KR20140146156A - Preparation of microbial oil containing polyunsaturated fatty acids - Google Patents

Abstract

The present invention provides a method for producing a microbial oil comprising culturing a microorganism in a two-stage fermentation process wherein, in a final step prior to termination of fermentation, the carbon source is consumed by the microorganism at a greater rate than added to the medium Being; Lt; = 0.30 M carbon / kg medium, or is rate-limiting for the growth of microorganisms. Therefore, microorganisms increase the ARA ratio in the cells by limiting the carbon source to preferentially metabolize lipids or lipids except arachidonic acid (ARA). Next, microbial oil having 50% or more ARA and 90% or more of triglyceride from the microorganism is recovered using hexane as a solvent.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a microbial oil containing polyunsaturated fatty acids,

The present invention relates to a method for selectively producing PUFAs in a microbial oil, comprising culturing the microorganism in a two-step process and continuously recovering the microbial oil from the microorganism. The present invention also relates to novel (e.g. microbial) oils produced from such processes. In oil, more than 50% lipid (or PUFA in oil) is arachidonic acid (ARA). The oil may have a low peroxide value (POV) of 2.5 or less than 2.0 and / or a low anisidine value (AnV) of less than 1.0. The present invention also relates to food and food additives comprising or made using the microbial oil of the present invention.

Polyunsaturated fatty acids, or PUFAs, are found naturally. A wide variety of PUFAs are produced by different single cell organisms (birds, fungi, etc.). Particularly important PUFAs are arachidonic acid (ARA), one of many long chain polyunsaturated fatty acids (LC-PUFAs). Chemically, the arachidonic acid is cis-5,8,11,14 eicosatetraenoic acid (20: 4) and belongs to the (n-6) family of LC-PUFAs.

Arachidonic acid is a major precursor of a variety of biologically active compounds, collectively known as the eicosanoids, including prostaglandins, thromboxanes and leukotrienes. Arachidonic acid is also one of the lipid components of human milk and is considered essential for optimal neurological development in infants and young children. Arachidonic acid has a variety of uses, including use in infant formula, food and animal feed.

Microorganisms and especially fibrous fungi Mortierella can be used to obtain arachidonic acid. However, the proportion of arachidonic acid in the obtained microbial oil is generally too low. There have been a number of attempts to Mortierella increase the yield of arachidonic acid and from party (Mortierella), were the various degrees of success. Many attempts to increase arachidonic acid levels have involved processes that can not be readily used in industrial settings.

For example, in Eroshin et al. [Process Biochemistry: (35) 2000, pp1171-1175], cultures were left for about a week after fermentation. The amount of ARA quoted is based on biomass (not based on the oil extracted from it) since this document does not describe the extraction of any oil. Totani et al., Industrial Applications of Single Cell Oils, American Oil Chemists' Society Campaign, 1992, Chapter 4, pp. 52-60 and Lipids, vol. 22 No. 12 (1987) 1060-1062] generally insists on the use of low fermentation temperatures, which means that fermentation is considerably retarded. The ARA content herein is based on extraction with a chloroform / methanol solvent mixture. Another document relating to the field of ARA production is WO 96/21037.

EP-A-1035211 (Suntory) discloses a method for obtaining ARA and DHGLA lipids from Mortierella alpina . The calculation of the ARA content, however, is not biomass (rather than the oil extracted therefrom) or primarily the polyunsaturated fatty acid (rather than primarily extracting the oil and then determining the ARA content based on the oil) Based on the results from an analytical method that is esterified and then extracted using a solvent.

A report on high yield ARA is presented in M. The concentration of almost 70% of the mycelium collected using strain 1S-4 of M. alpina is demonstrated (Shimizu S., Oils-Fats-Lipids 1995, Proc. World Congr. Int. Soc. Fat Res., 21 ^st (1996), Meeting Date 1995, Volume 1, pages 103-109 and Biochemical and Biphysical Research communications, Vol. 150 (1), 1988, pages 335-441). However, this ratio is cell-based and is not equal to the ARA ratio in the microbial oil. In fact, the oil produced shows only an ARA content of 39.0% (Table 27.2, page 105). (In the art, various methods for measuring the ARA content are used, which need not be the same unit as the values cited at the end of this specification or based on the same analytical protocol.) Moreover, M. It was obtained when M. alpina cells were left at room temperature for 6 days after fermentation, which is clearly not a viable option in industrial production processes.

Therefore, it is desirable to identify methods for increasing the proportion (and yield) of arachidonic acid in microbial oils, in particular, in a manner that can be used on an industrial scale.

