JPH0574350B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing a cocoa butter substitute using microbial cell oil. (Prior Art) Currently, the raw material oils for cocoa butter substitutes are southern vegetable oils such as shea butter and monkey butter, and these are used to produce cocoa butter substitutes. In recent years, the nutritional value or physiological importance of microbial cells themselves has attracted attention, and products for the food field of microbial cells have been developed. "Hard butter" or "hard fat" using microbial cells
One example of this is research on the production of a cocoa butter substitute known as ``Kokusho Sho 58-34114''. (Problem to be solved by the invention) However, oils and fats extracted and recovered from microbial cells are
The composition is far different from that of cocoa butter. For example, cocoa butter contains large amounts of 1,3-disaturated-2-
Contains unsaturated glycerides, in some cases 1.3
-Distearo-2-olein (hereinafter referred to as SOS)
24.2%, 1-stearo-2-oleo-3-palmitine and 1-palmito-2-oleo-3-stearin (hereinafter referred to as POS) 32.8%, 1,3-dipalmito-2-olein (hereinafter referred to as POP) 12.1%
(S ₂ U total 69.1%), but Rhodosporidium toruloides IFO413 in the above-mentioned application announcement
In the case of bacteria, the composition of oils and fats extracted and recovered from bacterial bodies is
SOS is 6.4%, POS is 22.2%, and POP is 19.3% (47.9% in total for _S2U ), and a separation process is required. For this reason, the cost is high, and it is difficult to say that this method is necessarily sufficient from an economic standpoint as a method for producing a cocoa butter substitute fat. Therefore, it is desired to obtain a microorganism with a high S ₂ U content and a glyceride composition close to that of cocoa butter, and to develop an industrially advantageous method for producing a cocoa butter substitute. (Means and Effects for Solving the Problems) The present inventors continued research on oil production by microorganisms and obtained microorganisms with metabolic specificity using various methods. Among these, we found that microbial fats and oils whose growth is particularly inhibited by sterculic acid or its derivatives have a higher S ₂ U component than previously reported microbial fats, and their triglyceride composition is similar to that of cocoa butter. Ta. The present invention relates to the production of a cocoa butter substitute, which is characterized by culturing microorganisms whose growth is inhibited by steruclic acid or derivatives thereof, accumulating fats and oils rich in S ₂ U components in the bacterial cells, and extracting and recovering the fats and oils. It is the law. The growth inhibition of microorganisms referred to in the present invention varies depending on factors such as the bacterial strain, medium composition, and concentration, but is defined as follows in the present invention. That is, in the presence of 1 ppm or more of sterculic acid or its derivatives in the medium, the growth rate is controlled to 80% or less compared to when no addition is made. Examples of microorganisms that can be used in the practice of the present invention include microorganisms belonging to the genus Candeida, which are microorganisms whose growth is inhibited by steruclic acid or derivatives thereof. To give a specific example of a species name, Candida
Candida humicola can be mentioned, and in particular, the cell oil of Candida humicola mutant strain (Candida humicola CBS 2822 KNK 286) has the following triglyceride composition,
POP18.6%, SOP39.0%, SOS14.6% (more than S ₂ U
72.2% in total) and can be used as is as a substitute for cocoa butter. This mutant strain was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and
It was entrusted as No. 783 (FERM BP-783). In order to select microorganisms such as those mentioned above that have been collected from the natural world or have been artificially mutated, it is preferable to use, for example, the following method. i.e. 1 ppm to 1% of steruclic acid or methyl sterculic acid;
Preferably, microorganisms are inoculated into a medium to which 5 ppm to 10 ppm is added and a medium which does not contain the substance,
Upon observation for 1 to 10 days, preferably 2 to 4 days, microorganisms are selected that do not grow in the former medium, or grow very slowly in the latter medium, but grow well in the latter medium without additives. do. The medium may be any medium in which microorganisms commonly grow, for example, a medium in which Candida genus can grow. In order to obtain microorganisms with the above-mentioned properties by artificial mutation, for example, ultraviolet irradiation, Co ⁶⁰ treatment,
