JP2012191875A - Psyllium brought to have low viscosity and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide psyllium brought to have low viscosity, having inherited biological activity, especially hyaluronidase inhibitory activity, and a method for bringing psyllium to have low viscosity while keeping the hyaluronidase inhibitory activity.SOLUTION: The method for producing psyllium brought to have low viscosity includes bringing psyllium into contact with a culture product derived from bacteria belonging to the genus Cellulosimicrobium.

Description

  The present invention relates to a low viscosity psyllium and a method for producing the same.

  Psyllium is a polysaccharide mainly composed of arabinoxylan obtained from seed hulls such as Plantago ovata Forskal, which is a kind of plantain. Psyllium has β-1,4 and β-1,3 linked xylan as the main chain, and the constituent sugars of the side chain are β-D-xylose, α-D-arabinose, α-L-rhamnose, α-D- It consists of galacturonic acid and has physicochemical properties such as water absorption and swelling due to gel formation. In addition, it has been reported to have physiological activities such as a bowel movement improving action, a cholesterol absorption inhibitory action, a blood sugar level rise inhibitory action, and an anti-inflammatory action by inhibiting hyaluronidase activity (Patent Document 1).

  Psyllium, when dissolved in water, forms a highly viscous gel and forms a starch syrup, so it is difficult to use psyllium as it is for liquid beverages. Thus, various methods for reducing the viscosity of psyllium have been studied.

  As a method for reducing the viscosity of psyllium, in addition to a method for reducing the viscosity by acid, alkali, or heating, several methods using an enzyme have been proposed. For example, Patent Document 2 describes a method for reducing viscosity using pectinase or hemicellulase. Patent Document 3 describes that the viscosity is lowered by xylanase or the like. Patent Document 4 describes a method for reducing the viscosity of psyllium using a protease.

JP 2002-145756 A Japanese Patent Laid-Open No. 11-75776 WO99 / 63053 pamphlet JP 2002-320464 A

  However, it is not easy to reduce the viscosity while maintaining the physiological activity inherent in psyllium, particularly hyaluronidase inhibitory activity.

  In the method of reducing the viscosity by acid, alkali, or heating, psyllium is exposed to a low pH, a high pH, and a high temperature, and there is a high possibility that physiological activity is impaired along with the decrease in viscosity. In addition, from the viewpoint of industrial production, manufacturing equipment that can withstand severe reaction conditions such as low pH, high pH, and high temperature is required, which causes an increase in cost.

  On the other hand, the enzyme-based low viscosity method is processed under mild reaction conditions close to normal temperature and normal pressure, so there is a possibility of reducing the viscosity of psyllium while maintaining the physiological activity with less equipment burden.

  However, for example, the methods for reducing viscosity using pectinase, hemicellulase, xylanase, etc. described in Patent Document 2 and Patent Document 3 mainly focus on the effect of improving bowel movement as physiological activity, and various other physiological activities, particularly hyaluronidase. It is unclear whether inhibitory activity is retained. Further, the method for reducing the viscosity of psyllium using the protease described in Patent Document 4 does not disclose the effect on the physiological activity.

  Therefore, the main object of the present invention is to provide a low-viscosity psyllium that retains physiological activity inherent in psyllium, in particular, hyaluronidase inhibitory activity, and a method for reducing psyllium while retaining hyaluronidase inhibitory activity. It is in.

  As a result of intensive studies in view of the problems of the prior art, the present inventor has found that the above object can be achieved by bringing a culture derived from a specific microorganism into contact with psyllium, and has completed the present invention. .

  That is, the present invention relates to a method for producing low-viscosity psyllium, which comprises bringing a culture derived from a cellulosimicrobium bacterium into contact with psyllium.

  Cellulosimicrobium bacteria, especially cellulosimicrobium sp. By bringing the culture derived from the SY502 strain into contact with psyllium, low-viscosity psyllium that retains physiological activity, particularly hyaluronidase inhibitory activity, can be produced.

  The low-viscosity psyllium obtained by the present invention has a low viscosity when dissolved in a solvent such as water, so that it can be easily ingested as a liquid beverage, and a functional food material that retains the physiological activity of psyllium. Can be widely used.

FIG. 1 is a diagram showing a size exclusion chromatogram of SY502 strain culture supernatant-treated psyllium. FIG. 2 is a diagram showing a size exclusion chromatogram of distilled water-treated psyllium. FIG. 3 is a diagram showing a size exclusion chromatogram of hydrochloric acid-treated psyllium.

  In the present invention, psyllium is reduced in viscosity by contacting a culture derived from the genus Cellulosimicrobium with psyllium.

