A novel strain of metschnikowia pulcherrima and uses of the novel strain

Abstract

The present invention provides novel strain of M. pulcherima, having accession number NCYC 4455. The strain can be cultured to produce a compound such as a lipid or an alcohol. The strain can also be used to produce a yeast biomass and for other uses.

Description

[0001] A NOVEL STRAIN OF METSCHNIKOWIA PULCHERRIMA AND USES OF THE NOVEL STRAIN

[0002] Field of Invention

[0003] The present invention relates to a novel strain of Metschnikowia pulcherrima, and to methods of producing compounds and yeast biomass using the novel strain.

[0004] Background to the Invention

[0005] Microbial lipids, produced from heterotrophic organisms, are a versatile feedstock that can be used to produce alternatives to fossil derived fuels, be used as a replacement for edible plant oils in food application and for oleochemicals. In comparison to higher plant oils, such as palm oil, microbial oils can be produced on non-arable land, that does not compete with virgin rainforest or food production. While the lipid profile from microalgae is highly variable, generally, oleaginous yeast produce lipid profiles akin to plant oils with elevated levels of oleic and palmitic acid. The oleaginous yeast Metschnikowia pulcherrima can be grown in non- sterile conditions, while having the ability to metabolise a range of oligosaccharide and monosaccharide carbon sources.

[0006] As described in Abeln and Chuck, 2020 (The role of temperature, pH and nutrition in process development of the unique oleaginous yeast Metschnikowia pulcherrima. J Chem Technol Biotechnol, 95: 1163-1172. https: / / doi.org / 10.1002 / jctb.6301), one strain of M. pulcherrima (NCYC 4331), which was evolved from the progenitor NCYC2580 has achieved lipid accumulation of >29.8% (w / w) and a maximum lipid production rate of 0.06 g L1h’1, productivity of 0.29 g L-1h-1and yield of 0.17 g g1glucose.

[0007] Summary of the Invention

[0008] The present inventors have recognised that there is still a need for faster growing strains of M. pulcherrima, preferably those strains also providing improved mycolipid and / or mycoprotein production. Accordingly, in a first aspect, the present invention provides a novel strain of M. pulcherrima, having accession number NCYC 4455.

[0009] In a second aspect, the present invention also provides methods for producing compounds and yeast biomass using the novel strain.

[0010] In a third aspect, the present invention also provides a method of producing a compound, the method comprising culturing the novel strain of M. pulcherrima having accession number NCYC 4455 with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells and lysing the cells to release the compound.

[0011] In certain embodiments, the method the compound is a lipid or an alcohol. In some examples, the lipid is one or more of a triacylglycerol (TAG), glyceride, free fatty acid, an ergosterol or a glycolipid; or wherein the alcohol is one or more of 2-phenylethanol, ethanol, arabitol or glycerol.

[0012] In certain further embodiments, the present invention also provides a lipid or alcohol obtained or obtainable by the method of the third aspect of the present invention.

[0013] In a fourth aspect, the present invention provides a method of producing a yeast biomass, the method comprising culturing the novel strain of M. pulcherrima having accession number NCYC 4455 with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells collecting and drying the cell.

[0014] In certain examples, the cells are cultured in non-sterile conditions.

[0015] In some examples, the cells are cultured in anaerobic conditions. In a fifth aspect, the present invention provides the use of the strain of M. pulcherrima having accession number NCYC 4455 in the preparation of a food ingredient; optionally wherein the food ingredient is a food ingredient selected from oils, yeast extracts, polysaccharides, such as yeast p- glucans, yeast proteins and emulsifiers.

[0016] In a sixth aspect, the present invention provides a method of preparing an oleogel, the method comprising treating a lipid obtained by the method of the third aspect of the present invention with a gelator.

[0017] In certain examples, the gelator is candelilla wax, rice bran wax or ethyl cellulose, preferably candelilla wax.

[0018] The present invention also provides an oleogel obtainable or obtained by the method of the sixth aspect.

[0019] In a seventh aspect, the present invention provides the use of the novel strain of M. pulcherrima having accession number NCYC 4455 in the preparation of food oils, cooking oils, confectionery, chocolate, creams, pastry, bakery, margarine and similar spreads, plant-based cheese and dairy-type products, meat analogues, cosmetic formulations, personal care products, cleaning products, candles and infant milk formula products.

