MXPA97005078A - Araquidonic acid and methods for the production and use of my - Google Patents

Abstract

The present invention relates to processes for the production of arachidonic acid containing oils, which are preferably substantially free of eicosapentenoic acid, the invention also relates to compositions containing oils having high variable amounts of arachidonic acid in the form of triglyceride, and to uses of said oils: in a preferred embodiment, Mortierella alpina is cultivated using conditions that produce triglyceride oil having particularly high levels of arachidonic acid residues, the biomass is harvested and the oil is extracted, recovered and used as an additive for infant formula

Description

ARAQUIDONIC ACID AND METHODS FOR THE PRODUCTION AND USE OF THE SAME CMPLE OF THE INVENTION This invention relates to the production of arachidonic acid, compositions containing arachidonic acid and uses thereof.

BACKGROUND OF THE INVENTION Araqui dona donate acid (ARA) is an acid or polyunsaturated long chain (PUFO) of class ornega-6 (acid 5, 8, 11, 14-e? Thing + aenoic, that is, 20? O. ARA is ol C20 PUFfl more abundant in the human body.Fs prevalent in particular in tissues of organ, muscle and blood, serving a major role as a lipid is + ructuraL associated predocu nanternento with phospholipids in the blood, liver, muscle and other systems of Main organs In addition to its main role as a structural lipid, ARA is also the direct precursor for a number of circulating eicosenoi such as prostaglandi to E3 (PGE2), prostacycline 1 to T2 (PGT2), thromboxane A2 (T "Fl2), and leucotpenes" (I.TB4) and O * (LTC "). These eicosenoids exhibit regulating effects in the me + abolition of 11 poprotein, blood rheology, vascular tone, leukocyte function and ac + Plate ation Despite its importance for human metabolism, ARA can not be sm + e + ao in human de novo hands ARA is synthesized by the lengthening and desaturation of aculeal l noleic acid (LOA), an essential fatty acid. This procedure requires the presence of the above Delta ñ-desa + urasa, an enzyme present in the human body at haos levels, Burre et al., L pids, 75: 354-356 (1990). Therefore, most ARA should be provided on the day, and this is especially important during times of very rapid body growth, such as the day. During the first year of your v? D < > , an infant can double or triple its weight. Therefore, high levels of ARA are required in the ie * a. To meet this increased demand, human breast milk contains high levels of ARA. Sanders and others, Arn. 3. Clin. Nutr., 31: 805-813 (1978). ARA is the C20 PUFA most prevalent in rna * erna milk. Of those mothers, especially vegetarians, who breastfeed their infants, many will benefit from the additional APA in the diet. However, many mothers do not breastfeed their infants, or do not breastfeed during the infant's rapid growth period, choosing instead to use a infant formula. No known commercial infant formula contains ARA in the form of an icicle. The Patent of E.U.A. No. 4,670,285 (Clandimn et al.), Incorporated herein by reference, describes the requirement for fatty acids by the infant, including ARA. To provide these fatty acids, Clandinm and others suggest a mixture of egg yolk, acei + or < he fish f stolipi two of red globules and vegetable oils as the fat component of a formula proposed for infants. However, fish oil contains large amounts of eicosapentaenoic acid (EPA). It is known that FPA reduces the synthesis of ARA in infants. Carl are, and others, INFQRH, 1: 360 (1990). In this way, it would be desirable to be able to provide ARA without also providing additional EPA. In addition, the egg yolks contain a relatively low concentration of ARA, so that the mixture of Cland nin and others is not viable econormiment. Because ARA is present in animal, but not vegetable oils, its production in commercial quantities has remained a desirable but elusive goal. Shinrnen, and others, flicrobiol. B otech ,. 31: 11-16 (1989), have reported the production of ARA by an isolated fungus, ior * iere1 a1 pina, using conventional fermentation in stirred tank. (See also Japanese Patent 1, 215,245 to Shinrnen and others). After cultivation, the organisms are harvested, dried and their lipids are extracted from fungal biogas with an organic solvent and the lipids are chemically modified (covalently). For example, the lipid mixture is hydrolyzed or converted to ethyl esters and then combined with cyclodextrin before being used as a dietary supplement. Shinmen et al. Do not describe or "suggest the administration of unmodified microbial oils.

Porphyp ium cruenturn, a rooting start-up, I can grow in swamps in large quantities and it has an immediate content that can contain up to 40% of ARA. Ahern and others, iotech, Bioeng. 75: 1057-1070 (19R3). Unfortunately, ol r- > ARA is mainly associated with galac + oli pidides, a complex polar lipid not present in breast milk. In this way, not only the total usable ARA produces a fraction of one percent of the biomass, but the ARA form is not suitable for use as an additive for the infant formula without additional modification. The Pa in * e of E.U.A. No. 4,870,011 (Suzu i et al.) Describes a method for obtaining lipids such as t-i noleruco acid from fungi of the genus Mortierella. The nolenic acid is purified from the mixture of lipids contained in the fungi. DE 3603000A1 (My Magnifying Glass) describes a mixture of highly polyaturated fatty acid and its use as the fatty compound of a infant formula. The fat mixture has a high content of ARA and docosahexaenoic acids (DHA) in a ratio of 2.5: 1 respectively, as well as a high cholesterol content. The sources of the fatty acids are listed as certain types of rnacroalga, vegetable oils, beef and pork organ fats or highly refined yolk oil. It is said that a source of DHA and ARA are rnacroalgae of the fecofite and rhodophyte types. There is no suggestion to use a microbe as a source of oil.