(1) Cellulosimicrobium-derived culture
(1-1) Bacterium belonging to the genus Cellulosimicrobium In the present invention, the bacterium used for reducing the viscosity of psyllium is a bacterium belonging to the genus Cellulosimicrobium. The genus Cellulosimicrobium is a new genus proposed by Schumann et al. In 2001 (Int. J. Syst. Evol. Microbiol., 2001, 50, 993-996).

  The bacterium used in the present invention may be any bacterium classified into the genus Cellulosimicrobium, but particularly preferred is Cellulosimicrobium sp. Examples include SY502 strain (Cellulosimicrobium sp. SY502). Cellulosimicrobium sp. The SY502 strain has been deposited as Deposit Number NITE P-1054 with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation.

(1-2) Bacterial culture The culture conditions are not particularly limited as long as bacteria belonging to the genus Cellulosimicrobium grow favorably.

  The culture temperature can be, for example, 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C, more preferably 30 ° C to 37 ° C, and if necessary, the temperature can be changed during the culture. Good.

  The culture time is not particularly limited, and may be, for example, 5 to 100 hours, preferably 10 to 90 hours, more preferably about 20 to 80 hours.

  The pH of the medium before or during the culture may be a value suitable for the growth of bacteria belonging to the genus Cellulosimicrobium, for example, it can be cultured at a pH of 5 to 9, preferably 6 to 8, Culturing can be performed while adjusting the pH as necessary. As the pH adjuster, an inorganic or organic acid, an alkaline solution, or the like can be used.

  Examples of the culture method include solid culture, liquid culture, stationary culture, shaking culture, and aeration and agitation culture. Among these, culture by a liquid culture method is preferable, and culture by shaking culture or aeration stirring culture is particularly preferable.

  Examples of the medium include one or more nitrogen sources such as yeast extract, tryptone, polypeptone, corn steep liquor, soybean or wheat bran leachate, sodium chloride, potassium chloride, monopotassium phosphate, dipotassium phosphate. Add one or more inorganic salts such as magnesium sulfate, magnesium chloride, sodium nitrate, ferric chloride, ferric sulfate or manganese sulfate, and add carbohydrate raw materials, vitamins, antibiotics, inducers, etc. Used.

  Moreover, in order to induce psyllium low-viscosity enzyme activity, it is preferable to add psyllium to the medium. The concentration of psyllium to be added is not particularly limited and can be, for example, 0.001 to 10% by mass, preferably 0.01 to 5% by mass.

(1-3) Culture In the present invention, the culture refers to a culture solution, a culture solution supernatant, a microbial cell, a microbial cell suspension, a microbial cell disruption solution, a crude enzyme solution, and a processed product thereof. Both those obtained by culturing bacteria belonging to the genus Microbium and those resulting therefrom are included. Cultures particularly preferably used in the present invention are a culture solution and a culture solution supernatant.

  The “culture solution” is a solution obtained after inoculating bacteria in a medium containing components suitable for growth and growing them under appropriate culture conditions. The “culture medium supernatant” is a fraction obtained by removing cells from the culture medium by means such as centrifugation or filtration after culturing. “Bacteria” means bacteria themselves, and includes not only those completely dried, but also those containing a small amount of water, buffer solution, culture solution, and other liquids. “Bacterial cell suspension” refers to a solution in which cells are suspended in water, buffer solution, or other liquid. “Bacteria cell disruption liquid” refers to bacteria obtained by crushing bacterial cells by ultrasonic treatment, high pressure treatment, grinding treatment, impact crush treatment, enzyme treatment, freeze-thaw treatment, hypotonic solution treatment, etc. A solution containing crushed material. The “crude enzyme solution” refers to a fraction obtained by removing insolubles from a cell disruption solution by means such as centrifugation or filtration. “Processed product” refers to a product obtained by subjecting the above-mentioned culture solution, culture solution supernatant, cells, cell suspension, cell disruption solution, or crude enzyme solution to physical / chemical treatment. . For example, an immobilized microbial cell obtained by immobilizing microbial cells on a carrier, and a fraction obtained by an operation such as ammonium sulfate precipitation or various chromatography from a culture supernatant or a crude enzyme solution are also included.

(2) Psyllium Psyllium is a natural polysaccharide obtained from seeds of the plant type Plantago ovata. Generally, it is marketed as psyllium husk and psyllium seed gum.

  In the present invention, particles of any particle size and grade can be used, but those having less complications such as dust are preferred. In this invention, it can manufacture by a well-known method (For example, the method as described in Unexamined-Japanese-Patent No. 01-197501), and can also use what is marketed. Examples of commercially available psyllium include Soalum PG200 and Soalum PG020MR (both manufactured by MRC Polysaccharide).

(3) Reducing the viscosity of psyllium In the present invention, bringing the culture into contact with psyllium means mixing a psyllium solution and a culture derived from a bacterium belonging to the genus Cellulomicrobium and allowing them to react for a certain period of time. In the present invention, a culture of microorganisms may be added to the psyllium solution containing psyllium, and conversely, psyllium or a psyllium solution may be added to the cultures of microorganisms. Here, the former method will be described as a representative.

