JP3600803B2 - Mannitol producing strain Candida magnolia and method for producing mannitol by fermentation using the same - Google Patents

Description

【００１４】
【表２】

【００１５】
また、形態学的特性、生理学的及び生化学的特性は表３に表し、新菌株の炭素源代謝の有無を表４に表した。
【００１６】
【表３】

【００１７】
【表４】

【００１８】
以上のような菌株の特性を同定した結果、本菌株はカンジダ・マグノリア種の菌株と判断され、新菌株カンジダ・マグノリア（Ｃａｎｄｉｄａ ｍａｇｎｏｌｉａｅ）を韓国微生物保存協会に２００１年３月５日付で寄託番号ＫＣＣＭ−１０２５２号で寄託した。
【００１９】
本発明のマンニトール発酵生成法をさらに具体的に説明すれば次の通りである。
【００２０】
本菌株の種培養のための増殖培地としては、ブドウ糖１５〜２５ｇ／Ｌ、酵母抽出物７〜１３ｇ／Ｌ及びペプトン１５〜２５ｇ／Ｌを含むＹＰＤ培地を使用し、フラスコの発酵培地としては炭素源でブドウ糖５０ｇ／Ｌ及び果糖１００ｇ／Ｌ、窒素源としては酵母抽出物及び硫酸アンモニウム、無機塩としては二リン酸カリウム、硫酸マグネシウムとから構成された培地を使用した。各成分の量はマンニトールの生産性を高めるために変更することができる。発酵槽で使用した培地は、流加式で総添加された濃度として３００ｇ／Ｌの果糖、ブドウ糖５０ｇ／Ｌ、酵母抽出物１０ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、硫酸マグネシウム０．４ｇ／Ｌとから構成された最適培地であった。
【００２１】
種培養は冷凍保存された菌株をＹＰＤ培地５０ｍｌが入っている２５０ｍｌのフラスコに接種して振とう培養器で２３０〜２７０ｒｐｍ、２７〜３３℃で菌体濃度が３〜４ｇ／Ｌ（約２４時間）で増殖するまで行った。フラスコ培養では培地の５％に該当される種培養液を発酵培地５０ｍｌが入っている２５０ｍｌのフラスコに接種して振とう培養器で２１０〜２３０ｒｐｍ、２７〜３３℃で６８〜７６時間培養した。
【００２２】
流加式発酵槽培養では、発酵初期に１００ｇ／Ｌの果糖が含まれた２Ｌの培地を含む５Ｌ発酵槽（韓国培養器（株））を使用した。発酵過程中に撹拌速度は２００〜５００ｒｐｍに調節し、通気量は０〜０．５ｖｖｍに調節した。ｐＨは発酵全前過程の間４．５〜５．５に調節し、培養温度は２７〜３３℃であり、維持される果糖の濃度は５０〜１２０ｇ／Ｌに調節した。
【００２３】
ブドウ糖、果糖、マンニトールの濃度は、Ｃａｒｂｏｈｙｄｒａｔｅ Ａｎａｌｙｓｉｓカラム（Ｗａｔｅｒｓ、ＵＳＡ）を装着したＨＰＬＣのＲｅｆｒａｃｔｉｖｅ Ｉｎｄｅｘ Ｄｅｔｅｃｔｏｒ（Ｗａｔｅｒｓ２４１０、ＵＳＡ）を使用して測定した。この時、溶媒はアセトニトリール（ａｃｅｔｏｎｉｔｒｉｌｅ）と水を８：２で混ぜて使用し、流速は１．５ｍｌ／分であった。菌体濃度は濁度計を使用して６００ｎｍで懸濁度を測定してあらかじめ測定した標準曲線を使用して乾燥重量に転換した。溶存酸素濃度はＩｎｇｏｌｄ社（Ｓｗｉｓｓ、ｐｏｌａｒｏｇｒａｐｈｉｃ ｔｙｐｅ）の溶存酸素電極を使用して特定した。
【００２４】
以上のような本発明の目的と別の特徴及び長所等は、以下に参照する本発明の好適な実施例に対する以下の説明から明確になるであろう。
【００２５】
【発明の実施の形態】
本発明を実施例によってより詳細に説明すれば次の通りである。
（実施例１）
種培養：キシリトール（ｘｙｌｉｔｏｌ）製造会社の発酵スラッジから分離した菌株カンジダ・マグノリア（ＫＣＣＭ−１０２５２号）をＹＰＤ培地５０ｍｌが入っている２５０ｍｌフラスコに接種して２５０ｒｐｍ、３０℃で２４時間培養した。
【００２６】
本培養：発酵培地５０ｍｌを含む２５０ｍｌフラスコに種培養液を接種した後、振とう培養器で２２０ｒｐｍで３０℃で果糖が完全に消費されるまで培養した。培地のｐＨは、発酵初期に５．０で調節し、その後の調節は行わなかった。この時、培地成分はブドウ糖５０ｇ／Ｌ、果糖１００ｇ／Ｌ、酵母抽出物５ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌであった。時間の経過によるマンニトールの生産を観察した結果を表５に表した。分離菌株の最大非増殖速度は０．２６ｈ^−１、最大菌体量は３．９ｇ／Ｌであり、４２ｇ／Ｌのマンニトールが生産された。
【００２７】
【表５】