The algae and fish oils also include t? P? N? Nen * e EPA that reduces the synthesis of ARA in < /? vo. In addition, the refined yolk oil is not an economical source of ARA. In addition, there is no description herein of a concentrated additive of ARA for supplemental pre-existing infant formula. Accordingly, a need remains for an economic method, as possible to produce ARA, preferably without the concomitant production of UPA. An object of the present invention is to satisfy said need. Another object of the invention is to provide an additive, and such an additive source, for use in a infant formula so that the levels of ARA in the formula approach those levels in human breast milk. A further object of this invention is to provide a fungicidal oil containing ARA for use in skin, skin or skin products.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to the production and use of fungal oil containing arachidonic acid (ARASCO) and to compositions containing said oils. The oil can be referred to as an individual cell oil. The fungi are grown under conditions that produce oil, harvested and the oil is extracted and recovered. The oil, without additional chemical modification, can be used directly to provide supplemental ARA to people who require it, including newborn infants, pregnant or breastfeeding women or people who exhibit APA deficiency pathologies. The advantages of the invention include its ease of production, and high purity, and the lack of amounts of EPA + ectables.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES "ARA" and "EPA" are also used herein to refer to residues of arachidonic acid and eicosapentaenoic acid, respectively, wherein the residues are esterified to glycerol with part of a fatty tipelic acyl or a phospholipid. As used herein, a composition is "free of EPA" when the residual amount of EPA in the composition is less than the amount that would reduce the synthesis of ARA when the composition is used as a nutritional supplement. The present invention succeeds in providing an economic source of arachidonic acid (ARA). In one embodiment, this invention relates to a method for the production of a fungal oil containing arachidonic acid (ARASCO) that is substantially free of eicosapentaenoic acid co (EPA). Co or used in this"Substantially free" means that the EPA is present in less than about one fifth of the amount of ARA in the oil. This oil, an individual cell oil, can be administered directly ?, in an unmodified form. As used in the present "unmodified" it means that the chemical properties of the fatty acids, or the oils themselves, have not been altered covelentemente., T) e this way, for example, a temporary modification to the ARASCO or ARA that can be inverted after oil absorption would not be beyond the scope of this invention. Unmodified fungic oils according to this invention provide plastics where a relatively high proportion of the fatty acid residues are ARA (preferably at least 40% of the fatty acid residues are ARA, preferably at least 50% of the waste is ARA), and the ratio of ARA waste to EPA waste is also + (at least 5: 1, preferably at least 20: 1 weight / weight). Said oil from natural sources has not been described before the present invention. While those with this composition can be chemically synthesized (for example, by mixing mixtures of free fatty acids with high ARA content or by transesterifying with ethyl esters of said fatty acid mixture), the manipulation of the fatty acid mixture (by example, purification, steping, etc.) can introduce unwanted side products. In contrast, the method of this invention provides products having the desired composition by means of extraction from natural sources. Table 1. Composition of Fatty Acid of Various Fungic Species. 0 0 (1 0 Of those fungi whose fatty acids have previously been characterized, it has been found that most do not ARA. Wee * e, 3.D., Fungal Lipid B ocheini s * ry, Plenu Press, N.Y. (1974). Piggy banks of those species that do not make ARAs, including all Pythium species previously characterized, produce significant amounts of eicosapent enoiic acid (EPA) in addition to ARA. Table 1 establishes the fatty acid profile of P. insid osurn as well as the fatty acid profile of other fungal species. Unexpectedly, it has been discovered that P. insidiosun produces ARA without concomitant production of FPA. Regarding fish oils, high levels of EPA in dietary supplements result in a reduced ability to form ARAs of dietary linoleic acid (LOA). Accordingly, while in the process of this invention, those fungic species that produce both ARA and EPA can be used, it is preferable to use species that do not produce significant amounts of EPA. Said preferred species include Pythium msidiosum and Mortierella alpina. Both species are commercially available and are on deposit with the American Type Culture Collective in Rocl-'Ville, Maryland, having accession numbers 28251 and 42430, respectively. P. insidiosum and M. alpina have been used as representative fungal species through this description. Of course, fungal species producing ARA containing triglyceride and reduced EPA as described in the present are also contemplated within this invention. One of the significant problems that one embodiment of the present invention overcome is the reduction of the bioeynthesis of ARA in infants caused by the presence of increased levels of EPA diet. This problem can be corrected by providing ARA for use in infant formula at levels substantially similar to those found in human breast milk. Typically in human breast milk, the ratio of ARA: EPA is approximately 20 il respectively. The present invention specifically contemplates any microbial oil that provides a sufficient amount of ARA to overcome the negative effects of EPA diet. Preferably, the use of the ARA-containing oil will result in an ARA: EPA ratio of at least about 5: 1. Very preferably, the ratio will be less than about 10: 1 and, very poorly, will be about 20: 1. As can be observed, as the amount of ARA is higher in the final product, with respect to the amount of EPA, the result will be more desirable. In a process of the present invention, the fungi are cultured under suitable culture conditions that produce ARA-containing oil. In general, the techniques of genetical culture are well known to those skilled in the art and those techniques can be applied to the method of the present invention. For example, the cultivation of a fungal inoculation amount may occur in culture submerged in stirred flasks. The bottles are provided with a growth medium, seeded with fungal mycelium, and grown on a reciprocating shaker for approximately three to four days. The composition of the growth medium may vary, but; It always contains carbon and nitrogen sources. A preferred carbon source is glucose, the amounts of which may vary from about 10-100 grams of glucose per liter of the growth medium. Typically, 15 grams / liter is used for the culture with the agitated flask. The amount may vary depending on the desired density of the final crop. Other carbon sources that can be used include molasses, high fructose corn syrup, hydrolyzed ally or any other low cost conventional carbon source used in fermentation processes. further, lactose can be provided as a carbon source for P. insidiosurn. In this way, the whey perrneado, is high in lactose and is a very low cost carbon source that can be used as a substrate. Suitable amounts of these carbon sources can be readily determined by those skilled in the art. In general, additional carbon needs to be added during the course of the crop. This is because organisms use so much carbon that it would be unmanageable to add everything in a one-time mode. Nitrogen is typically provided in the form of yeast extract at a concentration of from about 2 to about 15 grams of extract per liter of the growth medium. Preferably, four grams per liter are provided. Other sources of nitrogen, including peptone, tryptone, corn infusion, soybean meal, can be used. hydrolized vegetable protein, etc. The amount to be added to these sources can easily be determined by those skilled in the art. Nitrogen can be added in a one-time mode, that is, all at the same time before cultivation. After cultivation for 3-4 days at a suitable temperature, typically about 25-30 ° C, an amount of fungus has grown which is sufficient to be used as an inoculum in a conventional agitated ferrner (STF). Said Termenors are known to those skilled in the art and are available commercially. The fermentation can be carried out in one-off modes, by one-time feeding, or by continuous fermentation. Preferably, the STF is equipped with a marine impeller, although a turbine impeller i or Rushton can also be used. The ferrner is prepared by adding the desired carbon and nitrogen sources. For example, a 1.5 liter fermentor can be prepared by mixing 50 grams of glucose and about 15 grams of yeast extract per liter of drinking water. As previously discussed, other carbon or nitrogen sources or mixtures of the same can be used. The reactor containing the nutrient solution must be sterilized, for example, by heating before inoculation. After cooling to about 30oC, the inoculum can be added, and the culture initiated. Gas exchange is provided by spraying air. The air spray speed may vary, but is preferably adjusted from about 0.5 to about 4.0 VVM (volume of air per volume of ferrnentator per minute). Preferably, the dissolved oxygen level is maintained from about 10% to approximately 50% of the saturation value of the solution with air. Therefore, adjustments in sprinkling speed may be required during cultivation. agitation is desirable. Agitation is provided by the impeller. The maximum agitation speed is preferably adjusted within the range of about 50 crn / sec to about 500 cm / sec, preferably about 100 to 200 cm / sec. In general, the amount of inoculum may vary. Typically, about 2% to about 10% by volume inoculum can be used. Preferably, it can be used in a sowing train in the fennel, approximating 5% by volume of inoculum. The nutrient levels must be controlled. When glucose levels fall below 5 g / 1, additional glucose must be added. A typical culture cycle uses approximately 100 grams of glucose and approximately 15 grams of yeast extract per liter. It is desirable to deplete nitrogen during the course of cultivation as this improves the production of oil by fungi. This is especially true when M. alpí na is used as the production organism.

