JPH0418838B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing fats and oils, particularly triglyceride-rich fats, from microbial sources. It is known that fats and oils can be produced by culturing microorganisms that synthesize fats and oils, including algae, bacteria, molds, and yeasts. Such microorganisms synthesize fats and oils in the normal course of their cellular metabolism. Extensive research has been conducted in an effort to identify which microorganisms, media, and conditions will enable economical and practical oil production. One area of the production of fats and oils by fermentation that has received particular attention is that of producing cocoa butter substitutes.
Cocoa butter is a naturally occurring substance containing large amounts of 1,3-disaturated-2-unsaturated triglycerides. These triglycerides are 1-stearoyl-2-oleoyl-3-palmitoyl triglyceride and 1,3-dipalmitoyl-
Contains 2-oleoyl triglyceride. The method for producing triglycerides rich in the above compounds is
U.S. Patent No. 4032405 awarded June 28, 1977
It is stated in the number. As described in this patent, the cocoa butter substitute is made by aerobic microorganisms selected from the genera Endomyces, Rhodotorula, Lipomyces, or Rhodosporidium. It is produced by culturing under the following conditions, collecting the bacterial cells, and separating fats and oils rich in 1,3-disaturated-2-unsaturated triglycerides from the bacterial cells. The medium used in the fermentation process of the above patent generally contains an assimilable nitrogen source and a carbon source, preferably in the form of aldoses or disaccharides or polysaccharides. The resulting bacterial cells are collected, and a mixture of fats and oils rich in 1,3-disaturated-2-unsaturated triglycerides is separated from the bacterial cells. Improvements in the method described in the above patent were published in May 1979.
Co-pending Application No. 904099 filed on May 8th. As described in this co-pending application, the yield of fats and oils is increased when the fermentation medium contains a carbon source in the form of one or more fatty acids containing between 10 and 20 carbon atoms. It has been discovered that the distribution of specific fats and oils can be controlled. For example, it has been shown that the ratio of saturated to unsaturated acid groups in glyceryl oil can be controlled by using in the fermentation medium the acids that form the fatty acid moiety of cocoa butter, namely palmitic acid, oleic acid, and stearic acid. has been discovered. Although the process described in the above co-pending application shows clear improvements in both yield and acid distribution in fat fermentation processes, there is room for further improvement in the process. It would be desirable to study the factors that influence the production of the triglyceride oils thus produced, but also the control of the distribution of the acid component of the triglycerides themselves. Therefore, an object of the present invention is to provide a method for producing fats and oils by fermentation, in which the yield of fats and oils as described above is dramatically increased. A further particular object of the invention is a method for the production of fats and oils, especially triglyceride-rich fats, from microbial sources, in which the yield of desirable saturated fats and oils is increased in short fermentation times, while the yield of undesirable unsaturated fats and oils is reduced. The goal is to provide the following. The concept of the present invention is to produce fats and oils, in particular triglyceride-rich fats and oils, by culturing yeast cells capable of synthesizing fats and oils in a medium containing at least one emulsion of fatty acids containing 10 to 20 carbon atoms. It's in the law. The use of an emulsion of the fatty acid components of the fermentation medium increases the availability of fatty acids during assimilation by the yeast cells, thereby increasing the yield of fats and oils produced and controlling the acid distribution of the acid components forming triglycerides. It was discovered that. The process of the invention is particularly well suited for use in the production of fats and oils of the cocoa butter-based type. In accordance with one embodiment of the present invention, it has been found that the production of such oils can be greatly increased when the fermentation medium is formulated to include palmitic acid, oleic acid, and stearic acid. It's been found. In accordance with one embodiment of the present invention, it has been discovered that the use of a desaturase enzyme inhibitor promotes a higher ratio of stearate to oleate groups present in the resulting triglyceride oil. . Without limiting the invention to theory, it is believed that desaturase enzyme inhibitors minimize the action of intracellular desaturases and help prevent desaturation of stearic acid. The use of desaturase enzyme inhibitors thus results in increased stearic acid levels found in the resulting triglycerides. Hereinafter, the present invention relates to 1,3-distearoyl-2
-Oleoyl triglyceride, 1-stearoyl-2-oleoyl-3-palmitoyl triglyceride, and 1,3-dipalmitoyl-2-
Although described in relation to the production of fats and oils of the type based on cocoa butter, a triglyceride containing oleoyl triglycerides, it is understood by those skilled in the art that the concepts of the present invention can be similarly used in the production of other fats and oils by fermentation. This is understood by specialists. Microorganisms useful in the practice of this invention can be characterized as oleaginous yeasts. Such yeasts are well known in the art and are available. For example, many of these yeasts are described in US Pat. No. 4,030,2405, cited in the literature. Particularly suitable for use in the practice of the present invention are Rhodosporidium, Lipomyces, Candida,
