provided a degummed feedstock or degummed B. carinata oil as defined above, wherein the feedstock or oil is obtained or obtainable in a process of incubating a feedstock comprising B. carinata oil with a polypeptide having phospholipase A1 activity, as disclosed herein, wherein preferably the degummed feedstock or degummed B. carinata oil is a refined feedstock or refined B. carinata oil.

In one embodiment, a degummed feedstock or degummed B. carinata oil as defined above, is subjected to hydrotreatment, including e.g., at least one of both hydrodeoxygenation and hydroisomerisation, to prepare a hydrotreated vegetable oil (see e.g., WO2016/185046 and WO2016/185047), to be used as biodiesel or aviation biofuel.

In a third aspect, there is a provided a use of a polypeptide having phospholipase A1 activity as defined herein above, whereby the polypeptide having phospholipase A1 activity is used for degumming of a feedstock comprising oil from B. carinata, wherein preferably the feedstock comprises crude B. carinata oil, or wherein, more preferably the feedstock is crude B. carinata oil. In one embodiment, the polypeptide having phospholipase A1 activity is used for degumming in accordance with a process as herein disclosed.

In one embodiment, the polypeptide having phospholipase A1 activity is used in a process for refining a feedstock comprising oil from B. carinata, wherein preferably the feedstock comprises crude B. carinata oil, or wherein, more preferably the feedstock is crude B. carinata oil. In one embodiment, the polypeptide having phospholipase A1 activity is used for refining in accordance with a process as herein disclosed.

In one embodiment, the polypeptide having phospholipase A1 activity is used for reducing the level of phosphorus in a feedstock comprising oil from B. carinata, wherein preferably the feedstock comprises crude B. carinata oil, or wherein, more preferably the feedstock is crude B. carinata oil. In one embodiment, the polypeptide having phospholipase A1 activity is used for reducing the level of phosphorus in accordance with a process as herein disclosed.

In one embodiment, the polypeptide having phospholipase A1 activity is used for reducing the level of metals in a feedstock comprising oil from B. carinata, wherein preferably the feedstock comprises crude B. carinata oil, or wherein, more preferably the feedstock is crude B. carinata oil. In one embodiment, the polypeptide having phospholipase A1 activity is used for reducing the level of metals in accordance with a process as herein disclosed.

The present invention has been illustrated below by non-limiting examples. Modifications and alternative implementations of some parts or elements are possible and are included in the scope of protection as defined in the appended claims.

EXAMPLES

Example 1 : Composition of crude carinata oil

The composition of crude carinata oil was determined by analyzing the metal content with Inductively Coupled Plasma (ICP), by analyzing Free Fatty Acid (FFA) content with Gas Chromatography (GC), and by analyzing phospholipids profile, being intact phospholipid (PL), lysophospholipid (LPL) and glycerophosphate (GPL), by³¹P Nuclear Magnetic Resonance (NMR).

1. 1 Quantitative determination of Free Faty Acid (FFA) in oils

The oil is dissolved in pyridine, which contains pentanoic acid as an internal standard (ISTD). After addition of extra pyridine and N,0-Bis(trimethylsilyl)trifluoroacetamide (BSTFA)/ 1 % trimethylsilyl chloride (TMCS) the sample is heated. This results in the silylation of the hydroxygroups of the diacylglycerol (DAG), monoacylglycerol (MAG) and the carboxyl group of the free fatty acids (FFA’s). The samples are analyzed by Gas Chromatography (GC) with Flame Ionization Detector (GC-FID) using a Programmed Temperature Vaporizing (PTV) injector and equipped with a high temperature column with a DB-5 phase.

1.2 Quantitative determination of metals in oils

200-500 mg of sample was weighed accurately into a quartz digestion tube, afterwards a suitable amount of nitric acid (65%, suprapur) is added. The samples are digested using closed microwave digestion using the Milestone UltraWave. The digestion temperature is ramped up in multiple stages up to 250°C with the internal pressure limited to 110 bar. The digested sample is transferred to a Nunc tube with 2mL of Hydrochloric acid (30%, suprapur) and approximately 10 mL of MilliQ water. To this 0.5 mL of internal standard solution is added (100 mg NaNO3, 25 mL of 1000 mg/L Sc, Tb, and Au standard solutions. 30 mL of nitric acid (65%, suprapur) diluted to 1000 mL with MilliQ water). The digested sample is diluted further to 50 mL of end volume with MilliQ water.

The data is acquired with the Agilent 7700 ICP-MS equipped with a SPS-4 autosampler, a peltier cooled double pass spray chamber, and micromist nebulizer for sample introduction. Data acquisition parameters: standard plasma mode, He mode collision cell, 1 point peak pattern with 3 replicates per element, and 100 sweeps per replicate, integration time per element 0.20 sec for all elements masses except As (3.00 sec) and Hg (2.00 sec).