In a particularly preferred fashion, it can be cultivated in a fermentor-Mor with alpine with high oil content including high levels of ARA, using very high nutrient levels. that nutrient levels that contain nitrogen in excess of that provided by 15 grams / l or yeast extract can be added, at the beginning of the fermentation, as long as the total amount of nutrient containing added carbon is comparatively high during fermentation The total amount of carbon nutrient, preferably fed continuously or at a single time for the first 25-50% of the fermentation time course, or in aliquots at multiple time points on the same portion of the course of time, it will be preferably equivalent to 75-300 grams of glucose per liter of the culture medium (Ratio of C: N> 5: 1, expressed as weight / weight of glucose: yeast extrusion). In particular, the nitrogen nutrient is soy flour, added at a level of approximately 16 grams per liter of the medium, and the carbohydrate nutrient is present initially at a level equivalent to approximately 80 grams of glucose or more. When high levels of carbon and nitrogen nutrients are used, it is preferred to sterilize the solutions containing the two nutrient solutions separately. It has also been discovered that the yield of biornase can be improved for fermentations containing high levels of carbon-containing nutrients by retaining part of the nitrogen nutrient and feeding the remaining nitrogen-containing nutrient with only or in one or two parts. the icuo + as during the course of fermenting. Occasionally, the product will produce an excessive amount of foam. Optionally, an anti-foaming agent, such as those known to those skilled in the art, for example razu 310R or vegetable oil, can be added to prevent foam. The temperature of the culture may vary. However, those fungi that produce both ARA and EPA tend to produce less EPA and more ARA when grown at higher temperatures. For example, when it is grown Mor'tie she alpine at less than 18 ° C, begins to produce EPA. In this way, it is preferred to keep the temperature at a level that induces the production h > reference of ARA. Suitable temperatures are typically from about 25 ° C to about 3 (1 ° C.) Preferably, the culture is continued until a desired biodanse density is obtained.A desired bioinase is about 25 g / 1 of the organism. is achieved within 48-72 hours after inoculation.At this time, organisms typically contain about 5-40% complex lipids, ie, oil, of which approximately 10-40% is ARA or preferably at least less 40% of the ARA residues in the fraction of the t rigl icérido, most preferably at least 50% of ARA in the fraction of the tp gl icen do, and can be li ned.The fermentation fungí ca par-a the production of ARA in accordance with this invention can be carried out in a fermentation medium with a pH of between about 5 and 0. However, the yields of bio-handle, oil and ARA of the cultures of M. alpina can be created by making a per-fil of the pH of the medium, instead of allowing an uncontrolled pH increase. Yields can also be increased by maintaining high oxygen levels during fermentation. These modifications of the fermentation process are effective especially when high levels of the nutrient are used in the fermenter. When the level of the initial nitrogen nutrient exceeds the equivalent of about 15 grams of yeast extract per liter, and / or the level of the carbon nutrient exceeds the equivalent of approximately 150 grams of glucose per liter, the growth of fungi. This inhibition of growth can be overcome by single-feed fermentation, for example by dividing the nutrient for fermentation into aliquots that are fermented sequentially, once part or all of the nutrient has been metabolized. supplied by the previous aliquot. The benefit of overcoming growth inhibition can be achieved by feeding only the carbon nutrient (see Shinmen, et al.). It has been discovered that this benefit can also be obtained by dividing the total nutrient into aliquots and feeding the aliquots during fermentation, or by feeding the nutrient solution continuously. Similarly, it has been unexpectedly discovered that the benefit can be achieved by feeding the nitrogen nutrient only to a fermentation where the carbohydrate nutrient is initially present at a high level. It has also been unexpectedly discovered that the inhibition of growth can be mitigated by making a profile of the pH of the fermentation, keeping the oxygen tension high in the fermentation! , or both. It has been found that fermentation of M. alpina in a high nutrient medium at a low pH (pH = 5-6) results in improved biomass growth (and also an increased oil yield). However, oil produced under these conditions has lower levels of ARA residues in the oil. On the contrary, fermentation at a high pH (pH - 7-7.5) results in increased levels of ARA in the oil, but poorer growth. In a preferred mode, the fermentation method of this invention involves the pH profile wherein the pH is low during the early stages of the fermentation and is high during the latter stages. Early stages include periods of rapid (exponential) growth during which nutrients are rapidly metabolized; the last stages include the stationary phase, when cell division is stopped, usually due to IB (Insufficient amounts, one or more nutrients, and increases the production of oil rich in ARA.) The profile can be made by controlling the pH of the planer at levels that are adjusted in two or more separate steps during the period of fermentation. has discovered that maintaining the dissolved oxygen content of the medium (OD) at high levels (eg, >40% air saturation level) will result in a relief of growth inhibition by high nutrient levels and / or increase in the relative level of ARA residues in the oil. The O.D. it can be maintained at a high level by increasing the pressure of the container (forcing more air into the head space of the fertilizer), increasing the agitation (for example, increasing the maximum impeller speed), and increasing the aeration (ie, increasing the amount of air passing through the ferment in a given time, usually expressed as an increase in VVM, volume of air per volume of ferrator per minute) and / or increasing the O2 content of the spray gas. It has been discovered that fermentation under these conditions increases the use of car-bono, resulting in a higher final biomass concentration and higher productivity of ARA-rich oil in the fermenter. In particular, fermentations incorporating one or more of the above modifications result in a production of extractible t-glyceride oil having at least 40% ARA residues, and preferably at least 50% residues of ARA. In a particular preferred embodiment, the fermentation medium contains carbon nutrient equivalent to > 80 g / L of glucose and nitrogen nutrient equivalent to > 16 g / L of yeast extract, and the medium is adjusted to a pH between 5 and 6 subsequent to sterilization. After inoculation, the pH of the medium is controlled at or slightly above its initial level. Once the level of the carbohydrate nutrient has dropped to < 60 grams of the equivalent thing s / 111 or 'usually about 40 hours), the set point for the pH control is changed to a pH of about > 6. At or near the time at which the rate of oxygen uptake (and / or carbon dioxide release rate, CER) reaches its maximum (usually after approximately 7 hours), the set point is raised to a pH between 6.5 and 7 (usually in increments, for example, at a rate of about 0.1 pH unit per hour). The pH is then controlled to keep it below about pH 7-7, .5 for the final stages of the fermentation. For this modality, the level of oxygen dissolved in the medium (DO) is maintained near or above 40% of the saturation level with air, preferably by sequentially increasing the pressure of the container to 0.7733 kg / cm2, increasing the agitation to the equivalent of about 300 ern / sec of maximum impeller speed, and increasing aeration to approximately 0.5 volumes of air per volume in the per-minute fermenter. After a period of rapid growth and high absorption of O2 by fermentation, the growth (and absorption of 02) will decrease. Agitation / aeroation can be reduced at this time, as long as the O.D. at a high level, usually about 40% saturation with air. By optimizing the fermentation of M. alpina as it is desorbed in the present, it is possible to obtain very high yields of biomass containing 20-60% of oil in the biomass, where 25-70% by weight of the oil are ARA residues in the form of tp glycose. The biomass (and oil) can be harvested as described herein. Preferably, the biomass will be harvested from the ferrnentator within 48 hours after reaching maximum productivity, measured as grams of ARA / L / day. Harvesting can be done by any suitable method such as, for example, filtration, centrifugation, or spray drying. Due to the lower cost, filtration is preferred. After the harvest, the nicelial tor-ta can be removed. The icelial cake refers to the resulting biomass harvest after harvest. The cake can be loose or pressed, crumbled or not crumbled. Optionally, any residual water may be removed from the cake, for example, by vacuum drying, fluid bed drying, spray drying or lyophilization, <If the option is selected, it is preferred to use non-polar solvents to extract the oil containing ARA.Although any non-polar solvent is suitable, hexane is preferred.In a preferred embodiment, the oil is extracted. of the biomass dried by wet grinding or by percolation with virgin hexane.The solvent is generally added at a solvent to biomass ratio of approximately 5: 1 (w / w). After wet grinding, the solids are separated from the It is advantageous to keep the solvent-containing extract (mixture of mycelia) anaerobic enough to avoid