Summary of the Invention

The present invention provides a novel process for making (microbial) oils with increased ratios of arachidonic acid. This means that arachidonic acid can be obtained at reduced cost and increased rate. In addition, since the present invention does not rely on the genetic modification of the microorganisms involved in increasing the production of arachidonic acid, the present invention can help meet the increased need for natural, unmodified food ingredients. Moreover, oils have low oxidation potentials and are therefore suitable for incorporation into human food products where toxicity is particularly important, such as infant formulas.

Accordingly, a first aspect of the present invention is directed to a method of making a microbial oil or polyunsaturated fatty acid (PUFA). The method comprises fermenting (or cultivating) the microorganism in a fermentation vessel, suitably in a culture medium, whereby prior to (or at a preceding stage of) fermentation,

a) the carbon source is consumed by the microorganism at a greater rate than that added to the medium;

b) a carbon source is added at a rate of? 0.30M carbon / kg medium per hour;

c) the carbon source is rate-limiting for the growth of the microorganism or the carbon source is limited so that the microorganism metabolizes (its original) fat (s) and / or lipid (s);

d) the rate of addition of the carbon source is reduced to or below the consumption rate of the carbon source by the microorganism;

e) the carbon source is all used, or has a medium concentration of about zero at the end of fermentation or before termination;

f) the carbon source addition is discontinued but the fermentation is continued; And / or

g) The microorganism is placed in a state that metabolizes the fat (s) other than ARA (for example, intracellular such as PUFA) in preference to ARA.

A second aspect of the invention relates to a microbial oil comprising at least 50% (or at least 50.5, 51 or 52%) arachidonic acid (ARA). 55, 57, or 60% ARA. The oil

a) a triglyceride content of at least 90%;

b) POV less than 2.5;

c) AnV of less than 1.0; And / or

d) have a phospholipid content of less than 5%.

The oil may be prepared by the process according to the first aspect. The oil may be extracted with hexane.

Once the concentration of the carbon source is reduced or limited, the cell begins to metabolize fat or lipid in the cell. However, the cells primarily consume fat other than ARA. In this way, the ratio of ARA in the intracellular fat or lipid is increased. Thus, in this manner, the process according to the first aspect leads to a higher ARA content oil in the second aspect.

DETAILED DESCRIPTION OF THE INVENTION

microbe

The microorganism used may be bacteria, yeast, algae or fungi. Preferably, fungi are used and particularly fungi are used. A preferred fungus is the Mucorales tree. The fungi may be selected from the group consisting of Mortierella , Phycomyces , Entomophthora , Pythium , Thraustochytrium , Blakeslea , Rhizomucor or Asda will genus Aspergillus Scotland (Aspergillus). Preferred fungi are of the species Mortierella alpina . Preferred yeasts are Pichia < RTI ID = 0.0 > cipherii , such as Pichia or Saccharomyces . The bacteria may be selected from the group consisting of Propioni bacterium . Suitable birds parasite and can be and / or creep Te coordinated titanium Connie (Crypthecodinium cohnii) creep te coordination as a kind of uranium (Crypthecodinium), belongs in Fort pyridium (Porphyridium) or Chemnitz Kea (Nitschia).

The microbial strain used in the present invention may be a naturally occurring or commonly used industrial strain. The strain may not have been genetically modified, for example, not transformed into a vector or not containing a heterozygous gene (s). Given the current preference for foods that do not contain genetically engineered components, the microorganisms used may be strains that are not thus modified.

Polyunsaturated  fatty acid( PUFA )

The PUFA may be a single PUFA or two or more different PUFAs. Single or individual PUFAs may be of the n-3 or n-6 family. Preferably, it is a C18, C20 or C22 PUFA. It may be a PUFA having at least 18 carbon atoms and / or 3 or 4 double bonds. The PUFA (s) can be isolated in the form of free fatty acids, salts, as fatty acid esters (e.g., methyl or ethyl esters), phospholipids and / or mono-, di-, or triglyceride forms.

Suitable (n-3 and n-6) PUFAs are:

Docosahexaenoic acid (DHA, 22: 6? 3) derived from algae or fungi such as Crypthecodinium or Thraustochytrium , which is suitably (and bovine);

? -linolenic acid (GLA, 18: 3? 6);

α-linolenic acid (ALA, 18: 3 Ω3);

Conjugated linoleic acid (octadecadienoic acid, CLA);

Dihomo-gamma-linolenic acid (DGLA, 20: 3? 6);

Arachidonic acid (ARA, 20: 4? 6); And

Eicosapentaenoic acid (EPA, 20: 5? 3).

Preferred PUFAs include arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and / or gamma-linoleic acid (GLA). In particular, ARA is preferred.