[0020] In an eighth aspect, the present invention also provides a food oil, cooking oil, confectionery product, chocolate, cream product, pastry product, bakery product, margarine, non-dairy or partially non-dairy spread, plantbased cheese, plant-based dairy-type product, meat substitute or infant milk formula product comprising a lipid or an oleogel obtained or obtainable by the methods of the first and sixth aspects of the present invention.

[0021] In a ninth aspect, the present invention also provides a cosmetic formulation, personal care product, cleaning product or a candle comprising a lipid or an oleogel obtained or obtainable by the methods of the first and sixth aspects of the present invention.

[0022] The present inventors have demonstrated that the novel strain of M. pulcherrima, having accession number NCYC 4455 has a 30% faster growth rate and can produce more lipid per cell than previous strains, over 50% of the cell. The yeast is also capable of withstanding higher osmotic pressures, demonstrating reasonable growth on glucose at 400g / L concentrations, producing higher biomass and lipid under these conditions. The cell line also appears to produce lower extracellular material, reducing the viscosity allowing for higher oxygen uptake rates under high cell density. This has resulted in a 18% increase in cell density under semicontinuous cell recycle conditions and a maximum oxygen uptake rate of 60 mmol / L / hr. These are significant improvements that will allow a more efficient scaled up processes for the production of lipids, other compounds and yeast biomass.

[0023] Description

[0024] The novel strain of M. pulcherrima having accession number NCYC 4455 can be used to produce compounds, such as lipids or alcohols, by culturing the yeast with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells and lysing the cells to release the compound.

[0025] Lipids produced by the method may be one or more of a triacylglycerol (TAG), a glyceride, free fatty acid, ergosterol or a glycolipid. In embodiments of the invention the lipids produced by the method are a lipid mixture comprising about 80 wt% to about 99 wt%, or about 90 wt% to about 99 wt%, or about 95% wt% TAGs, with the remainder of the mixture (i.e., made up to 100 wt%) comprising one or more of glycerides, free fatty acids, ergosterols and glycolipids. The lipid mixture may comprise about 0.5 wt% to about 5 wt%, or about 1 wt % to about 4 wt%, preferably about 3 wt% of glycerides. The lipid mixture may comprise about 0.5 wt% to about 4 wt%, or about 1 wt % to about 3 wt%, preferably about 2 wt% of free fatty acids. The lipid mixture may additionally comprise about 0.5 wt% ergosterols and / or about 0.1 wt% glycolipids.

[0026] Preferably the lipid is a saturated lipid suitable as a palm oil substitute. The lipid may be a C16:0 fatty acid and / or a C18: l fatty acid. The lipid profile may contain between 5-60 wt% or 10-40 wt% or 20-30 wt% 16:0 fatty acid. In preferred embodiments of the invention the lipid profile may contain 20-26 wt% 16:0 fatty acid. The lipid profile may contain between 20-80 wt% or 30-75 wt% or 50-65 wt% 18: 1 fatty acid. In preferred embodiments of the invention the lipid profile may contain 48-62 wt% 18: 1 fatty acid. Optionally, the lipid profile also includes one or more of 14:0, 16: 1, 18:0 and 18:2 fatty acids.

[0027] Alcohols produced by the method may be one or more of 2-phenylethanol, ethanol, arabitol or glycerol. Preferably the alcohol is 2-phenylethanol.

[0028] The novel strain of M. pulcherima having accession number NCYC 4455 can also be used to produce a yeast biomass, by culturing the yeast with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells, collecting and drying the cells. Alternatively, yeast biomass can be harvested from cells previously used to produce compounds such as lipids or alcohols, as described above. In such methods, the yeast cells which have produced the compounds can be collected and dried after the compounds have been collected.

[0029] The term 'yeast biomass' refers to accumulated yeast cells, and is typically rich in proteins and vitamins, including vitamin D2, D3, vitamin Bs, and Vitamin E. Yeast biomass may also contain carbohydrates and / or residual lipid (which may remain after lipids produced by the cells have been collected). The term 'saccharide feedstock' refers to substances containing glucose, fructose and / or sucrose. For example, the saccharide feedstock may be molasses or glucose syrup.