oxidation of the unsaturated fatty acid residues in the oil.The mixture of mycelia is dissolved to produce a crude fungal oil. The crude oil extracted from the fungal biomass with non-polar solvents must be turbid, particularly when the mass is crushed, because the crushing can liberate particulate fi such as fragments of the cell wall and soluble polysaccharides. The clarification of this turbid oil can be effected by dissolving the crude oil in more polar solvents, such as acetone or alcohol. In a preferred embodiment, the crude oily extract of fungal mycelium is subsequently clarified by means of acetone extraction and precipitation. A mixture of acetone mycelia is prepared by adding acetone to the cloudy crude oil extract (preferably at a level of approximately 20% of the oil; 00 that is, about 4 volumes of acetone per volume of crude oil), mixing uniformly and allowing the mixture to stand for a sufficient period for precipitation of the fine particles (usually about one hour at room temperature). The mixture of acetone mycelia containing the oil is clarified by centrifugation and / or filtration and then separated from the solvent to produce acetone-clarified fungic acid. The fungic acid clarified with acetone is preferred for later or later (for example, separation of gums, bleaching and deodorization by means of conventional techniques) because the fines produced during the extraction of the fungal biogeny interfere with the treatments of refining if they are not removed in the acetone step. Another preferred embodiment includes the countercurrent extraction of dry biomass, which can be carried out in commercially available extraction units, for example those manufactured by Crown (Crown Mark IV) or French, Inc., which are not generally used to extract oils. vegetables, but they were designed to extract dirt and stains. Although the extraction efficiencies are not as high without the bio-phase re-capture, the countercurrent ex + reaction procedure has the advantage of producing fewer "fines" thereby reducing the technical difficulty in recovering a clear retina oil. Alternatively, the wet cake (typically containing about 30 to 50% solids) can be comminuted and extracted directly using polar solvents such as ethanol or isopropyl alcohol, or superential fluid extraction with solvents such as CO2 or NO. Preferably, the cakes are shredded before extraction. Conveniently, the present invention allows the economical use of supercritical fluid extraction techniques. rloHugh, et al., Supereriti cal Fluid Extration, B? tterworth (1986). These techniques are known to those skilled in the art and include those that are currently applied, for example, to desirafe 1 nar coffee beans. A preferred method of aqueous extraction includes the mixing of the mycelial biomass with the polar solvent isopropyl alcohol in a suitable reaction map. Such kettles are known. It is convenient to use 3 to 6 pairs of solvent per part of biomass. Preferably, the mixture is made under nitrogen or in the presence of antioxi dantes to avoid oxidation of the ARA in the lipid extract. As used in the foregoing, "lipid extract", "aceLte", "lipid complex" and "füngic oil" are used interchangeably. After extraction, the mixture can be filtered to separate the biornase from the solvent containing the extrac or lipid. At this point, the biomass can be recovered and used as a food supplement. As used in the foregoing, "food supplement" means food or an additive to be mixed with typical food, such as grain, etc., which may be offered to children. The solvent is repaired from the lipid extract and can also be recovered to reuse, tai < orno by evaporation to a suitable collector, leaving what is referred to herein as "crude oil". The use of alcohol and LCO as a solvent conveniently results in the elimination of any residual water from the crude oil, since evaporation removes the water / isopropyl alcohol azeotrope that has formed spontaneously. Although the crude oil can be used without additional treatment, it can also be further purified. Processes such as those used in the preparation of lecithin from plant products, and known to those skilled in the art, can be used in this additional purification step. These procedures do not modify chemically or covalently between the lipids containing ARA nor ARA itself. Yields vary, but typically are about 5 grams of phospholipid containing ARA per 100 grams of dry mycelium. In the case of M. alpina, an additional 10 to 50 grams of triglyceride can be obtained per 100 grams of dry mycelium. The crude oil or the refined product can be used for administration to humans. Both are included within the definition of ARASCO, as used herein. A preferred object of the invention is to provide a 75 additive to be used with human infant formulas, since the ARA concentration in said formula closely approximates the concentration of ARA in human breast milk. Table 2 compares the composition of the fatty acids in ARASCO with those of breast milk and infant formula with and without ARASCO. TABLE 2 Fatty acid composition of fungal oil and loche maternal products Fatty acid ARASCO Formula Formul .eche i nfant 11 + ace? * E in erna 8. 0 24.1 23.6 0.35 10: 0 17.3 1.39 12: 0 14.9 14.6 6.99 14: 0 4.6 5.8 5.8 7.96 16: 0 16.0 6.8 7.0 19.80 16: 3.2 0.2 0.3 3.20 18: 0 - 7.3 5.91 18: 1 26.4 no 10.3 34.82 18 : 2n6 9.9 17.4 17.3 16.00 18: 3n3 4.1 0.9 1.0 0.62 20: 1 2.2 0.1 0.14 1.10 20: 2n6 0.61 20: 3n6 1.4 0.03 0.42 20: 4n6 32.0 0.64 0.59 20: 5n3 0.03 22: 1 0.10 76 22: 4n6 - - - 0.21 ? 2: 5n6 - - - 0.22 22: 6n3 • - - - 0.19 1 Sunopoulis, A., O? Nega-3 Fatty Acids m Health and isease, p. 115-156 (1990) As can be seen, the amount of ARA present in infant formula supplemented with ARASCO closely approximates the levels of ARA in human breast milk. Adi cyonally, the total fatty acid composition of? Infant formula has not been significantly altered with the addition of ARASCO. Typically, about 50 to about 1000 mg of ARASCO per liter of infant formula can be used. The specific amount of ARASCO required depends on the content of ARA. This can vary from approximately 10 minutes to approximately 70% of the fatty acids in the oil. However, typically the content of ARA is approximately 30 to 50%. Preferably, the oil used for supplemental-infant formula contains at least 40% of the fatty acid residues of ARA, preferably, at least 50% of the ARA residues. When the content of ARA is about 30%, an especially preferred supplement regimen is about 600 to 700 mg of ARASCO per liter of infant formula. Said regimen dilutes the pre-existing fat components of a infant formula such as Sirnilac® (Ross Laboratories, Colurnbus, Ohio) with only one part of ARASCO for 50 parts of formula oils. Similar lilution regimes can be calculated for oils that have higher ARA contents. Preferably, the ARASCO is substantially free of EPA. When Pythiurn insidiosurn is used in the process described, the oil that has been excreted with ARA is predominantly phospholipid. However, it has been found that a significant amount of tpglypephed that have high ARA residue content can also be recovered from P. ínsidiosum, cultured as described herein. When used in this Mortierella alpina procedure, the oil containing ARA is predominantly glyceryl. Both forms of ARASCO are useful as additives to infant formula. These not only provide ARA to the formula, but also an ernulsificant, that is, phosphatilyl-filler, which is added concomitantly to commercial formulas. M. alpina oil is probably cheaper to produce. The ARA-containing oil of the present invention has many uses in addition to its use as an additive for infant formula. As experts in the field know, there are many pathologies associated with ARA deficiencies, such as marasmus (Vajreswari, and others Metabolis 39: 779-782 (1990)), atopic diseases (Melni, B., Monatsschr. Kinderheilta, 138 : 162-166 (1990)), hepatic disease, phenylketonupa, skiing ofrenia, disq? Ines to late or different peroxy somali disorders. In one embodiment of the present invention, these pathologies are treated by administering an amount Pharmaceutically effective of the oil of the present invention. Typically, the amount of effective armamentarium is the amount required to normalize the serum level of ARA in the patient. Particularly preferred for supplement in the treatment of said pathologies are the above ARA oils described above, especially oils that have at least 40% ARA, or preferably 50% ARA residues. The oil may be administered topically, topically, or parenterally, as selected by the health service provider. As is known to those skilled in the art, encapsulation is an effective method of enteral administration. Capsules containing the fungal oil can be administered to people who require or want a dietary supplement from ARA. Said method is particularly effective to adhere the ARA to pregnant or lactating women. In cases in which Je is administered ARASCO to combat the ARA deficiency associated with pathologies, a pharmaceutically effective amount must be administered. This amount can be determined by experts in the field with due experimentation. Typically this amount is 0.5 to 2.0 g / day, which usually normalizes the serum level of ARA. Another embodiment of the present invention encompasses cosmetic compositions containing ARASCO, such as the higher ARA oils described herein. The cosmetic compositions refer to those compounds applied as cosmetics. A preferred example of said composition is a wrinkle cream. Said cosmetic compositions provide an effective means of applying ARA t to the skin to help maintain skin tone. Having described the invention in general, the following non-limiting specific examples are set forth to further illustrate the invention.

