JP5366114B2 - Triglyceride-producing yeast - Google Patents

Description

  The present invention relates to a yeast that produces triglycerides, a method for producing triglycerides using the yeast, and a triglyceride produced by this method.

  When oil crops such as soybeans are deficient due to abnormal weather on a global scale, attempts have been made for a long time to put microorganisms, particularly yeast, into practical use as an alternative method for producing triglycerides. Some yeast species accumulate high concentrations of lipid components including triglycerides. These are called lipid-producing yeasts, and typical yeast species include Rhodosporidium toruloides. In Germany during the First and Second World Wars, an attempt was made to use lipid-producing yeast as a triglyceride resource as a means of supplementing food shortages (Prog. Ind. Microbiol., Vol.1, p .181 (1959)). However, due to the background of the times, the equipment, operating costs, and the price of the carbon source were high, so that they could not be put to practical use. Later, in the United States, Europe and Japan, various fungi were separated and grown for the purpose of using lipid-producing yeast as highly nutritious food and livestock feed, mainly on an industrial scale using sulfite pulp as a culture raw material. Manufactured by. However, production was discontinued after it was found that the cells contained the carcinogen 3,4-benzpyrene derived from sulfite pulp ("Applied Microbiology" Baifukan, 1996, p. 140). Even now, various lipid-producing yeasts are being studied while maintaining the possibility as a raw material for triglycerides, but none has been put into practical use for economic reasons.

Methods for producing polyunsaturated fatty acid-containing triglycerides by the yeast Yarrowia lipolytica and the filamentous fungus Mortierella alpina are known (Patent Documents 1 and 2). A preferred cultured carbon source is glucose. Moreover, the extraction method (patent document 3) of the fats and oils from baker's yeast and beer yeast Saccharomyces cerevisiae is known.
International Publication WO 2004/101757 Pamphlet JP 2007-319161 A JP-A-5-30981

    The biggest problem in putting triglyceride production by yeast into practical use is that the cost is higher than the conventional case where oil crops such as soybeans and rapeseed are used as raw materials. However, if a yeast type or strain is selected and culture conditions suitable for the selected strain can be established, various problems may be eliminated. Needless to say, when culturing yeast, it is necessary to select a culture raw material that is inexpensive, can be supplied in large quantities, and is safe.

  Beet (sugar beet), the main field crop in Hokkaido, and the beet sugar industry that produces sugar from it are the key industries in the region. The total amount of beet waste molasses produced as a by-product during the production of beet sugar is as large as 15,600 t per year. Beet molasses is mainly used as a culture raw material in the manufacture of baker's yeast, but since its use is limited and its added value is low, it is desired to be used in an industrially advanced manner.

  Since beet waste molasses is rich in sugars such as sucrose, it can be manufactured at low cost by culturing lipid-producing yeast using this as a raw material and extracting triglycerides from the cultured cells. Yeast Rhodosporidium toluroides and its related species are also called red yeasts, and produce a large amount of carotenoid pigments such as carotene. Therefore, a large amount of carotenoid pigments are mixed with the triglyceride in the extract from the microbial cells, so that it takes a cost to remove them.

  On the other hand, since the optimal culture carbon source of yeast Yarrowia lipolytica and filamentous fungus Mortierella alpina is glucose, sucrose contained in beet molasses cannot be used. Needless to say, in order to use the cultured cells as edible fats and oils by a simple extraction / purification method, the yeast must be safe. One such yeast that can be mentioned first is the yeast Saccharomyces cerevisiae, which is widely used in breadmaking and brewing, but accumulates only a small amount of triglycerides.

  The inventors of the present invention have made extensive studies in order to achieve the above object. As a result, the yeast Kluyveromyces lactis strain TYC-269 isolated from dairy products can be cultured with beet molasses, We found that the body is rich in triglycerides. Criveromyces lactis has a long history of eating and is recognized as a safe yeast. Furthermore, yeast cells having a high triglyceride content can be obtained by culturing at an initial pH of 7.0 in a medium containing soybean-derived proteolysate as a nitrogen source to be added and ammonium acetate as a factor that dramatically increases the triglyceride content. The present invention has been completed.

