JP2010041972A - Method for enriching sphingolipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably enriching a large amount of a safe sphingolipid without passing through a complicated process in malted rice that is directly used as a food composition as it is without carrying out an organic solvent extraction, concentration, etc. <P>SOLUTION: The method for enriching a sphingolipid in malted rice comprises producing malted rice in the presence of one or more kinds of inorganic salts selected from the group consisting of a potassium salt, a sodium salt, a magnesium salt, a calcium salt and an iron salt in the production of malted rice by inoculating a steamed raw material for producing malted rice with Aspergillus oryzae and carrying out culture. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for enriching sphingolipids in sputum.

  A sphingolipid is a general term for complex lipids having a sphingoid base (long-chain base) as a structural skeleton. The sphingolipid skeleton of animal cells is sphingosine, but it is mainly phytosphingosine in plants and fungi (Non-patent Document 1).

  Sphingolipids include ceramides in which an acyl group is amide-bonded to the amino group of a sphingoid base, and complex sphingolipids in which various polar groups such as sugar, phosphorus, sulfur, and amino acids are added to ceramide. Among them, glycosphingolipids to which sugar is bound are universally present in eukaryotes and part of bacteria, and it is known that the structures of sugar chain portions are various. Glucosylceramide to which glucose is bound is also called cerebroside and is a typical glycosphingolipid commonly found in plants, fungi and animals (Non-patent Document 2).

  Ceramide accounts for about 50% as the main component of intercellular lipids in the stratum corneum of human skin, and is said to be involved in skin moisture retention and flexibility. It decreases with aging, causing wrinkles, dry skin and rough skin, and is also thought to be involved in atopic dermatitis. Traditionally, bovine brain-derived sphingolipid, which is an inexpensive source, was used as a method for supplementing ceramide, but prions that cause BSE (bovine spongiform encephalopathy) onset have accumulated in the brainstem region. Since then, it has become impossible to use it for food and cosmetics. Synthetic ceramides have limited use as food materials from the viewpoint of safety.

  As an alternative supply source for cattle brain, soybean oil sesame and beer lees, which are agricultural processing by-products, such as cereal germs such as rice and wheat, and konjac koji are currently used. Since it is difficult to use as a food as it is, extraction and concentration with an organic solvent or the like is performed. For this reason, there was a problem that the production cost was high and the product was very expensive (Patent Documents 1 and 2).

  Cerebroside is also extracted from fungi such as yeast and mushrooms. Since the species in which cerebroside is present in yeast is limited (Non-patent Document 3), various cerebroside-producing bacteria such as Saccharomyces kluyveri and Kluyveromyces lactis are used in liquid culture. A manufacturing method for increasing the production amount and the accumulation amount by adding environmental stress and adding salts to the medium has been studied (Patent Documents 3 and 4).

  For example, there has been a report on the addition of salt to the medium as application of environmental stress, which increases the cerebroside content per cell weight (Patent Document 3). In addition, methods of adding ammonium sulfate to the medium as a nitrogen source and methods of adding various sodium compounds as a sodium source have been studied, both of which have been reported to increase the amount of cerebroside per cell (patent document) 4). Therefore, in yeast, it is considered that environmental stress is applied or nutrient sources are replenished by adding salts to the medium, whereby cerebroside accumulates in the cells.

  Thus, in yeast, since the amount of sphingolipid per amount of bacterial cells varies depending on the culture conditions, various high production methods have been studied. However, even in these yeasts enriched with sphingolipids, organic solvent extraction, freezing treatment, etc. are used to recover cerebroside from the cells, and the process is complicated and costly. There was a problem such as concern about the load on

  On the other hand, there are reports that koji molds that have been used in the production of traditional fermented foods in Japan also produce sphingolipids such as ceramide and cerebroside during liquid culture (Non-Patent Documents 4 and 5). However, if we examine the culture conditions in the liquid culture of Aspergillus oryzae and increase the sphingolipid content of Aspergillus oryzae, there is no experience of eating by filtering and recovering only the cells, so organic solvent extraction etc. Processing is required, and the above-described problems occur.

  On the other hand, a solid culture of Aspergillus oryzae traditionally has a dietary habit of eating the whole as “Acupuncture”, so it is very suitable for using Aspergillus sphingolipid as a food.

  However, there are no reports on the production capacity and molecular species of sphingolipids during solid culture, that is, when they are made into cocoons, and not only examination of culture conditions for high production, but also studies on using cocoons as a sphingolipid supply source. It has not been done.

  Solid culture (i.e., koji making) using koji molds has generally been performed for the purpose of enzyme production and spore growth as seed pods. As salt addition at the time of koji making, wood ash addition in seed koji production is generally known. Addition of wood ash is considered to have effects such as antiseptic action due to alkalinity of ash, pH adjustment, physical effects, and nutritional effects (Non-patent Document 6). In addition, in the production of miso and sake lees, some additions of salt at the time of koji making are being studied, and it is known that the addition of calcium carbonate, calcium sulfate, and magnesium sulfate increases the production of proteases and amylases. Yes. In addition, there is a report that the production of alkaline protease in rice bran is promoted by adding and kneading nitrogen-containing compounds such as nitrates and acidic amino acid sodium, and is further enhanced by the coexistence of potassium dihydrogen phosphate (non-patent document). 6). Regarding addition of salts, there is a report that in the production of shochu, the addition of sodium nitrate and calcium carbonate suppresses the production of fatty acids. (Non-patent document 6).