  The concentration of psyllium in the psyllium solution is not limited as long as psyllium is reduced in viscosity. For example, 0.001 to 10% by mass, preferably 0.01 to 5% by mass, and more preferably 0.1 to 1% by mass. can do.

  Water can be exemplified as the solvent of the psyllium solution, but it is possible to add an organic solvent such as ethanol or an inorganic / organic salt having a buffering action as long as the reduction in viscosity is not hindered.

  The buffer solution to be used is not particularly limited, and examples thereof include buffer component concentrations of 5 to 500 mM, preferably about 5 to 150 mM, and pH of about 5 to 9. Examples of inorganic / organic salts having a buffering action include tris (hydroxymethyl) aminomethane (Tris), sodium or potassium phosphate, citrate, acetate, and the like.

  The reaction conditions may be any conditions as long as the viscosity of the psyllium can be reduced. The reaction temperature can be 10-60 ° C, preferably 20-50 ° C, more preferably 30-40 ° C. The pH may be 4 to 10, preferably 5 to 9, and more preferably 6 to 8.

  If necessary, the reaction solution can be stirred. This is because the reaction can proceed efficiently. The rotation speed can be, for example, 5 to 500 rpm (rotation / min), preferably 10 to 200 rpm, and more preferably 20 to 100 rpm.

  The amount of the cellulosimicrobium-derived culture to be contacted is not limited as long as low viscosity can be achieved, and it is arbitrary considering the low viscosity activity and other reaction conditions contained in the culture. Can be set. For example, when the culture supernatant is used directly, the mass is 0.001 to 10 times that of the psyllium solution, preferably 0.01 to 5 times the mass, more preferably 0.1 to 2 times the mass. can do.

  After performing the low viscosity reaction, inactivation treatment for eliminating the low viscosity activity contained in the culture can be performed to stop the reaction. The deactivation treatment may be any treatment as long as it can eliminate the low-viscosity activity and does not cause excessive thinning or decomposition of psyllium. For example, heat treatment can be given. As heat processing, it can be set as 50-100 degreeC, for example, Preferably it is 60-100 degreeC, More preferably, it can be set at 70-100 degreeC.

  Moreover, the low viscosity psyllium obtained by the low viscosity reaction can be refine | purified as needed. As a purification method, any method can be used as long as a desired low-viscosity psyllium solution can be obtained. For example, methods such as centrifugation, filtration, various types of chromatography, and alcohol precipitation can be used.

  The conditions for the centrifugation may be any conditions as long as unnecessary impurities or the like are precipitated and the supernatant containing low-viscosity psyllium can be recovered. For example, the conditions are 1,000 to 50,000 × g, preferably 5,000. ˜20,000 × g.

  Filtration can be performed using filter paper, filter cloth, membrane or the like. Examples of the filter paper include purified cotton fibers (cellulose) or glass fiber filter paper. For example, filter paper for qualitative analysis and quantitative analysis according to JIS P3801 can be used.

  As the filter cloth, a filter cloth made of polypropylene, polyester, polyamide (nylon), vinylidene chloride, vinylon, cotton or the like can be used. As the form of the filter cloth, for example, a spun yarn (short fiber yarn), a multifilament yarn (long fiber yarn), a monofilament yarn (long single fiber yarn), a split ultrafine fiber yarn, or the like can be used. . As the structure of the filter cloth, plain weave, twill, double weave, felt, satin weave, or the like can be used.

  When filtering with a filter paper or a filter cloth, a filter aid can be used in combination. Examples of filter aids include diatomaceous earth and pearlite (pearlite), activated carbon, and cellulose filter aids.

  As the membrane, a microfiltration (MF) membrane or an ultrafiltration (UF) membrane can be used. The material of the MF film includes an organic polymer film and an inorganic ceramic film such as alumina. As the organic polymer film, polyethylene, polypropylene, polysulfone, polyvinylidene fluoride, or the like is used. As the pore size of the MF membrane, for example, a pore size of about 0.1 to 0.5 μm can be used. The material of the UF film includes an organic film using a polymer material and an inorganic film such as a ceramic film. Examples of the polymer film include cellulose acetate, polyacrylonitrile, and polysulfone. As the UF membrane, a membrane having a fractional molecular weight according to the purpose can be selected and used. For example, a UF membrane of about 1,000 to 300,000 can be used.

  As the chromatography, any method that can obtain a desired low-viscosity psyllium solution may be used. For example, size exclusion chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, anion chromatography, etc. Can be mentioned.