【００２８】
（実施例２）
培養方法は実施例１と同様であり、ブドウ糖５０ｇ／Ｌ、果糖１００ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌ、酵母抽出物５ｇ／Ｌの窒素含有量に準じるそれぞれの窒素源とから構成された発酵培地で発酵時間７２時間後、各種の窒素源がマンニトールの生産に及ぼす影響を観察した。各種の窒素源で培養した結果を表６に表した。マンニトールは酵母抽出物と酵母窒素（ｙｅａｓｔ ｎｉｔｒｏｇｅｎ ｂａｓｅ）を除く他の有機窒素源では低い濃度を表して、酵母抽出物が最も良い窒素源であった。
【００２９】
【表６】

【００３０】
（実施例３）
培養方法は実施例１と同様であり、本培養で発酵培地をブドウ糖５０ｇ／Ｌ、果糖１００ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、酵母抽出物、二リン酸カリウム２．５ｇ／Ｌとして、このうち酵母抽出物の濃度を変化させて７２時間培養した結果を表７に表示した。
【００３１】
【表７】

【００３２】
（実施例４）
培養方法は実施例１と同様であり、本培養で発酵培地をブドウ糖５０ｇ／ｌ、果糖１００ｇ／Ｌ、酵母抽出物１０ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、二リン酸カリウムとして、このうち二リン酸カリウムの濃度を変化させながら７２時間培養した結果を表８に表示した。
【００３３】
【表８】

【００３４】
（実施例５）
培養方法は実施例１と同様であり、本培養で発酵培地を果糖１００ｇ／Ｌ、酵母抽出物１０ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、硫酸マグネシウムとして、このうち硫酸マグネシウムの濃度を変化させながら３０時間培養した結果を表９に表示した。
【００３５】
【表９】

【００３６】
（実施例６）
種培養は実施例１と同様であり、本培養で最適発酵培地（ブドウ糖５０ｇ／Ｌ、果糖１００ｇ／Ｌ、酵母抽出物１０ｇ／Ｌ、硫酸アンモニウム２．５ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌ、硫酸マグネシウム０．４ｇ／Ｌ）が３Ｌである５Ｌ発酵槽を使用して培養した。培養温度３０℃でいろいろなｐＨ別の実験を行った。通気量は０．５ｖｖｍ、撹拌速度は２００〜５００ｒｐｍに調節して、ｐＨを変化させて果糖が完全に消費されるまで培養した結果を表１０に表示した。
【００３７】
【表１０】

【００３８】
（実施例７）
培養方法と発酵培地は実施例６と同様であり、本培養で培養ｐＨは５．０として培養温度別実験を行い、その結果を表１１に表示した。
【００３９】
【表１１】