Fermentation

Fermentation / culture will typically be performed in a suitable fermentation tank (or fermentation vessel) containing medium (lipid, generally aqueous). The main fermentation vessels will generally be aseptically inoculated from small source fermentors. Typically, liquid and / or aerobic fermentation processes are used. This is done in a deep tank fermenter. The fermenter may have a device for monitoring and / or changing pH and temperature. The container may be further adapted to perform cell or lipid mixing, such as aeration and / or agitation of the solution, or may be performed. This can be achieved, for example, using mechanical means.

The volume of the fermenter is suitably at least 10, 20, 40, or 60 m ³ . A volume of 100 or 150 cm < ³ > may be used.

Fermentation will typically last for 10 days or less, preferably 9 days or less, more preferably 8 days or less. At least 4, 5, 6, or 7 days.

Optionally, the fermentation can be carried out for 150 to 200 hours, for example 160 to 190 hours, 170 to 180 hours. The fermentation termination is generally a time point when stirring and / or venting is stopped. This may be when the fermentation vessel and / or the component device is (effectively) switched off. Then, the microorganism can be removed from the fermentation tank.

The fermentation may be at a temperature of 20 to 40 占 폚.

Carbon source  And nitrogen source

For example, any suitable medium, such as a medium suitable for microorganisms, may be used for fermentation. The carbon source may include maltodextrin, oat flour, oatmeal, molasses, vegetable (e.g., soybean) oil, malt extract, starch, ethanol or soybean oil. The preferred (non-complex) carbon source is selected from the group consisting of fructose, maltose, sucrose, xylose, mannitol, glucose or lactose or glycerin (e.g. vegetable source), citrate, acetate, glycerol, ethanol or ) Carbohydrates such as ascorbate or sugars. In a preferred embodiment of the invention, the carbon source is or comprises glucose, and in particular glucose syrup.

Suitable nitrogen sources include yeast extract, urea and peptone. The medium can exclude agar. Preferred nitrogen and / or carbon sources are water soluble or water miscible.

Each component of the medium (such as nitrogen and / or carbon source) may be present at the beginning of fermentation (all), continuously added during fermentation, or added stepwise or batchwise. In particular, the amount of carbon source present in the medium will typically be controlled by adjusting the rate of addition of the carbon source, as described below.

The nitrogen and / or carbon source may be separately supplied (or added), or simultaneously fed, or supplied as a combined preparation. Thus, they may be present in the same composition, which is preferably liquid (if deemed necessary). The carbon and / or nitrogen source may be added before the fungal cells are added (to the vessel), i.e. before the inoculation, or during the fermentation (to the fermentation vessel). Alternatively, they can be added before fermentation and during fermentation.

culture badge

The culture medium is preferably an aqueous solution. The medium can be, for example, a chelating agent (e.g., citric acid), a defoaming agent (e.g., soybean oil), a vitamin (e.g., thiamine and / or riboflavin) (E.g., phosphate) and / or sulfur (e.g., sulphate), as well as other materials to aid fermentation, such as, for example, calcium, calcium, or other metals such as zinc or iron and / or cobalt and copper May be added. The medium may, if necessary, contain additional oils such as olive or soybean oil, but preferably the medium does not contain such oils.

(Optimal) growth (or fermentation) temperature may vary depending on the microorganism used. However, it is preferably 20 to 40 캜, and more preferably 22 to 30 or 32 캜. In particular, the temperature at which the fermentation is carried out may be? 22 ° C or? 25 ° C, for example 22-30 ° C, for example 23-28 ° C. The pH of the aqueous solution during fermentation can be from 4 to 10, such as from 5 to 8, optimally from 6 to 7.

To aid venting, the medium will typically be stirred during fermentation. The aqueous solution and the cells are suitably mixed or stirred. This can be done, for example, when venting is provided by venting a gas, such as air, in an aqueous solution. This can help further the purpose of providing oxygen to fungal cells. Therefore, the fermentation is preferably aerobic. Other means of stirring or mixing include stirring using, for example, an impeller. This may be a hydrofoil axial flow design, or the aqueous medium may be designed to point radially outward from the impeller (such as a turbine). It is desirable to provide oxygen to the microbial cells during fermentation even without stirring, and such aeration (for example, by venting air, oxygen or other oxygen-containing gas) is advantageous. The vent may be from 0.1 to 2.0 vvm, such as from 0.5 to 1.0 vvm.

Preferably, the volume of the fermenter is at least 2 or 5 L, preferably at least 10 L. However, in the case of a fermenter used on an industrial or industrial scale, the vessel volume is preferably at least 50, 100, 500 or 1,000 liters.