  As alcohol used for alcohol precipitation, ethanol, isopropanol, or the like can be used. The concentration of the alcohol may be any concentration as long as the precipitation of low-viscosity psyllium can be obtained. For example, the concentration is 50 to 99% (V / V), preferably 60 to 99%, more preferably 70. A concentration of ˜99% may be used. If necessary, the obtained psyllium precipitate can be rinsed with an aqueous alcohol solution having a similar concentration. The obtained psyllium precipitate can be dried by vacuum drying, freeze drying or the like.

(4) Low viscosity psyllium
(4-1) Reducing viscosity In the present invention, reducing viscosity means reducing the viscosity of the psyllium solution. The method for measuring the viscosity is not particularly limited. For example, when measured as an aqueous solution at 25 ° C., it is preferably 50% or less, more preferably 20% or less, and even more preferably 10%, compared with the viscosity of the psyllium solution before the low viscosity reaction (raw material). The following means to reduce the viscosity.

  The viscosity of the psyllium solution can be measured using various viscometers. Examples of the viscometer include a vibration viscometer, a rotary viscometer, a capillary viscometer, a falling body viscometer, and a cup viscometer. Any viscometer may be used as long as it can appropriately measure the viscosity of the psyllium solution, but a rotary viscometer can be preferably used in the present invention. The rotary viscometer includes a coaxial double cylinder type, a single cylinder type rotary viscometer (B type viscometer), a cone plate type (E type viscometer), etc. In the present invention, the B type viscosity is used. A meter can be used suitably.

  In the present invention, the strength of the ability to reduce the viscosity of psyllium is referred to as the activity of reducing viscosity. The low-viscosity activity in the present invention can be measured and compared by the viscosity after mixing a test sample and a psyllium solution to make a reaction solution, and contacting (reacting) for a predetermined time under a predetermined condition.

(4-2) Size Exclusion Chromatography The low viscosity psyllium of the present invention has two molecular weight peaks in the size exclusion chromatogram. One of them has a peak top molecular weight of a peak on the high molecular weight side of 1 million or more, and the other has a peak top molecular weight of a peak on the low molecular weight side of 100,000 or less. A preferred peak top molecular weight of the high molecular weight peak is about 1,000,000 to 2,000,000, more preferably about 1,200,000 to 1,500,000. Moreover, the preferable peak top molecular weight of the low molecular weight side peak is about 1,000 to 100,000, more preferably about 5,000 to 50,000.

  In the present invention, the size exclusion chromatogram means the optical signal (absorbance, suggested refractive index, fluorescence, etc.) intensity reflecting the retention time or elution volume on the horizontal axis and the solute concentration on the vertical axis in size exclusion chromatography. Refers to the electrochemical signal intensity. Size exclusion chromatography refers to chromatography in which substances can be separated by a size exclusion mechanism between pores on the surface of the filler and a polymer (high molecular weight body) in a column packed with a porous filler.

  In size exclusion chromatography, polymers with large molecular weight (high molecular weight) are not able to reach the depth of the porous packing, so they are eluted quickly, while polymers with small molecular size (low molecular weight) can reach the depth. Since it is eluted slowly, the polymer can be separated by the difference in molecular size (molecular weight).

  Various packing materials (columns) for size exclusion chromatography are commercially available. In the present invention, any column can be used as long as it can appropriately measure the molecular weight of psyllium. For example, Shodex OHpak SB-806M HQ (Showa Denko KK) can be suitably used.

  In size exclusion chromatography, the molecular weight of psyllium, which is the test polymer, can be determined by using a polymer having a known molecular weight (standard polymer) and obtaining in advance a calibration curve of retention time or elution volume and molecular weight. The standard polymer is not particularly limited as long as it can appropriately calculate the molecular weight of psyllium as a test polymer, and in particular, pullulan (eg, STD P-82, STD P-250, Showa Denko KK) is preferably used. Can do.

  The average molecular weight such as number average molecular weight (Mn), weight average molecular weight (Mw), z average molecular weight (Mz), peak top molecular weight (Mp) can be determined by size exclusion chromatography. The low viscosity psyllium in the present invention can be specified by the peak top molecular weight.

  The peak top molecular weight is determined as a molecular weight corresponding to the apex (peak top) of a molecular weight peak (hereinafter also simply referred to as a peak) in a size exclusion chromatogram. The peak top is a portion corresponding to the peak of the “mountain” on the size exclusion chromatogram, and is a data point in which the first-order differential value of the output value in the peak region is 0 and the second-order differential value is negative. it can. Any method can be used to detect the peak in the chromatogram. For example, the differential value of the chromatogram is obtained, and when the differential value exceeds a predetermined level of + and −, the peak start point and end point are used, and the interval between the start point and the end point is determined. The method of peaking this part is mentioned.