[0030] The term 'lignocellulosic feedstock' refers to plant dry matter and can be broadly classified into virgin biomass, waste biomass and energy crops. Virgin biomass includes all naturally occurring terrestrial plants such as trees, bushes and grass. Waste biomass is produced as a low value byproduct of various industrial sectors such as agriculture (corn stover, sugarcane bagasse, straw etc.) and forestry (saw mill and paper mill discards). Energy crops are crops with high yield of lignocellulosic biomass produced to serve as a raw material for production of second generation biofuel; examples include switch grass (Panicum virgatum') and Elephant grass.

[0031] Food waste feedstock is typically rich in carbohydrates, nitrogen, and fats. The food waste feedstock may be food waste hydrolysate, which can be produced by enzymatic or chemical hydrolysis of solid food waste.

[0032] The M. pulcherrima having accession number NCYC 4455 can be cultured at temperatures between about 15°C and about 40°C, preferably between about 15°C and about 30°C or between about 15°C and about 25°C. The yeast cells may be cultured at a temperature of about 20°C.

[0033] Typically, the yeast cells are cultured in non-sterile conditions. The yeast cells may be cultured under limited oxygen or under anaerobic conditions.

[0034] The above and other aspects of the present invention will now be described in further detail, by way of example only, with reference to the following examples and the accompanying figures, in which: Figure 1 is a bar chart comparing oil-binding capacity of oleogels prepared from an embodiment of a yeast oil in accordance with the present invention with a rapeseed oil, using Candelilla wax as the gelator;

[0035] Figure 2 is a bar chart comparing oil-binding capacity of oleogels prepared from an embodiment of a yeast oil in accordance with the present invention with a rapeseed oil, using rice bran wax as the gelator;

[0036] Figure 3 is a bar chart illustrating oil-binding capacity of oleogels prepared from an embodiment of a yeast oil in accordance with the present invention using ethyl cellulose as the gelator; and

[0037] Figure 4 is a temperature ramp plot of embodiments of oleogels of the present invention formed from a yeast oil of the present invention with Candelilla wax as the gelator

[0038] EXAMPLES

[0039] Except where otherwise indicated or the context indicates otherwise, weight ratios are given as w / w ratios.

[0040] The first aspect of the present invention is a M. pulcherrima cell line named CFG1106 NCYC 4455. It was created through the directed evolution of a progenitor M. pulcherrima cell line known CFG1001 (NCYC 4331, which was itself created through directed evolution of NCYC 2580). To this end the yeast was put under suitable conditions for growth and the most robust cells separated from the rest, re-cultured and the evolution continued for multiple generations. The CFG1106 cell line that was produced has a number of stable changes compared with CFG1001, as demonstrated by comparison of the genomes. This has resulted in the advantages listed below. Table 1: Comparison of M. pulcherrima strains CFG1106 and CFG1001 Materials & Methods

[0041] 1. CFG1001 vs CFG1106 (NCYC 4455) kinetic experiment

[0042] The strains were kept as a glycerol stock at -80C was revitalised on MEA agar plates (g / L: soy peptone 5, malt extract 30, agar 15, pH: 5) for 3 days at 25C, then inoculated O / N in 20 ml of SMB media (g / L: soy peptone 30, malt extract 25, pH 5). The day after, 3.2% v / v was taken, washed 3 times in Mcllvaine buffer pH 5, inoculated in 20 ml of NLB media (g / L: KH2PO4 7, (NH^SC 2, NaH2PO4 1, yeast extract 1, MgSC * 7H2O 1.5, Glucose 80, pH 5) and cultivated overnight to make sure the cells are acclimatised to the right media. This large inoculum size was chosen to avoid a long lag phase and low cell bottleneck problems. The day after, 3.2% v / v was taken and inoculated in new 20 ml NLB shake flasks for the lOh kinetic experiment (triplicate). A 1 ml sample was taken hourly and the OD was measured spectrophotometrically (600 nm).

[0043] MEA and SMB media were autoclaved whereas NLB media was prepared as follows. A 5x concentrated stock solution called "NLB-base" was prepared with KH2PO4, (NH4)2SO4, NaHPCU and yeast extract and autoclaved. A 5x concentrated stock solution of MgSCU * 7H2O was also prepared and autoclaved separately to avoid precipitation. The glucose stock was also prepared as a 5x concentrated stock and filter sterilised with cellulose acetate 0.22 pm filters. The stocks were merged to obtain the final desired concentration and the media was topped up with sterile autoclaved water to the desired final volume.