At the end of fermentation (or second ) step

The fermentation process can be divided into at least two stages. The second or last step, which can be preceded by the end of the fermentation, is characterized by the reduction of available carbon pellets for the microorganism or any of the features (a) to (g) obtained in the first aspect. Typically, this step can be initiated at 15-2 hours prior to the end of fermentation, preferably within 10 hours after the end of fermentation, more preferably at 3-5 hours after fermentation. Preferably, this step will typically be initiated within 10 days after initiation of fermentation, more preferably within 9 days of fermentation termination, and even more preferably within 8 days of fermentation termination.

During the first or early stages of fermentation, the carbon source may be present in excess. Thus, the amount of carbon source available will not be limited to microbial growth. The rate of addition of the carbon source will exceed its consumption rate by microorganisms. In the second or last stage of fermentation, the amount of carbon source added will be reduced or completely stopped. This means that the amount of carbon source available to the microorganism will decrease during the second or last stage of fermentation. Typically, at the second, last or last stage, or at the end of fermentation,

- is consumed by microorganisms at a greater rate than added to the medium (e.g., the rate of addition is less than the rate of consumption);

- a proportion of ≤ 0.30 M carbon / kg medium per hour, excluding at least 0.01, 0.02 or 0.05 M carbon / kg medium / hr (units related to the molar or molar amount of carbon in the carbon source than the weight or mole of the carbon source itself) Lt; = 0.25 or < = 0.20;

Rate of growth of microorganisms and / or production of (PUFAs).

Typically, the concentration of the carbon source during the second step is < 10 g carbon source / kg, preferably 0.01 or 0.1-8 or 10 g / kg, more preferably 0.5-5 g / kg of medium, 2 to 4 or 5 g / kg. This means that on average during the last stage of fermentation there will be ≤0.30M carbon per kg of medium, preferably between 0.003M and 0.3M carbon per kg. Advantageously, it is from 0.015 M to 0.17 M carbon and more preferably from 0.03 M to 0.17 M carbon per kg.

If the carbon source comprises glucose, typically the concentration of glucose (at the last step) will be on average ≤10 g / kg of medium, preferably 0.01 or 0.1 to 8 or 10 g / kg. Advantageously, this is from 0.5 to 5 g / kg, even more preferably from 1 or 2 to 4 or 5 g / kg of medium. In this sense, the medium comprises cells and aqueous medium, i.e. "juicy" (cells and surrounding liquid).

The rate of addition of the carbon source in the last step is preferably 0.03 M or less carbon per kg, preferably 0.025 or 0.02 M carbon per kg (medium). Preferably, the addition rate is about 0.015 M carbon per kg. If the carbon source is glucose then preferably the glucose addition rate is less than 1.0 per kg of medium per hour, for example less than 0.8, for example less than 0.5 g.

Preferably, the addition rate of the carbon source in the last step is about half of the consumption rate of the carbon source by the microorganism. However, the ratio of addition rate: consumption rate varies from 1: 1 to 3, such as from 1: 1.5 to 2.5, alternatively from 1: 1.8 to 2.2. Alternatively, the ratio of the addition rate is 30 to 70%, such as 40 to 60%, alternatively 45 to 55% of the consumption rate.

The appropriate concentration of carbon source during the second stage of fermentation can be obtained by carefully controlling the rate of addition of the carbon source. Typically, this will be appropriately reduced during the last step or to facilitate the initiation of the last step. Periodic sampling and analysis of the culture is used to determine the concentration of the carbon source and adjustments necessary for the rate of addition of the carbon source. This can be done automatically using a computer system.

Pasteurization process

Pasteur sterilization will normally be performed after fermentation is terminated. In a preferred embodiment, Pasteur sterilization will terminate fermentation since heat during Pasteur sterilization kills the cells. Thus, Pasteur sterilization can be performed on fermentation broth (or cells in a liquid (aqueous) medium), but it can be performed on the microbial biomass obtained from the broth. In the former case, Pasteur sterilization can occur while the microbial cells are still in the fermenter. Preferably, the Pasteur sterilization is effected prior to any further treatment of the microbial cells, for example by granulation, pulverization or kneading (for example by extrusion).

Preferably, the Pasteur sterilization protocol is sufficient to inhibit or inactivate one or more enzymes, such as lipases, that adversely affect or degrade PUFAs or microbial oils.

Once the fermentation is over, the fermentation broth is filtered or otherwise treated to remove water or aqueous liquid. After removing moisture, a biomass "cake" can be obtained. If the Pasteur sterilization has not occurred, the dehydrated cells (or biomass cake) can be pasteurized.

Oil extraction

Preferably, for example, after fermentation is terminated, the microorganism may be killed or pasteurized. This is to deactivate any undesirable enzymes, for example enzymes that can degrade the oil or reduce the yield of PUFAs.