  As described above, the addition of salts to the medium at the time of koji making has been performed for the purpose of spore formation and enzyme production. In other words, in terms of increasing the sphingolipid content in the solid culture of Aspergillus, no study of the koji-making conditions (solid culture conditions) has been made.

JP-A-4-282317 JP-A-11-279586 JP-A-2005-185126 JP 2006-55070 A Chem. Phys. Lipids, 5, 6-43 (1970) CMLS, Cell. Mol. Life Sci. , 60, 919-941 (2003). FEMS Yeast Research, 2, 533-538 (2002) Japan Agricultural Chemistry, Vol. 49, No. 4, 205-212 (1975) Biochimica et Biophysica Acta, 486, 161-171 (1977) Akatsuki, published by Japan Brewing Association, edited by Hideya Murakami (1986)

  Microorganisms such as fungi are suitable for efficiently obtaining sphingolipids, but yeasts and mushrooms have the above-mentioned problems, and because of their form and flavor, both are used as foods. difficult. Accordingly, the present invention provides a method for stably and safely enriching sphingolipids in large quantities without going through complicated steps in a cocoon that can be used as it is as a food composition without performing organic solvent extraction or concentration. The purpose is to do.

  As a result of repeated studies to solve the above-mentioned problems, the present inventors are manufactured by performing ironmaking in the presence of one or more of potassium salt, sodium salt, magnesium salt, calcium salt and iron salt. The present inventors have found that the sphingolipids in the cocoons are efficiently enriched, and have completed the present invention.

That is, the present invention includes the following features.
(1) One or more selected from the group consisting of potassium salt, sodium salt, magnesium salt, calcium salt and iron salt in the koji making inoculated with koji mold on the koji making raw material A method for enriching sphingolipids in koji, characterized in that koji making is carried out in the presence of an inorganic salt.
(2) The method according to (1), wherein the ironmaking is performed in the presence of a potassium salt or a sodium salt.
(3) The method according to (1) above, wherein the raw material for koji is rice.
(4) The method according to any one of (1) to (3) above, wherein the inorganic salt is added before or after the inoculation of the koji mold after the koji raw material is steamed.
(5) The method according to (4) above, wherein the added concentration of the inorganic salt is 1 to 360 mmol in terms of ions per 1 kg of the raw material for koji making.
(6) The method according to any one of (1) to (3) above, wherein an inorganic salt is added at the time of dipping the raw material for koji before steaming.
(7) The method according to (6) above, wherein the addition concentration of the inorganic salt is 50 to 500 mmol in terms of ions per 1 kg of the raw material for koji making.
(8) The method according to any one of (1) to (7) above, wherein the sphingolipid is cerebroside.
(9) The method according to any one of (1) to (8) above, wherein the koji mold used for koji making is Aspergillus oryzae or Aspergillus sojae.
(10) The method according to any one of (1) to (9) above, wherein the moisture content of the steamed koji making raw material is 30 to 36% by weight.

  ADVANTAGE OF THE INVENTION According to this invention, the sphingolipid in the cocoon which can be eaten as it is for food can be enriched simply and cheaply.

  As used in this specification, the term “koji making” refers to a koji making method known to those skilled in the art (for example, the lid koji method, the box koji method, the floor koji method), in which koji molds are inoculated and cultured in steamed koji making materials. Law, machine dredge, etc.). These koji making methods include the steps of washing koji raw materials, immersing them in water, steaming them, inoculating and culturing koji molds, during which, in general, “retraction”, “pick”, “ It includes processes such as “middle work” and “end work”.

  “Prime” refers to an operation of making the temperature control easier by dividing the portion of the fungus that is about to grow vigorously after “flooring” (that is, inoculating seeds) into small sections. Generally, in the ironmaking, the first part is called the “floor era” and the second part is called the “shelf era”. The floor era is a process where the steamed raw material is deposited after wrapping the floor and wrapped in a cloth to maintain humidity and prevent drying. Incubate by simply turning over.

  The koji-making temperature in the floor age is generally 28 to 42 ° C., for example, 30 to 37 ° C., depending on the optimum temperature for cell growth of the koji mold used. However, when the temperature of the ironmaking in the bed age is set to a relatively high temperature of about 38 ° C. or more, the koji mold may be inhibited from growing. The iron making temperature means the product temperature of the rice cake. The koji-making temperature can be adjusted by controlling the heat of fermentation accompanying the growth of koji mold or by controlling the ambient temperature in koji-making.

  The time required for the floor age is generally 18 to 20 hours. If it is left for a long time in a state where growth is vigorous, the heat of fermentation cannot be removed, the temperature of the product cannot be controlled, and the growth of koji making is suppressed because the raw materials are dense, so this At the time of the event, it moves to the shelf era.

  On the other hand, the shelf era is a process in which the cocoons are thinly stacked and the moisture of the cocoons is evaporated, and in this culturing process, maintenance work called “middle work” and “end work” is performed. According to the said nonpatent literature 6, it is described that 75% of the total water | moisture content evaporated by tapping will evaporate by the middle work.

  For details of koji making, see, for example, Non-Patent Document 6 and the Fermentation Handbook (edited by Fermentation and Metabolism Research Association, 2001).