【００４０】
（実施例８）
選定された最適培地（酵母抽出物１０ｇ／Ｌ、二リン酸カリウム２．５ｇ／Ｌ、硫酸マグネシウム０．４ｇ／Ｌ）で果糖の濃度を３００ｇ／Ｌで増加させて発酵槽で流加式培養を行った。この時、流加式培養を行った理由は、カンジダ・マグノリアの場合１００ｇ／Ｌ以上の果糖ではマンニトール生産速度が低下したためであった。培養は、発酵初期に炭素源として５０ｇ／Ｌのブドウ糖を含む２Ｌの培地を含む５Ｌ発酵槽（韓国発酵器（株））を使用した。発酵過程中に２２５ｇの果糖が含まれた２５０ｍｌの溶液を４回（２０ｈ，３６ｈ，６２ｈ，９０ｈ）追加して、最終培養液の体積が３Ｌ（総添加された果糖の濃度は３００ｇ／Ｌに該当）になる流加式培養をした。溶存酸素は撹拌速度を５００〜６００ｒｐｍで調節して培養初期には２０％以上維持させて菌体濃度が約５ｇ／Ｌになる時点で撹拌速度を２００〜３５０ｒｐｍに変化させて溶存酸素を制限した。ｐＨは発酵全過程の間５．０、培養温度は３０℃、通気量は０．５ｖｖｍで調節し、時間によるマンニトールの生産は表１２に表した。このように流加式培養方法により３００ｇ／Ｌの果糖から１２０時間後に２４８ｇ／Ｌのマンニトールを得た。このような結果は、果糖に対するマンニトールの収率８３％とマンニトールの生産性２．０７ｇ／Ｌ^−ｈに該当するものである。
【００４１】
【表１２】