EXAMPLE 1 Preparation of P. insidiosum lipid and addition to infant formula In an 80 liter fermenter (crude volume), 51 liters of tap water, 1.2 g of glucose, 240 g of yeast extract and 15 ml of amphora were combined. espuinante MAZU 210SR.

The ferrner-was sterilized at 1? 1 ° C for 45 minutes. They added an additional 5 liters of condensed rinse during the sterilization procedure. The pH was adjusted to 6.2 and then approximately one liter of inoculum (at a cell density of 5-10g / l) of Pythiurn msid osurn (ATCC) was added. # 28251). The stirring speed was adjusted to 125 rpm (maximum speed 250 ern / sec) and the aeration rate was set at 0.028 standard cubic meters per normal minute. At 24 hours of operation, the aeration rate was increased to 0.084 normal cubic meters per minute. At hour 28, an additional 2 liters of 50% glucose syrup (one kilogram of glucose) was added. At 50 o'clock the fermentor is harvested, resulting in a yield of approximately 7.2 kg wet weight (approximately 15 g dry weight) per liter. the cultured bio-cheese was compressed to make a cake of high solids content (50% solids) on a suction filter before freeze drying. The dry biomass was triturated with a mortar and pestle and extracted with one liter of hexane per 700 g of dry biornase at room temperature under continuous agitation for 7 hours. After the mixture was filtered and the filtrate evaporated to yield about 5 to 6 g of crude oil per 100 g of dry biomass. The biornase was then extracted again with one liter of otanol per 20 g of dry biornase for one hour at room temperature, filtered and the solvent evaporated to yield 2? additional g of crude oil per 100 g of dry biomass. The second fraction was predominantly phospholipids while the first fraction contained a mixture of tospholipids and t riglicep two. The combined fractions produced an oil containing approximately 30 to 35% arachidonic acid and no detectable EPA. This oil was added dropwise to the commercial infant formula product Si ilac "(Ross Laboratories, Colurnbus, Ohio) at a feed rate of 60 ing per liter of prepared medium.

EXAMPLE 2 Preparation of M. alpina lipid and addition to infant formula Mortierella alpi a (ATACC # 47430) was developed in a stirred 2 liter volume containing one liter of tap water and 20 g of potato rose dex medium. The mat az remained under constant orbital agitation and was maintained at 25 ° C for 7 days. After harvesting-by means of centrifugation, the mass was dried on freezing yielding approximately 8 g of lipid-rich mycelium. The mycelium was extracted using hexane co or in example # 1 and approximately 2.4 g of crude oil was originated. This oil contains approximately 23% arachidonic acid and is added to the commercial formula Sirnilac ", drop by drop, in concentrations of 1000 rng per liter.

EXAMPLE 3 Large-scale production of arachidonic acid with M. alpina An inoculation fermenter containing GYE medium (50 g / 1 dextrose and 6 g / 1 of Tastone 154) was inoculated with M_.alpine. The fermentation temperature was set at 28 ° C, initial agitation at 130-160 ern / sec, and initial aeration rate at 0.42 kg / ern2. The pH was adjusted to 5.0 before sterilization, and the initial fermentation pH was set to 5.5 after sterilization. The medium pH was maintained at pH > 5.5 with 8N NaOH. The oxygen level was maintained at O.D. > 40% adjusting the agitation / aeration in the following sequence: Increase the container pressure to 0.77 kg / cm2; Increase agitation to 175 cm / sec of maximum impeller speed, and increase aeration to 0.5 VVM. Foaming was controlled by the addition of Dow 1520 -US antifoam, as needed. (Approximately 0.1 rnl / 1 of the antifoam should be added to the medium before sterilization to help avoid foaming). The inoculum is transferred from the seed fermentor to the main fermenter within 12 hours after the pH rises above 6.0. The main fermentor contains GYE medium (50 g / 1 dextrose and 6 g / 1 of Tastone 154); the glucose is sterilized separately and added to the main fermenter after sterilization. The temperature of the ferrntentador is established to 29 ° C, initial agitation to 160 crn / seg, initial pressure of the container to 0.47 kg / c? N2, and initial aeration rate of 0.15 VVM. The initial pH is set to 5.5 after sterilization, and is maintained at pH > 5.5, with 8N NaOH. The pH should be increased during the stationary phase (starting approximately 24 hours after the inoculation), but keeping below pH 6.8 with the addition of H2SO4. The oxygen level is maintained at O.D. > 40% sequentially increasing the pressure of the vessel to 7.7 kg / cm2, increasing the agitation to 175 cm / sec of the impeller maximum speed, and increasing the aeration to 0.5 VVM. Foaming is controlled by the addition of Dow 1520-US antifoam, as needed. (Approximately 0.1 rnl / l of antifoam should be added to the medium before sterilization to help prevent foaming). Samples of the culture are taken every 17 hours for analysis of biornasa and fatty acids and the culture is started 3 to 4 days after the pH rises to 6.5. The density of the dry biornase should be > 8.5 g / 1. The concentration of glucose in the broth should have fallen from 50 g / 1 to < 25 g / 1. In the harvest, all the culture broth is passed through a basket centrifuge to separate the mycelium from the spent medium, and the biornase is dried.