  In the method for enriching sphingolipids in the soot according to the present invention (hereinafter also referred to as the method of the present invention), unless otherwise specified below, koji making is related to time, humidity, temperature conditions, etc. Can be carried out in the same manner as known iron making. That is, after inoculation with koji mold, the humidity is maintained high (for example, 85% or more) in the early stage of culture (ie, bed age), and the humidity is decreased in the latter half of the culture (ie, after about 20 hours after inoculation) For example, ironmaking can be performed at 30 to 40 ° C. for 43 to 48 hours.

  Specifically, the method of the present invention is characterized in that the ironmaking is performed in the presence of one or more inorganic salts selected from the group consisting of potassium salts, sodium salts, magnesium salts, calcium salts and iron salts. And

  The raw material for koji used in the method of the present invention is not particularly limited as long as it can be made into koji by growing koji molds, and for example, cereals, pseudo-cereals, beans, sesame, potatoes and the like can be used. Specifically, examples of cereals include white rice, brown rice, black rice, red rice, barley, and wheat, examples of pseudo-cereals include buckwheat, amaranth, and quinoa, and examples of beans include soybeans, red beans, black beans, and chickpeas. However, it is not limited to these. It is preferable to use cereals, for example, gramineous plants as raw materials for straw, and it is particularly preferable to use white rice and barley such as barley as gramineous plants. In the present invention, it is most preferable to use rice as the raw material for koji.

  Particularly preferred salts in the method of the present invention are potassium salt and sodium salt. Examples of the potassium salt include potassium phosphate, potassium chloride, potassium nitrate, potassium sulfate, potassium acetate, potassium citrate and the like, and most preferred are potassium nitrate and potassium citrate. Examples of the sodium salt include sodium nitrate, sodium citrate, sodium tartrate, sodium phosphate, sodium chloride and the like, and sodium phosphate is most preferable.

  In the method of the present invention, the timing of adding the inorganic salt is not particularly limited as long as it is during the culture period. Or after the inoculation or immediately after the inoculation. More preferably, it is before the inoculation of the koji mold after steaming the koji-making raw material, or until after the inoculation or immediately after the inoculation. Here, “before the inoculation of koji molds after steaming the koji raw material” means during or after the steaming of the steamed raw material but before the addition of the seed koji. “Up to the post-inoculation” refers to the end of the preparation process after the addition of the seed meal. The term “immediately after inoculation” refers to the situation before bed culture is started after inoculation (inoculation). Moreover, “at the time of dipping the raw material for koji before steaming” means at the time of the water absorption process to the raw material cereal.

  In the method of the present invention, the addition concentration of inorganic salts can vary depending on the inorganic salts used, the raw material for making koji, the timing of addition of inorganic salts, and the like. For example, in the present invention, when rice is used as a koji-making raw material and is added before gonococcal inoculation after steaming inorganic salts, or until after the inoculation or immediately after inoculation, depending on the inorganic salts used The following addition concentrations can be obtained: potassium salt, 1 to 360 mmol in terms of potassium ion per kg of rice, preferably 4 mmol to 240 mmol, more preferably 12 mmol to 120 mmol; sodium salt, 1 to 1 in terms of sodium ion per kg of rice 200 mmol, preferably 4 mmol to 160 mmol, more preferably 12 mmol to 120 mmol; magnesium salt, 1 to 100 mmol in terms of magnesium ion per kg of rice, preferably 4 mmol to 40 mmol; calcium salt, 1 to 1 in terms of calcium ion per kg of rice 00mmol, preferably 1mmol~20mmol; iron salts, iron ions in terms of per US 1kg 0.01~1mmol, preferably 0.03mmol~0.3mmol.

  Moreover, in this invention, when using rice as a koji raw material and adding inorganic salt at the time of immersion of the koji raw material before steaming, it can be set as the following addition density | concentrations according to the inorganic salt to be used: Potassium salt, 50 to 500 mmol, preferably 80 to 250 mmol in terms of potassium ion per kg of rice.

  It should be noted that the above concentration range is an exemplification, and those skilled in the art can appropriately select an appropriate concentration depending on the inorganic salt used, the raw material for making koji, the timing of adding the inorganic salt, and the like. .

  Aspergillus oryzae that can be used in the method of the present invention is not particularly limited as long as it can produce sphingolipids, and examples thereof include Aspergillus oryzae and its closely related Aspergillus sojae. be able to. A. Specific examples of oryzae that can be used include commercially available seed potatoes and specific strains designated by registration numbers RIB40, NBRC4214, JCM2228, or ATCC36261. A. As the sae, a commercially available seed meal or a specific strain designated by the registration number JCM2226, NBRC4239 or NBRC5241 can be used. A. NBRC4214, JCM2228 as Orize, and A. JCM2226 is particularly suitable for use in the present invention because of its high cerebroside production in both white rice and barley straw. In addition, as a koji mold other than the koji mold, A. Orize registered the registration numbers NBRC4255, ATCC11494, ATCC16868, NBRC5786, JCM2245, NBRC5240, etc. in the case of rice bran. Orize can use registration numbers JCM2227, JCM2173, JCM2229, NBRC4261 and the like.

  In the method of the present invention, the moisture content of the steamed koji making raw material is preferably 30 to 36% by weight. If the moisture content of the raw material for koji making after steaming is less than 30% by weight, germination and cell growth may be suppressed, or if it exceeds 36% by weight, germination will be accelerated but cell growth will be slow. It is because there is a possibility of becoming.

  In the method of the present invention, it is preferable to perform the shelf age under high humidity conditions (for example, 85% or more). Thereby, the transpiration of cocoon moisture in the shelf era can be suppressed, and as a result, sphingolipid can be efficiently enriched.