After the incubation or fermentation is complete or terminated, the fermentation broth (cells and aqueous solution) is removed from the fermentor and the liquid (usually water) is removed from the fermenter if necessary. Any suitable high solution separation technique may be used. This (dehydration) can be centrifuged and / or fruited. For example, an aqueous solution (such as water) can be used to wash cells, for example, to remove extracellular water soluble or water dispersible compounds. The oil can then be recovered from the microorganism using, for example, a solvent and the oil can be solvent extracted, preferably hexane extracted.

The oil may not contain GLA and / or DGLA (or substantially lack GLA and / or DGLA).

PUFA  Extraction process

PUFAs (or microbial oils generally containing PUFAs) can be extracted from (e.g., dried) granules (e.g., extrudates) containing cells. Extraction can be carried out using a solvent. Preferably, for example, a non-polar solvent such as a C ₁ -C _8, C _{2 -6} alkanes such as hexane are used. In general, carbon dioxide can be used (for example, in a liquid phase that is in an over-critical state).

Thus, cells can be extracted with an organic solvent, preferably under a nitrogen flow. Other usable solvents include ether, methanol, ethanol chloroform, dichloromethane and / or petroleum ether. Extraction into methanol and petroleum ether and / or extraction into a single layer solvent system consisting of chloroform, methanol, and water may also be used. Evaporation of the organic solvent (s) from the extract under reduced pressure can provide microorganisms containing arachidonic acid at a high concentration.

Preferably, the solvent may be such that the solvent exudes against the dried granules. Suitable microbial granulation and extrusion techniques and extraction of subsequent microbial PUFA containing oils are described in WO-A-97/37032.

The solvent allows the person skilled in the art to obtain the crude PUFA containing oil. This oil can be used in that state without further processing, or it can undergo one or more refining steps. Suitable purification protocols are described in International Patent Application No. PCT / EP01 / 08902, the contents of which are hereby incorporated by reference in their entirety. For example, the oil may be removed by acid treatment or removal of rubber, alkali treatment or removal of free fatty acids, bleaching or dye removal, filtration, cooling (or cooling to remove saturated triglycerides, for example), deodorization ) And / or abrasive machining (or removal of oil-insoluble materials).

The resulting oil is particularly suitable for nutritional purposes and can be added to food (for humans) or feed (for animals). Examples include milk, infant formulas, health drinks, bread and animal feed.

Refining / Refining

The microbial oil can be refined and purified. This includes the following components; (S), phospholipids, trace metals, pigments, carbohydrates, proteins, FFA, oil insoluble substances, water-insoluble substances, soap or saponification substances, oxides, sulfur, mono- or diglycerides, And removing one or more of them. Tablets can reduce or eliminate "tasteless " and / or improve the stability of the oil.

To perform this, the process (e.g., tablet) may be carried out by removing rubber (or acid treatment), neutralization (or alkali treatment), water washing, bleaching, filtration, deodorization, polishing and / . Preferably, the tablet comprises an acid treatment and / or an alkali treatment (rubber removal and neutralization). Alternatively, the purification process can include bleaching and / or deodorization. Preferably, however, the tablet will comprise bleaching and / or deodorization, and optimally will further comprise an acid and / or an alkali treatment.

oil

A second aspect of the present invention provides a microbial oil comprising 35 or 40% of one or more PUFAs such as ARA. The oil may comprise at least 50, 55, or 60% or more of PUFAs such as ARA. And may have a triglyceride content of 90% or more. Preferably, the microbial oil comprises 50, 55, or 60 to 90% arachidonic acid, more preferably 60 to 80% and even more preferably 60 to 70% arachidonic acid.

Preferably, the microbial oil has a triglyceride content of 90 to 100%, such as 90 or 96% or more, preferably 98% or more, more preferably 99% or more, and optimally 99.5% or more. Typically, the microbial oil will contain less than 5%, preferably less than 1%, and more preferably less than 0.5% eicosapentaenoic acid (EPA). The oil may comprise less than 5%, less than 2%, less than 1% of each of C ₂₀ , C _{20 : 3} , C _{22 : 0} and / or C _{24 : 0} polyunsaturated fatty acids (PUFAs). The free fatty acid (FFA) content may be? 0.4, 0.2, or 0.1%.

Among the triglycerides, preferably at least 40%, such as at least 50%, and more preferably at least 60%, of the PUFAs present are present at the [alpha] -position of the glycerol (also present in the triglyceride backbone) also known as the 1 or 3 position. It is preferable that 20% or more, for example, 30% or more, more preferably 40% or more of the PUFA (s) is present at the? (2) position.