[0044] The OD values were plotted in a semilog graph against the time and the linear phase (where p = pmax) was identified. Tmid was finally calculated as follows: where To exp and Tf exp are respectively the beginning and end time of the exponential phase h). p max was also calculated as the slope of the exponential phase of the semilog OD graph VS time, whereas the doubling time (T2) was calculated as the ratio between In2 and p max.

[0045] 2. Fed batch

[0046] The strains were kept at 20°C on malt extract agar plates (MEA: agar 15 g L-1; malt extract 30 g L-1; mycological peptone 5 g L-1) and sub-cultured every fortnight. A single colony was used to inoculate soy-malt broth (SMB: soy peptone 30 g L-1; malt extract 25 g L-1; pH 5) in 250 ml Erlenmeyer flasks, incubated for 24 hr to an OD600 of around 24 (preculture). Fed batch operation mode the yeast was inoculated with 2.5% (v / v) preculture to the media KH2PO4 7 g L-1; (NH^zSCU 2 g L-1; NaHPCU 1 g L-1; MgSO4-7H2O 1.5 g L-1; yeast extract 1 g L-1; glucose 8% w / v; pH 4). Fresh media, including additional yeast extract, was added on glucose depletion to return the glucose concentration to 8% throughout the run.

[0047] 3. Batch, 400 g / L glucose cultures

[0048] The strains were kept at 20°C on malt extract agar plates (MEA: agar 15 g L-1; malt extract 30 g L-1; mycological peptone 5 g L-1) and sub-cultured every fortnight. A single colony was used to inoculate soy-malt broth (SMB: soy peptone 30 g L-1; malt extract 25 g L-1; pH 5) in 250 ml Erlenmeyer flasks, incubated for 24 hr to an OD600 of around 24 (preculture). Stirred tank reactor (STR.) cultivations were performed in a 2 L FerMac 320 STR (Electrolab) equipped with off-gas analyzer. With a working volume of 1 L fermentation broths were maintained at pH 4 and dissolved oxygen (DO) at 50% In batch operation mode the yeast was inoculated with 2.5% (v / v) preculture to the media KH2PO4 7 g L-1; (NH4)2SO4 2 g L-1; NaHPO41 g L-1; MgSO4-7H2O 1.5 g L-1; yeast extract 1 g L-1; glucose 40% w / v; pH 4.

[0049] 4. Semi-continuous culture with cell recycle.

[0050] The strains were kept at 20°C on malt extract agar plates (MEA: agar 15 g L-1; malt extract 30 g L-1; mycological peptone 5 g L-1) and sub-cultured every fortnight. A single colony was used to inoculate soy-malt broth (SMB: soy peptone 30 g L-1; malt extract 25 g L-1; pH 5) in 250 ml Erlenmeyer flasks, incubated for 24 hr to an OD600 of around 24 (preculture). Stirred tank reactor (STR) cultivations were performed in a 2 L FerMac 320 STR (Electrolab) equipped with off-gas analyzer. With a working volume of 1 L fermentation broths were maintained at pH 4 and dissolved oxygen (DO) at 50% In batch operation mode the yeast was inoculated with 2.5% (v / v) preculture to the media KH2PO4 7 g L-1; (NH4)2SO4 2 g L-1; NaHPO41 g L-1; MgSO4-7H2O 1.5 g L-1; yeast extract 1 g L-1; glucose 8% w / v; pH 4. On depletion of the glucose the reactors were put into semi-continuous mode, 25% (v / v) of the fermentation broth was removed, centrifuged, the pellet resuspended in an equal amount of media to bring the total glucose content back to 8%.

[0051] 5. Oxygen uptake rate

[0052] The strains of M. pulcherrima were kept as 20% glycerol stock at -80 °C. One full loop from the stock was plated out on malt extract agar (MEA: agar 15 g L-1; malt extract 30 g L-1; mycological peptone 5 g L-1) plates, incubated at 20 °C for a minimum of 3 days and maximum of 2 weeks. For the preculture, one colony from the agar plate was placed in 20 mL soy-malt broth (SMB: soy peptone 30 g L-1; malt extract 25 g L-1; pH 5) in 100 mL Erlenmeyer flasks and incubated at 20 °C for 24 h.