【００４２】
【発明の効果】
本発明の効果は天然スラッジから分離された新菌株カンジダ・マグノリア（Ｃａｎｄｉｄａ ｍａｇｎｏｌｉａｅ）を使用してブドウ糖５０〜１００ｇ／Ｌ、果糖５０〜１２０ｇ／Ｌ、酵母抽出物５〜２０ｇ／Ｌ、二リン酸カリウム１．０〜５．０ｇ／Ｌ及び硫酸マグネシウム０．１〜０．５ｇ／Ｌを含有する培地で培養して、マンニトールを高収率かつ高生産性で生産することができる。これは従来の化学合成及び発酵方法に比べて選択的、経済的、効果的にマンニトールを発酵生産できるものであり、製品品質及び国際競争力でも比較優位を占めることができる。
【００４３】
以上では本発明を実施例によって詳細に説明したが、本発明は実施例によって限定されず、本発明が属する技術分野において通常の知識を有するものであれば本発明の思想と精神を離れることなく、本発明を修正または変更できるであろう。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel strain Candida magnoliae which produces mannitol in high yield and high productivity, isolated from nature, and a method for producing mannitol from fructose using the same. Things.
[0002]
[Prior art]
Mannitol is naturally contained in brown algae, mushrooms, fungi, and the like as a hexose sugar alcohol. Mannitol has a sweetness of 30 to 40% of sugar, and is used not only as an alternative sweetener in the production of foods in which the use of sugar is restricted, but also as a cold drink, low moisture absorption, fluidity, etc. Due to its excellent properties, it is widely used in confectionery additives, pharmaceutical fillers, surfactants and waterproofing agents. In addition, it is widely used in the food and pharmaceutical industries, such as being used as an intermediate of antihypertensive agents and as a coating agent for reducing bitterness in the manufacturing process of various pharmaceutical preparations.
[0003]
Mannitol occurs naturally in various fruits and vegetables, but in this case it is present in very small amounts, so extracting it from fruits and vegetables is not economically industrial. Therefore, the commercial production method of mannitol separates fructose produced by hydrolysis from sugar and the like, and produces the fructose by chemically adding hydrogen under a catalyst under high temperature and high pressure. However, since sorbitol is produced as a by-product, not only a separate purification step is required, but also the conversion yield is very low and the production cost is high. In addition, since the reaction is performed at a high temperature and a high pressure, there are disadvantages that there is a problem of danger and waste disposal.
[0004]
In order to solve such disadvantages, many studies on a method for producing mannitol by a microorganism have been made. Examples of microorganisms related to mannitol production include, in the case of yeast, Candida zelanoid, Candida lipolytica, Cryptococcus neoformans (Cryptococcus neoformis antos entropi ntropis trossip), Cryptococcus neoformis anthropis trophy, and the like. In the case of bacteria, there are Lactobacillus brevis (Lactobacillus brevis), Leuconostoc mesenteriode (Leuconostoc mesenteriode), Mycobacterium smegmatis (Mycobacterium smegmatis), etc. xii), Aspergillus nidulans (Aspergillus nidulans), there is such as Penicillium Kaburosamu (Penicillum scabrosum). Microbial methods are economical, can produce only mannitol specifically from glucose or fructose, and can greatly facilitate the process of separating and purifying mannitol after the reaction, but its productivity or yield is low. Therefore, there is difficulty in industrialization.
[0005]
[Problems to be solved by the invention]
The technical problem to be solved by the present invention is to isolate and identify a high-yield mannitol-producing new strain Candida magnoliae from fermented sludge of a xylitol manufacturing company (Bolak Co., Ltd. Karasuyama). A method for producing mannitol with high productivity and high yield by optimizing culture conditions has been developed.
[0006]
[Means for Solving the Problems]
The present invention provides a seed culture of a new strain Candida magnoliae (Deposit No. KCCM-10252) isolated from natural sludge, and converts the seed-cultured strain into a medium containing glucose, fructose, a nitrogen source and inorganic salts. The present invention provides a high-yield, high-productivity mannitol production method characterized by separating and purifying mannitol produced by fermentation cultivation in the above, and mannitol produced by the method.
[0007]
The fermentation medium contains 50-100 g / L of glucose, 50-120 g / L of fructose, 5-20 g / L of yeast extract, 1-5 g / L of ammonium sulfate, 1.0-5.0 g / L of potassium diphosphate and sulfuric acid. It is characterized by containing 0.1 to 0.5 g / L of magnesium, the stirring speed during the fermentation culture is 200 to 500 rpm, the aeration is 0 to 0.5 vvm, and the pH is 4.5 to 5.5. 5, and the culturing temperature is 27 to 35 ° C.
[0008]
On the other hand, seed culture of the new strain Candida magnolia is performed in a YPD medium containing glucose 15 to 25 g / L, yeast extract 7 to 13 g / L and peptone 15 to 25 g / L, and the cell concentration is 3 to 5 g. / L, which is characterized by culturing with shaking until it grows at / L.
[0009]
Also, after diluting a sample of natural sludge, it contains glucose 10-20 g / L, fructose 18-22 g / L, peptone 15-25 g / L, yeast extract 7-15 g / L, agar 12-17 g / L. A single colony was selected mainly on strains growing rapidly at 27-33 ° C. using a flat plate, and these selected strains were separated into glucose 30-70 g / L, fructose 50-100 g / L, yeast extract 5 -20 to 20 g / L, ammonium sulphate 1 to 5 g / L, potassium diphosphate 1 to 5 g / L, and a fermentation time of 68 to 76 hours after fermentation time. Method for isolating strain Candida magnoliae (Accession No. KCCM-10252) and isolated mannitol-producing new strain Candida magnolia ( Candida magnoliae (Accession No. KCCM-10252).
[0010]
Hereinafter, the method for isolating the novel strain Candida magnolia of the present invention will be described as follows.
[0011]
After appropriately diluting a fermented sludge sample from a xylitol manufacturer, glucose 200-400 g / L, peptone 15-25 g / L, yeast extract 7-15 g / L, agar 12-17 g / L. Using the plates contained, a single colony is selected, centering on strains that grow rapidly at 27-33 ° C., and these selected strains are screened for 30-70 g / L glucose, 50-100 g / L fructose, yeast extract In a medium composed of 5 to 20 g / L, 1 to 5 g / L of ammonium sulfate, and 1 to 5 g / L of potassium diphosphate, a bacterium producing the most mannitol was finally selected after a fermentation time of 68 to 76 hours. .
[0012]
The strain was identified according to the method of analyzing the base sequence of 26s rRNA (Kurtzman & Robnett, 1998) and the morphological, cultural, and physiological characteristics according to the method of Yarrow (Elsevier Science Publishers, Amsterdam, 1998). (Yeasts: Characteristics and identification. Cambridge University Press, London, 1983). Tables 1 and 2 show the results of the analysis of the base sequence of the 26s rRNA of the selected strains and the base sequence with the similar species, respectively.
[0013]
[Table 1]

