JP2014223027A - Novel amino polysaccharide which filamentous fungus produces - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel amino polysaccharide which has a similar structure to conventional chitosan, while has a high solubility and a low viscosity to water and the like, as well as is easy to handle and has various applications.SOLUTION: Provided is an amino polysaccharide having a structure bound in a structure formed by alternately linking glucosamine by β1 → 3 binding and β1 → 4 binding. The amino polysaccharide having a molecular weight of 300,000-500,000 produced by Molecular Peyronellaea calorpreferens NITE P-01575 strain or a natural or artificial variant thereof is available as a waste fluid purification agent for purifying waste fluid by coagulation-sedimentation method.

Description

    The present invention relates to a novel aminopolysaccharide having a low viscosity and easily dissolved.

Conventionally, chitosan has been mainly used as an aggregating agent, but since then, various uses have been developed in the medical field, industrial field, agricultural field, and food field, and in recent years, attention has been focused on anti-cholesterol and anti-hypertension. Due to the use of health foods and glucosamine, which is a constituent sugar, as health foods for joint pain, the demand as a supply source is increasing, and various chitin / chitosan-like substances have been reported (Patent Documents 1 and 2). Non-patent documents 1, 2). However, it is known that conventional chitin / chitosan-like substances have a wide molecular weight distribution, high viscosity, and insolubility in water.

Japanese Patent No. 3797851 JP 2004-081106 A

Journal of Bioscience and Bioengineering Vol89, No. 1, 40-46, 2000 Biotechnology Letters (2006) 28: 241-245

  An object of the present invention is to provide a novel aminopolysaccharide which has a low viscosity and a high solubility even when dissolved in water and is easy to use.

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by the following items, and have completed the present invention.
That is, the present invention is as follows.
[1] An aminopolysaccharide having a structure in which glucosamine is bound in a structure in which β1 → 3 bonds and β1 → 4 bonds are alternately linked.
[2] Infrared absorption ^{spectrum: 891 ± 10 cm -1, 3300} ± 100cm -1, 2900 ± 50cm -1, 3300 ± 100cm -1, peak to 1550~1650cm ^-1 - having click [1] Amino according Polysaccharides.
[3] The aminopolysaccharide according to [2], wherein the elemental analysis value (% by weight) is as follows.
C: 38-40
H: 6-9
N: 6-9
O: 38-40
[4] The amino polysaccharide according to [2] or [3], which has a molecular weight of 300,000 to 500,000.
[5] The aminopolysaccharide according to any one of [1] to [4], which is produced by Peyronellaea calorpreferens NITE P-01575 strain or a natural or artificial variant thereof.
[6] An aqueous solution containing the aminopolysaccharide according to any one of [1] to [5] and having a viscosity of 5 cP or less.
[7] An aqueous solution of 15% by weight or less containing the amino polysaccharide according to any one of [1] to [5].
[8] A waste liquid purifying agent comprising the amino polysaccharide according to any one of [1] to [5].
[9] A purification method, wherein the waste liquid purifying agent according to [8] is purified by a coagulation precipitation method.
[10] A food containing the aminopolysaccharide according to any one of [1] to [5].
[11] A feed comprising the amino polysaccharide according to any one of [1] to [5].
[12] A medicament comprising the amino polysaccharide according to any one of [1] to [5].

  The present invention makes it possible to provide a novel aminopolysaccharide having a unique physical property and physiological activity that has low viscosity and high solubility even when dissolved in water.

The present invention makes it possible to provide a purification agent containing a novel aminopolysaccharide, and to easily purify a waste liquid using a coagulation precipitation method.

Colony formation on potato dextrose agar agar plates Mycelium Changes over time in Example 1 Changes over time in Examples 2-1 and 2-2 Results of GPC chromatography in Example 3 Results of GPC chromatography in Example 4 1H-NMR spectrum of the hydrolyzate of the present invention 13C-NMR spectrum of the hydrolyzate of the present invention 1H-NMR spectrum of glucosamine preparation 13C-NMR spectrum of glucosamine preparation 1H-NMR spectrum of chitosan 7B hydrolyzate 13C-NMR spectrum of chitosan 7B hydrolyzate Infrared absorption spectra of the product of the present invention and chitosan preparation 1H-NMR spectrum of the product of the present invention 1H-NMR spectrum of chitosan preparation 13C-NMR spectrum of the product of the present invention Two-dimensional NMR spectrum of the product of the present invention GPC chromatogram of this product and standard sample pullulan MW344,000 Viscosity change at each concentration of the product of the present invention and chitosan preparation

As shown in the general formula (1), the aminopolysaccharide of the present invention has a structure in which glucosamine is alternately linked in a β1 → 3 bond and a β1 → 4 bond.

