JP2008245607A - Yeast and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel yeast containing cerebroside, a method for producing cerebroside from the yeast and food containing cerebroside. <P>SOLUTION: The yeast containing cerebroside and belonging to the genus Pseudozyma, particularly the yeast containing cerebroside in ≥1 mg/g-dried cell weight, the yeast belonging to the genus Pseudozyma aphidis, particularly Pseudozyma aphidis Y178 strain (FERM P-21005), and a method for producing cerebroside by culturing the yeast, separating the same and purifying the cultured product are provided. Furthermore, food containing cerebroside obtained from the yeast or by the method is provided. In addition to nutrient supply with the yeast, cerebroside having various useful physiological activities can simultaneously be imparted to the food. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel yeast containing cerebroside, a method for producing cerebroside from the yeast, and a food containing cerebroside.

  In recent years, ceramide, which is a sphingolipid having various physiological activities, has attracted attention. The sphingolipid is a general term for lipids having sphingosine or a similar long-chain amino alcohol, and includes sphingophospholipid and glycosphingolipid. Sphingophospholipids include sphingomyelin, and glycosphingolipids include cerebroside and ganglioside. Ceramide is a general term for a part in which a fatty acid is amide-bonded to sphingosine. Sphingolipids are widely distributed in other organs and tissues including the brain and nerve tissues of animals, and in the epidermis, they are deeply involved in the keratin moisturizing function as the main component of intercellular lipids between horny cells (Non-patent Document 1). ). Ceramide is said to be effective in preventing skin aging or preventing carcinogenesis, and its use is increasing not only as a pharmaceutical and cosmetic material but also as a food material such as general foods and beverages. Cerebroside, which is one of ceramides, is a sphingolipid in which a sphingoid base in which a monosaccharide or oligosaccharide is ether-bonded to the hydroxyl group at the 1-position of sphingosine and a fatty acid. As the effect of cerebroside, for example, antiemetics (Patent Document 1), antiallergic agents (Patent Document 2) and the like have already been reported.

So far, natural cerebroside has been produced mainly from bovine brain, but recently, due to its safety problem, production methods using cereals, seafood and the like as raw materials have been studied. For example, cerebroside extracted from a plant body has been commercialized, but the cerebroside is very expensive because the plant body contains only a very small amount of cerebroside. Thus, since these contain only a very small amount of cerebroside, much labor and cost are required to extract, separate and purify cerebroside. In addition, microorganisms have recently attracted attention as raw materials for efficiently obtaining cerebroside. In particular, it is considered to use yeast from the viewpoint of safety. In recent years, yeast that accumulates a high content of cerebroside, such as Zygosaccharomyces fermentati or Kluyveromyces lactis (Patent Document 3) that accumulates 1.3 mg of cerebroside per 1 g of dried cells, or sphingo. A mutant of Pichia ciferrii that produces an id base (Patent Document 4) has been reported. In addition, as a method for producing cerebroside, a method for fermenting cerebroside characterized by culturing Kluyveromyces lactis in a medium containing whey or a derivative thereof (Patent Document 5), and Kluyveromyces marxianas whey. Alternatively, a method for fermentative production of cerebroside characterized by culturing in a medium containing a derivative thereof (Patent Document 6), and without nitrogen source and / or carbon source during and / or after culturing cerebroside-producing yeast or filamentous fungus A method of culturing in a stress environment such as in the presence of a medium, sodium chloride and / or alcohol (Patent Document 7), a method of extracting ceramide by drying fungi such as yeast, mold, mushroom and the like by a specific drying method (Patent Document 8) )It has been known.

Japanese Patent Laid-Open No. 6271598 JP 2003-155231 A JP 2004-89047 A Special Table 2002-519070 JP 2006-55070 A JP 2006-55071 A JP 2005-185126 A JP 2006-67842 A Edited by Masato Suzuki, “Cosmedic”, CMC, December 1988, Volume 2, p. 4 (193)

  An object of the present invention is to provide a novel yeast containing cerebroside, a method for producing cerebroside from the yeast, and a food containing the yeast and / or the cerebroside.

In light of the above situation, the present inventors have sought for a novel microorganism containing cerebroside and, as a result of intensive search, found for the first time a yeast belonging to the genus Pseudozyma isolated from seafood that contains a large amount of cerebroside, and completed the present invention. It came to do. Accordingly, the present invention is a yeast belonging to Pseudozyma (Pseudozyma) genus containing cerebrosides, more particularly said yeast containing cerebrosides cell dry weight per 1 mg / g or more. The present invention also relates to the yeast belonging to Pseudozyma aphidis , and more particularly to the yeast which is Pseudozyma aphidis Y178 strain (FERM P-21005). The present invention also relates to a method for producing cerebroside, characterized in that the yeast is cultured, separated and purified. Moreover, it is related with the foodstuff containing the cerebroside obtained by this yeast and / or this method.