[0014]
[Table 2]

[0015]
Table 3 shows the morphological characteristics, physiological and biochemical characteristics, and Table 4 shows the presence or absence of carbon source metabolism of the new strain.
[0016]
[Table 3]

[0017]
[Table 4]

[0018]
As a result of identifying the characteristics of the above strains, the strain was determined to be a strain of Candida magnolia sp. No. 10252.
[0019]
The mannitol fermentation production method of the present invention will be described more specifically as follows.
[0020]
A YPD medium containing 15 to 25 g / L of glucose, 7 to 13 g / L of yeast extract and 15 to 25 g / L of peptone is used as a growth medium for seed culture of the present strain. A medium composed of glucose 50 g / L and fructose 100 g / L as sources, yeast extract and ammonium sulfate as nitrogen sources, and potassium diphosphate and magnesium sulfate as inorganic salts was used. The amounts of each component can be varied to increase mannitol productivity. The medium used in the fermenter was 300 g / L of fructose, 50 g / L of glucose, 10 g / L of yeast extract, 2.5 g / L of potassium diphosphate, 2.5 g of ammonium sulfate in a fed-batch system. / L and 0.4 g / L of magnesium sulfate.
[0021]
Seed culture is performed by inoculating a cryopreserved strain into a 250 ml flask containing 50 ml of YPD medium and shaking incubator at 230 to 270 rpm and a cell concentration of 3 to 4 g / L at 27 to 33 ° C (about 24 hours). ) Until growth. In the flask culture, a seed culture solution corresponding to 5% of the medium was inoculated into a 250 ml flask containing 50 ml of fermentation medium, and cultured in a shaking incubator at 210 to 230 rpm at 27 to 33 ° C for 68 to 76 hours.
[0022]
In the fed-batch fermenter culture, a 5 L fermenter (Korean Incubator Co., Ltd.) containing a 2 L medium containing 100 g / L fructose at the beginning of fermentation was used. During the fermentation, the stirring speed was adjusted to 200-500 rpm, and the aeration was adjusted to 0-0.5 vvm. The pH was adjusted to 4.5-5.5 during the entire pre-fermentation process, the culture temperature was 27-33 ° C, and the fructose concentration maintained was adjusted to 50-120 g / L.
[0023]
Glucose, fructose, and mannitol concentrations were measured using a HPLC Refractive Index Detector (Waters 2410, USA) equipped with a Carbohydrate Analysis column (Waters, USA). At this time, the solvent used was a mixture of acetonitrile and water at a ratio of 8: 2, and the flow rate was 1.5 ml / min. The cell concentration was converted to dry weight using a standard curve measured in advance by measuring the suspension at 600 nm using a turbidimeter. The dissolved oxygen concentration was determined using a dissolved oxygen electrode from Ingold (Swiss, polarographic type).
[0024]
The above and other objects, features and advantages of the present invention will be apparent from the following description of preferred embodiments of the present invention with reference to the accompanying drawings.
[0025]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in more detail with reference to examples.
(Example 1)
Seed culture: Candida magnolia (KCCM-10252) isolated from fermented sludge of a xylitol manufacturing company was inoculated into a 250 ml flask containing 50 ml of YPD medium, and cultured at 250 rpm at 30 ° C. for 24 hours.
[0026]
Main culture: A seed culture solution was inoculated into a 250 ml flask containing 50 ml of fermentation medium, and cultured in a shaking incubator at 220 rpm at 30 ° C. until fructose was completely consumed. The pH of the medium was adjusted to 5.0 at the beginning of the fermentation and no further adjustment was made. At this time, the medium components were glucose 50 g / L, fructose 100 g / L, yeast extract 5 g / L, ammonium sulfate 2.5 g / L, and potassium diphosphate 2.5 g / L. Table 5 shows the results of observing the production of mannitol over time. The maximum non-growth rate of the isolated strain was 0.26 h ^-1 , the maximum bacterial mass was 3.9 g / L, and 42 g / L of mannitol was produced.
[0027]
[Table 5]