High-energy radiation treatment such as X-ray irradiation, or sodium sulfite, hydroxyamine, 4-methyl-N'-nitrosoguanidine, acriflavine,
A conventional mutation treatment using a chemical inducer such as nitrogen mustard may be used as appropriate. Some of the bacteria collected in this way are also endowed with physiological and morphological variations such as auxotrophy for vitamins, amino acids, and other nutrients, and respiratory deficiencies; However, it goes without saying that any extracted oil or fat that is inhibited in production by steruclic acid or its derivatives and has a high S ₂ U component can be used in the present invention. Culture may be carried out either by standing still or by shaking, but deep culture with aeration and agitation is generally advantageous. The medium used for culturing is usually a liquid medium and contains carbon sources, nitrogen sources, and other nutrient sources that can be utilized by the bacteria. For example, carbon sources include pentose (xylose, etc.), hexose (glucose, fructose, etc.),
Examples include disaccharides (sucrose, maltose, etc.), but there are no particular limitations as long as they can be used by microorganisms. Further, highly assimilated industrial wastes such as sulfite pulp waste liquid, waste molasses, and wood saccharification waste liquid may also be used. As the nitrogen source, in addition to ammonium salts, nitrates, amino salts, etc., natural products such as yeast extract, meat extract, fish meal, corn steep liquor, and soybean meal can be used. In addition, in addition to this,
If necessary, metal salts such as iron, magnesium, calcium, manganese, and other organic or inorganic salts may be added, or if the improved bacteria used has auxotrophic properties, the necessary nutrients may be added as appropriate. An appropriate amount can be added. The various nutrient sources mentioned above are contained in the medium in an optimal formulation for the bacteria used. These conditions vary depending on the bacterial species, but the C/N ratio in the medium is important, especially for bacterial growth and oil accumulation.
The decision should be made after careful consideration. Such a medium usually has a pH of 2 to 9, preferably
PH4~7, temperature 20℃~40℃, preferably 25℃~33
Maintain at ℃ and culture aerobically. During this time,
If necessary, an antifoaming agent that does not inhibit fermentation may be added. In this way, fats and oils rich in S ₂ U components are accumulated in the cultured bacterial cells. To extract fats and oils from the bacterial cell growth product obtained as described above, the culture solution is first filtered or centrifuged to collect wet bacterial cells. Wet bacterial cells can be ground as they are using a colloid mill, ball mill, etc., and then extracted with a solvent such as hexane, or once frozen or spray-dried, extracted using a gauge press, expeller, etc., or extracted using a gauge press, expeller, etc. Extract by or
Alternatively, there is also a method of extraction using both.
The waste bacterial cells after oil and fat collection are a rich protein resource and can be used as feed. On the other hand, the extracted crude oils and fats contain impurities such as mono- and di-glycerides and free fatty acid sterols.
Must be removed by purification. Refining may be carried out in accordance with the refining of ordinary fats and oils. As described above, according to the present invention, S ₂ U can be extracted from microorganisms.
It is effective in industrially advantageously producing a cocoa butter substitute fat with a high content and a glyceride composition similar to that of cocoa butter, and this will be concretely clarified in the Examples below. (Example) The present invention will be described in more detail with reference to Examples. Example Candida humicola
A malt extract agar slant culture of CBS 2822 (original strain) was scraped and suspended in sterile physiological saline to give a number of viable cells of approximately 10 ⁷ to ^{10 8} cells/ml, and 2 ml of the cell number was treated with NTG to determine the mutation. Glucose 12.5 after
g/, yeast extract 3.75g/, malt extract 3.75
g/, polypeptone 6.25g/, agar 25g/
Spread 0.2ml on Hirasaka medium A consisting of
After culturing for 48 to 72 hours, this was transferred to flat medium B containing 3 ppm of methyl sterculate using the replica method (transfer).