  According to the present invention, a novel yeast containing cerebroside is provided. Moreover, the manufacturing method of the cerebroside using this yeast, the cerebroside obtained by it, and the foodstuff containing the obtained cerebroside are provided. Thereby, in addition to nutritional supplementation with yeast, cerebroside having various beneficial physiological activities can be simultaneously imparted.

The present invention is a yeast belonging to Pseudozyma (Pseudozyma) genus containing cerebrosides relates yeast containing particular cerebrosides cell dry weight per 1 mg / g or more among them. The present invention yeast is a yeast belonging to Pseudozyma (Pseudozyma) genus containing cerebrosides, cerebrosides dry cell weight per 1 mg / g or more, but preferably is not particularly limited as long as yeast containing more than 1.5 mg / g, Preferably, yeast belonging to Pseudozyma aphidis ( Pseudozyma aphidis ), particularly preferably Pseudozyma aphidis Y178 strain (FERM P-21005). The yeast of the present invention can be separated from the fish intestinal tract according to a general method for separating yeast. For example, the intestinal tract of Kuma Sasa Hanamuro, a fish of the sea bass scorpion family, is suspended in sterilized seawater, and the suspension is smeared on a medium and cultured. At this time, the medium to be used is not particularly limited as long as it is a solid medium. However, a medium used for separating bacteria from fish, seawater or the like may be used, and a potato dextrose agar medium is particularly preferably used. The formed colonies are picked and suspended in a liquid medium and cultured. This culture solution is further inoculated into another liquid medium and cultured with shaking. At this time, the medium to be used is not particularly limited as long as it is a liquid medium, but a medium used for separating bacteria from fish, seawater or the like may be used, and a potato dextrose liquid medium is particularly preferably used. Moreover, the conditions for the above-mentioned culturing may be the same as the conditions for separating bacteria from fish, seawater, etc., but the culture temperature is preferably around 30 ° C., for example. Bacteria are separated from this medium, extracted with an organic solvent or the like, and a strain is selected using sphingosine production as an index. At this time, the organic solvent to be used is not particularly limited. For example, alcohols such as methanol, ethanol, propanol, isopropanol, and butanol, methyl acetate, ethyl acetate, acetone, chloroform, toluene, etc. are used alone or in combination of two or more. Can be used. By comparing the bacteriological properties and the rDNA homology of the strains thus selected, a strain belonging to the genus Pseudozyma, which is the yeast of the present invention, can be obtained. This strain exhibits the following mycological properties. The strain of the present invention was deposited as Pseudozyma aphidis Y178 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and deposited on August 25, 2006 as deposit number FERM P-21005.

Carbon source utilization
D-glucose +
D-galactose +
L-Sorbose S
D-ribose +
D-xylose +
L-arabinose S
L-rhamnose +
Sucrose +
Maltose +
α, α-trehalose +
α-Methyl-D-glucose +
D-cellobiose +
Salicin S
Melibiose +
D-Raffinose +
Merezitose +
Soluble starch S
Glycerol S
Mesoerythritol +
Ribitol L
D-sorbitol +
D-mannitol +
D-galactitol −
Myo-inositol +
D-Gluconic acid S
Glucuronic acid +
DL-lactic acid +
Citric acid S
Ethanol W /-

Sugar fermentability
D-glucose −

Vitamin requirement <br/> No vitamin −

Nitrogen source assimilation <br/> Potassium nitrate +
Ethylamine +
L-lysine +
Cadaverine +

Resistance test
37 ℃ + / W
40 ° C −
10% NaCl −
(+; Positive,-; negative, W; weak positive, S; gradually positive, L; positive rapidly after time)