[0028]
(Example 2)
The culturing method is the same as in Example 1, and is based on the nitrogen content of 50 g / L glucose, 100 g / L fructose, 2.5 g / L ammonium sulfate, 2.5 g / L potassium diphosphate, and 5 g / L yeast extract. After a fermentation time of 72 hours in a fermentation medium composed of each nitrogen source, the effects of various nitrogen sources on mannitol production were observed. Table 6 shows the results of culturing with various nitrogen sources. Mannitol represented a low concentration of yeast extract and other organic nitrogen sources except yeast nitrogen base, and yeast extract was the best nitrogen source.
[0029]
[Table 6]

[0030]
(Example 3)
The culturing method is the same as that in Example 1. In the main culture, the fermentation medium was changed to 50 g / L glucose, 100 g / L fructose, 2.5 g / L ammonium sulfate, yeast extract, and 2.5 g / L potassium diphosphate. Table 7 shows the results of culturing for 72 hours while changing the concentration of the yeast extract.
[0031]
[Table 7]

[0032]
(Example 4)
The culturing method is the same as that in Example 1. In the main culture, the fermentation medium was changed to glucose 50 g / l, fructose 100 g / L, yeast extract 10 g / L, ammonium sulfate 2.5 g / L, and potassium diphosphate. Table 8 shows the results of culturing for 72 hours while changing the concentration of potassium phosphate.
[0033]
[Table 8]

[0034]
(Example 5)
The culturing method is the same as that in Example 1. In the main culture, the fermentation medium was changed to fructose 100 g / L, yeast extract 10 g / L, potassium diphosphate 2.5 g / L, ammonium sulfate 2.5 g / L, and magnesium sulfate. Table 9 shows the results of culturing for 30 hours while changing the concentration of magnesium sulfate.
[0035]
[Table 9]

[0036]
(Example 6)
Seed culture was the same as in Example 1. In the main culture, the optimal fermentation medium (glucose 50 g / L, fructose 100 g / L, yeast extract 10 g / L, ammonium sulfate 2.5 g / L, potassium diphosphate 2.5 g / L) (L, magnesium sulfate 0.4 g / L) was cultured using a 5 L fermenter having 3 L. Various pH-dependent experiments were performed at a culture temperature of 30 ° C. Table 10 shows the results of culturing until the fructose was completely consumed by changing the pH while adjusting the aeration rate to 0.5 vvm and the stirring speed to 200 to 500 rpm.
[0037]
[Table 10]

[0038]
(Example 7)
The culture method and the fermentation medium were the same as in Example 6. The main culture was performed at a culture pH of 5.0, and experiments were performed at different culture temperatures. The results are shown in Table 11.
[0039]
[Table 11]