[0053] Fermentations were performed in 2-5 L STRs equipped with gas analyser, temperature and foam sensors as well as pH and DO sensor. Standard operation conditions were pH 4, 20 °C, DO 40-50%

[0054] The fermentation medium consisted of glucose syrup, with a dextrose equivalent of 80 g / L, and between 3-12 g L-1yeast extract and was inoculated with 4% (v / v) preculture. Fermentations were controlled at pH4, DO 40%

[0055] Oleogelation

[0056] Oleogelation is an approach for converting liquid oil into semi-solid gel without any chemical modification. Gelling of oil is achieved through the addition of an oleogelator. Oleogels can provide wide range of applications in food, cosmetics and pharmaceutical industry as fat replacer, texture modifier, shelf-life extension, bioactive delivery, clean and natural foods, low-calorie foods, and functional foods Oleogels prepared from commercial oils have been widely studied for food application as fat replacer in industry as well, as it is becoming increasingly apparent that plant-based food products can only utilize tropical oils such as palm oil and coconut oil, that have large sustainability issues to replace the solid fat from animal products because of their plasticity effect. Plant-based oils nevertheless raise various environmental sustainability issues and plant oils from temperate climates such as rapeseed oil, still have large greenhouse gas (GHG) impact. Hence, replacement of vegetable oils in the preparation of oleogels with an unsaturated microbial oil is highly advantageous.

[0057] Oleogels are viscoelastic mixtures which are mainly composed of an oil as a solvent phase and a gelator agent (oleogelator) (typically at a concentration of 10% of lower). Gelators are classified into two types - low molecular weight (LMW) gelators and polymeric or high molecular weight (HMW) gelators. The properties and performance of the resultant oleogel differ based on their chemical structure, concentration, mechanism, and formulation conditions. LMW gelators include waxes having a natural origin (such as candelilla wax, carnauba wax, carnauba wild wax, beeswax, sunflower wax, fruit wax), lecithin, monoglycerides, phytosterols, ceramides. Examples of HMW gelators include proteins (soy, zein, casein) and polysaccharides (ethyl cellulose, xanthan gum, pectin, chitin and chitosan). The gelling ability of oleogelators mainly depends on two aspects: (i) the balance between solubility and insolubility in the oil phase; and (ii) self-assembly and self-organization into higher-ordered structures such as liquid crystals, crystal lattice, fibrils, bilayers, micelles, and agglomerates to form the structuring network.

[0058] Materials and Methods

[0059] Oils Vegetable oils were purchased from a local grocery store. The labelled fatty acid composition of rapeseed oil used in the following comparisons, was stated as 7.3g saturated fats, 58g MUFA, 27g PUFA.

[0060] Degummed / bleached oil from M. pulcherrima cell of strain type CFG1106 was prepared as above.

[0061] Gelators

[0062] Candelilla wax (CLW) and rice bran wax (RBW) were obtained from Makers Ingredients. Ethyl cellulose (EC), was purchased from Sigma Aldrich (viscosity of 46 cP in 5% (w / v) in an 80:20 solution of toluene / ethanol and 48.9% ethoxy content). All the gelators were used without any purification.

[0063] Preparation of oieogeis

[0064] Oleogels were prepared by direct dispersion of gelators in the oil. Initially, screening was performed to find the critical gelling concentration (CGC) of candelilla wax, rice bran wax and ethyl cellulose for gelling rapeseed oil (RO). The minimum oleogelator concentrations were identified using tilt test (formation of self-standing gels upon inversion of tubes). The concentration of CLW and RBW was used at 1%, 2%, 3% & 5% and 3%, 5% & 6% for ethyl cellulose.

[0065] For preparation, a known volume of oil was weighed and heated to a temperature above the melting point of the oleogelator on a hot plate coupled with a thermometer. DSC analysis was performed for all gelators to evaluate the Tm. The oleogelator was added to the oil with stirring (with a magnetic stirrer at a speed of 200 rpm): for 15 minutes at 68°C for CLW, 15 minutes at 80°C for RBW, and for 30 minutes at 170°C for EC. When completely melted, the mixture was transferred to a glass vial with cap for subsequent analyses. Oleogels were prepared using commercial oils such as rapeseed oil, sunflower oil, and olive oil utilizing candelilla wax, rice bran wax at a concentration of 5%, 10%, 15% and ethyl cellulose at 8%, 10% 12% concentration. Based on visual observation by tilt test and oil binding capacity CLW, RBW and EC were selected to make oleogels from yeast oil at concentrations of 1%, 2%, 3% and 5% (CLW and RBW) and 2%, 3%, 5% and 6% (ethyl cellulose). In the tilt test, all the samples showed selfstanding gels in all oils and OBC data shows no significant difference between the samples.