EXAMPLE 4 Improved yield of biomass from M. alpma-first run M. alpina in ferment do e of stirred tank of 20 1, inoculated from the stirred flask culture, according to the procedure of example 3. M. alpina in 65 g / 1 of glucose (Staieydex), and 6 g / 1 of yeast extract (Tastone 154), dLeron as a result the production of 12 g / 1 of biomass. The addition of an additional 6 g / 1 of Tastone 154 at 16 hours resulted in the production of 18 g / 1 of biornase. 1 EXAMPLE 5 Improved yield of biomass from M.alpina, second run Experiments were carried out in an attempt to increase the biomass also by means of fastone additions 154. These experiments consisted of fermentation of 2 X 20 1 of 168 hours of residence. For both of these fermentations, the initial glucose concentration was 100 g / 1 (compared to 65 g / L pair-to example 4). One fermenter received additions of 3 X 6 g / 1 of Tastone 15, and the other recollected additions of 4 X 6 g / 1. The yeast extract was made as a concentrated solution, autoclaved? and added to the ferrator at different times after sterilization. To prepare the inoculum, working seeds (1 ml of macerated mycelium) were inoculated in two flasks, each containing 50 i of GYE medium (100 g / l of Staleydex). , 6 g / 1 of Tastone 154), and were developed during 4 days at 28 ° C and 150 rprn. After 4 days of growth, the broth contained pelleted biomass, the pellets were 2 to 5 nm in diameter. The growth in these flasks was slower than expected, possibly due to the concentration with the highest glucose content. The biomass was macerated 2 x 3 seconds in a Uar ing mixer, and 25 ml of macerate was used to inoculate each of two subjects of F: ernbach inoculum of 2.8 l of 800 net volume. (In previous experiments, 10 ml of mash was used). The amount of inoculum increased, due to the inenor density of biomass in the seed flask, and because growth was expected to be slower in the Fernbachs, due to the concentration with higher glucose content). The medium in the Fernbach flasks was dextrose (Staleydex) 100 g / 1, and yeast extract (Tastone 154), 8 g / 1. The dextrose and the yeast extract were autoclaved septately for 40 minutes. The sowing fermentation temperature < , and kept at 28 ° C and agitation at 100 r pin up to 150 rpm. After 44 hours of cultivation in the ina * races Fernbach, the inoculum was transferred to 2 femients of 70 1. The inoculum was in the form of loose and loose aggregates, and the biomass density was approximately 5.2 g / 1. The fermenters at stations 14 and 15, which contained 1.6 kg (10%) dextrose (Staleydex), and antispuinan + e MAZU 204 (1.6 g, dissolved in 12.5 l of H2O RO), were sterilized for 45 minutes at 127 ° C. C. Then 8U0 rnl inoculum (5%) was added to each fermenter (at time 0). The operating parameters of the ferrnentador were: tempe ra-i ur: 78 ° C., PH: controlled at 5.5 with 2N NaOH and H2 S0 «2N. Ae reaction: 0.5 VV. With rap res i on: 0. 2 bari as. Agitation (initial): 80 cm / sec, and O.D .: with rolling above 40%.

Station 14: X 6 g / 1 Tas * one 154 Yeast extract (Tastone 154) was dissolved until a? -centration of 95 g / 1 and autoclaved for 1 hour. Loads of yeast extract were made in amounts of 3 X 11 (1.8%) at 0, 20 and 26 hours. At 15 hours, the low DO below 40% and the stirring was gradually increased to 175 cm / sec from 15 to 22 hours. Then DO was controlled by correcting the flow of air-e with oxygen; Oxygen was added to the air flow from 73 to 72 hours. Beginning at 36 hours, the stirring was further increased to ensure proper mixing. At 48 hours, the agitation had increased to 200 crn / sec; at 72 hours, at 250 c / sec; and at 80 hours, at 280 cm / sec. At 120 hours, stirring was increased to 290 cm / sec to promote adequate temperature control. At 144 hours the agitation was reduced to 280 cm / sec.

Fstation 15: 4 X 6 g / 1 Tastone 154 Yeast extract (Tastone 154) 384 g was dissolved in 96 g / 1 and autoclaved for 1 hour. Additions of yeast extract in amounts of 4 x 11 (2.4%) were made at 0, 20, 26 and 3? hours. At 4 o'clock, the OD decreased below 40% and the agitation was gradually increased to 175 cm / sec in 73 hours. Then, DO was controlled above 40% by correcting the air flow with oxygen; oxygen was added to the airflow from 23 to 72 hours. Starting at 36 hours, the agitation is increased further to ensure proper mixing. At 48 hours the agitation had increased to 210 cm / sec; at 72 hours, at 260 cm / oeg; and at 80 hours at 290 cm / sec. To the < - 90 hours, the agitation was reduced to 280 crn / sec, and the 144 hours, it was reduced to 260 cm / sec.

Ob ervione In the inoculation, the biomass in both fermentors was in the form of hypha, feathery, very loose aggregates. At 24 hours began to form pellets. The pellets were small (1-3 mrn) with small central nuclei and large loose peripheries. At 48 hours, the pellets were larger, and better defined. At 72 hours, the peripheries were narrower, and the presence of many fragments of loose hypha indicated that the pellets were ragmentando. At 168 hours, the pellet nuclei were 0.5 to 2 mm in diameter, the per *? fairs were reduced with the aggregates of hypha in thick filaments, and there were many condensed hypha aggregates. Fernentators foamed only slightly during the first 24 hours. The amount of foaming was then increased, and controlled by the manual addition of antispray when the head of the foam was greater than 2 to 4 cm. The spur under a little at 48 hours, although there were sporadic outbreaks. Both fermenters foamed in the outlet filters once during the course of fermentation. The fermentations required approximately 150 ml of antifoam e. Both termenors accumulated a considerable amount of increased biomass in the upper space. This is not a common problem with fermentation of mycelium in small fermenters with a large surface area / volume ratio. the amount of biomass increased at station 15 seemed to increase during the last 74 hours, when the volume level decreased caused a considerable amount of splash (the liquid level approached the top impeller). The final volume in the fertilizers after 168 hours was approximately 13 1. The microscopic examination showed that, at 72 hours, much waste was present in the tuitive broth, and there was some evidence of damaged and atrophied fungal tips. . The presence of oil droplets in the cytoplasm was demonstrated by means of staining with my red at 168 hours. The drops of oil were very small and numerous, in contrast to the large drops of oil sometimes observed. Table 3 shows the biomass and oil yield, together with the use of carbon and nitrogen.

TABLE 3 Fermentation Time Course 1 70 to EXAMPLE 6 Improved biomass yield from M.alpina - third run This group of experiments tried to increase the amount of product obtained by increasing the levels of phosphate and minerals. The procedure was essentially that of Example 5, except that dextrose and Mazu 204 defoamer were dissolved in 11.5 L of H2O R.O .., more than 12.5 L, to make room for saline solutions 40 which were added in 30 hours. Station 14 received additional Fe, Zn, and Cu; station 15 reo ib LO additional phosphate, as well as Fe, Zn and Cu. Station 14; X 6 g / L of Ta tone 154 Yeast extract was dissolved at 96 g / l, in amounts of 3 X 1 L, and autoclaved for 1 hour.

Aliquots of 1 liter of the yeast extract solution were added in 0, 22 and 28 hours. At 22 and 28 hours, the rate of production of carbon dioxide (CER, an indication of the metabolic rate in the ferrnentator) was increasing exponentially, and fermentation had barely begun to require a base. The salts fed contained: FeCi3 BH2O 4 8 mg ZnS? 4 7H20 2 4 (1 m Cu So * 5H2O 1 6 rng FeCl3 was dissolved in 1 L of citric acid at 5 g / L. The remaining salts were added, and the pH adjusted with NaOH to 4.5 The solution was autoclaved for 1 hour.The feed salts were added in 30 hours.The initial agitation speed for the fermenter was 50 cm / sec, more than 80 crn / sec, as originally planned, because the initial level of liquid in the ferrnentator (13 L) resulted in the upper impeller,? it was barely submerged, and the higher speed of the agitation gave rise or resulted in more splash. In 16 hours, the O.D. fell below 40%, and agitation was increased incrementally to 175 crn / sog per-78 hours. Fl O.D. it was then controlled above 40% by amending the air flow with oxygen. At 46 hours, the stirring was increased to 190 cm / sec to allow mixing. Agitation was further increased to 200 cm / sec for 48 hours, to 770 cm / sec for 51 hours, to 235 cm / sec for 53 hours, to 250 crn / sec for 56 hours, to 260 crn / sec for 57 hours and 280 cm / sec to 70 hor-as. Even at this agitation speed (450 rpm), the mixture was poor. While a minimum criterion of "some movement" was maintained, the production of bioassa was very low, and some areas approached stagnation. The addition of a few drops of antisplash reduced the foam gap, and removed the stagnant cavities. In 116 hours, the agitation was reduced to 265 kin / sec and in 120 hours, it was further reduced to 250 cm / sec. The ferrnentador began foaming at approximately 18 hours. Foaming was controlled by manual addition of antifoam. The anti foaming agent was added first in 70 hours. For 24 hours, the fermentation was foaming significantly, and required the regular addition of anti-scalding. For- 72 hours, the foaming had, for the most part, decreased. However, the fermentation still required the occasional addition of antifoam.