[0040]
(Example 8)
Fructose concentration is increased to 300 g / L in the selected optimal medium (10 g / L for yeast extract, 2.5 g / L for potassium diphosphate, 0.4 g / L for magnesium sulfate), and fed-batch culture is performed in a fermenter. Was done. At this time, the reason why the fed-batch culture was performed was that in the case of Candida magnolia, the production rate of mannitol was reduced with fructose of 100 g / L or more. The cultivation used a 5 L fermenter (Korean Fermenter Co., Ltd.) containing a 2 L medium containing 50 g / L glucose as a carbon source at the beginning of fermentation. During the fermentation process, 250 ml of a solution containing 225 g of fructose was added four times (20 h, 36 h, 62 h, 90 h) to make the final culture volume 3 L (total concentration of added fructose was 300 g / L). Fed-batch culture was performed. The dissolved oxygen was regulated at a stirring speed of 500 to 600 rpm, and maintained at 20% or more in the initial stage of the culture. When the bacterial cell concentration reached about 5 g / L, the stirring speed was changed to 200 to 350 rpm to limit the dissolved oxygen. . The pH was adjusted to 5.0 during the entire fermentation process, the culture temperature was adjusted to 30 ° C., the aeration rate was adjusted to 0.5 vvm, and the production of mannitol over time is shown in Table 12. In this way, 248 g / L of mannitol was obtained after 120 hours from 300 g / L of fructose by the fed-batch culture method. Such a result is that fall in productivity 2.07 g ^{/ L -h} yield 83% mannitol mannitol for fructose.
[0041]
[Table 12]

[0042]
【The invention's effect】
The effect of the present invention is to use a new strain Candida magnoliae isolated from natural sludge, using glucose 50-100 g / L, fructose 50-120 g / L, yeast extract 5-20 g / L, diphosphate, By culturing in a medium containing 1.0 to 5.0 g / L of potassium and 0.1 to 0.5 g / L of magnesium sulfate, mannitol can be produced with high yield and high productivity. It can selectively, economically and effectively ferment produce mannitol as compared with conventional chemical synthesis and fermentation methods, and can occupy a comparative advantage in product quality and international competitiveness.
[0043]
In the above, the present invention has been described in detail by examples, but the present invention is not limited by the examples, without departing from the spirit and spirit of the present invention as long as the person has ordinary knowledge in the technical field to which the present invention belongs. The invention could be modified or changed.

Claims (4)

Strain Candida magnolia  ( Deposit number KCCM- No. 10252 ) A method for producing mannitol with high yield and high productivity using
  i) The following fermentation conditions
a) The medium composition for high yield and high production of mannitol is glucose 50-100 g / L , Fructose 50-120 g / L , Yeast extract 5-20 g / L , Ammonium sulfate 1-5 g / L , Potassium diphosphate 1.0-5.0 g / L And magnesium sulfate 0.1 to 0.5 g / L Including
b) pH Is 4.5-5.5,
c) the culture temperature is 27-35 ° C;
d) Aeration rate is 0 to 0.5 vvm And
e) The stirring speed during fermentation culture is 200 to 500. rpm Is
Fermenting a glucose or fructose medium with the strain by adjusting
  ii) removing cells and other debris from the fermentation medium;
  iii) separating and recovering mannitol from the fermentation medium of step ii);
Including, methods. The seed culture of the new strain Candida magnolia was performed in a YPD medium containing glucose 15 to 25 g / L, yeast extract 7 to 13 g / L and peptone 15 to 25 g / L, and the cell concentration was 3 to 5 g / L. The method for producing mannitol according to claim 1, wherein the culture is carried out with shaking until the cells are multiplied. After diluting a sample of natural sludge, glucose 10-20 g / L, fructose 18-22 g / L, peptone 15-25 g / L, yeast extract 7-15 g / L, agar (agar) 12-17 g / L Using the resulting plate, single colonies are selected, centering on strains that grow rapidly at 27-33 ° C., and these selected strains are screened for 30-70 g / L glucose, 50-100 g / L fructose, yeast extract 5 to 20 g / L, ammonium sulfate 1 to 5 g / L, potassium diphosphate 1 to 5 g / L, and fermenting for 68 to 76 hours in a medium composed of: A method for isolating a novel strain Candida magnolia (deposit number KCCM-10252). A new mannitol-producing strain Candida magnolia isolated by the method of claim 3 (Deposit No. KCCM-10252).