The element concentrations in measuring solution are measured with 3-point calibration lines: 0, 125, and 250 pg/L for all elements except As (0, 10, and 20 pg/L), Sb and Tl (0, 25, and 50 pg/L), Hg (0, 2.5, and 5 pg/L), and Pb (0, 5, and 10 pg/L).

Internal standard correction is applied with Sc for element masses up to 97 AMU, and with Tb for the higher element masses.

1.3 Quantitative determination of phospholipids using³¹ P NMR

500-1000 mg oil was weighed accurately into a suitable vial, and approximately 10 g cold acetone was added and mixed thoroughly. The oil-acetone mixture was kept at 4°C for at least 30 min, and then centrifuged for 10 min at 3000 rpm, after which the liquid phase was discarded. The pellet is resuspended in 500 pl buffer (containing 25 g L-1 deoxycholic acid, 5.84 g L-1 EDTA, and 10,9 g L-1 TRIS, buffered using KOH at pH 9.0), and 50 pL of an internal standard solution (containing 10 g L-1 triisopropylphosphate in extraction buffer) was added.

1 D³¹P NMR spectra were recorded on a Bruker Avance III HD spectrometer, operating at a³¹ P frequency of 161 .97 MHz equipped with a nitrogen cooled cryoprobe, at sample temperature of 300K. An inverse gated pulse program (ZGIG) with Waltz16 proton decoupling was used, recording 4 dummy scans, and 128 scans per spectrum, using a 90-degree pulse. An acquisition time of 3.37s, and a relaxation delay of 11 ,5s was used.

The analyte concentrations were calculated relative to triisopropylphosphate. A correction factor was applied to correct for the incomplete relaxation of cholinephosphate and ethanolaminephosphate.

1.4 Results

The determination results are shown in Table 1 .

Table 1 . Composition of crude carinata oil

Example 2: Deep-degumming/refining of crude carinata oil using a fungal phospholipase A1

For each individual experiment, 10 g crude carinata oil was weighed into a vial, and heated to 80°C, acid (phosphoric or citric acid) was added to the oil, and incubated for 30 min, after which the oil is allowed to cool down to 60°C. The acid type and dosage are shown in Table 2. Purifine® PLA1 is a phospholipase A1 from Aspergillus niger having the amino acid sequence of SEQ ID NO: 1 (Table 7), which phospholipase can be produced as described in WO2019/215078 or can be obtained from DSM (www.dsm.com/foodspecialties). Purifine® PLA1 is diluted with demi water, and added to the oil, which was immediately followed by high-shear mixing with Ultra Turrax. The enzyme and water dosage are shown in Table 2. After high-shear mixing, the vial was kept at 60°C for 3 hours, while mixing with a magnetic stirrer at 150 rpm. Table 2. Purifine PLA1 deep-degumming experimental setup.

* PLAU = phospholipase A1 units. 1 PLAU is defined as the amount of enzyme that liberates one micromole of free fatty acid per minute under the conditions of the PLA1 assay described in WO 2019/215078.

** 2500 PLAU corresponds to 0.6 mg active protein.

After 3 hours incubation, temperature was increased to 85°C and kept for 30 minutes to inactivate the enzyme. At the end of the inactivation, homogenous samples were taken for the phospholipid content analysis, i.e., phosphatidic acid (PA), phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), and phosphatidyl inositol (PI), and their lyso-form (lyso-phospholipids, LPL), glycerol-form (glycerol phospholipids, GPL), using³¹P-NMR as described above. Oil samples were taken for FFA analysis using GC method described above. The results are shown in Table 3.

Table 3. Purifine PLA1 deep-degumming FFA and phospholipids composition before centrifugation.

After inactivation, the samples were centrifuged in a Sigma 2-15 Howe Centrifuge to separate the oil and gum: the oils were kept at 65-85 °C during the entire process. Centrifugation was for 5 minutes at a max. RCF (Relative centrifugal force) of 2000 G. After centrifugation, oil samples were taken for metal analysis with the ICP method as described above. The results are shown in Table 4.

Table 4. Purifine PLA1 deep-degumming oil metal composition after centrifugation

Table 4 shows that enzymatic degumming using Purifine PLA1 reduces levels of phosphorus and metals to less than 10 ppm.

The experiment C-2 was repeated at 250 g scale, and oil obtained after centrifugation was washed with 10% hot water by mixing at 85 °C for about 30 min. A separation was done with lab centrifuge at 2000 g for 5 min. The centrifuged oil was then adsorbed using 1 % bleaching earth (Tonsil 9192F from Clariant), by mixing the bleaching earth with the centrifuged oil at 105 °C for 30 min. The adsorbed oil was then separated from the bleaching earth by filtration. The resulting oil was analyzed by³¹P-NMR for phosphorus content, ICP for the metal content and by titration for acid value. The results are shown in Table 5.

