JP4005677B2 - Production method of iron-containing yeast - Google Patents

Description

【００１８】
実施例２ 菌体内に含まれる鉄含量
実施例１で得られた各々の培養液５０ｍｌを遠心分離（５０００ｒｐｍ、１０分間、日立工機製、ＳＣＲ１８Ｂ）し、沈澱物を蒸留水で１回洗浄した（これを初発菌体という。）。ついでこれを１％塩酸溶液４０ｍｌに懸濁してよく攪拌後５分間放置した。これを５０ｍｌにメスアップし、その中の２５ｍｌを取り、遠心分離し、沈澱物を１％塩酸溶液２５ｍｌ、蒸留水２５ｍｌでそれぞれ菌体を洗浄し、沈澱物を５０ｍｌにメスアップした（これを塩酸処理菌体という。）。各洗液を集めて１００ｍｌにメスアップした。
初発菌体、塩酸処理菌体、洗液は常法に従って湿式分解して鉄濃度を測定した。
測定結果を表２に示す。
表２から明かなように、鉄を徐々に添加することにより菌体内含量が向上するが、これら菌体内の鉄は塩酸抽出液でも菌体外に流出しない、良質のものが多いことが分かる。
【００１９】
【表２】

【００２０】
【発明の効果】
以上説明してきた通り、本発明によると、鉄を徐々に、特に一定の流速で添加することにより、鉄由来の沈澱物を抑えるとともに、添加した鉄の菌体への取り込み効率も改善され、菌体中の鉄も極めて良好な状態である、鉄を２％以上含有した酵母が製造できる。[0001]
[Industrial application fields]
The present invention suppresses iron-derived precipitates by culturing edible yeast belonging to the genus Candida, particularly in the presence of an iron compound, and contains 2% or more of iron content in the yeast cells for health food applications It is related with the manufacturing method of yeast.
[0002]
[Prior art]
In recent health booms, interest in minerals and trace elements is increasing along with vitamins as nutrients. Under such circumstances, iron is one of the minerals that are said to be deficient alongside calcium in the case of Japanese. In particular, growing children and pregnant women are said to be more susceptible to iron deficiency anemia due to increased demand. Therefore, iron in a form that is easy to drink and absorb is required.
[0003]
Iron has already been actively added to processed foods, and ferrous sulfate, ferrous fumarate, ferrous succinate, etc. are usually used. However, these compounds originally have an iron odor, so the addition amount must be limited, and there is a problem in absorption into the body. Therefore, attention is recently focused on attempts to supply yeast in a form that absorbs iron to reduce the odor of iron in the form of organic iron, or to increase the absorbability into the human body.
[0004]
As a method for producing iron-containing yeast, edible microorganisms are cultured in a medium containing an iron compound, and a method for producing a microbial cell containing a large amount of iron (Japanese Patent Publication No. 61-9835), in the presence of an iron compound, A method for producing an organic ferrous iron-containing hematopoietic composition for culturing yeast in a saccharide-containing nutrient medium (Japanese Patent Laid-Open No. 58-101686), a microorganism containing an iron compound added to a medium containing a carbon source and a nitrogen source In general, a method for producing a microbial cell that needs to contain approximately 1% iron and that the concentration of magnesium in the medium is 10 mg / l or less (JP-A-62-134083). No.) is known. Furthermore, a method for producing a bivalent iron-containing yeast cell in which cells obtained by culturing yeast are dispersed in an aqueous solution containing an organic compound and a divalent water-soluble iron compound to accumulate iron (JP-A-5-176758). No.) is known.
[0005]
[Problems to be solved by the invention]
As described above, the conventional method for obtaining iron-containing cells is to obtain an iron-containing cell by culturing and growing a microorganism in a medium in which an iron compound is previously added to the medium, or a medium in which no iron compound is added in advance. Then, the microorganism is cultured and propagated, and then the cells are collected, an iron compound is added, and the cells are incorporated into the cells to obtain iron-containing cells. The former method is generally unable to add high concentration of iron because of the growth inhibition by iron. Furthermore, the added iron compound is oxidized within a relatively short time by aeration stirring during the culture, and insoluble compounds such as iron phosphate, especially with phosphate ions added as a normal essential medium component. In addition to inhibiting the iron uptake of microorganisms, there is no significant difference in the sedimentation rate between the insoluble matter and the cells in the separation of the cells by centrifugation after completion of the culture, making it difficult to separate them from the cells. It is. The presence of this insoluble matter becomes an obstacle in terms of iron odor, color and absorbency.
On the other hand, the latter method is considered to be a means for avoiding the growth inhibitory effect of iron itself on microorganisms. However, since the compound of phosphoric acid and iron increases depending on the concentration and contact time of both, iron is added in advance. In the method of collecting microbial cells cultured in a medium not added and then bringing it into contact with an iron compound to take up iron, in order to facilitate iron uptake, high concentrations of phosphoric acid and iron Since the compound is used, the production and removal of iron phosphate and the like became a problem.
[0006]
The present inventors previously proposed a method for producing an iron-containing yeast in which these drawbacks have been eliminated (Japanese Patent Application No. 8-82998). In order to further increase the iron content to, for example, 2 to 3% or more, When a large amount of iron is added to the medium, yeast with a high iron content is obtained, but iron is not sufficiently taken into the cells, and iron-derived compounds precipitate as fermenter tube walls, especially at the gas-liquid interface. There was a problem of sticking to the pipe wall.
[0007]
[Means for Solving the Problems]
The present inventors, as a result of intensive studies for solving the above problems, Candida-Uchirisu (Candida utilis ) CS-7529 (FERM BP-1656) yeast, when iron is gradually added and cultured, the degree of iron uptake increases and cells with a high iron content are obtained, and iron-derived precipitates It has been found that the amount of the precipitate is extremely small, and even if the precipitate is deposited, it is difficult to adhere to the fermenter, and the present invention has been completed.
That is, this invention provides the manufacturing method of the yeast which contained iron more highly 2% containing the precipitate derived from iron.
[0008]
The present invention is described in detail below.
Yeast used in the present invention, Candida-Uchirisu (Candida utilis ) CS-7529 (FERM BP-1656) is a high RNA-containing yeast. The high RNA-containing yeast, for example as disclosed in Japanese Patent Publication 56-46824 Patent Publication, it had a yeast having the ability to RNA to be produced and accumulated more than 12 wt% per cell weight.