The phospholipid content of the oil may be at most 5%, 3% or 2% and / or at least 0.1, 0.5 or 1.0%.

Typically, the microbial oil can be obtained by the process according to the first aspect of the present invention. Preferably, the oil is separated from the fungus, more preferably the oil is separated from Mortierella , And is isolated from M. alpina . The oil is suitably extracted with hexane.

ARA  content

In particular, since the literature often calculates the ARA content based on different principles, for purposes of clarity only, the calculation of the percentage of ARA content will be described. The ARA percentage is based on oil (extracted from biomass) and is not based on biomass itself. It is based on weight. It is based on the oil extracted by hexane and is therefore based on hexane extractable lipids (HEL). It is based on the total amount of oil and is not based on the total amount of fatty acids (which sometimes indicates a bad large number). The ARA content is determined by the well-known FAME assay protocol (using fatty acid methyl esters) detailed in AOCS Cel b89. Different solvents will extract different lipids. In the present invention, first note that the oil is extracted with hexane and then the ARA content of the oil is determined by FAME analysis. First, it is possible to obtain different results by esterifying the arachidonic acid (while still in the cell) and then extracting the resulting methyl ester for further analysis.

Peroxide value POV )

Preferably, the POV of the microbial oil is 3.0, 2.5 or 2.0 or less. However, much lower POV values can be obtained using the method of the present invention, and these values can be 1.5 or less than 1.0. Values less than 0.8 and even less than 0.4 can be obtained.

Anisin ( Anv )

Preferably, the anisidine value of the microbial oil is no greater than 1.0, for example, no greater than 0.6, no greater than 0.3, or even no greater than 0.1.

Uses and products

A third aspect of the invention relates to a composition comprising the oil of the second aspect, wherein one or more (additional) materials are used. The composition is a food and / or food additive for humans or animals. In an embodiment of the present invention for human consumption, the oil may be provided by refining or purifying an oil, suitably for human consumption, typically from a microorganism.

The composition may be an infant formula or a (human) food. Wherein the composition of the formula can be adjusted to have a lipid or PUFA in an amount similar to normal breast milk. This involves combining the microbial oil of the present invention with other oils to obtain a suitable composition.

The composition may be an animal or aquaculture feed composition or additive. Such feeds and additives may be provided to certain farm animals, particularly sheep, cows and poultry. Feeds or additives can also be provided to marine aquatic organisms such as fish and shellfish. The composition therefore contains one or more feed materials or ingredients for such animals.

The oil of the present invention can be sold as an oil directly sold or sold in a suitable package, typically an aluminum bottle that has been internally coated with an epoxy phenolic lacquer and washed with nitrogen. The oil may contain one or more antioxidants (e.g., tocopherol, vitamin E, palmitate) at a concentration of, for example, 50 to 800 ppm, such as 100 to 700 ppm.

Suitable compositions include, for example, oral pharmaceutical or veterinary compositions or cosmetic compositions. The oil may be ingested directly or encapsulated, for example, in the shell, and thus may be in the form of a capsule. The shell or capsule may contain gelatin and / or glycerol. The composition may contain other ingredients, such as spices (e. G., Lemon or lime flavor) or a pharmaceutically or veterinarily acceptable carrier or excipient.

The preferred nature and features of one aspect of the invention apply equally well to the other aspects with the necessary modifications.

The present invention provides a novel process for making (microbial) oils with increased ratios of arachidonic acid. This means that arachidonic acid can be obtained at reduced cost and increased rate. In addition, since the present invention does not rely on the genetic modification of the microorganisms involved in increasing the production of arachidonic acid, the present invention can help meet the increased need for natural, unmodified food ingredients. Moreover, oils have low oxidation potentials and are therefore suitable for incorporation into human food products where toxicity is particularly important, such as infant formulas.

The present invention will now be described by way of example only and with reference to the following examples, which are intended to illustrate and not to limit the scope of the invention.

compare Example  1 and 2 and Example  3 and 4

Of arachidonic acid (ARA)  Produce

M. Alpina (Mortierella alpina ) strain CBS 168.95 (Netherlands 2600 MA Delft DSM NV (DSM NV granted the applicant the right to receive the deposited biological material) was transferred to the Netherlands by CBS (Prentice Hall) 3508 E. Utrecht POBox 85167 Centraven Borsch Merkur Tres (CBS) 0.0 > DS 30340) < / RTI > was stored at-80 C and opened in sterile conditions.