Table 5. Purifine PLA1 deep-degumming oil C-2, metal composition after centrifugation and bleaching earth absorption.

Reference Example 3: Conventional chemical deep-degumming/refining of crude carinata oil

For each individual experiment, 250 g crude carinata oil was weighed into a conical flask, and heated to 75°C, 500 ppm citric acid was added to the oil, and mixed for 30 min with a magnetic stirrer at 150 rpm. Then 160 ppm sodium hydroxide was added to the oil and mixed at the same condition for 60 min. Afterwards the samples were centrifuged with Sigma 2-15 Howe Centrifuge to separate the oil and gum: the oils were kept at 65-75 °C during the entire process, the spinning was for 5 minutes at a max. RCF (Relative centrifugal force) of 2000 G. After centrifugation, oil samples were taken for metal analysis with ICP method as described above. After degumming, the oil was mixed with 1 % adsorbent Tonsil 9192F from Clariant for 30 min, and then filtered. The filtered oil were taken for metal analysis with ICP method as described above.

The results are shown in Table 6. Table 6. Traditional deep-degumming oil metal composition after handling.

Table 7. Amino acid sequence of the mature A. niger phospholipase A1 .

Claims

1 . A process for reducing an amount of at least one of intact phospholipids and metals in a feedstock comprising oil from Brassica carinata, the process comprising the step of incubating the composition with a polypeptide having phospholipase A1 activity, wherein the polypeptide comprises an amino acid sequence having at least 50%, more preferably at least 75%, identity to SEQ ID NO: 1 .

2. A process according to claim 1 , wherein the polypeptide reduces at least 85% of the amount of intact phospholipids originally present in a crude B. carinata oil when the polypeptide is incubated with the crude B. carinata oil in an amount of 2500 PLAU / kg oil in the presence of 300 - 500 ppm citric acid, at a temperature of 60°C for 3 hours.

3. A process according to claim 1 or 2, wherein the polypeptide is an Aspergillus phospholipase A1 , preferably an A. niger phospholipase A1 , more preferably the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 .

4. A process according to any one of claims 1 - 3, wherein the feedstock is incubated with the polypeptide having phospholipase A1 activity in the presence of an acid, wherein, preferably the acid is an acid selected from the group consisting citric acid, phosphoric acid, acetic acid, tartaric acid, succinic acid, and mixtures thereof.

5. A process according to claim 4, wherein the acid is present in an amount of 100 - 1000 ppm, preferably 200 - 800 ppm, more preferably 400 - 600 ppm, and most preferably 400 - 600 ppm citric acid.

6. A process according to any one of claims 1 - 5, wherein the feedstock is incubated with the polypeptide having phospholipase A1 activity at a temperature in the range of 50 - 70 °C, preferably at temperature in the range of 55 - 65 °C.

7. A process according to any one of claims 1 - 6, wherein the process comprises a further step of separating the phosphorous-containing components from the processed feedstock as obtained in a process according to any one of claims 1 - 7, preferably by centrifugation.

8. A process according to any one of claims 1 - 7, wherein the process comprises a further step of combining a processed feedstock as obtained in any one of claims 1 - 8 with an adsorption medium to generate a slurry and separating the adsorbed processed feedstock from the adsorption medium.

9. A process according to claim 8, wherein the adsorption medium is a bleaching earth.

10. A process according to any one of claims 1 - 9, wherein the process is a process for degumming and/or refining the feedstock.

11. A process according to any one of claims 1 - 10, wherein the feedstock comprises crude B. carinata oil, or wherein preferably the feedstock is crude B. carinata oil.

12. A degummed feedstock comprising B. carinata oil, wherein the feedstock is characterized by at least one of: a) a phosphorus content of less than 10, 5, 2 or 1 ppm, preferably as determined by³¹P NMR; and, b) a total metals content of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method, and wherein preferably the degummed feedstock is degummed B. carinata oil.

13. A degummed feedstock or degummed B. carinata oil according to claim 12, wherein the feedstock or the oil is characterized by at least one of: i) a content of calcium of less than 10, 5, 2 or 1 ppm, preferably as determined by an ICP spectroscopic method; ii) a content of iron of less than 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method; and, iii) a content of magnesium of less than 5, 2, 1 , 0.5 or 0.2 ppm, preferably as determined by an ICP spectroscopic method.

14. A degummed feedstock or degummed B. carinata oil according to claim 12 or 13, wherein the feedstock or oil is obtainable in a process according to any one of claims 1 - 11.

15. Use of a polypeptide having phospholipase A1 activity as defined in any one of claims 1 - 3, for at least one of: a) degumming of; b) refining of; c) reducing the level of phosphorus in; and, d) reducing the level of metals in, a feedstock comprising oil from B. carinata, wherein preferably the feedstock comprises crude B. carinata oil, or wherein, more preferably the feedstock is crude B. carinata oil.