That is, the present invention is as follows.
1. A yeast belonging to the genus Kluyveromyces, containing at least 5 mg / g of triglyceride per dry weight;
2. The yeast according to 1 above, wherein the yeast is Kluyveromyces lactis,
3. The yeast, chestnut Vero Mrs. lactis (Kluyveromyces lactis) TYC-269 strain (consignment number: NITE P -525) is a yeast described above 2,
4). A method for producing triglyceride, comprising culturing the yeast according to any one of 1 to 3 in a culture solution containing beet waste molasses,
5. The method for producing triglycerides according to 4 above, wherein the culture solution contains beet waste molasses corresponding to 1 to 2% in terms of sugar.
6). The method for producing triglycerides according to 4 or 5 above, wherein the culture solution further contains a soybean-derived proteolysate,
7). The method for producing triglycerides according to any one of the above 4 to 6, wherein the culture solution further contains ammonium acetate,
8). The method for producing triglycerides according to any of 4 to 7, wherein the initial pH of the culture solution is 7.0,
9. Triglycerides obtained from the yeast cell according to any one of 1 to 3 above,
10. A method for producing triglycerides, comprising using the yeast according to any one of 1 to 3 above,
11. A food or feed containing the yeast cell according to any one of 1 to 3 above,
12 10. A food containing the triglyceride according to 9 above.

  According to the present invention, instead of extracting triglycerides from oil crops such as soybeans and rapeseed, using a means of fermentation production using beet waste molasses that can be stably supplied in large amounts, it is industrially inexpensive. Triglycerides can be obtained. In other aspects, beet waste molasses by-produced in a large amount during sugar production can be industrially useful and can be used for advanced new applications to increase added value.

The yeast referred to in the present invention refers to a yeast belonging to the genus Kluyveromyces that produces triglycerides and accumulates them at a high concentration. Preferably, the yeast is Kluyveromyces lactis. Particularly preferably, the yeast is Kluyveromyces lactis TYC-269. It should be noted that this strain, the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center March 12, 2008 with, as consignment number NITE P -525, has been deposited. Moreover, the kind of triglyceride is not specifically limited, You may contain various triglycerides. For example, triolein is applicable.

  The method for producing triglycerides of the present invention comprises culturing the yeast of the present invention in a culture solution containing beet waste molasses. In the method for producing triglycerides of the present invention, the culture solution preferably contains beet molasses corresponding to 1 to 2% in terms of sugar. In some cases, the culture solution may further contain a soybean-derived protein degradation product. The culture solution can further contain ammonium acetate. Moreover, in the manufacturing method of the triglyceride of this invention, it is preferable that the initial pH of a culture solution is 7.0.

  Moreover, as a manufacturing method of the triglyceride from the microbial cell of this invention, conventionally well-known arbitrary extraction methods can be used. For example, it can include the collection of cells from the culture, extraction of triglycerides from the collected cells, and purification of the extracted triglycerides.

  More specifically, first, yeast cells are grown. When a small amount of yeast cells are inoculated into a liquid medium and cultured with shaking at 200 rpm at 30 ° C. for 30 hours, the cells grow and become cloudy. Next, the clouded cells on the liquid medium are collected by centrifugation, and the total lipid components in the living cells immediately after the culture are extracted with an organic solvent. And a crude triglyceride can be obtained by concentrating and drying an extraction solution.

  Next, the obtained crude triglyceride is purified. A refinement | purification process may refine | purify as a triglyceride (including the triglyceride which consists of various fatty acid chain lengths), and may refine | purify a single component among triglycerides. Applicable to industrial triglyceride purification methods such as removal of gums precipitated by adding water or acid to the crude triglyceride obtained in the separation step, removal of free fatty acids by alkali addition and decolorization using activated clay it can. Further, the components may be further separated and purified by repeating silica gel chromatography.

  A liquid medium is used for culturing the yeast, and the medium can contain beet waste molasses as a carbon source. The concentration of beet waste molasses in the medium is preferably 1 to 2% in terms of sugar. As a medium component, it is desirable to include a nitrogen source useful for the growth of the yeast in addition to the carbon source.

  In order to obtain the desired triglyceride with high productivity, a predetermined amount of a nitrogen source should be used as a medium component in addition to the carbon source. When the amount of triglyceride accumulated was tested in a culture experiment with various nitrogen sources added, the most accumulated nitrogen source was defatted soybean-derived proteolysate (trade name: Polypeptone-S, manufactured by Nippon Pharmaceutical Co., Ltd.). )Met. The amount of polypeptone-S added to the medium may be determined as appropriate, but is usually preferably 1%. Furthermore, when changes in the amount of triglyceride accumulated were examined by the addition of various acetates, it was found that the accumulation of triglycerides increased rapidly by the addition of ammonium acetate. The amount of ammonium acetate added to the medium may be determined as appropriate, but is usually preferably 1%.