[0009]
The present invention is carried out by adding 20 ppm (as magnesium) or more of magnesium to the culture medium and adding 700 to 2000 ppm of iron as the total iron amount to the culture solution.
Examples of magnesium used in the present invention include magnesium sulfate and magnesium chloride. The concentration of magnesium is required to be 20 ppm or more, and if it is less than that, it will adversely affect the yield of microbial cells and further the accumulation of iron.
The iron compound used in the present invention may be any organic or inorganic substance as long as it has an appropriate water solubility. Specific examples of such a source include ferrous citrate, ferrous fumarate, ferrous lactate and the like. In addition, ferrous sulfate, ferrous chloride and the like used as medium components in normal microorganism culture can be used as a matter of course. As described above, various iron compounds can be used, but ferrous sulfate, which is inexpensive and has standards for food addition, is most preferable in view of production on an industrial scale. As these addition amounts, the total iron amount needs to be 700 ppm to 2000 ppm. If the amount is less than 700 ppm, the amount of accumulated iron becomes less than 2%. On the other hand, if the amount exceeds 2000 ppm, growth inhibition starts to appear, and the accumulated efficiency of added iron decreases.
[0010]
In the present invention, iron is gradually added. Any addition method may be used, but a method of adding at a constant flow rate is preferable from the viewpoint of easy work. The addition rate as iron is 10 to 150 mg / L / hour, preferably 20 to 100 mg / L / hour as iron. If the added amount is larger than this, the inorganic iron compound tends to adhere to the inner wall of the fermenter, which is not preferable.
[0011]
As the medium composition of the strain, glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, etc. that are usually used for culturing microorganisms are used as the carbon source, and urea, ammonia, ammonium sulfate, ammonium chloride, nitrate as the nitrogen source Etc. are used. As the phosphoric acid and potassium source, potassium phosphate, ammonium phosphate, potassium chloride and the like are used. As other trace metals, inorganic salts such as zinc, copper, manganese and iron ions are effective. Further, if necessary, organic substances such as corn steep liquor, casein, yeast extract and peptone may be added.
[0012]
The cells used in the present invention can grow at a culture temperature of 20 to 33 ° C, but a relatively low temperature of 21 to 24 ° C is preferable for the accumulation of iron.
The culture pH is 3.5 to 7.5, preferably 4.0 to 6.0, and the culture time is usually 20 to 30 hours, although it varies depending on the concentration of the carbon source.
[0013]
In the present invention, Candida-Uchirisu (Candida utilis ) CS-7529 (FERM BP-1656), a high RNA-containing yeast, has a high phosphate uptake capacity, solubilizes precipitates such as iron phosphate and also absorbs iron, so high concentration Even when culturing in a medium supplemented with an iron compound, growth inhibition is hardly observed.
Further, as described in, for example, JP-A-62-134083, it is known that the magnesium concentration in the medium needs to be lowered in order to obtain a high iron-containing microorganism. And by making magnesium concentration higher than a well-known example, the yeast containing 2% or more of iron can be manufactured easily.
[0014]
A feature of the present invention is that iron is gradually added, particularly at a constant flow rate, to suppress iron-derived precipitates. The present inventors presume that this can be avoided by controlling the concentration of iron to the extent that phosphoric acid and iron in the medium are mixed to cause precipitation.
Furthermore, by gradually adding iron, an unexpected effect of improving the uptake efficiency of the added iron into the microbial cells is achieved, and the microbial cells contain almost no low-molecular iron compounds and are extremely good. A high iron content yeast can be produced.
[0015]
【Example】
Hereinafter, the present invention will be described with reference to examples.
The bacterial cell concentration was determined from the weight after taking a certain amount of the culture solution, washing the bacterial cells twice with a centrifuge, and taking a portion of them and drying them at 105 ° C. overnight. The iron content incorporated into the cells was measured by the atomic absorption method after ashing the cells by a wet decomposition method. In the following Examples and Reference Examples, the bacterial cell concentration and the concentration of iron incorporated into the bacterial cell were all measured by the same method.
[0016]
Example 1 Yeast Culture and Iron Accumulation YPD medium (glucose 2%, polypeptone 2%, yeast extract 1%) 100ml was dispensed into a 500ml Erlenmeyer flask and sterilized at 121 ° C for 15 minutes, and then Candida utilis. One platinum ear of CS7529 was inoculated and cultured at 30 ° C. with shaking for 24 hours to form an inoculum. All of this was inoculated into a 30 liter fermenter. The culture medium is glucose 6.5%, monoammonium phosphate 0.26%, ammonium sulfate 0.15%, magnesium sulfate 0.09%, potassium chloride 0.2%, manganese sulfate 2ppm, zinc sulfate 2ppm, copper sulfate 0.4ppm Was used for batch culture. The culture conditions were as follows: the amount of liquid in the tank was 15 L, the culture temperature was 30 ° C., the aeration rate was 15 L / min, the stirring was 400 rpm, and the pH was 4.0 (automatic control by adding ammonia). Iron was dissolved in 5000 ppm (1000 ppm as iron) content of ferrous sulfate as a total concentration in another Erlenmeyer flask (7.5% ferrous sulfate concentration), sterilized at 121 ° C. for 15 minutes, The mixture was added at a rate of 20 to 60 mg / L / hour with a peristaltic pump (manufactured by Ato, SJ1211). As a control, ferrous sulfate 5000 ppm (1000 ppm as iron) was first added to the medium, and the same culture was performed.
The culture was terminated when glucose was completely consumed.
The results are shown in Table 1.
As is clear from Table 1, it can be seen that the method of gradually adding has a higher iron content in the cells and the amount of the inorganic iron compound adhering to the tube wall is greatly reduced.
[0017]
[Table 1]