  In the present invention, the peak and peak top molecular weight for specifying low-viscosity psyllium are essentially the peak derived from psyllium and the peak top molecular weight, and impurities other than psyllium appearing on the chromatogram, etc. The peak derived from and its peak top molecular weight are not the object. Whether or not the peak is derived from psyllium can be easily determined by comparing the chromatogram of the psyllium solution, which is the test sample, with the chromatogram of the same solution in which the components other than psyllium are the same as the test sample. it can.

(4-3) Hyaluronidase Inhibitory Activity In the present invention, hyaluronidase inhibitory activity refers to an activity that inhibits the activity of hyaluronidase, an enzyme that degrades hyaluronic acid. Hyaluronic acid is a linear macromolecular polysaccharide formed by alternately binding β-DN-acetylglucosamine and β-D-glucuronic acid, and is distributed in a large amount in mammalian connective tissue. It is also present in chicken crowns and streptococcal capsules.

  Hyaluronidase is a general term for enzymes that depolymerize hyaluronic acid, and is present in higher animal semen, snake venom, pneumoniae, and streptococci. In mammals, it is considered that during fertilization, a viscous gel containing hyaluronic acid covering the surface of an egg cell is removed to assist invasion of sperm or to facilitate invasion of pathogenic bacteria. It is also known that it is activated during inflammation, destroys the matrix of connective tissue, and mediates infiltration into inflammatory tissues, increased vascular permeability, and histamine release from mast cells in type I allergy.

  Known substances having hyaluronidase inhibitory activity include anti-allergic agents such as sodium cromoglycate and tranilast, and anti-inflammatory agents such as aspirin and indomethacin.

  Hyaluronidase inhibitory activity can be determined by examining how much hyaluronidase activity is inhibited when a test substance is allowed to coexist in a hyaluronidase activity measurement system as compared to when the test substance is not allowed to coexist. Hyaluronidase activity can be measured, for example, by quantifying the amount of N-acetylglucosamine released from hyaluronic acid by hyaluronidase by the Morgan-Elson method (J. Biol. Chem., 1955, 217, 959-966).

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1: Preparation of cellulosimicrobium-derived culture and confirmation of psyllium-thinning activity]
Cellulosimicrobium sp. The SY502 strain (Cellulosimicrobium sp. SY502; hereinafter referred to as “SY502 strain”) was cultured as follows.

  Glycerol stock 0.2 mL of SY502 strain stored at −80 ° C. was prepared in a 100 mL pre-culture medium prepared in a 500 mL Erlenmeyer flask (1 g of sodium nitrate, 0.1 g of dipotassium hydrogen phosphate per 1 L of medium, sulfuric acid Magnesium heptahydrate 0.5 g, potassium chloride 0.5 g, ferric sulfate heptahydrate 0.01 g, yeast extract 0.5 g, polypeptone fine granules 2 g, psyllium (Soarium; manufactured by MRC Polysaccharide Co., Ltd.) 0 0.5 g and adjusted to pH 7 with an aqueous sodium hydroxide solution, inoculated into autoclave sterilized (121 ° C., 20 minutes), and cultured with shaking at a temperature of 30 ° C. and a rotation speed of 200 rpm for 72 hours.

  25 mL of the obtained preculture solution was added to 3 L of 2.5 L main culture medium (1 g of sodium nitrate, 0.1 g of dipotassium hydrogen phosphate, 7 mg of magnesium sulfate per liter of the medium) prepared in a 3 L mini jar fermenter. Hydrate 0.5g, potassium chloride 0.5g, ferric sulfate heptahydrate 0.01g, yeast extract 0.5g, polypeptone fine granules 2g, psyllium 15g, Pluronic L-61 (manufactured by ADEKA Corporation) 0 .2g, prepared to pH 6, 7 and 8 with aqueous sodium hydroxide solution and then inoculated into autoclave sterilized (121 ° C, 20 minutes), temperature 30 ° C, rotation speed 200rpm, pH control value 6 , 7 or 8 (controlled with 12.5 mass% sodium hydroxide aqueous solution and 12 mass% sulfuric acid aqueous solution), air flow rate 1.25 L / min (change to 0.6 L / min after 26 hours) ), Pressure was aerated agitation culture at atmospheric pressure.

  After culturing 48 hours or 72 hours, the OD660 value (turbidity at a wavelength of 660 nm) reflecting the bacterial concentration was measured. Moreover, a part (40 mL) of the culture solution was subjected to centrifugation (7,500 × g, 20 minutes), and the resulting supernatant was designated as “culture solution supernatant”.

Using each of the obtained culture supernatants, the low-viscosity activity was measured as follows. 30 mL of psyllium solution (1.2 g of psyllium dissolved in 20 mM KH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 8) and dissolved by autoclave (121 ° C., 20 minutes)) and 30 mL of each culture supernatant or control Then, 30 mL of distilled water was mixed and reacted at a temperature of 37 ° C. and a rotation speed of 160 rpm for 24 hours (96 hours in the case of distilled water), and then the viscosity was measured with a B-type viscometer. The viscosity was measured at a temperature of 25 ° C. using a B-type viscometer (Tokyo Keiki Co., Ltd.). Table 1 shows the measured OD660 values and reaction liquid viscosity.