Normally, glucose has a cyclic structure as shown in the general formula (2), and can take two types of chemical structures depending on the orientation of the OH group at the C-1 position. The case where the OH group is opposite to the carbon atom at the C-6 position with respect to the cyclic surface (axial) is distinguished as α-glucose, and the case where it is on the same side (ectorial) is distinguished as β-glucose. It is in an equilibrium state in an aqueous solution, and the abundance ratio is about 36% α and about 64% β.

In the case of polysaccharides, there are two types of linkage between monosaccharides, α-type and β-type. The polysaccharide in which α-glucose is bonded is called α-glucan, and the polysaccharide in which β-glucose is bonded is called β-glucan. The carbon atom at the C-1 position of glucose can be bonded to five types of carbons other than the C-5 position of other glucoses. There are five types of combinations of coupling positions: (1 → 1), (1 → 2), (1 → 3), (1 → 4), (1 → 6). 3), (1 → 4), (1 → 6). Chitosan has glucosamine β (1 → 4) and is very similar to the aminopolysaccharide of the present invention.

The molecular weight of the aminopolysaccharide of the present invention is 300,000 to 500,000 as determined by high performance liquid chromatography.
Conventional chitosan and other high molecular weight polymers usually show high viscosity even when dissolved in water, and the solubility shows high viscosity as the concentration of the solution increases. Regardless of this, it has a very low viscosity of 5 cP or less and is well soluble in water, acid and alkali. At room temperature, it can be dissolved in an aqueous solution up to 15% by weight, particularly 5% by weight. Among aqueous solutions, an acidic aqueous solution can be dissolved up to 10% by weight, and an alkaline aqueous solution can be dissolved up to 2% by weight. That is, the amino polysaccharide of the present invention can be freely added up to a concentration equivalent to 15% by weight in an aqueous solution, regardless of the application form, and can be used in a wide range of applications.

The elemental analysis value (% by weight) of the aminopolysaccharide of the present invention is in the following range.
C: 38-40
H: 6-9
N: 6-9
O: 38-40

The aminopolysaccharide of the present invention is produced by a microorganism and is isolated from a culture of the microorganism and a processed product thereof. A preferred microorganism is Peyronellaea calorpreferens, and MGC130L was deposited as NITE P-01575 on March 18, 2013 at the Patent Evaluation Center for Microorganisms of the National Institute of Technology and Evaluation. Yes.

Bacteriological properties A new strain that lost the original trait (Studies in Mycology, 65: 31-32 2010) without spore formation on agar potato dextrose (hereinafter abbreviated as PDA) .
That is, the growth was fast in a PDA agar plate medium adjusted to pH 5.6 at 25 ° C., and a white-green-brown, wooly colony was formed by culturing for 14 days as shown in FIG. Observation using a microscope is shown in FIG. The vegetative mycelium was colorless to brown and had a partition wall and had a feature such that a part of the mycelium swelled, but no conidia were formed.

Culture Conditions The microorganisms for producing the aminopolysaccharide of the present invention can be cultured by a conventional aerobic culture method. The pH is 3 to 8, preferably 4 to 7, and the temperature is 20 to 35 ° C., preferably The culture time is 25 to 30 ° C., and the culture time is 72 to 168 hours, preferably 120 to 144 hours.

As a nutrient source, any of a natural medium and a synthetic medium in which commonly used carbon source, nitrogen source, inorganic salt, etc. are mixed and dissolved in an appropriate ratio can be used. Glucose, sucrose, molasses, etc. can be used as the carbon source, and these may be added at a concentration of 2 to 10%, preferably 3 to 5%. Peptone, yeast extract, corn steep liquor, meat extract, ammonium sulfate, etc. can be used as the nitrogen source. You may use these 0.1 to 2% individually or in combination. Further, Mg and Ca are added as inorganic salts. Magnesium sulfate · 7 hydrate is used as Mg and calcium carbonate is used as Ca, and 0.1 to 1%, preferably 0.3 to 0.6%, is added respectively.