The species is selected from the genus Endomyces, the genus Saccharomyces, the genus Rhodotorula, the genus Trichosporon, or the genus Torulopsis. This kind of fat-synthesizing yeast is well known,
and can be isolated by conventional techniques from natural sources such as leaves, plant stems, etc. However, for example, the American Type Culture Collection
It is generally convenient to obtain such yeast from a variety of culture repositories, including the Microbial Culture Collection. For economic reasons, it is generally preferred to use oleaginous yeasts that tend to synthesize and store large amounts of oil. Yeasts that have the ability to accumulate 20% oil, preferably at least 30% oil in standard media (such as glucose, ammonium salts and minerals) are generally suitable. Culture of the particular yeast species used. The growth and/or fermentation medium that provides nutrients for the growth and/or fermentation medium will depend somewhat on the particular yeast selected for use in the method of the present invention. A dilute aqueous basic solution containing carbon and nitrogen sources in an amount of less than 6% by weight.Suitable media generally have a PH of about 4.0, as is customary for optimal yeast culture. and 9.0, preferably between 5 and 8.5
adjusted or buffered to be within the range of As a nitrogen source, various common nitrogen-containing compounds that are often used as nutritional sources for the growth of microorganisms can be used. Suitable nitrogen compounds include asparagine, glutamine, peptone, and the like. Additionally, other nitrogen-containing compounds such as ammonium salts and urea can be used as well. While nitrogen sources generally serve to enhance yeast growth, the nitrogen sources described above serve to enhance fat accumulation in yeast cells. Thus, while high nitrogen:carbon ratios are useful in promoting yeast cell growth, high carbon:nitrogen ratios maximize fat accumulation. One nitrogen-containing nutrient source that is particularly well suited for use in the practice of the present invention is a corn steep, an aqueous solution formed in a conventional corn wet milling process in which dry corn is soaked in heated dilute sulfurous acid. It is a liquid. Corn steep consists of approximately 25% by weight crude protein (8% nitrogen) and small amounts of ash, sugars and other useful culture ingredients. Corn steep is an inexpensive and complete nitrogen source that can be used alone or in combination with other conventional nitrogen sources well known to those skilled in the art. can. The medium should also contain one or more of the known important metabolizing inorganic salts, including salts of potassium, sodium, calcium, magnesium, iron, and the like. In addition, secondary nutritional sources such as vitamins and amino acids are desirable as well, especially if the culture period for the yeast is extended. The fermentation medium used in the practice of the invention also contains a carbon source, in accordance with the key concepts of the invention. As briefly mentioned earlier, the fermentation medium was 10
It should contain a predominant amount of one or more fatty acids containing between 1 and 20 carbon atoms. Without limiting the invention with respect to theory, it is believed that yeast cells utilize fatty acids during their metabolism, so the fatty acid content of the fermentation medium should be at least 10% of the total carbon source, and preferably 40% of the total carbon source. % or more. In this way, the fatty acids are not masked by the presence of other carbon sources in the fermentation medium to the extent that they serve to alter the metabolism of the yeast cells to produce triglyceride oils with specific fatty acid contents. The fatty acid or fatty acids used as a carbon source in the practice of this invention can be obtained from a variety of known sources. For example, palmitic acid (C ₁₆ :0), stearic acid (C ₁₈ :0) or oleic acid (C ₁₈ :1) are commercially available in the form of the free acid or in the form of a salt such as the sodium salt. Obtainable. These common fatty acids can be used alone or mixed with other fatty acids. Linoleic acid ( _C18 :2),
Polyunsaturated fatty acids such as linoleic acid (C ₁₈ :3) and natural fatty acids containing 16 to 20 carbon atoms can also be obtained in pure form, but commercially available mixtures such as soap ingredients more readily available in cheaper forms. The composition of the fatty acids used is important to the extent that each fatty acid produces unique changes in the oil synthesis metabolism of the yeast species used. When a mixture of fatty acids is used, the combined action is an interaction resulting in a metabolic mixture of triglycerides containing the various fatty acids present in the fermentation medium. However, accurate prediction of the exact yield in the composition of the oil to be obtained from a particular fatty acid carbon source is highly empirical. Conventional analytical tools allow for the determination of yields in the composition of oils made from a particular carbon source, and routine experiments therefore allow rapid identification of a suitable fatty acid carbon source for the production of a particular