  The SY502 strain grew well even if the pH during the main culture was any of pH 6, 7, and 8. Moreover, even when any culture supernatant was used, the viscosity of the reaction solution was greatly reduced as compared with the case where the target distilled water was used. Therefore, it was confirmed that these culture supernatants contain psyllium-lowering activity.

［実施例２：ＳＹ５０２株培養液上清による低粘化反応］
実施例１で得られたＳＹ５０２株培養液上清（本培養時ｐＨ７）をサイリウム溶液と接触させて低粘化反応を行った。

[Example 2: Low-viscosity reaction with culture supernatant of SY502 strain]
The SY502 strain culture supernatant obtained in Example 1 (pH 7 during main culture) was brought into contact with a psyllium solution to perform a low viscosity reaction.

500 mL of psyllium solution (10 g of psyllium added to 500 mL of 25 mM KH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 7) and autoclaved (121 ° C., 20 minutes)) and SY502 strain culture supernatant (pH 7 during main culture) ) 500 mL was mixed, reacted at a temperature of 37 ° C., pH control value 7 (controlled with 12.5 mass% sodium hydroxide aqueous solution and 12 mass% sulfuric acid aqueous solution), rotation speed of 100 rpm for 2 days, and the reaction solution (= “SY502 strain” The viscosity of the culture supernatant treated psyllium ") was measured.

  The results are shown in Table 2. The viscosity was 40 mPa · s, and it was confirmed that the viscosity of the reaction solution was greatly reduced.

[Comparative Example 1: Reducing viscosity with distilled water]
50 mL of psyllium solution (1 g of psyllium added to 50 mL of distilled water and autoclave (121 ° C., 20 minutes)) and 50 mL of distilled water are mixed and reacted at a temperature of 37 ° C. for 4 days. The viscosity of psyllium ") was measured.

  The results are also shown in Table 2. The viscosity of the distilled water-treated psyllium was 5700 mPa · s, which was confirmed to be very high compared to the SY502 strain culture supernatant-treated psyllium reaction solution described above.

[Comparative Example 2: Reducing viscosity with hydrochloric acid]
To 100 mL of a psyllium solution (1 g of psyllium added to 100 mL of distilled water and autoclaved (121 ° C., 20 minutes)) is added 0.25 mL of 2N hydrochloric acid (pH about 2.7 after the addition), and the temperature is 30 ° C. After reacting for a minute, the viscosity of the reaction solution (= “hydrochloric acid-treated psyllium”) was measured.

  The results are also shown in Table 2. The viscosity of the hydrochloric acid-treated psyllium was 8 mPa · s, and it was confirmed that the viscosity of the reaction solution was greatly reduced.

[Comparative Example 3: Low viscosity reaction by Novozyme 188]
Psyllium solution (10 g of psyllium is added to 1000 mL of 25 mM KH ₂ PO ₄ -Na ₂ HPO ₄ buffer, 5 mL of Novozyme 188 (manufactured by Novozyme) is added to 1000 mL of autoclave (121 ° C., 20 minutes), temperature 40 ° C., pH The reaction was carried out at a control value of 7 (controlled with a 12.5 mass% aqueous sodium hydroxide solution and a 12 mass% sulfuric acid aqueous solution) at a rotational speed of 100 rpm for 7 days, and the viscosity of the reaction solution (= “Novozyme 188 treated psyllium”) was measured.

  The results are also shown in Table 2. The viscosity of Novozyme 188 treated psyllium was 11 mPa · s, and it was confirmed that the viscosity of the reaction solution was greatly reduced.

［実施例３：ＳＹ５０２株培養液上清処理サイリウムの分子量測定］
実施例２の反応液を８０℃で４０分加熱処理を行い、低粘化活性を消失させた。遠心分離（７，５００×ｇ、２０分間）を行い、得られた上清の一部を用いて、ＳＹ５０２株培養液上清処理サイリウムの分子量をサイズ排除クロマトグラフィーにより測定した。プルラン（ＳＴＤ Ｐ−８２及びＳＴＤ Ｐ−２５０；昭和電工株式会社製）を標準ポリマーに用いて較正曲線を作成した。分析条件を以下に示す。

[Example 3: Measurement of molecular weight of SY502 strain culture supernatant treated psyllium]
The reaction solution of Example 2 was heat-treated at 80 ° C. for 40 minutes to eliminate the low-viscosity activity. Centrifugation (7,500 × g, 20 minutes) was performed, and the molecular weight of SY502 strain culture supernatant-treated psyllium was measured by size exclusion chromatography using a part of the obtained supernatant. A calibration curve was prepared using pullulan (STD P-82 and STD P-250; Showa Denko KK) as a standard polymer. The analysis conditions are shown below.