[0018]
Example 2 Iron content contained in bacterial cells 50 ml of each culture solution obtained in Example 1 was centrifuged (5000 rpm, 10 minutes, manufactured by Hitachi Koki, SCR18B), and the precipitate was washed once with distilled water ( This is called the first cell.) This was then suspended in 40 ml of 1% hydrochloric acid solution and allowed to stand for 5 minutes after stirring. This was made up to 50 ml, 25 ml of it was taken and centrifuged, the precipitate was washed with 25 ml of 1% hydrochloric acid solution and 25 ml of distilled water, respectively, and the precipitate was made up to 50 ml. This is called hydrochloric acid-treated cells.) Each wash was collected and made up to 100 ml.
The initial cells, hydrochloric acid-treated cells, and the washing solution were subjected to wet decomposition according to a conventional method, and the iron concentration was measured.
The measurement results are shown in Table 2.
As is apparent from Table 2, the content of the bacterial cells is improved by gradually adding iron, but it can be seen that there are many good quality irons that do not flow out of the bacterial cells even with a hydrochloric acid extract.
[0019]
[Table 2]

[0020]
【The invention's effect】
As described above, according to the present invention, by gradually adding iron, particularly at a constant flow rate, the iron-derived precipitate is suppressed, and the efficiency of incorporating the added iron into the cells is improved. Yeast containing 2% or more of iron, in which the iron in the body is also in a very good state, can be produced.

Claims (2)

In a method for culturing yeast by adding 20 ppm (as magnesium) or more of magnesium to a medium to produce an iron-containing yeast, the yeast is produced by Candida utilis ( Candida utilis ) CS-7529 (FERM BP-1656), a high RNA-containing yeast, wherein iron is gradually added to the culture medium, and 700-2000 ppm is added as the total iron content, and this is an iron-derived precipitate. A method for producing yeast cells that contain 2% or more of iron as the iron content in which the substances are suppressed. The process according to claim 1, wherein the amount of iron added is 10 to 150 mg / L / hour.