For 74 hours, the biomass was in the form of very loose pellets (1-7 in) and loose hi-fi aggregates. There was a considerable amount of cellular waste. For 48 hours, the biomass was in the form of very loose hyphal aggregates, very small pellets (1-2 rnrn) with small and small nuclei and loose peripheries, and small compact pellets (1-3) with loose peripheries. For 96 hours, the biornase was in the form of compact round pellets (1-2 inm), needle-shaped pellets (less than 0.5 mm) and loose aggregates. The stain with ro or even in 144 hor-as showed the presence of many tiny droplets of oil in the mycelia. Station 15: 3 X 6 g / L of Tastone 154 Yeast extract was dissolved at 96 g / L, and autoclaved for 1 hour. The solution of yeast extract was added in amounts of 3 x 1 L at, 22 and 76 hours. At 22 and 76 hours, the CER was exponentially increased, and the fermentation had begun to require-base.

A feed salt containing: KH2 P0 «7 7 was prepared FeCl3 6H20 rng Zn SO * 71- O rng CuSO * 5H20 1 6 rng FeCl3 was dissolved in 500 rnl of citric acid at 5 g / L. The remaining salts were added, and the pH adjusted with NaOH to 4.5. The KH2 PO4 was dissolved in 500 rnl of water R.O. Both solutions were autoclaved for 1 hour, and then cooled to 23 ° C, before being combined and added to the ferrntent in 30 hours. The initial rate of agitation in the fermentor was 50 cm / sec, more than 80 cm / sec, as originally planed, because the initial level of liquid in the fermenter (13 L) resulted in the top impeller as soon as it was submerged, and the higher speed of agitation resulted in significantly more splash. In 16 hours, the O.D. fell below 40%, and agitation was increased incrementally up to 175 cm / sec for 27 hours. The O.D. It was then controlled over 40% by amending the air flow with oxygen. At 41 hours, the stirring was increased to 200 cm / sec to allow at least a minimum amount of mixing. The agitation was also increased to 220 kin / sec in 42 hours, to 230 cm / sec in 46 hours, to 235 crn / sec in 51 hours, and to 240 cm / sec in 70 hours. At this agitation speed (410 rpm), the mixture was hardly poor to regulate. A minimum level of biomass movement was maintained. At 80 hours, the agitation was reduced to 205 cm / sec. The ferrnentator started foaming at approximately] 8 hours. Foaming was controlled by manual addition of anisomer. p amp and foaming r, he added first in 17 hours. For 20 hours, the fermentation was foaming, if necessary, and required the regular addition of anti-scalding. The foaming had diminished in large part by 7? hours. However, fermentation still required the occasional addition of antispuminant. For 24 hours, the biornasa was in the form of very loose pellets (l-2mm) and loose hyphae aggregates. There was a considerable amount of cellular waste. For 48 hours, the biomass was in the form of very loose head aggregates, very small pellets (1-? Rnm) with very small nuclei and loose peripheries, and small compact pellets (1-3) without loose peripheries. For 96 hours, the biomass was in the form of round pellets 1-2 mm in diameter, many with loose and hairy peripheries, and many loose liver fragments. Staining with ro or mine at 144 hours showed the presence of many very small droplets of oil in some mycelia, and also the presence of large droplets of oil in all parts of other mycelia. Zone 15, which differed from station 14 only by the addition of phosphate, showed better mixing throughout the fermentation, at generally lower agitation speeds. Station 15 also exhibited a "loose" biomass morphology. The production of bioinase and oil, as well as the use of carbon, are shown in quad 4. Increased glucose utilization (82 g / L for station 15 compared to 64 g / L for station 14) , increased accumulation of biomass, and the presence of large drops of oil in mycelial potions, characterized the fermentor containing a greater amount of phosphate.

TABLE 4 Fermentation time course F tac ion 14 + Sales Log Glucose Weight Content Content Produc i vi-dry ARA oil (/ L) (g / L) (% wt% (% of ac te / dry) acei e) L / d) 0 116.0 1.1 74 101.0 1.8 1.2% 22. 27. 0.02 48 84.0 14.3 6.2% 24.7% 0.44 72 60.0 24.5 10, .6% 24.2% 0.8? 96 45.0 28.2 15.5% 25.3% 1.09 120 34.0 28.9 18.1% 26.6% 1, .05 144 27.0 30.8 20.8% 27.2% 1.07 Station 15 + Sales + Fos ttos Log Glucose Weight Contents Producti VL - dry oil oil (g / L) (g / L) (% weight (% of oil g / L) dry) oil) L / d) 0 113.0 0.4 24 101.0 2.1 1.1% 24.0% 0.02 15 48 74.0 21.7 8.1% 24.7% 0.88 72 51.0 26.7 19"9% 76.5% 1.74 20 96 31.0 30.1 25.5% 78.6% 120 18.0 33.8 31.7% 31.4% 2.14 144 6.0 34.5 36.0% 32.9% 7.07? EXAMPLE 7 Large scale production of M. alpina biomass containing arachidonic acid A seed warmer containing GYE medium (50 g / L of dextrose and 6 g / L of Tastone 154) is inoculated from the fermentation promoter. A temperature of 28"C 35 is maintained and the initial agitation is set at 130-160 cm / sec (almost 43 rrnp).