  In the method of the present invention, it is preferable to carry out the shelf age until the iron making time reaches 46 hours or more. Thereby, the sphingolipid in the produced koji can be enriched with time.

  The koji produced as described above is rich in sphingolipids (especially cerebroside). Specifically, the method of the present invention is different from rice bran produced by koji making without adding inorganic salts or commercially available koji koji produced by conventional koji making. In response, the total amount of sphingolipids in rice bran could be enriched up to about 2.2 times and 10.6 times, respectively (Example 5, FIG. 6). The amount of sphingolipid in the cocoon can be measured by using thin layer chromatography, high performance liquid chromatography, or the like.

  As described above, the method of the present invention does not require a step of applying stress under complicated conditions and can be used as it is without being extracted or concentrated using an organic solvent, and has high safety. Sphingolipids can be efficiently enriched.

  The sputum containing sphingolipids enriched by the method of the present invention may be used as a part of a raw material for food and drink, or may be added or blended after the production process or production of the food or drink, or used as it is as a food or drink. May be. Such foods and drinks can also be provided as health foods, functional foods, foods for specified health use, dietary supplements, and the like.

  The form of the food or drink is not particularly limited, and for example, but not limited to, yogurt, drink yogurt, juice, milk, soy milk, alcohol (alcoholic beverage), coffee, tea, sencha, oolong tea, Various beverages such as sports drinks, baked confectionery such as cookies, bread, cakes, rice crackers, Japanese confectionery such as sheep candy, frozen confectionery such as pudding, jelly and ice cream, confectionery such as chewing gum and candy, crackers, chips, etc. Noodles such as snacks, noodles, soba noodles, fish paste products such as kamaboko, ham, fish sausage, seasonings such as miso, soy sauce, dressing, mayonnaise, sweeteners, tofu, konnyaku, other boiled fish, salad, soup Examples of various side dishes such as

  Moreover, the intake of sphingolipid-enriched koji as an active ingredient in foods and drinks is usually 0.7 to 1.4 g / day, preferably about 1.1 to 2.2 g / day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  A. Oryze is known to produce sphingolipids such as glucosylceramide and ceramide during liquid culture. In addition, there is a report that A. niger also produces galactosylceramide (Non-patent Document 2). In other words, koji molds are thought to produce different molecular species depending on the species. Currently, it is unclear what kind of complex sphingolipids other than the above are produced by various koji molds. The total amount of sphingolipid was evaluated by selecting oryzae and measuring the amount of glucosylceramide, that is, cerebroside, which has many analysis examples among sphingolipids.

[Example 1] Purification and structural analysis of a rice bran cerebroside standard product A method for preparing a rice bran cerebroside standard product was shown below. A. 5.4 kg of rice bran kneaded using A. oryzae was pulverized, extracted twice with 5 times the amount of chloroform-methanol (2: 1), and the extract obtained by filtration was concentrated. After drying, the mixture was shaken with a 0.2 M KOH-methanol solution at 37 ° C. for 2 hours to decompose glycerolipids to obtain alkali stable lipids. After neutralization, the mixture was concentrated and dried, and redissolved in chloroform-methanol-0.8% NaCl (8: 4: 3). After shaking and mixing, the lower layer was concentrated after centrifugation.

  This was subjected to silicic acid column chromatography, and free fatty acids and the like were removed using chloroform-methanol. After being subjected to silicic acid column chromatography again, 407.9 mg of a white solid oily substance was obtained as a cerebroside fraction. Thereafter, in order to remove slightly mixed free fatty acids, purification was further performed using an HPLC-differential refraction detector (manufactured by Shimadzu Corporation). The column was YMC Pack ODS-A; 20 × 250 mm, the column oven was 40 ° C., and the mobile phase was methanol for isocratic elution to a flow rate of 6 ml / min. The obtained purified product was subjected to thin layer chromatography to become a single spot, and as shown below, the main molecule was identified as glucosylceramide, so that cerebroside was quantified using this as a standard product.

  Structural analysis of major molecular species was performed. That is, using HPLC-differential refraction detector (manufactured by Shimadzu Corporation), main molecules contained in purified cerebroside were collected under the above-mentioned conditions. Structural analysis was performed on the obtained three main molecules (retention times 28.7 minutes, 32.1 minutes, 37.4 minutes).

Molecules with a retention time of 28.7 minutes were dissolved in methanol, and HRESI-MS (+) was measured. As a result, (M + Na) ⁺ ion was observed at m / z 776.557456, and the molecular formula was determined as C ₄₃ H ₇₉ NO ₉ (C ₄₃ H ₇₉ NO ₉ Na cald 776.556525 (Δ + 4.57 mmu)).

Next, this molecule was dissolved in heavy pyridine (C ₆ D ₅ N) and subjected to nuclear magnetic resonance (NMR) analysis. This molecule was identified as cerebroside from the characteristics of the ¹ H and ¹³ C NMR spectra. First, from the ¹ H, ¹³ C chemical shift value and J _HH value of the sugar moiety, the constituent sugar is glucose, which is bound to a long-chain base via a β-bond (J _{1 ″, 2 ″} = 7.8 Hz). (= Β-glucosylceramide) was found. Next, ¹ H- ¹ H COSY, HMQC, and HMBC spectra were measured, and the structures of fatty acids and long-chain base moieties were analyzed.