When an agar medium is used as the synthetic medium, the medium is not particularly limited as long as it can grow on a normal agar medium or the like, and a PDA agar medium often used as a mold growth medium is suitable.

Separation method The method for separating the aminopolysaccharide produced by the cells is not particularly limited, and for example, ethanol precipitation, which is a very common method, can be used. After adding 2 to 3 times the amount of ethanol to the culture supernatant after centrifugation, the precipitate settles down. After removing the upper part with a skewer, the precipitate is centrifuged at about 1000 to 3000 rpm. When separated, it is obtained as an off-white cake.

As a method for preserving the strain of the present invention, storage by drying such as freeze-drying is not preferred because it does not form spores, and it can be stably stored in -80 ° C. or liquid nitrogen using a glycerin stock.

As described above, the aminopolysaccharide of the present invention has a β-1,4 bond of glucosamine similar to chitosan and has a similar structure. On the other hand, polysaccharides collectively called β-glucan are also known to have β-1,4 bonds. It has been reported that β-glucan has various molecular species depending on the presence / absence of branching, the molecular weight, and the type of sugar to be composed. Thus, chitosan, the product of the present invention and β-glucan all have β-bonds and have similar basic structures.
A component containing β-glucan has been reported to have various physiological activities such as an action to enhance immune activity, an immune function activation action, an antitumor activity, an intestinal bacterial growth action, a moisturizing action, and a heavy metal excretion action. Many similar physiological activities have been reported for chitosan, and it is easily assumed that the physiological activity is due to a basic structure having a β-bond.
Furthermore, barley-derived β-glucan has been reported as having a binding mode similar to that of the present invention. (Saitama University: Development of barley foods focusing on the health maintenance and promotion function of barley β-glucan) Barley β-glucan has a non-branched linear structure and consists of two types of β1 → 3 bonds and β1 → 4 bonds. It is shown that β1 → 3 bonds are present at a ratio of 1 to 2 of consecutive β1 → 4, and β1 → 3 bonds are not continuously present, with -D-glucose as a constituent sugar. It has been reported that the physiological function of this substance is cholesterol lowering action, blood glucose level improvement, immune function promotion, etc., and the US Food and Drug Administration (FDA) has recognized that it has the action of lowering serum cholesterol level as a health claiming substance. Authorized.
Thus, it can be easily assumed that the product of the present invention has the same effect as chitosan from the accumulation of knowledge on the physiological activities of β-glucan and chitosan.

That is, the aminopolysaccharide of the present invention can be used in various applications such as foods such as health foods, food additives, feeds, pharmaceuticals, cosmetics, wastewater purification / sludge flocculants, and the like, similar to chitosan.
Examples of health foods include cholesterol lowering, blood pressure lowering, fat absorption inhibition, intestinal bacteria activation, or glucosamine raw materials for improving knee joint pain, and pharmaceuticals such as immune cell activation, cancer Examples of cell growth suppression applications include cosmetics such as moisturizers such as lotions, and examples of food additives include antibacterial and antifungal applications.

Regarding wastewater purification / sludge flocculant, radioactive substances and heavy metals in waste liquid can be easily purified. As a purification method, it is possible to use an agglomeration precipitation method in which an aqueous solution or a powder is dispersed and adsorbed and precipitated. When spraying with an aqueous solution, depending on the scale of the waste liquid, it is more efficient to add it at a high concentration. For example, in the case of an acidic aqueous solution, it is preferably added at a concentration of 10% by weight or higher. Specific examples of the heavy metal include Zn, Cu, Fe and the like. Conventionally, as disclosed in JP-A-8-68893 and the like, it has been known that chitosan is packed into a column and the waste liquid is passed through and adsorbed and removed. However, since the amino polysaccharide of the present invention is water-soluble, it is packed into the column. The waste liquid can be easily purified without performing the process of passing the waste liquid.

Next, the present invention will be described more specifically with reference to examples and comparative examples. However, the present invention is not limited only to these examples.