[0066] Characterization of oieogei

[0067] OH binding capacity (OBC)

[0068] The oil binding capacity of oleogels was determined using a centrifugation method. A known volume of liquid mixture (1ml) was taken in a previously weight (Wl)1.5 mL of centrifuge tube. The liquid sample in the tube was allowed to set at the same conditions of oleogelation and stored at 20°C for 48 hours. The sample with tube (W2) was weighed and centrifuged at 13000 rpm for 15 minutes in a centrifuge (VWR Micro Starl2). Then, the tube was inverted for 1 hour to remove excess oil and the decanted tube (W3) was measured. All the measurements were performed in triplicates at room temperature (Sivakanthan, S., Fawzia, S., Mundree, S., Madhujith, T. and Karim, A., 2023. Optimization and characterization of new oleogels developed based on sesame oil and rice bran oil. Food Hydrocolloids, 142, p. 108839.). The OBC was calculated using the equation below.

[0069] Temperature ramp test

[0070] Oleogels exhibit a viscoelastic behaviour, which has both viscous and elastic properties. The elastic portion (solid-like behavior) is expressed as G' and loss modulus (liquid-like behaviour) is expressed as G". Dynamic rheological measurements were used to measure the structural stability of the oleogel samples when a small deformation is applied (Sivakanthan, et al. ,2023) and allow differentiation between weak and strong gels. An oleogel can be classified as a strong gel if G" / G' < 0.1, or a weak gel if 0.1 < G" / G' < 1. A viscous sol has a value of (G" / G' > 1). A gel state is defined as a point at which the G' value is higher than G". The formation of final gel state can be described during sol-gel transition process. Information about the oleogels structural strength is obtained by amplitude sweep test and frequency sweep test (Sivakanthan, et al., 2023).

[0071] Viscosity was measured at a range of temperatures within the range of 5°C to 75°C for CLW, 85°C for RBW and 170°C for EC within the strain value in the LVR region. The experiments were conducted at a fixed gap of 1mm the gap size. The gel point / crossover temperature was determined as the crossover point of G' and G" (G' = G") (Sivakanthan, et al., 2023).

[0072] Results and Discussion

[0073] OH binding capacity

[0074] The oil binding capacity of an oleogel is one of the major properties that describes the functional quality of a food product and reflects the degree of liquid oil entrapment in the 3D network of gel by the oleogelator. The results at a range of gelator concentrations are illustrated in Figures 1 to 3.

[0075] Figure 1 shows the OBC values of both the yeast oil and rapeseed oil oleogels reached 100% at 2%, 3% & 5% wax. This indicates these levels are sufficient for the formation of stable gel network. Oleogel with more than 99% OBC indicates the strength of gel network is sufficient to prevent oil leakage. The oleogels produced in this study were translucent at low concentrations and the opaqueness of gel increased with the increase in wax concentration.

[0076] The OBC of corresponding samples formulated with RBW are shown in Figure 2, which shows a lower OBC for both the Metschnikowia pulcherrima oil and the rapeseed oil oleogels compared with those formulated with CLW.

[0077] The oil binding capacity of oleogel formulated using Metschnikowia pulcherrima oil and ethyl cellulose as the gelator is illustrated in Figure 3, showing an increase in OBC with increasing EC content, reaching 100% at 6% EC concentration. The OBC of EC based oleogel at 3% and 5% concentration is higher (at 65% and 99% respectively) compared with the values obtained for the RBW-based oleogel (Figure 2).

[0078] Temperature ramp test

[0079] Data from the temperature ramp test for Metschnikowia pulcherrima oil (MP) at 2% w / w CLW concentration and at 3% w / w CLW concentration is presented in Figure 4, illustrating the behaviour of the oleogels at different temperature transitions.

[0080] The graphs showed an increase and decrease in moduli G' and G" during the heating / cooling process from 5°C to 75°C under constant frequency and strain value. As can be seen, in both the samples the value of G' & G" is constant and linear with increasing temperature from 5°C to around 25°C for 2% CLW (circles) and 5°C to 28°C for 3% CLW (squares). Within this range, both samples solidified and gelled well.