oil. do. However, some general rules in the form of general standards have been determined. For example, the presence of a fatty acid of a given carbon length in a carbon source usually results in an increase in the proportion of triglyceride esters containing the fatty acid as a component of the triglyceride. Similarly, the degree of saturation and/or unsaturation (and especially polyunsaturation) in the oil produced is directly related to the corresponding saturation level of the fatty acid composition used as carbon source. The use of palmitic acid, oleic acid and stearic acid as carbon sources thus promotes the formation of an oil that closely resembles the oil present in cocoa butter. The conditions for culturing yeast to produce fats and oils according to the method of the invention do not differ from those commonly used in conventional fermentation systems. in general,
The yeast used in the practice of the present invention to produce the oils and fats described above is generally the same as in conventional processes using the same type of yeast species. The fermentation medium is determined by the amount of nitrogen present in the medium.
Carbon sources present in the fermentation medium generally range from 0.1 to 5% carbon (by weight), while nitrogen-containing nutrient sources range from 0.005 to 1% nitrogen (by weight).
is within the range of The temperature at which fermentation takes place is generally about
Within the range of 20 to 40°C, temperatures higher than this range favor the production of saturated oils and temperatures lower than this range favor the production of unsaturated oils. Similarly, enzymes have some influence on the growth of yeast cells. In general, it has been discovered that aerobic cultivation of yeast cells increases the final yield of oil produced by the microorganism. Once the fermentation is carried out for the desired period, generally from 1 to 7 days, preferably from 2 to 5 days,
The yeast cells are separated from the fermentation medium by conventional means and the oil is recovered. For example, the yeast cells are first destroyed, for example by freeze-drying or hydrolysis;
The oil can then be extracted from the disrupted material with a suitable solvent, preferably a volatile solvent to facilitate subsequent removal of the solvent from the oil. As mentioned above, an important concept of the present invention is that the fatty acids present in the fermentation medium are in emulsified form. This is suitably done by adding to the fermentation medium emulsifiers which are compatible with the fatty acids used and which do not adversely affect the metabolism of the yeast cells. Generally, the emulsifiers used in the practice of this invention are ionic and nonionic emulsifiers with an HLB of 15 or greater. Suitable emulsifiers for this purpose are those in the form of fatty acid derivatives of sorbitol and sorbitol anhydride. A particularly suitable emulsifier is a polyoxyethylene derivative of a fatty acid partial ester of sorbitol anhydride sold by Atlas Chemical Industries, Inc. under the trademark "Tween".
emulsifiers marketed by Industries Inc.),
and nonionic emulsifiers, such as the emulsifier sold under the trademark "Span," which is a fatty acid partial ester of sorbitol anhydride. These two types of emulsifiers are suitable for food use.
It is approved by the FDA. It has also been discovered that these emulsifiers do not adversely affect the metabolism of yeast cells in the formation of fats and oils. Generally, only an amount of emulsifier is used that is sufficient to emulsify the fatty acids present in the fermentation medium. Generally, this amount will range from 0.0001 to 1% based on the weight of the fermentation medium. Emulsions are preferably made by adding an emulsifying agent to a fatty acid or fatty acids to create a substantially homogeneous fermentation medium, with or without the addition of other components of the fermentation medium that are then added during agitation. It is made by stirring thoroughly. In a preferred practice of the invention, the emulsion is prepared by heating fatty acids with a buffer solution to obtain a
It is formed by autoclaving the fatty acids to bring them to pH and then sterilize them if necessary. An emulsifier is then added to this and the resulting mixture is homogenized. The emulsion is then rapidly cooled at a rate sufficient to crystallize the very small sized stearic acid particles. In accordance with the practice of the present invention, it has been discovered that particle sizes of less than 10 microns are particularly suitable for ensuring that the fatty acid or fatty acids are effectively utilized in the fermentation process. Carbohydrates include aldoses (such as glucose, hexoses, pentoses, etc.), disaccharides such as maltose, sucrose, etc., and oligosaccharides,
Carbohydrates selected from the group consisting of oligosaccharides, preferably obtained from the hydrolysis of starch, are preferably used. Glycerin can likewise be used advantageously. In accordance with other embodiments of the invention, it has been discovered that it is often desirable to include a desaturase enzyme inhibitor in the fermentation medium. As mentioned above,
Without limiting the invention as to theory, desaturase enzyme inhibitors serve to minimize the action of the desaturase enzyme during fermentation, thus tending to increase the ratio of saturated to unsaturated oil. One such desaturase enzyme inhibitor used is sterculic acid, a cyclopropanone derivative of stearic acid found in cottonseed oil.