Example 1
In an Erlenmeyer flask, dissolve in ion-exchanged distilled water at a ratio of 3% glucose, 1.5% peptone, 0.1% yeast extract, 0.5% ammonium sulfate, 0.1% sodium chloride, 0.3% magnesium sulfate, 0.4% calcium carbonate. 400 ml of the prepared medium was adjusted to pH 7, sterilized by autoclave, inoculated with NITE P-01575 strain, and cultured for 7 days at a culture temperature of 25 ° C. and a rotation speed of 180 rpm.

Qualitative colorimetric coloration of amino sugar by indole-hydrochloric acid method with reference to Chitin Chitosan Experiment Manual (Gihodo Publishing Co., Ltd.) edited by Chitin Chitosan Experimental Manual The analysis was performed according to the following procedure, which was used as a guideline for production.
Qualitative colorimetric analysis of amino sugars 1) 1 ml of the culture supernatant after nitrous acid degradation centrifugation was put in a test tube and 10 μl of acetic acid was added. To this was added 1 ml of a 10% aqueous sodium nitrite solution and 1 ml of a 5% aqueous potassium hydrogen sulfate solution, and the mixture was allowed to react overnight at room temperature. The next day, 2 ml of 12.5% ammonium sulfamate aqueous solution was added and shaken for 10 minutes.
2) Indole-HCL method The nitrous acid decomposition product obtained after completion of shaking was allowed to stand for 30 minutes, after which 0.1 ml of a 1% indole-ethanol solution was added and reacted in a boiling water bath for 5 minutes. After sufficiently cooling in ice water, 1 ml of ethanol was added. The OD (λ = 492 nm) of this color developing solution was measured.

FIG. 3 shows the changes over time, with the horizontal axis representing the number of culture days and the vertical axis representing OD _{λ = 492 nm} . As the graph shows, production started from the beginning of the culture and increased with the passage of the day, reaching a maximum at about 7 days. On the seventh day after the start of the culture, the culture was stopped, and after centrifugation, 300 ml of the culture supernatant was obtained. When 3 times the amount of ethanol was added thereto and allowed to stand, precipitation gradually progressed, and after removing the supernatant by decantation, the precipitate was centrifuged at 3000 rpm, washed twice with ethanol and dried in vacuo. It was 0.28 g as a crude product.
This confirmed that the product of the present invention was an aminopolysaccharide.

Examples 2-1 and 2-2
The culture temperature was 30 ° C., and the medium composition was cultured using the same method as in Example 1 except that the carbon source was glucose (Example 2-1) or sucrose (Example 2-2). Compared. FIG. 4 shows changes over time. Compared to Example 1, the growth was slightly faster, and the upper limit of production was reached in 4-5 days. There was no difference in production due to differences in carbon sources, and both were successfully assimilated.

Refill ion-exchange distilled water in a timely manner while concentrating by ultrafiltration using a filter for filtration having a molecular weight cut off of 50000 (Adiacon Diaflow Membrane XM50) in Millipore stirring cell 8400 → Concentration → Ion-exchange distilled water The replenishment was repeated, and the concentrated solution was monitored by GPC analysis by HPLC of Example 3 every time of concentration, and extraction and purification were performed when the peak of the low molecular fraction was not recognized as a guideline for the end of the ultraconcentration. Since the product of the present invention has a very low viscosity, the filtration rate does not decrease due to the progress of concentration, and the concentration and desalting proceeded very smoothly simultaneously. By using an ultrafilter (Millipore stirring type cell 8400), if the amount of liquid was about 300 ml, concentration desalting proceeded simultaneously in one day (not at night), and a purified concentrated solution was obtained in a short time. After concentration to an appropriate amount, the product of the present invention was obtained by lyophilization. Yields were 0.29 g (Example 2-1) and 0.25 g (Example 2-2), respectively.

Example 3
Culture was performed for about 1 week under the same medium composition and culture conditions as in Example 1, and 350 ml of the supernatant was obtained from 400 ml of the culture solution. The supernatant was subjected to ultraconcentration by Millipore stirring cell 8400. The filter used at this time had a molecular weight cut off of 50000 (Diaflow Membrane XM50 manufactured by Amicon).

Concentrated desalting was performed by the following steps. Concentrate 350 ml of the supernatant to 70 ml by ultrafiltration, add 250 ml of ion-exchanged distilled water, repeat the operation of concentration and desalting again to 70 ml for a total of 6 times, and the 6th time, concentrate to 50 ml and freeze-dry it. did. The yield was 0.36 g and the culture supernatant was 0.1%.