Analytical column: Shodex OHpak SB-806M HQ 8 mm x 300 mm (Showa Denko KK)
Guard column: Shodex OHpak SB-G 4.6 mm × 100 mm (Showa Denko KK)
Flow rate: 1 mL / min Mobile phase: 50 mM aqueous sodium sulfate detection: Suggested refractive index Column temperature: 40 ° C.
Sample concentration: about 0.25 mg / mL
Injection volume: 20 μL.

  The size exclusion chromatogram of the SY502 strain culture supernatant-treated psyllium is shown in FIG. 1, and the calculated peak top molecular weight is shown in Table 3.

  Unlike the chromatogram of distilled water-treated psyllium (Comparative Example 4; FIG. 2) and hydrochloric acid treatment (Comparative Example 5; FIG. 3) described later, the size exclusion chromatogram of the SY502 strain culture supernatant treated psyllium is clearly separated. Two molecular weight peaks observed. The peak top molecular weight of the high molecular weight peak was about 1.4 million, and the peak top molecular weight of the low molecular side peak was about 7500.

［比較例４： 蒸留水処理サイリウムの分子量測定］
比較例１で得られた反応液の一部を用いて、蒸留水処理サイリウムの分子量を実施例３と同様の分析条件により測定した。蒸留水処理サイリウムのサイズ排除クロマトグラムを図２に、算出されたピークトップ分子量を表３に併せて示す。

[Comparative Example 4: Molecular weight measurement of distilled water-treated psyllium]
Using a part of the reaction solution obtained in Comparative Example 1, the molecular weight of distilled water-treated psyllium was measured under the same analysis conditions as in Example 3. The size exclusion chromatogram of distilled water-treated psyllium is shown in FIG. 2, and the calculated peak top molecular weight is also shown in Table 3.

  Unlike the chromatogram of the SY502 strain culture supernatant-treated psyllium of Example 3, the size exclusion chromatogram of the distilled water-treated psyllium showed two adjacent molecular weight peaks. The peak top molecular weight of the peak on the high molecular weight side was about 1.6 million, and the peak top molecular weight of the peak on the low molecular weight side was about 200,000.

[Comparative Example 5: Molecular weight measurement of hydrochloric acid-treated psyllium]
After adjusting the reaction solution of Comparative Example 2 to pH 6 using an aqueous sodium hydroxide solution, the molecular weight of hydrochloric acid-treated psyllium was measured under the same analytical conditions as in Example 3 using a part of the reaction solution. FIG. 3 shows the size exclusion chromatogram of the hydrochloric acid-treated psyllium, and Table 3 shows the calculated peak top molecular weight.

  Unlike the chromatogram of the SY502 strain culture supernatant treated psyllium of Example 3, and the chromatogram of the distilled water-treated psyllium of Comparative Example 4, the size-exclusion chromatogram of the psyllium treated with hydrochloric acid is also two adjacent chromatograms. A molecular weight peak was shown. The peak top molecular weight of the high molecular weight peak was about 900,000, and the peak top molecular weight of the low molecular weight peak was about 130,000.

[Example 4: Measurement of hyaluronidase inhibitory activity of SY502 strain culture supernatant treated psyllium]
The centrifugal supernatant of Example 3 was subjected to pressure filtration at 0.2 MPa using a membrane filter (ADVANTEC, pore diameter 0.45 μm, material cellulose acetate). The obtained filtrate was subjected to pressure filtration at 0.15 MPa using an ultrafiltration membrane (MILLIPORE, fractional molecular weight 10,000, material regenerated cellulose), and the solution concentrated on the ultrafiltration membrane was recovered.

  To 10 mL of the collected solution, 0.5 g of ion exchange resin (AG501-8X, Bio-Rad Laboratories, Inc.) was added, and the mixture was shaken overnight at 25 ° C. for desalting. The resulting solution was frozen and then lyophilized.

  Using the obtained dried product, the hyaluronidase inhibitory activity of SY502 strain culture supernatant-treated psyllium was measured as follows. Lyophilized SY502 strain culture supernatant treated psyllium was dissolved in 0.1 M acetic acid buffer (pH 3.5, hereinafter referred to as acetic acid buffer) to a final concentration of 0.08 to 0.3 g / L to prepare a psyllium solution. .

  0.2 mL of psyllium solution is taken in a test tube, and 0.1 mL of hyaluronidase solution (containing hyaluronidase (from bovine testis; type IV-S, Sigma) at a concentration of 0.7 g / L in an acetate buffer) And incubated at 37 ° C. for 20 minutes. Compound 48/80 solution (compound 48/80 (manufactured by Sigma) at a concentration of 0.1 g / L in acetate buffer) 0.2 mL was added, and further incubated at 37 ° C. for 20 minutes.