Glucose, 20;

페이스트(고형부 80%, 단백질(N x 6.25) 46%, NaCl 16%, pH (2% 용액) 5.6, 회분 22%, 총 플레이트수 10⁴/g, 엔테로박테리아케에(Enterobacteriaceae) 〈10/g, 이. 콜라이(E. coli) 〈1/g, 효모 및 곰팡이〈100/g, DSM N.V. 에서 구입, 세이버리(Savory) 성분 PO 박스 1,2600 MA Delft), 12.5;Yeast extract (Gistex

Paste (and male part 80%, protein (N x 6.25) 46% NaCl 16%, pH (2% solution) 5.6, ash content of 22%, a total plate number of 10 ⁴ / g, the Enterobacter bacteria Kane (Enterobacteriaceae) <10 / g, E. coli <1 / g, yeast and mold <100 g, purchased from DSM NV, Savory ingredient PO Box 1,2600 MA Delft), 12.5;

(Basildon 86 / 013K silicon / non-silicon defoamer compound used according to the manufacturer's instructions of British OX14 IRZ, Oxford, Abingdon, Kimberleide, Basilon Chemical Company), 0.2 ml (g / The medium was inoculated into a 500 ml flask. The pH of the medium was adjusted to 7.0 before autoclaving.

paste), 25; 소포제(Basildon 86/013K), 0.2를 함유하는 500ml 의 배지를 사용하여 4개의 2000ml 플라스크의 접종에 사용하였다. 멸균전의 pH 는 7.0 이였다. Cultured at 25 DEG C for 48 hours with shaking at 250 rpm, glucose, 20; Yeast extract (Gistex

paste), 25; The cells were used for inoculation of four 2000 ml flasks using 500 ml of medium containing defoamer (Basildon 86 / 013K), 0.2. The pH before sterilization was 7.0.

These cultures were incubated at 25 ° C for 24 hours and seeded into a 5 m ³ inoculation fermentor containing 2400 liters of the same composition as used in the 2000 ml flask (pH prior to sterilization was 6.0).

The fermentation temperature was fixed at 25 ° C, stirred at 150 rpm, the vessel pressure was 0.5 bar, and the venting rate was 0.5 VVM.

The culture of the inoculation fermenter was transferred to the main fermenter after about 36 hours (oxygen uptake was> 3 mmol / kg / h).

The main fermenter

Glucose, 35;

분말, 저 나트륨 양조 효모, 펩톤(추출물), 고형분 ≥96%, 총 N 〉10%, 아미노 N 6-7%, NaCl〈1%, pH (2% 용액 5.3-6.3), 회분 〈12.5%, DSM N.V. 로부터 구입가능한 균질의 분말, 세이버리 성분), 5.0;Yeast extract (Expresa 2200

NaCl <1%, pH (2% solution 5.3-6.3), ash <12.5%, ash, low sodium brewing yeast, peptone (extract), solids ≥96% Homogeneous powder available from DSM NV, Sabour component), 5.0;

NaH ₂ PO ₄ .2H ₂ O, 1.0;

KH ₂ PO _4, 2.0;

MgSO ₄ .7H ₂ O 0.5;

Basildon 86 / 013K, 0.3; Citrate, 1H ₂ O, 0.6;

ZnCl ₂ , 0.010;

Fe ₂ (SO ₄ ) ₃ 20% H ₂ O, 0.025;

MnSO ₄ .1H ₂ O, 0.010; (G / l) before and after sterilization (pH 5.0 before sterilization).

Glucose was separately sterilized and added to the main fermentor after sterilization.

The fermentation was continued for 175 hours. The pH of the medium was maintained at about pH 6 (+/- 0.1) with aeration (air flow) of 0.5 VVM, air pressure of 0.8 bar and agitation at 70 rpm. The oxygen level was maintained at D.O. &gt; = 30% by successively increasing the agitation speed to 100 rpm and the air flow to 0.9 VVM.

A sterile glucose solution of about 50% (w / w) was injected into the fermenter to maintain the glucose concentration above 10 g / l and 625 kg of the 25% yeast extract solution for about 30-78 hours adjusted to an ammonia concentration of <30 mg / And fed into the fermenter.

Experiments were performed 4 times (Examples 1 to 4) and the glucose concentration of the medium was monitored over time. A graph of glucose concentration versus fermentation time is presented in Table 1 at g / kg. This table shows that the final value of Example 4 was 2.2 g / kg at 172 hours. This was three hours before the end of fermentation (EoF). Glucose levels were 0 before about one hour before EoF.

In Comparative Examples 1 and 2, the concentration of the carbon source (glucose) was 5 g / kg or more at the end of fermentation. Moreover, 10 hours before EoF, the glucose concentration was about 20 g / kg. Thus, in Examples 1 and 2, the concentration of glucose was not limited to the production of the microorganism group, or ARA.