FIG. 5 shows a chromatographic result obtained by taking a part of a 50 ml concentrated purified solution in a microsyringe and analyzing it with a GPC column by HPLC under the following conditions. It was confirmed that the peak in the latter half 14 minutes was derived from the eluent. As the figure shows, no peaks other than the product of the present invention were detected, and the product was purified to a purity of 90% or more.
Column: Shodex Asahipack GF510HQ7.6 × 300mm (exclusion limit molecular weight 300,000)
Temperature: 35 ° C
Solvent: 1% acetic acid Flow rate: 1 ml / m
Detection: RI
Injection volume: 20 μl

Example 4-1 Solubility test Culture was carried out under the same conditions as in Example 1 except that sucrose was used as a carbon source. The culture supernatant at this time was 250 ml. After the cultivation, it was subjected to ultraconcentration and freeze-dried in the same manner as in Example 3. The yield was 0.28 g and the culture supernatant was 0.11%.
Furthermore, 20 mg of the freeze-dried product was taken into a small test tube, 0.2 ml of water was added, and the dissolved state was visually observed in a water bath of an ultrasonic cleaner as a 10% water suspension. When the whole could not be dissolved, water was successively added to the solution and the state of dissolution was observed. When water was added to the equivalent of 5%, the whole was dissolved and became a light brown solution.

FIG. 6 shows the chromatographic results when a 0.23% / 1% acetic acid solution of a lyophilized product was prepared and analyzed using a GPC column by HPLC under the same conditions as in Example 3. It was a peak of the aminopolysaccharide of the present invention alone, the ratio by area ratio was 95%, and it was eluted at the same retention time as the peak of FIG.

Example 4-2
5 mg of the freeze-dried product obtained in the same manner as in Example 4-1 was taken in a small test tube, 0.2 ml of 1% acetic acid was added thereto, and the dissolved state was visually observed. Because it dissolved, an additional 5 mg was added. Since it was dissolved, 5 mg each was added two more times, and it was dissolved. It was confirmed that at least 10% equivalent was dissolved in 1% acetic acid. Furthermore, it was speculated that further dissolution was possible.

Example 4-3
10 mg of the freeze-dried product obtained in the same manner as in Example 4-1 was put in a small test tube, 0.5 ml of 2% sodium hydroxide was added thereto, and the dissolved state was visually observed. Since it melt | dissolved, it confirmed that 2% equivalent melt | dissolved.

Example 5 Component Analysis Elemental analysis was performed on the amino polysaccharide obtained in Example 1 and chitosan preparation (Chitosan 7B Lot No. TB25-M: Funakoshi Co., Ltd.) as Reference Example 1. The results are shown in Table 1. Elemental analysis showed a component content very similar to that of chitosan 7B.
In addition, protein and lipid are not included, and in the colorimetric analysis of sugars by the phenol sulfate method “quantitative method for reducing sugars, Fukui Sakuzo (Tokyo University Press)”, the same level as chitosan, ie, the same level as the control (water). OD (λ = 490 nm) was indicated, and it was confirmed that hexoses were not contained.

Example 6 Confirmation of Constituent Sugar Next, the constituent sugar of the aminopolysaccharide of the present invention was confirmed by colorimetric analysis using the Elson-Morgan method “Quantitative method for reducing sugar, Sakuzo Fukui (University of Tokyo Press)”. . The Elson-Morgan method is a colorimetric analysis for monosaccharides, and in the case of a high molecular weight such as the aminopolysaccharide of the present invention, it needs to be hydrolyzed in advance, so that non-patent literature (Biotechnology Letters 2005 Vol. 27. With reference to the hydrolysis conditions of chitosan described in 13-18), a 1% solution in 6N hydrochloric acid was prepared, and hydrolysis was performed at 115 ° C. for 3 hours.