The present invention also relates to a method for producing cerebroside, which comprises culturing, separating and purifying the yeast of the present invention. The method for selecting and culturing the yeast of the present invention is as described above. The present invention also relates to a method for producing cerebroside, characterized by further culturing the yeast of the present invention thus cultured and selected, and separating and purifying cerebroside from the cells. As a method for extracting cerebroside from the bacterial cells, extraction may be performed according to a conventional method. For example, the bacterial cells are added to an appropriate organic solvent and stirred for several hours, and this solution is filtered. This may be repeated several times, and the filtrate obtained may be concentrated and then distributed by an appropriate distribution method. At this time, the organic solvent described above is used. Further, the concentration method is not particularly limited as long as it is a conventional method, and examples thereof include concentration under reduced pressure. The distribution method is not particularly limited as long as it is a commonly used method, and examples thereof include high performance liquid chromatography, affinity chromatography, gel filtration, and silica gel chromatography. In this way, the desired cerebroside can be extracted.
The present invention also relates to a food containing the yeast of the present invention or cerebroside obtained by the method. By adding, mixing, and applying the yeast of the present invention as it is or as a food material to food, it is possible to simultaneously provide cerebroside having various beneficial physiological activities in addition to supplementing with yeast. Similarly, the extracted cerebroside can be used as a food material, and various physiological activities useful for food can be imparted. In addition to food, it can also be used for pharmaceuticals, cosmetics, feeds, and the like.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

Selection of Cerebroside Production Strain The intestinal tract of Kumasa Sanamuro, a fish of the perch family, was collected and suspended in sterile seawater. The suspension was diluted and smeared on potato dextrose agar medium (0.4% potato starch, 2% dextrose, 1.5% agar, 2% NaCl) supplemented with sodium chloride, and cultured at 30 ° C. The formed colony was scraped off by 1 platinum ear, suspended in 2 ml of potato dextrose medium (0.4% potato starch, 2% dextrose, 2% NaCl) and pre-cultured at 30 ° C. for 2 days. Further, the cells were inoculated into a 500 ml baffled flask containing 100 ml of potato dextrose liquid medium, and cultured with shaking at 25 ° C. to 30 ° C. for 5 to 7 days (rotation speed: 140 rpm). In this way, 155 fungi were obtained. As positive controls, yeasts Pichia farinosa NBRC1163, Candida tenuis NBRC10315, and Kluyveromayces aestuarii NBRC10597 obtained from the National Institute for Product Evaluation Technology were also cultured in the same manner. Methanol was added to 100 ml of the culture and extracted with ultrasound for 30 minutes, and further 200 ml of chloroform was added and extracted with ultrasound for another 30 minutes. The above extract was placed in a 500 ml separatory funnel and divided into two layers. After leaving still overnight, the lower layer was separated and concentrated on a rotary evaporator to obtain an extract of several tens of mg. Weigh 5 to 20 mg of this extract into a sealed test tube, add 1 ml of aqueous-hydrochloric acid methanol reagent (add 8.6 ml of concentrated hydrochloric acid to 9.4 ml of water and dilute to 100 ml with methanol) at 70 ° C. overnight. Hydrolysis was performed. After cooling, the volume was adjusted to a 5 ml volumetric flask. Of these, 500 μl was taken into a test tube and 500 μl of 0.5N sodium hydroxide was added to adjust the pH to 10. 2.5 ml of ethyl acetate was added and shaken vigorously and centrifuged at 3000 rpm for 5 minutes. The lower layer was removed, 1 ml of water was added, the ethyl acetate layer was washed, and then centrifuged at 3000 rpm for 5 minutes. This washing operation was repeated twice. After adding 1 ml of 0.01M sodium acetate buffer (pH 3.65, washed with ethyl acetate) and 0.1 ml of methyl orange reagent (0.5% solution, washed with chloroform) to the ethyl acetate layer after washing, shake for 1 minute. Centrifuged at 3000 rpm for 5 minutes. Thereafter, the absorbance of the ethyl acetate layer at 412 nm was measured. The sample blank was treated in the same manner and a methyl orange reagent-free sample was used. Further, 5 to 25 μg of sphingosine was dissolved in 2.5 ml of ethyl acetate, and then a sodium acetate buffer solution and an acetate buffer were added in the same manner to cause color development, and a calibration curve was prepared from the absorbance values. The sphingosine content contained in the extract and the sphingosine production amount per 1 L culture were calculated from the calibration curve. The results are shown in Table 1.

As a result, 7 strains were found that had sphingosine production equal to or higher than those of yeasts Pichia farinosa NBRC1163, Candida tenuis NBRC10315, and Kluyveromayces aestuarii NBRC10597 that have been reported as microorganisms containing cerebroside.

Identification of Cerebroside Production Strain Nucleic acids were extracted from each of the seven strains obtained in Example 1, and the nucleotide sequence of 28srDNA was examined and homologous with the database. Among the 7 strains obtained, No. 178 strain for which no pathogenicity report was found was identified. Table 2 shows the biochemical properties of this strain.

As a result, from the above properties and the results of 28SrDNA and ITS-5.8SrDNA, this strain was identified as Pseudozyma aphidis and confirmed to produce cerebroside for the first time as a strain of the genus Pseudozyma . This strain was named Pseudozyma aphidis Y178, deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, and deposited on August 25, 2006 as deposit number FERM P-21005.