H-2 '(δ5.10) -H -3' at ¹ H- ¹ H COZY spectra for fatty (δ6.10, J3 ', 4' = 15.7Hz) -H-4 '(δ6.16) That is, it was clarified that it is a straight-chain 2-hydroxy, 3-trans fatty acid, since a terminal spin is observed as a triplet at δ0.85 by ¹ H NMR. Next, in order to clarify the carbon number of the fatty acid, this molecule was decomposed (methanoly) by refluxing at 100 ° C. for 18 hours with methanolic 0.9N HCl to obtain a fatty acid methyl ester. After the fatty acid methyl ester was purified by hexane extraction, it was derivatized with trimethylsilyl (TMS) and subjected to GC / MS analysis. As a result, a peak of (M-15) + was observed at m / z 369, and the number of fatty acid carbons constituting this molecule was determined to be C18.

In ¹ H- ¹ H COZY spectra for the long chain base H-1 (δ 4.23, δ4.69 ) -H-2 (δ4.79) -H-3 (δ 4.74) -H-4 ( δ 5.99, J _4,5 = 15.8 Hz) -H-5 (δ 5.92) -H-6 (δ 2.14) -H-7 (δ 2.14) -H-8 (δ 5.24) And a ¹ H- ¹³ C remote spin coupling from 9-CH ₃ (δ1.60) to C-8 (δ123.3) and C-9 (δ13.5) in the HMBC spectrum. From the observation, it was clarified that they have 9-methyl-4trans and 8-trans-sphingadienin structures. Moreover, since the terminal methyl was observed as a triplet at δ0.85 by ¹ H NMR, it was also clear that the alkyl chain portion was a straight chain. Since the carbon number of the fatty acid is already known at this point, the carbon number constituting the long-chain base was determined to be C18 obtained by subtracting the carbon number of sugar (C6) and fatty acid (C18) from the molecular formula. As a result, the structure of this molecule was uniquely determined to be 18h: 1-9Me d18: 24t ^{, 8t-} Glc as shown in FIG. Moreover, this structure was a characteristic structure for fungi (Non-Patent Document 2).

Molecules with a retention time of 32.1 minutes were dissolved in methanol and HRESI-MS (+) was measured. As a result, (M + Na) ⁺ ion was observed at m / z 778.58403, and the molecular formula was determined as C ₄₃ H ₈₁ NO ₉ (C ₄₃ H ₈₁ NO ₉ Na cald 778.558090 (Δ + 3.13 mmu)).

Next, this molecule was dissolved in heavy pyridine (C ₆ D ₅ N) and subjected to nuclear magnetic resonance (NMR) analysis. ¹ H and ¹³ C NMR spectra were very similar to 18h: 1-9Me d18: 24t ^{, 8t-} Glc (retention time 28.7 minutes). Since the ¹ H, ¹³ C chemical shift value and J _HH value of the sugar moiety are almost the same, the constituent sugar is glucose, which is a β-bond (J _{1 ″, 2 ″} = 7.8 Hz) and a long chain base (= Β-glucosylceramide) was found to be bound. Next, ¹ H- ¹ H COSY, HMQC, and HMBC spectra were measured, and the structures of fatty acids and long-chain base moieties were analyzed.

¹ H- ¹ H COSY H-2 in the spectrum '(δ4.56) -H-3' (δ2.12) that vicinal spin - binding was observed that for fatty acid, also has terminal methyl at ¹ H NMR [delta] 0. It was clarified as a triplet at 85 that it was a straight-chain 2-hydroxy fatty acid. Next, in order to clarify the carbon number of the fatty acid, methanolysis was performed to obtain a fatty acid methyl ester. After the fatty acid methyl ester was purified by hexane extraction, it was derivatized with trimethylsilyl (TMS) and subjected to GC / MS analysis. As a result, a peak of (M-15) ⁺ was observed at m / z 371, and the number of fatty acid carbon atoms constituting this molecule was determined to be C18 (= 2-hydroxystearic acid).

18h in ¹ H- ¹ H COZY spectrum and HMBC spectra for the long chain ^base: 1-9Me ^{d18: 2 4t,} since the observed exactly the same correlation with 8t -Glc, long chain base of the molecule also 9-methyl - It was clarified that it has a 4-trans, 8-trans-sphingadienin structure. Further, since the terminal methyl was observed as a triplet at δ0.85 by ¹ H NMR, it was also clear that the alkyl chain portion was also a straight chain. Since the number of carbon atoms of the fatty acid has already been clarified, it was determined that the number of carbon atoms constituting the long-chain base was C18 obtained by subtracting the carbon number of sugar (C6) and fatty acid (C18) from the molecular formula. As a result, the structure of this molecule was uniquely determined to be 18h: 0-9Med18: 24t ^{, 8t-} Glc as shown in FIG. Moreover, this structure was a characteristic structure for fungi (Non-Patent Document 2).

Molecules with a retention time of 37.4 minutes were dissolved in methanol and HRESI-MS (+) was measured. As a result, (M + Na) ⁺ ion was observed at m / z 792.60158, and the molecular formula of F5 was determined to be C ₄₄ H ₈₃ NO ₉ (C ₄₄ H ₈₃ NO ₉ Na cald 792.59655 (Δ + 0.94 mmu)).