[0081] It can be observed that softening of both 2% CLW and 3% CLW begins at temperatures around 26°C and 29°C and complete melting of sample starts at-30°C and-35°C. In both samples, the gel completely melts below 37°C, forming a viscous sol. This melting behaviour of the inventive oleogels at a temperature which corresponds with human body temperature provides a very important advantage for the inventive oleogels as it mimics a 'fat-like' melt-in-mouth effect, providing excellent mouthfeel to food products using the inventive oleogels in place of conventional fats.

[0082] Conclusions

[0083] M. pulcherrima is a viable organism for biotechnology capable of being cultured in non-sterile conditions under industrially relevant conditions. However, previously reported strains are slow growing which limits the applicability for industrial production. In comparison to CFG1001 (NCYC 4331) the described strain CFG1106 (NCYC 4455) has a 30% faster growth rate and can produce more lipid per cell than previous strains, over 50% of the cell. The yeast is also capable of withstanding higher osmotic pressures, demonstrating reasonable growth on glucose at 400g / L concentrations, producing higher biomass and lipid under these conditions. The cell line also appears to produce lower extracellular material, reducing the viscosity allowing for higher oxygen uptake rates under high cell density. This has resulted in a 18% increase in cell density under semicontinuous cell recycle conditions and a maximum oxygen uptake rate of 60 mmol / L / hr. These are significant improvements that will allow a more efficient scaled up process.

[0084] As discussed above, the novel strain of M. pulcherrima of the present invention is useful for numerous purposes, including in the production of food ingredients, particularly functional food ingredients, such as oils, yeast extracts, polysaccharides, such as yeast p-glucans, yeast proteins and emulsifiers. Oils extracted from the novel strain of M. pulcherrima of the present invention may be prepared as oleogels, using oleogelators appropriate to the desired end product, and may be subjected to further processing. More specifically, the novel strain of M. pulcherrima of the present invention is useful in the preparation of food oils, including cooking oils, confectionery, chocolate, creams, pastry, bakery, margarine and similar spreads, plant-based cheese and dairy-type products, meat analogues, cosmetic formulations, personal care products, cleaning products, candles and infant milk formula products.

Claims

CLAIMS1. A novel strain of M. pulcherima, having accession number NCYC 4455.

2. A method of producing a compound, the method comprising culturing the novel strain of M. pulcherrima according to claim 1 with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells and lysing the cells to release the compound.

3. The method of claim 2, wherein the compound is a lipid or an alcohol.

4. The method of claim 2, wherein the lipid is one or more of a triacylglycerol (TAG), glyceride, free fatty acid, an ergosterol or a glycolipid; or wherein the alcohol is one or more of 2-phenylethanol, ethanol, arabitol or glycerol.

5. A method of producing a yeast biomass, the method comprising culturing the novel strain of M. pulcherrima according to claim 1 with a saccharide, lignocellulosic or food waste feedstock, isolating the cultured cells collecting and drying the cell.

6. The methods of any of claims 2 to 5, wherein the cells are cultured in non-sterile conditions.

7. The methods of any of claims 2 to 6, wherein the cells are cultured in anaerobic conditions.

8. A lipid or alcohol obtained by the method of claim 2.

9. Use of the novel strain of M. pulcherrima of claim 1 in the preparation of a food ingredient; optionally wherein the food ingredient isa food ingredient selected from oils, yeast extracts, polysaccharides, such as yeast p-glucans, yeast proteins and emulsifiers.

10. A method of preparing an oleogel, the method comprising treating a lipid obtained by the method of claim 2 with a gelator.

11. A method as claimed in claim 2 wherein the gelator is candelilla wax, rice bran wax or ethyl cellulose, preferably candelilla wax.

12. An oleogel obtainable or obtained by the method of claim 10 or claim 11.

13. Use of the novel strain of M. pulcherrima of claim 1 in the preparation of food oils, cooking oils, confectionery, chocolate, creams, pastry, bakery, margarine and similar spreads, plant-based cheese and dairy-type products, meat analogues, cosmetic formulations, personal care products, cleaning products, candles and infant milk formula products.

14. A food oil, cooking oil, confectionery product, chocolate, cream product, pastry product, bakery product, margarine, non-dairy or partially non-dairy spread, plant-based cheese, plant-based dairy-type product, meat substitute or infant milk formula product comprising a lipid as claimed in claim 8 or an oleogel obtained or obtainable by the method of claim 10 or claim 11.

15. A cosmetic formulation, personal care product, cleaning product or candles comprising a lipid as claimed in claim 8 or an oleogel obtained or obtainable by the method of claim 10 or claim 11.

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