  The initial pH of the medium is preferably adjusted to 7.0. As the pH adjuster, sodium hydroxide or hydrochloric acid can be used. This liquid medium is inoculated with Crivellomyces lactis TYC-269 strain having triglyceride accumulation ability, and preferably cultured with shaking at 30 ° C. The culture time is up to a stationary phase where triglycerides are sufficiently accumulated, and preferably 30 hours after inoculation.

  The present invention also relates to a triglyceride obtained from the yeast of the present invention and a food containing the same, and a food or feed containing the yeast cell of the present invention.

  For example, the yeast of the present invention can be contained in food and livestock feed. Containing means having a step of fermentation using yeast that accumulates high concentration of triglyceride in the production process of food and livestock feed, or adding yeast that accumulates high concentration of triglyceride to food and livestock feed as it is Say. In either case, the food and livestock feed can contain a high concentration of triglyceride. At the same time, vitamins, minerals, proteins, dietary fiber and the like of yeast itself can be contained in food and livestock feed. Examples of foods that are fermented using yeast that accumulates high concentrations of triglycerides include yogurt and bread. Alternatively, yeast that accumulates high concentration of triglyceride may be used as a dry powder so that the yeast itself can be ingested as food and livestock feed. In this case, components such as vitamins may be added in addition to yeast.

  Moreover, the triglyceride obtained by the manufacturing method of the triglyceride of this invention can be utilized for various foodstuffs. As described above, triglyceride is a main component of edible oil, and thus can be expected to be used as an alternative edible oil. In addition, since yeast can grow in a short time, triglycerides can be easily produced as compared with conventional extraction from plants.

<Preparation of sample>
One platinum ear scrape of the yeast Kluyveromyces lactis strain TYC-269 isolated from the dairy product (Yeast extract 1%, polypeptone 2%) in a test tube (diameter 1.8 cm, length 18 cm) Inoculated into 1 ml of glucose 2%) and seed culture was performed. 400 μl of the seed culture solution in which the cells were grown was transplanted to 40 ml of YPD medium in a 200 ml Erlenmeyer flask, and cultured with shaking (200 rpm) at 30 ° C. for 30 hours. Centrifugation was performed on the culture solution in which the bacterial cells grew and became cloudy, and 7 ml of chloroform-methanol-distilled water (5: 10: 4, v / v) was added to the collected bacterial cells, followed by sonication for 3 minutes. The body was suspended. After adding 2 ml of chloroform and 2 ml of distilled water to this cell suspension, the lower layer obtained by centrifugation was concentrated and dried to obtain a total lipid as sample 1. Moreover, the standard strains NBRC1090, NBRC0433, NBRC0648, and NBRC1267 of Criveromyces lactis, which are also owned by the National Institute of Technology and Evaluation, which has been separated from dairy products, are treated in the same manner to obtain total lipids. Comparative samples 1 to 4 were used.

<Analysis of triglyceride content in bacterial cells by TLC>
Commercially available triglyceride preparation (triolein) as the above sample and comparative sample 1 was dissolved in a small amount of chloroform-methanol (2: 1, v / v), and hexane-diethyl ether-acetic acid (80: 20: 1, v / V) was applied to silica gel thin layer chromatography (TLC) as a developing solvent. And the state of the brown spot which appears after spraying and heating 25% sulfuric acid was observed. The image of the spot was taken in by an image analyzer, and the content per dry cell was calculated from the intensity of the spot.

<Calculation of triglyceride content>
The calculation results of the triglyceride content of these cells are shown in Table 1.

  The triglyceride content was calculated by taking the spot image with an image analyzer and calculating the content per cell from the intensity of the spot. From these results, it was found that Crivellomyces lactis TYC-269 of the present invention produced 5 mg or more of triglyceride per 1 g of dried cells.

  One platinum ear scrape of yeast Kluyveromyces lactis strain TYC-269 isolated from dairy products, YPD medium (yeast extract 1%, polypeptone 2%) in a test tube (diameter 1.8 cm, length 18 cm) Inoculated into 1 ml of glucose 2%) and seed culture was performed. 400 μl of the seed culture solution in which the cells were grown was transplanted to 40 ml of the test medium in a 200 ml Erlenmeyer flask, and cultured with shaking (200 rpm) at 30 ° C. for 24 hours. Subsequent operations and triglyceride quantification were carried out according to Example 1.