  0.5 mL of potassium hyaluronate solution (potassium hyaluronate (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in acetic acid buffer and adjusted to a final concentration of 0.4 mg / mL) was added, and the mixture was added at 37 ° C. for 40 minutes. Reacted. The reaction was stopped by adding 0.2 mL of 0.4 M sodium hydroxide aqueous solution, and then ice-cooled, boric acid solution (50 mL of water was added to 4.95 g of boric acid, pH = 9. 1 with 1N sodium hydroxide solution). 1 and adjusted to 100 mL by adding water) 0.2 mL was added. After heating in boiling water for 3 minutes, dissolve 10 g of DMAB solution (p-dimethylaminobenzaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) in a mixture of 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid with acetic acid immediately before use. After adding 0.2 mL) and incubating at 37 ° C. for 20 minutes, absorbance (Abs585) at a wavelength of 585 nm was measured.

Abs585 in the system to which the test sample was added was Abs585 (T), Abs585 value in the system to which acetate buffer was added instead of the test sample was Abs585 (C), and the hyaluronidase activity inhibition rate IR (%) was calculated by the following formula: .
IR (%) = {Abs585 (C) −Abs585 (T) / Abs585 (C)} × 100.

  The test sample concentration (%) in the reaction system is plotted on the horizontal axis, IR (%) is plotted on the vertical axis, and the test sample concentration such that IR = 50 (%) is IC50 (%), and an index of hyaluronidase inhibitory activity did. IC50 (%) values are shown in Table 4.

  The SY502 strain culture supernatant-treated psyllium has an IC50 (%) value of 0.00582, which clearly has higher hyaluronidase inhibitory activity compared to distilled water-treated psyllium, hydrochloric acid-treated psyllium, and Novozyme 188-treated psyllium described below. It was confirmed that

［比較例６：蒸留水処理サイリウムのヒアルロニダーゼ阻害活性測定］
比較例１の反応液を凍結乾燥し、得られた乾燥品を用いて、蒸留水処理サイリウムのヒアルロニダーゼ阻害活性を、実施例４と同様の方法により測定した。ＩＣ５０（％）値を表４に併せて示す。

[Comparative Example 6: Measurement of hyaluronidase inhibitory activity of distilled water-treated psyllium]
The reaction solution of Comparative Example 1 was freeze-dried, and the hyaluronidase inhibitory activity of distilled water-treated psyllium was measured by the same method as in Example 4 using the obtained dried product. IC50 (%) values are also shown in Table 4.

  The IC50 (%) value of distilled water-treated psyllium was 0.00920, and it was confirmed that the hyaluronidase inhibitory activity was low as compared with SY502 strain culture supernatant-treated psyllium.

[Comparative Example 7: Measurement of hyaluronidase inhibitory activity of psyllium treated with hydrochloric acid]
The neutralization reaction solution obtained in Comparative Example 5 was fractionated, purified and dried in the same manner as in Example 4. Using the obtained dried product, the hyaluronidase inhibitory activity of hydrochloric acid-treated psyllium was measured by the same method as in Example 4. IC50 (%) values are also shown in Table 4.

  The IC50 (%) value of psyllium treated with hydrochloric acid was 0.00982, and it was confirmed that the hyaluronidase inhibitory activity was low as compared with psyllium treated with SY502 strain culture supernatant.

[Comparative Example 8: Measurement of hyaluronidase inhibitory activity of Novozyme 188-treated psyllium]
The reaction solution obtained in Comparative Example 3 was subjected to heat treatment and centrifugation as in Example 3, followed by fractionation, purification and drying in the same manner as in Example 4. Using the resulting dried product, the hyaluronidase inhibitory activity of Novozyme 188-treated psyllium was measured by the same method as in Example 4. IC50 (%) values are also shown in Table 4.

  The IC50 (%) value of Novozyme 188-treated psyllium was 0.00912, and it was confirmed that the hyaluronidase inhibitory activity was low as compared with SY502 strain culture supernatant-treated psyllium.

Claims (4)

  A method for producing low-viscosity psyllium, comprising bringing a culture derived from a bacterium belonging to the genus Cellulosimicrobium into contact with psyllium.   A bacterium belonging to the genus Cellulosimicrobium is known as Cellulosimicrobium sp. The method of Claim 1 which is SY502 strain | stump | stock (Cellulosimicrobium sp.SY502) (NITE P-1054).   A low-viscosity psyllium having two molecular weight peaks in a size exclusion chromatogram, wherein the peak on the high molecular weight side has a peak top molecular weight of 1 million or more and the peak on the low molecular side has a peak top molecular weight of 100,000 or less.   Cellulosimicrobium sp. SY502 strain (Cellulosimicrobium sp. SY502) (NITE P-1054).

Images