Next, this molecule was dissolved in heavy pyridine (C ₆ D ₅ N) and subjected to nuclear magnetic resonance (NMR analysis). ¹ H and ¹³ C NMR spectra were very similar to 18h: 0-9Me d18: 24t ^{, 8t-} Glc (retention time 32.1 minutes). Since the ¹ H, ¹³ C chemical shift value and J _HH value of the sugar moiety are also the same as 18h: 0-9Me d18: 24t ^{, 8t-} Glc, the constituent sugar is also glucose, which is β-linked (J _{1 ″, 2 ″} = 7.8 Hz) was found to be bound to a long-chain base (= β-glucosylceramide). Next, ¹ H- ¹ H COSY, HMQC, and HMBC spectra were measured, and the structures of fatty acids and long-chain base moieties were analyzed.

18h in ¹ H- ¹ H COZY spectra for ^{fatty: 0-9Me d18: 2 4t, vicinal} spin coupling of 8t -Glc as well as H-2 '(δ4.56) -H -3' (δ2.12) Was observed, and terminal methyl was observed as a triplet at δ0.85 by ¹ H NMR, which revealed that it was a straight-chain 2-hydroxy fatty acid. Next, in order to clarify the carbon number of the fatty acid, methanolysis was performed to obtain a fatty acid methyl ester. After purification, it was derivatized with trimethylsilyl (TMS) and subjected to GC / MS analysis. As a result, a peak of (M-15) ⁺ was observed at m / z 399, and the number of fatty acid carbon atoms constituting F5 was determined to be C20 (= 2-hydroxyarachidic acid).

For long chain bases ¹ H- ¹ H COZY spectrum H-1 (δ4.23, δ4.69) -H-2 (δ4.79) -H-3 (δ4.74) -H-4 (δ5. 99, J _4,5 = 15.8 Hz) -H-5 (δ5.92) -H-6 (δ2.16) -H-7 (δ2.16) -H-8 (δ5.46) -H— 9 (δ5.46) is observed, and the 9-CH3 signal observed at 18h: 0-9Me d18: 2 ^{4t, 8t-} Glc is not observed. -It was found to have a sphingadienin structure. Moreover, since the terminal methyl was observed as a triplet at δ0.85 by ¹ H NMR, it was also clear that the alkyl chain portion was a straight chain. Since the number of carbon atoms in the fatty acid has already been clarified, the number of carbon atoms constituting the long-chain base is determined to be C18 obtained by subtracting the carbon number of the sugar (C6) and the fatty acid (C20) from the molecular formula. 1 was uniquely determined to be 20h: 0 d18: 24t ^{, 8t-} Glc as shown in FIG. Moreover, this structure is a characteristic structure for fungi and plants (Non-patent Document 2), and it is considered that cerebroside derived from rice is also included.

Subsequently, the purified product of cerebroside was analyzed by LC / MS (LCMS-2010EV Shimadzu Corporation), and the amount ratio of the main three molecules was examined. The ionization mode is ESI (−), the column is Cadenza CD-C18 150 × 2 mm, the mobile phase is THF / methanol (1/9 v / v), and the flow rate is 0.2 ml / ml. did. As a result, it was suggested that the amount ratio of each molecular species is as follows. From this, it was shown that 18h: 0-9Me d18: 24t ^{, 8t-} Glc, a molecule characteristic of fungi, occupies most of cerebroside in rice bran.

[Example 2] Method for measuring cerebroside content in rice bran and barley koji Quantitative determination of cerebroside in Examples 3 to 6 was carried out by extracting cerebroside from each sample with an organic solvent and then using cerebroside obtained in Example 1 as a standard product. And was performed by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). It was confirmed that the quantitative values were almost the same in both TLC and HPLC (FIG. 2).

  In addition, extraction of cerebroside from koji was performed by single or repeated extraction as shown below. A single extraction was performed in order to compare cerebroside content in each experimental group by a simpler method, and repeated extraction was performed for the purpose of quantifying the cerebroside content actually contained in the straw. An example of comparison data for quantitative values obtained by each extraction method is shown in FIG. As a result of the examination, the extraction efficiency of single extraction varies depending on the cerebroside content in the koji, and the cerebroside content in the rice koji obtained by repeated extraction is about 1.5 to 2 times the quantitative value obtained by the single extraction. It was shown that it corresponds to the degree.

  A method for measuring the amount of cerebroside by single extraction is shown below. After freeze-drying the koji, 3 ml of a chloroform-methanol solution (2: 1) was added to 1 g of the pulverized product, and sonicated at 40 ° C. for 30 minutes. 250 μl of 0.8% NaCl solution was added to 1 ml of the supernatant obtained by centrifuging, and the mixture was shaken and mixed. The lower layer obtained by centrifuging this was recovered, and a certain amount was used for measurement.

  The method for measuring the total cerebroside content by repeated extraction is shown below. After the rice bran was freeze-dried, 3 ml of a chloroform-methanol solution (2: 1) was added to 1 g of the pulverized product, sonicated at 40 ° C. for 30 minutes, and a supernatant was obtained after centrifugation. After a total of three extractions, a quarter amount of 0.8% NaCl solution was added to the total amount of the supernatant and mixed by shaking. The lower layer obtained by centrifuging this was recovered. Chloroform was added to the supernatant, and extraction was further performed twice. The total amount of the lower layer was measured according to a certain amount, and the total amount of cerebroside contained per 1 g of dried koji was calculated.

  A method for quantifying cerebroside by TLC-densitometry is shown below. The extracted sample was subjected to silicic acid thin layer chromatography (Merck, trade name: HPTLC silica-60 F254). After developing with chloroform-methanol (95:15), spray with orcinol sulfate reagent (orcinol: 0.2% w / v, sulfuric acid: 11.4% v / v) and spot of reddish purple cerebroside that appears after heating Was quantified using densitometry.