In Examples 3 and 4, at the last stage of fermentation, just prior to EoF, the glucose concentration was adjusted so that the concentration of glucose was about 5 g / kg before 10 hours before EoF. During this last step, over 10 hours glucose was added at an addition rate of 0.5 g / kg / hr. The glucose concentration in EoF was actually zero. During this period, the consumption rate of glucose was about twice the addition rate, i.e. about 1 / g / kg / hr.

The glucose concentration (g / kg) in the medium over several hours during fermentation is shown in Tables 1 to 4 (each corresponds to Examples 1 to 4).

Time (hrs) Glucose concentration g / kg 0 48.7 24 57.6 28 47.8 54 46.4 78 62.0 102 62.5 126 48.2 150 42.5 167 20

Time (hrs) Glucose concentration g / kg 0 47.8 28 32.5 54 32.2 78 41.3 102 49.5 126 46.8 150 29.9 165 17.1

Time (hrs) Glucose concentration g / kg 0 49.2 28 60.1 54 40.8 78 34.0 102 37.3 126 23.2 150 16.7 172 7

Time (hrs) Glucose concentration g / kg 0 45 28 34.1 54 34.7 78 29.2 102 38.1 126 45 150 23.5 172 2.2

At the end of fermentation, the microorganisms and the surrounding aqueous solution (fermentation broth) were removed from the fermentation tank. Remove some of the water by separating the high solution from the juice. The remaining cells were then extruded and solvent-extracted using hexane. Thus, the ARA containing microbial oil (hexane extractable lipid) was obtained from the cells that carried out each of the four different fermentation protocols.

The percentage of ARA content (by weight) in the oil was determined using the well known FAME analysis protocol (detailed in AOCS Celb89). In Example 3, the ARA concentration in the microbial oil was 508 g / kg (50.8%). The equivalent value for Example 4 was 545 g / kg (54.5% ARA). By comparison, the microbial oils extracted from cells in Comparative Examples 1 and 2 were much lower, 36.8% and 36.7%, respectively.

Centrabory Borsch Merkur Tres DS30340 19950220

Claims (10)

A method for producing a polyunsaturated fatty acid (PUFA), comprising culturing a microorganism in a culture medium in a fermentation vessel,
In the preceding stage of fermentation termination,
a) the carbon source is consumed by the microorganism at a rate greater than that added to the medium;
b) the carbon source is added at a rate of? 0.30M carbon / kg medium per hour;
c) the carbon source is rate-limiting for the growth of the microorganism or the carbon source is limited so that the microorganism metabolizes the fat and / or lipid;
d) the rate of addition of the carbon source is reduced or less than the rate of consumption of the carbon source by the microorganism; or
e) the carbon source has all been used, or has a medium concentration of zero or more at the end of fermentation or before termination;
f) the carbon source addition is discontinued but the fermentation is continued and / or;
g) the microorganism is in a condition that metabolizes or consumes one or more fats or lipids in preference to arachidonic acid (ARA)
A method for producing a polyunsaturated fatty acid (PUFA). The method according to claim 1,
h) the concentration of the carbon source in said step averages? 10 g / kg and / or? 0.17 M carbon / kg medium;
i) the carbon source is glucose and / or;
j) A method for producing a polyunsaturated fatty acid, wherein the polyunsaturated fatty acid is present in the microbial oil. 3. The method according to claim 1 or 2,
k) A process for the production of polyunsaturated fatty acids, wherein the second stage is the beginning of the end of fermentation within 15 to 2 hours before the end of fermentation or 10 days after the start of fermentation. 4. The method according to any one of claims 1 to 3,
l) fermentation is performed at &gt; = 22 [deg.] C and / or &lt; = 30 [deg.] C;
m) fermenting in the absence of additional oil and / or;
n) The fermentation is continued so as not to exceed 9 days. 5. The method according to any one of claims 1 to 4,
n) the PUFA comprises arachidonic acid (ARA) and / or;
o) the container has a volume of at least 10 liters. 6. The method according to any one of claims 1 to 5,
If the microorganism is Mortierella , optionally Mortierella alpina ) and / or is inherently modified. The method according to claim 1,
Wherein the carbon source is continuously added to the culture medium during fermentation or is added batchwise to the culture medium. 8. The method of claim 1 or 7,
A method for producing a polyunsaturated fatty acid wherein a nitrogen source is added to a culture medium. 9. The method of claim 8,
Wherein the nitrogen source is added to the culture medium continuously or continuously in the culture medium during fermentation. 10. The method according to any one of claims 1 and 7 to 9,
Wherein the carbon source and the nitrogen source are added separately or simultaneously, or as a combination.