After culturing at 30°C for 48 hours, colonies that did not appear in B were collected from A to obtain the mutant strain CBS 2822 KNK-286 (FERM BP-783).
I got it. Next, the original strain and the mutant strain were mixed with blackstrap molasses (sugar concentration of about 35
%) 142.85g/, urea 0.3g/, 85% phosphoric acid
After sterilizing the medium consisting of 0.3 ml/ml, each pure culture seed was inoculated and cultured with shaking at 30°C for 4 days. After the culture was completed, the cells were collected by centrifugation, dried, and the yield of cells was measured. In addition, after treating the remaining wet bacterial cells with sulfuric acid, crude oils and fats in the bacterial cells were extracted using ethyl ether, and the fatty acid composition and triglyceride composition were measured. The results are described below. The mutant crude oil has an acid value of 40.4, an iodine value of 38.3,
The melting point was 28.5°C. The growth rate of this mutant strain was 68% in the presence of 4 ppm of methyl sterculate in the medium compared to that without the addition of methyl sterculate. No inhibition of growth of the original strain was observed even when 5% methyl sterculate was added to the medium. Original strain KNK286 (FERM BP-783) Oil content: 30.5% 29.8% Fatty acid composition C16: 0 29.6% 25.8% C18: 0 12.0 33.8 C18: 1 46.5 32.7 C18: 2 11.9 6.0 C18: 3 Trace Trace C20: 0 Trace 0.7

【table】

[Table] The total of S ₂ U for POP, SOP, and SOS is 31.3 for the underlying stock.
%, whereas mutant strain KNK 286 (FERM
BP-783), it was 72.2%. Mutant strain KNK 286 (FERM BP−783) extraction
As shown in the cooling curve of FIG. 1, it is clear that the physical properties are similar to cocoa butter. This KNK 286 (FERM BP−783) extracted oil 13
%, a mixture of 10% cocoa butter, 16% cocoa mass, 18% whole milk powder and 43% powdered sugar, KNK 286
(FERM BP-783) Extracted oil and fat 7%, palm oil medium melting point part (dissolved in twice the amount of normal hexane as the oil and fat, stirred at -13℃ and 5℃ for 60 minutes each, and fractionated) Chiyocolate was prepared in a conventional manner using a mixture of 6% cacao butter, 16% cacao mass, 18% whole milk powder, and 43% powdered sugar. As a result, the allowable tempering temperature difference for any combination series is ±
The temperature was 2.0°C, the temperature was high, and the release from the mold was good. Also,
in each 12-hour cycle in the anti-bloom test.
When temperature changes of 18°C and 32°C were repeated 10 times, no bloom was observed, indicating excellent bloom resistance.

[Brief explanation of the drawing]

FIG. 1 is a cooling curve of the extracted oil and fat of Candida humicola CBS 2822 KNK-286 used in the present invention.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Candida whose growth is inhibited by steruclic acid or its derivatives is cultured in a medium, and S ₂ U (where S ₂ is glycerin 1
and 3-position hydroxyl groups are C ₁₆ , C ₁₈ and/or C ₂₀
Due to the saturated fatty acid residue, U is 2 of the same glycerin.
The hydroxyl group at position is due to C ₁₄ to _{C 20} unsaturated fatty acid residue,
means a respectively substituted disaturated-monounsaturated triglyceride. ) A method for producing a cocoa butter substitute, which comprises collecting microbial cells that have accumulated a significant amount of the component, and recovering fats and oils rich in S ₂ U components from the collected microbial cells. 2 The microorganism is Candida humicola.
humicola) CBS 2822 KNK 286. 3. The production method according to claim 1, wherein the growth inhibition is such that the growth rate is controlled to 80% or less in the presence of 1 ppm or more of sterculic acid or its derivative in the medium, compared to when no addition is made.