  The cerebroside quantification method by HPLC-corona CAD detector was shown below. The quantitative method is based on the HPLC-evaporative light scattering detector method (J. Oleo Sci., Vol. 53, No. 3, p. 127-133, 2004). I went there. That is, the mobile phase was gradient elution using A liquid: chloroform, B liquid: 95% methanol (Table 2). The column used was a silica column: Inertsil SIL-100A (4.6 × 150 mm, 5 μm), a column oven: 40 ° C., and a flow rate: 1 ml / min. As the detector, a corona CAD detector, which is considered to have better sensitivity, quantitative range, and reproducibility than the evaporative light scattering detector, was used. The corona CAD detector was gas: nitrogen gas, gas pressure during operation: 35 psi.

[Example 3] Production amount of cerebroside of various koji molds 0.15% of various koji molds (A. oryzae, A. sojae) obtained from 10 kinds of commercially available koji molds and strain storage organizations for steamed rice and steamed barley The inoculation was carried out at a rate of 35 ° C. and a relative humidity of 80% or more, and koji for 46 hours. The obtained koji was freeze-dried, and after crushing, the amount of cerebroside in the dried koji was measured by single extraction and TLC in Example 1.

  FIG. 3 shows the amount of cerebroside produced by various koji molds (A. oryzae, A. sojae) contained in barley koji and rice koji. Whether the raw material is rice or barley, the name of the strain and the identification number of the source strain storage organization are described for the strains with high cerebroside content. A. In Oryzae, NBRC 4214, JCM 2228, ATCC 36261 are As for sojae, JCM2226 was shown to have particularly high cerebroside production in any medium of rice and barley. Moreover, when the raw material was barley in all the strains, the production amount was higher (about 1.1 to 4 times) than that of rice.

[Example 4] Examination of various salt additions in rice bran The change of cerebroside content when various types of salts were added and koji making was evaluated. Steamed rice having a water content of 30% was prepared using Koshihikari (water content 12.5%) as raw rice. Various salt solutions (potassium phosphate, potassium chloride, potassium nitrate, dipotassium sulfate, potassium acetate, tripotassium citrate, sodium phosphate, magnesium chloride, calcium chloride, iron sulfide, sulfide) at a rate of 40 ml per 1 kg of this steamed rice Zinc, manganese sulfide) solution was added and mixed to be uniform. Potassium phosphate was prepared using monopotassium phosphate and dipotassium phosphate so as to have a pH of 5.8. The obtained steamed rice is inoculated with 0.1% of a powder type koji mold (A. oryzae: NBRC4214), the product temperature is 35 ° C., the relative humidity is 80% or more, and the product temperature is 40 ° C. after 32 hours. Smelting time. The amount of cerebroside in the prepared rice bran was measured by single extraction and TLC in Example 1.

  The amount of cerebroside of the prepared rice bran is shown in FIG. 4 (various salt amounts in the graph indicate the amount added to 1 kg of white rice). As a result, the amount of cerebroside was significantly increased in salts such as potassium citrate, potassium nitrate, sodium phosphate, and potassium phosphate, and the amount of cerebroside was increased in a concentration-dependent manner.

[Example 5] Examination of concentration of potassium salt in rice bran In Example 4, since remarkable effects were observed in potassium salts such as potassium citrate, potassium nitrate, and potassium phosphate, optimum addition of these potassium salts The amount was examined. That is, steamed rice having a water content of 30% was prepared using Koshihikari (water content 12.5%) as raw rice. To 1 kg of this steamed rice, each potassium salt solution (tripotassium citrate, potassium nitrate, potassium phosphate) was added at a rate of 40 ml and mixed uniformly. Potassium phosphate was prepared using monopotassium phosphate and dipotassium phosphate so as to have a pH of 5.8. The addition concentration of each potassium salt was made into 4-40 mmol of potassium phosphate, 40-160 mmol of potassium nitrate, and 20-120 mmol of tripotassium citrate per 1 kg of white rice. The obtained steamed rice is inoculated with 0.1% of a powder type koji mold (A. oryzae: NBRC4214), the product temperature is 35 ° C., the relative humidity is 80% or more, and the product temperature is 40 ° C. after 32 hours. Smelting time.

  The amount of cerebroside in the prepared rice bran was measured by single extraction and TLC in Example 1. The amount of cerebroside of the brewed sample is shown in FIG. 5 (the amount of salts in the graph is the amount added to 1 kg of white rice). In any experimental group, the amount of cerebroside was increased as compared with the control (water addition). The amount of cerebroside was maximized when 20 mmol of potassium phosphate was added, 80 mmol of potassium nitrate was added, and 40 mmol of potassium citrate was added. The increase in the amount of cerebroside was 1.3 times when potassium phosphate was added, 2.0 times when potassium nitrate was added, and 2.1 times when potassium citrate was added, compared with rice bran without additive.

  Furthermore, the total concentration in each concentration (potassium phosphate 20 mmol, potassium nitrate 80 mmol, potassium citrate 40 mmol addition), control (water addition), and general koji conditions (46 hours: commercial rice bran) in which each potassium salt was found to be most effective. The amount of cerebroside was measured by repeated extraction of Example 1 and HPLC to determine the total amount of cerebroside per gram of straw. The results are shown in FIG. As a result, with the most effective addition of potassium citrate, the total amount of cerebroside reaches 2.2 times that of the control, and further reaches 10.6 times that of general koji making conditions (46 hours: commercial rice bran). It has been shown.