It will be understood by those skilled in the art that other inhibitors may be used as well. Generally, the amount of such inhibitor is sufficient to inhibit the desaturase enzyme, typically between 0.001 and
0.5% by weight, preferably in the range 0.01 to 0.2% by weight. Having described the basic concept of the invention, an example will now be provided. These examples are provided to illustrate the practice of the invention and are not intended to limit the practice of the invention. In these examples, all percentages are by weight unless otherwise indicated. Example 1 This example illustrates the practice of the invention utilizing stearic acid. First, 1g of stearic acid is heated to about 80℃,
An emulsion was then prepared by mixing this with a solution of potassium phosphate having a pH of 6 to 7.
The mixture thus obtained was heated to 120° C. for 15 minutes to sterilize the fatty acids. Thereafter, 0.01% of 1 g of emulsifier Tween 20 was added and the resulting mixture was rapidly cooled to precipitate crystals of stearic acid with a size in the range of 1 to 10 microns. The milky suspension thus obtained is 6
It had a pH of 7 to 7, was stable, and showed no coalescence. Next, this emulsion was mixed with a fermentation medium to obtain a fermentation medium having the following overall composition. Peptone 0.5% Yeast extract 0.1% Glyose 2.0% K ₂ HPO ₄ 0.1% Antibiotics 10 g/ml Emulsifier (Tween 20) 0.01% Stearic acid 1.0% Water 100 ml This composition had a pH of 5.5 to 6.0. Next, this fermentation medium (sample) was inoculated with yeast cells of R. toruloides grown in a nutrient medium containing 5% glucose, 5% peptone, and 1% yeast extract at 28°C. . At the same time, a second medium (sample) was prepared in the same manner except that the amount of glucose was increased to 4%.
Other media, or samples, were prepared in a similar manner, but without glucose and with a stearic acid level of 4% by weight. Samples and were inoculated with the same inoculum as above at 28°C. Fermentation of samples and was carried out in shake flasks at 200 rpm and 28° C. for 6 days. Other samples (samples) were prepared, inoculated, and fermented similarly to the sample, but in this case the PH
was adjusted to 7.5 after 2 1/2 days. Yeast cells were collected from each sample, oil was collected, and analyzed. The results obtained were as follows.

The above results show that the presence of carbohydrates in the fermentation medium increases palmitic and oleic acid levels at the expense of stearic acid levels.
Furthermore, adjusting the pH of the fermentation medium to a pH of 7 or higher results in increased conversion. Example 2 This example illustrates the importance of emulsifiers in the fermentation medium used in accordance with the practice of this invention. In this example, several fermentation media were prepared in the same manner as described in Example 1, except that the procedure was modified as follows. The fermentation medium used in this example was 0.02% potassium phosphate, 0.02% Tween 20 or Tween 80 used with stearic acid as follows:
I have become more mature. Sample A Stearic acid (no homogenization, no rapid cooling,
B Tween 20 0.02% (without homogenization or rapid cooling) C Stearic acid homogenized (without emulsifier or rapid cooling) D Stearic acid homogenized with Tween 80 (without rapid cooling) E Homogenized with Tween 20 Stearic acid (without rapid cooling) F Stearic acid homogenized with Tween 20 and with rapid cooling G Stearic acid homogenized with a PH of 4.5 but without rapid cooling All of the above samples, 48 at 28 °C in a fermenter of 5
Time-propagated Rhodotorula toruloides (R.
toruloides). Culture in a shaking incubator
It was carried out at 32°C and 260 rpm. 1 g of Rhodotorula toruloides was added to 100 ml of lipid medium.