The initial pressure in the vessel is 0.4218 l-g / cm2, and the initial aeration rate was set at 0.25 VVM. The pH is adjusted to 5.0 pre-sterilization, and then the initial pH of the ferrnentator is set at 5.5 post-sterilization. The level of or igene in the middle is maintained as O.D. > 40% by the following sequence: (T) increase in pressure in the vessel to 0.7733 g / cm2, iii) increase in torque agitation * of the maximum impeller speed of 156 to 175 cm / sec, and ( IIL) increase of aeration to 0.5 VVM. Foaming is controlled by the addition of Dow 1520-US antifoam, as needed. (Approximately 0.1 mL / L of the antispuminant should be added before the sterilization to help prevent sputtering). After inoculation, the culture is maintained at pH > 5.5 with NaOH at 8N. Within 12 hours after the pH increases above 6.0, the contents of the seed fermentor are transferred to the main fermenter. The medium of the main fermentor contains 80 g / L of pink dexf (ADM) 16 g / L of soybean meal (nutri soya ADM) 30 rng / L of FeCl3-6H2? (Next to / Al dp ch) 1.5 rng / L of 7nSO,? - 7H2? (igma / Aldp ch) 0.1 rng / L of CuSO «-5H2? (i gma / Al drich) 1 rng / L of biotma (Sigrna / Aldp ch) 2 rng / L of tiamma-HCl (Sigma / Al dp ch) 2 rng / L of pantoté acid co (hercylic salt) (Si gma / To the drich). (Adjust to pH 4.8-5.0 pre-sp ec ization) Inoculate the main ferrnentator with the seed ferrner (11.8%). The fermenter temperature is maintained at? 8 ° C. The initial agitation is set at 162 cm / sec (approximately 23 rprn), l < ? Initial pressure in the vessel at 0.4218 kg / cm2, and the initial aeration rate at 0.15 VVM. The level of oxygen in the medium is maintained at O.D. > 40% to i) increase the pressure in the vessel to 0.7733 l / crn2, ii) increase the agitation in the naxuna speed of the impeller to 300 cm / sec (in increments of approximately 30 cm / sec), and 1 1) increase aeration to 0.5 VVM. The pH is outlined according to the following pH control protocol: Set the initial pH at 5.5 post-sterilization. Maintain the pH at .5.5. with NaOH at 8N. In 24-36 hours after inoculation, add the following: 2 g / L of KH2 P0¿ (110 kg in approximately P00 L of H2O). In 48 hours, if the concentration of dextrose is _ < _ 60 g / L, change the pH fixing point to _ > .6.1. In 72 hours, begin to slowly raise the pH fixation point to > 6.6 at a rate of approximately 0.1 pH units per hour. Keep the pH below 7.3 with the addition of H2 SO * if necessary. The fermenter is tested every 7 hours for the analysis of biomass and fatty acid, and the harvest is started approximately 3 days after raising the pH to > _ 6.6 (almost 6 days after the inoculation). The dry biodanse density should be > _ 24 g / L. The dextrose concentration in the broth should have dropped from 80 g / L to < _ 14 g / L. the harvest is carried out by passing all the culture broth through a vacuum rotary filter to separate the mycelia from the medium used. The results of two typical fermentation courses according to the procedure of this example are shown in Tables 5 and 6. TABLE 5 Progress of fermentation of M. alpina TABLE 6 Progress of fermentation of M. alpina

Claims (13)

NOVELTY OF THE INVENTION CLAIMS

1. - An unmodified fu tglicépdo oil comprising at least about 40% of ARA in the icépdo tngl and not more than one tenth as much of EPA as ARA.

2. A triglyceride or fungicidal oil not modified in accordance with claim 2, wherein the oil comprises at least 40% of ARA in the tpglicep or and essentially none of EPA.

3. The unmodified fu triglyceride oil of claim 1 or 2, wherein the oil has at least 50% ARA.

4. An oil according to any of claims 1 to 3, wherein the fungus is Mortierella sp.

5. The oil of claim 4, wherein the fungus is M. alpina.

6.- A method for the production of an oil containing arachidonic acid, containing said oil glycides in which at least 25% of the fatty acid residues are ARA, and the amount of EPA residues in the oil it is no more than a fifth of the amount of ARA residues, which comprises (a) growing Mortierella sp. in an aerated fermentor containing culture medium where a carbon source is added in an amount equivalent to at least 80 g / 1 of glucose and a source of nitrogen in an amount equivalent to at least 15 g / L of extract yeast, to the culture medium dur-ante-) the course of the fermentation; (b) maintaining the pH between 5 and 6 at the beginning of the culture; (c) maintain the pH between 7 and 7.5 at the end of the crop; (d) harvest- the biomass from fermented! ' and recovering said oil containing arachidonic acid from said biodanse,

7. The method of claim 6, wherein the level of dissolved oxygen in the culture medium is at least 35% of the saturation level with a? r ~ e.

8. The method of claim 6, wherein the source of nitrogen is divided into two or more aliquots, which are fed into the fermenter at different times, with at least one aliquot being fed into the fermenter at one hour. different from the time when the carbon source is placed in the fertilizer.

9. The method of any of claims b to 8, wherein Mortierella sp. it's M. alpina.

10. The method according to claim 6, wherein, in addition, crude oil containing arachidonic acid is recovered from the biornase by extraction with a non-polar solvent and the crude oil is clarified by extraction with a solvent. polar organic.

11. The method of claim 10, wherein the non-polar solvent is hexane.

12. The method of claim 10, wherein the polar solvent is selected from the group consisting of acetone, ethanol and isopropyl alcohol. 13.- A method to provide tri glycept that contains ARA to a infant formula, which comprises the addition of an unmodified triglyceride fungic acid comprising at least 40% of ARA and comprising no more than a fifth part as much of EPA as ARA to a infant formula in an amount enough to provide an ARA content which corresponds to the amount of ARA in human milk. 14. A method according to claim 13, wherein said oil is produced by a species of Mor-t i erel a. 15.- A method in accordance with the claim 14, wherein said oil is produced by Mortierella alpina. 16. A method of conformance with any of the foregoing provisions 13 to 15, wherein said oil comprises no more than one-tenth as much EPA as ARA. 17. A method according to any of claims 13 to 16, wherein said oil does not comprise essentially EPA. 18. A method according to any of claims 13 to 17, wherein said oil comprises 50% of ARA. 19. Infant formula comprising triglyceride containing ARA in a < ant? lad comparable to the amount in l to human milk, wherein ARA is provided by the addition, to a infant formula, of a sufficient amount of an unmodified fungal oil comprising glycerol tp containing at least 40% ARA and not more than one fifth as much as FPA as ARA. 20, .- Infant formula according to claim 19, wherein the unmodified fungal oil comprises no more than a top d part as much of FPA as ARA. 71.- Infant formula according to claim 20, wherein said fungic oil is essentially free of FPA. 72. Infant formula according to any of claims 19 to 21, wherein the unmodified fungal oil comprises triglyceride containing at least 50% of ARA. 23. The use of an effective amount of an unmodified fungal oil containing ARA in the form of triglyceride, said oil containing at least 40% of ARA and not more than one fifth as much oil of eicosapentenoic acids ( EPA) as ARA, in the preparation of compositions to treat pathologies of ARA deficiencies in a human, providing it with complementary arachidonic acid (ARA). 24.- The use of regiment 23, where the <It contains at least 50% of ARA. 25.- r-l use of claim 73 or 74, wherein the prepared composition is administered to the human in an amount that supplies n.7-0.8 g of ARA / di. 26. The use of conformity with any of claims 73 to 25, wherein said composition is fully illustrated. 27.- The use according to any of claims 73 to 25, wherein said prepared composition is administered in accordance with "28.- The use according to any of claims 23 and 25, wherein said composition prepared adni nistra topi canent or "29. The use according to any of claims 23 to 25, wherein said human is a pregnant woman or nurse. 30. The use according to any of claims 23 to 25, wherein said human requiring complementary ARA suffers from a neurological disorder. 31.- The use according to claim 30, wherein the neurological disorder is tardive dyskinesia, squizophrenia or a t-scare but i sarna. 32. The use according to any of claims 23 to 25, wherein said human requiring complementary ARA suffers from a disease associated with reduced level of serum ARA.

13. - The use according to claim 32, wherein the disease is a liver disease, feni 1 cetonupa or cystic fibrosis. 34.- A cosmetic composition comprising unmodified fungicidal oil containing ARA in the form of a p-glyceride, said oil containing at least 40% of ARA and not more than one fifth as much of the petrosolenoic acid (EPA) as ARA, wherein said oil is present in said cornposj < . > n in an effective amount to help maintain skin tone when said prepared composition is applied topically. 35.- The composition of claim 34, wherein the oil contains at least 50% of ARA.