[Example 6] Examination of concentration of potassium salt added at the time of immersion In Examples 4 and 5, salts were added at the time of inoculation. It was. Koshihikari (moisture 12.5%) was used as the raw material rice, and a steamed rice was prepared by immersing and absorbing a potassium salt solution (potassium phosphate, potassium nitrate, tripotassium citrate) that had a remarkable effect in Example 4. . Potassium phosphate was prepared using monopotassium phosphate and dipotassium phosphate so as to have a pH of 5.8. The steamed rice moisture content was 30 to 33%, and the potassium salt addition concentration calculated from the steamed rice moisture content was 1 kg of white rice, potassium phosphate: 250 mmol, 470 mmol, potassium nitrate: 200 mmol, tripotassium citrate: 80 mmol. The obtained steamed rice is inoculated with 0.1% of a powder type koji mold (A. oryzae: NBRC4214), the product temperature is 35 ° C., the relative humidity is 80% or more, and the product temperature is 40 ° C. after 32 hours. Smelting time. The amount of cerebroside in the prepared rice bran was measured by single extraction and TLC in Example 1.

  The amount of cerebroside of the prepared rice bran is shown in FIG. 7 (the amount added in the graph indicates the amount added per 1 kg of white rice). As a result, the amount of cerebroside increased in the rice bran added with potassium salts as compared to the control (no added) rice bran. From this, it was confirmed that the amount of cerebroside was increased in the same manner as the addition at the time of inoculation even when the addition of the potassium salt was performed when the raw material was immersed. Compared with the control rice bran, the increase in the amount of cerebroside was 1.9 times when the potassium salt during immersion was potassium phosphate, 2.2 times with potassium nitrate, and 2.2 times with potassium citrate.

[Comparative Example 1] Amount of cerebroside per cell in various liquid media In order to evaluate the effect of the difference in medium conditions on the amount of cerebroside per cell, CZAPEK DOX medium (2% Glucose, 0.3% NaNO ₃ , 0.2% KCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O), dextrin-peptone medium (2% dextrin, 1% peptone, 0.5% KH ₂ PO ₄ , 0 0.1% NaNO ₃ , 0.05% MgSO ₄ .7H ₂ O), malt extract medium (2% malt extract) and potato dextrose medium (2.4% potato dextrose) with a spore count of 10 ⁵ / ml Inoculated with a powder type koji mold (A. oryzae: NBRC4214) and cultured at 30 ° C. and 120 rpm for 72 hr. After culturing, the cells were collected and washed, then lyophilized, and the amount of cerebroside per dry cell was determined by single extraction and TLC in Example 1.

  The amount of cerebroside per dry cell in each medium is shown in FIG. As can be seen from FIG. 8, it was found that the amount of sphingolipid per amount of microbial cells varied depending on the culture conditions in the koji mold as well as the report in yeast. This suggests that sphingolipid does not increase simply when the amount of bacterial cells increases.

  The method for producing a sphingolipid-enriched koji according to the present invention does not require complicated steps, and it is also necessary to perform extraction and concentration using an organic solvent, as conventionally performed in the production of sphingolipids by microorganisms. Therefore, not only can labor and cost be reduced, but also a highly safe sphingolipid-enriched soot can be provided as a food composition.

FIG. 1 shows the structure of the main molecular species of glucosylceramide derived from rice bran. FIG. 2 shows a comparison between thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) in the measurement of cerebroside. FIG. 3 shows cerebroside production amounts of various koji molds. FIG. 4 shows a comparison of the amount of cerebroside in rice bran prepared by adding various salt solutions in koji making. FIG. 5 shows the relationship between the amount of potassium salt added and the amount of cerebroside in ironmaking. FIG. 6 shows a comparison of the total amount of cerebroside in rice bran when koji making was performed in the presence of each potassium salt at the concentration at which the effect was most recognized in the examples. FIG. 7 shows the amount of cerebroside when various salts are added during immersion in the raw material for koji making. FIG. 8 shows the amount of cerebroside per dry cell in various liquid media.

Claims (10)

  One or more inorganic salts selected from the group consisting of potassium salt, sodium salt, magnesium salt, calcium salt and iron salt in koji making inoculated with koji mold on steamed koji making raw material A method for enriching sphingolipids in koji, characterized by performing koji making in the presence of   The method according to claim 1, wherein the ironmaking is carried out in the presence of a potassium salt or a sodium salt.   2. The method according to claim 1, wherein the raw material for making koji is rice.   The method according to any one of claims 1 to 3, wherein the inorganic salt is added before the koji mold is inoculated or before the koji mold is inoculated after the koji raw material is steamed.   The method according to claim 4, wherein the addition concentration of the inorganic salt is 1 to 360 mmol in terms of ions per kg of the raw material for koji making.   The method according to any one of claims 1 to 3, wherein an inorganic salt is added at the time of dipping the raw material for koji before steaming.   The method according to claim 6, wherein the addition concentration of the inorganic salt is 50 to 500 mmol in terms of ions per 1 kg of the raw material.   The method according to claim 1, wherein the sphingolipid is cerebroside.   The method according to any one of claims 1 to 8, wherein the koji mold used for koji making is Aspergillus oryzae or Aspergillus sojae.   The method according to any one of claims 1 to 9, wherein the moisture content of the steamed raw material is 30 to 36% by weight.

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