Pseudozyma spp cerebrosides content present invention yeast and a congener Pseudozyma yeasts 6 strains of yeast (Pseudozyma tsukubaensis NBRC1940, Pseudozyma aphidis NBRC10182 , Pseudozyma fusiformata NBRC10225, Pseudozyma antarctica NBRC10260, Pseudozyma flocculosa NBRC10875, and Pseudozyma rugulosa NBRC10877; National Institute product evaluation In the same manner as in Example 1, sphingosine was extracted after cultivation and the sphingosine content was measured. The results are shown in Table 3.

As a result, it was confirmed that all yeasts of the genus Pseudozyma contained sphingosine, and in particular, it was confirmed that the amount of sphingosine produced by Pseudozyma aphidis NBRC10182, which is the same yeast as the yeast strain Y178 of the present invention, was the highest.

Identification of cerebroside In order to identify cerebroside contained in the yeast strain Pseudozyma aphidis Y178 of the present invention, the strain was cultured in large quantities as follows, followed by extraction, separation and purification, followed by structural analysis. That is, 1 yeast colony is collected from potato dextrose agar medium and inoculated into 5 ml of liquid medium (2% peptone, 1% yeast extract, 2% glucose, 2% NaCl) in a test tube. The seed culture was performed at 30 ° C. for 2 days (preculture (1)). 5 ml of the above preculture solution (1) in which the cells were grown was transplanted to 100 ml of the above liquid medium in a 500 ml Erlenmeyer flask and cultured at 30 ° C. for 1 day (preculture (2)). The preculture liquid (2) was dispensed in 25 ml portions into 240 ml of the above liquid medium in four 1 L Erlenmeyer flasks and cultured at 30 ° C. for 2 days (preculture (3)). To 4 jar fermenters, 6 L of a liquid medium and a foam-absorbing agent adecanol were added, and 240 ml each of the grown preculture solution (3) was added, followed by shaking culture at 30 ° C. for 4 days. After culturing, the cells were collected by continuous centrifugation (15,000 rpm), and 950 g (wet weight) of cells were obtained. Of these, about 1.5 L of chloroform-methanol (2: 1, v / v) was added to 386 g of the bacterial cells, stirred for 3 hours, and filtered through filter paper (Advantech Toyo Co., Ltd .; No. 2). About 1.5 L of chloroform-methanol (2: 1, v / v) was added to the residue, extraction and filtration were performed in the same manner, and extraction operation was performed three times in total. After the filtrate was concentrated, methanol with the same volume of the remaining water and 2 volumes of chloroform were added to partition the two layers. After separation, the lower layer was concentrated to obtain 6.92 g of extract. 6.2 g of this extract was dissolved in 55 ml of chloroform-methanol (10: 1, v / v), then subjected to silica gel chromatography (diameter 6 × 24.5 cm), and eluted sequentially with a mixed solvent of chloroform-methanol. 10 fractions were drawn. When the fraction containing cerebroside on TLC was further fractionated with a silica gel column and ODS column, 135.3 mg of cerebroside was obtained. The cerebroside per dry cell weight was 1.6 mg / g. As a result of analyzing this cerebroside by one-dimensional NMR and two-dimensional NMR, at least the following two ceramides were obtained among the ceramides. One of them is sugar or hydrogen bonded to the 1-position hydroxyl group of sphingoid base 9-methyl-4,8-sphingadienine, and fatty acid or 2-hydroxy fatty acid bonded to amino group (Chemical Formula 1) It became clear that. In addition, it was confirmed that the sphingoid base was also sphingosine (Chemical Formula 2).

  According to the present invention, a novel yeast containing cerebroside is provided. Moreover, the manufacturing method of the cerebroside using this yeast, the cerebroside obtained by it, and the foodstuff containing the obtained cerebroside are provided. Thereby, in addition to nutritional supplementation with yeast, cerebroside having various beneficial physiological activities can be simultaneously imparted.

Claims (7)

Containing cerebrosides Pseudozyma (Pseudozyma) yeast belonging to the genus. The yeast of Claim 1 which contains a cerebroside 1 mg / g or more per dry cell weight. The yeast according to claim 1 or 2, which belongs to Pseudozyma aphidis . Pseudozyma Afidisu (Pseudozyma aphidis) is Y178 strain (FERM P-21005), according to claim 1 to 3 yeast according. A method for producing cerebroside, comprising culturing, separating and purifying the yeast according to claim 1. Cerebroside obtained by the method according to claim 5. A food containing the yeast according to claim 4 and / or the cerebroside according to claim 6.