After the emulsion was prepared, visual observation was made and the report was as follows. Sample Observation A Large chunks of stearic acid B Same as A C Small particles of stearic acid D Same as C E Same as C F No particles observed GC Same as C Observation using a microscope just before collecting bacterial cells from lipid medium was carried out, and the results were as follows. Sample Observation A and B There is little or no accumulation of lipids. C-E 10-20% lipid accumulation F 20-40% lipid accumulation G 10-20% lipid accumulation Visual observation of the above sample before inoculation shows that A
-E and G were shown to contain visible particles, and these particles were shown to be larger than 1 to 2 microns in size. Microscopic observation immediately before bacterial collection revealed that the larger the particle size, the smaller the lipid accumulation. Samples A and B were not homogenized and thus had the largest particles. As the particle size decreased (ie, from samples C to E), larger intracellular lipid droplets were observed. The results of the analysis are shown in the table below. Sample Natural oil (mg) A- (no homogenization) 50.2 mg B- (no homogenization - Tween 20 0.02%) 5.47 C- (homogenization - no emulsifier) 103.0 D- (homogenization - Tween 80) 100.1 E- (Homogenized - Tween 20 - No Rapid Cooling) 106.9 F- (Homogenized - Tween 20 - Rapid Cooling) 139.7 G- (Homogenized - Tween 20 - PH4.5) 93.7 The results shown in the above example are: This illustrates that a good emulsifier is important for effective utilization of stearic acid. Factors that contribute to the formation of a good emulsion are proper homogenization, appropriate levels of emulsifier, neutral pH,
and rapid cooling in an ice pack. Experimental data shows that using emulsifiers without heating or melting the fatty acids, homogenizing and rapid cooling is not particularly effective for fatty acid carbon sources with high melting points. This is why the method of the present invention using emulsions was developed. The degree of unsaturation in the B-position is important to the structure and melting of cocoa butter. Table 2 shows the B-position data for a given sample. This is based on a stereospecific method using pancreatic lipase. Luddy FE et al.: JAOCS, Vol.41, p.693,
See 1964.

[Table] This information shows that the fatty acid composition can be almost compared to cocoa butter and that triglycerides with almost the same properties of SuS in cocoa butter are biosynthesized. It is to be understood that various changes and modifications may be made in the details of procedures and techniques without departing from the spirit of the invention, particularly as set forth in the claims.

Claims (1)

[Scope of Claims] 1. A yeast cell capable of synthesizing fats and oils is cultured in a medium containing at least one emulsion of a fatty acid having 10 to 20 carbon atoms, and from the cultured yeast cell A method of producing fats and oils by separating them. 2 The emulsified liquid heats fatty acids together with an emulsifier,
A method according to claim 1, which is produced by subsequently homogenizing the resulting mixture. 3. The method of claim 2, wherein the emulsion of fatty acid and emulsifier is rapidly cooled before being added to the fermentation medium. 4. The method according to claim 1, wherein the yeast cells are cultured aerobically. 5. The method according to claim 1, wherein the yeast cells are cultured in the presence of a desaturase inhibitor. 6. The method according to claim 1, which comprises the step of increasing the pH to a level of 7 or higher after the fermentation reaction has partially taken place. 7 Yeast cells are Rhodosporidium, Lipomyces,
Candeida, Endomyces, Satucharomyces,
2. The method of claim 1, wherein the species is a species of the genus selected from the group consisting of Rhodotoral, Trichosporon, or Torulopsis. 8. The method according to claim 1, wherein the emulsion also contains carbohydrates. 9. Fatty acids account for at least 50% of the carbon source of the fermentation medium.
9. A method according to claim 8, which corresponds to % by weight. 10 Cultivating yeast cells capable of synthesizing fats and oils found in cocoa butter in a medium containing at least one emulsion of fatty acids having 10 to 20 carbon atoms, and culturing the cultured yeast cells. The method according to claim 1, wherein the oil and fat are separated from the oil. 11. The method of claim 10, wherein the fatty acid is a mixture of stearic acid and palmitic acid. 12 Claim 1 in which the fatty acid is a mixture of stearic acid, palmitic acid and oleic acid
The method described in Section 1.