<Effects of the type of nitrogen source>
Culturing was performed with the concentration of various nitrogen sources added to the liquid medium being 1% for the organic nitrogen source and 0.1% for the inorganic nitrogen source. As a carbon source, 2% of beet waste molasses was added in terms of sugar. Triglyceride content was analyzed for cultured cells 24 hours after inoculation. The results are shown in Table 2.

    From the above results, it was revealed that triglyceride yield per medium increased most in the addition of defatted soybean-derived proteolysate (trade name: Polypeptone-S, manufactured by Nippon Pharmaceutical Co., Ltd.) with enzymes derived from animals and plants.

<Effect of adding acetate>
Culture was carried out by adding 1% of various acetate concentrations to a liquid medium containing 2% of beet waste molasses in terms of sugar and 1% of polypeptone-S. Triglyceride content was analyzed for cultured cells 24 hours after inoculation. The results are shown in Table 3.

  From the above results, it was clarified that by adding ammonium acetate, the amount of triglyceride accumulated per cell increased and the yield of triglyceride per medium further increased.

<Effect of culture temperature>
The beet waste molasses was cultured at various temperatures in a liquid medium containing 2% in terms of sugar, 1% polypeptone-S and 1% ammonium acetate. Triglyceride content was analyzed for cultured cells 24 hours after inoculation. The results are shown in Table 4.

  From the above results, it was revealed that the culture temperature is preferably 30 ° C.

<Effect of initial pH>
Sodium hydroxide or hydrochloric acid is added to a liquid medium containing 2% beet waste molasses in terms of sugar, polypeptone-S1% and ammonium acetate 1% to adjust the initial pH of the medium to 4.0 to 8.0. Then, culture was performed. Triglyceride content was analyzed for cultured cells 24 hours after inoculation. The results are shown in Table 5.

  From the above results, it was revealed that the initial pH is preferably 7.0.

<Effects of incubation time and carbon source concentration>
Culture was performed by adjusting the concentration of beet waste molasses added to the liquid medium to 1 to 16% in terms of sugar. To the liquid medium, 1% each of polypeptone-S and 1% each of ammonium acetate were added to adjust the initial pH to 7.0. Triglyceride content was analyzed for cultured cells at 15 hours (logarithmic growth phase), 30 hours (early stationary phase) and 45 hours after inoculation. The results are shown in Table 6.

  From the above results, it was clarified that the triglyceride yield per sugar was the highest when the culture was performed for 30 hours and the concentration of the carbon source was 1%.

<Composition analysis of triglyceride>
Furthermore, the calculation result of the composition of the triglyceride produced by this microbial cell is shown in Table 7.

  After subjecting Sample 1 to TLC, the triglyceride spot portion was scraped off and the triglyceride was eluted from the silica gel with a small amount of hexane. By analyzing the eluted triglycerides by gas chromatography, the composition of the constituent fatty acids was calculated from the separation of the molecular species of the constituent fatty acids of the triglycerides and their peak areas. From these results, the main constituent fatty acids of the triglyceride produced by Criveromyces lactis TYC-269 of the present invention are unsaturated fatty acids such as oleic acid (36.6%) and linoleic acid (30.2%). I found out.

  According to the present invention, instead of extracting triglycerides from oil crops such as soybeans and rapeseed, using a means of fermentation production using beet waste molasses that can be stably supplied in large amounts, it is industrially inexpensive. Triglycerides can be obtained. In other aspects, beet waste molasses by-produced in a large amount during sugar production can be industrially useful and can be used for advanced new applications to increase added value.

Claims (6)

Kluyveromyces lactis TYC-269 strain (reception number: NITE AP-525) containing 5 mg / g or more of triglyceride per dry weight. A method for producing triglycerides, comprising culturing Criveromyces lactis according to claim 1 in a culture solution containing beet waste molasses. The method for producing triglycerides according to claim 2 , wherein the culture solution contains beet waste molasses corresponding to 1 to 2% in terms of sugar. The method for producing triglycerides according to claim 3 , wherein the culture solution further contains soybean-derived proteolysate. The method for producing triglycerides according to claim 4 , wherein the culture solution further contains ammonium acetate. The method for producing triglycerides according to claim 5 , wherein the initial pH of the culture solution is 7.0.