The resulting strongly acidic hydrolyzate is diluted with water to an extent that does not interfere with color development, and diluted to a final hydrochloric acid concentration of 0.12N (hydrolyzate amount 60 μg / 0.12N hydrochloric acid 1 ml). Colorimetric analysis was performed by the N-Morgan method. For comparison, a hydrolyzate of chitosan preparation and a glucosamine preparation were prepared and analyzed in the same concentration as a control. The results are shown in Table 2.
As shown by the value of OD (λ = 530 nm), the OD of the hydrolyzed chitosan is approximately similar to the OD of the glucosamine preparation within an error range, but the amino polysaccharide of Example 6 is about 86% of the glucosamine preparation. It became the value of. This difference is presumed that the hydrolysis conditions for the aminopolysaccharides of the present invention are not necessarily the optimum conditions, so that some of them were left uncut or the purity was inferior to that of chitosan preparation due to some impurities (elemental analysis values). Thus, it was speculated that the product of the present invention would be a homopolymer having amino sugar as a constituent sugar.

Example 7 Identification of Constituent Sugar Next, it was confirmed what kind of amino sugar the constituent sugar was. 1H-NMR and 13C-NMR measurements were performed on the hydrolyzate, glucosamine preparation, and chitosan hydrolyzate obtained in Example 6.
For NMR, JEOL RESONANCE ECA-500 manufactured by JEOL Ltd. was used, and 3- (trimethylsilyl) -2,2, '3,3'-tetradeuteropropionic acid (TSP-d4) was used as a reference substance for chemical shift in heavy water as a solvent. What added 0.1% was used.
As shown in Table 3, each sample was dissolved at 0.3 to 0.5 wt% and measured at room temperature. The results are shown in FIGS. The spectra of each sample were consistent and it was found that the constituent sugar was glucosamine.

Example 8 Enzyme reaction Assuming a structure equivalent to that of chitosan, it should be decomposed by chitosanase to liberate glucosamine. To verify this, an enzyme reaction using chitosanase was carried out, and then the reaction mixture was subjected to the Elson-Morgan method. Colorimetric analysis was performed. In this case, quantification was not the purpose, and the difference with chitosan was verified qualitatively by the presence or absence of color development (pink) in response to the Elson-Morgan method.

A 0.5% solution of the amino polysaccharide obtained in Example 3 in 0.1M acetate buffer (pH 5.6) and chitosanase (Bacillus pumilus BN-262: Wako Pure Chemical Industries, Ltd.) powder in 0.1M What was dissolved in an acetate buffer (pH 5.6) and prepared at a ratio of 0.04 mg / 1 ml was reacted at 40 ° C., a certain amount was sampled over time, and colorimetrically analyzed by the Elson-Morgan method.

As a control, a chitosan preparation made into a 0.1% solution in 1% acetic acid and adjusted to pH 5.6 was reacted with chitosanase as a reaction solution at 40 ° C. A certain amount was sampled over time, and the ratio was measured by the Elson-Morgan method. Color analysis was performed.

The chitosan preparation developed a strong color (pink) by the Elson-Morgan method over the course of the reaction time, and it was revealed that glucosamine was released over time in response to chitosanase. That is, chitosanase showed the natural result that the β-1,4-bond of β-1,4-polyglucosamine (chitosan) was hydrolyzed to the endo type. On the other hand, the product of the present invention did not respond at all to the Elson-Morgan method, and the reaction solution after any time passed was colorless and transparent. That is, although the constituent sugar of the aminopolysaccharide of the present invention is glucosamine, the result suggests that the binding mode is different from that of chitosan.

Example 9 Binding mode Enzyme analysis revealed that the aminopolysaccharide glucosamine binding mode of the present invention was different from that of conventional chitosan, and the following structural analysis was performed in order to clarify this binding mode.

Measurement of infrared absorption spectrum The product of the present invention and the chitosan preparation were subjected to Fourier transform infrared spectroscopy. The results are shown in FIG. Both show similar absorption spectra, and are derived from the amide group, OH stretching vibration 3300 ± 100 cm ⁻¹ , CH stretching vibration 2900 ± 50 cm ⁻¹ , NH stretching vibration 3300 ± 100 cm ⁻¹ , which are absorption spectra peculiar to chitin and chitosan. Absorption was observed at 1550-1650 cm ⁻¹ .

It is known that the α ¹ type has 844 ± 10 cm ^−1, and the β type has 89 ¹ ± 10 cm ⁻¹ C ₁ -H bending vibration as absorption characteristics of sugar. The chitosan preparation was found to be in the vicinity of 896 cm ⁻¹ , and the product of the present invention was also found to be in the β-type because absorption was seen at the same position.

Example 10 Coupling position
NMR measurement
Although the binding mode was found to be β-type, it was speculated that the bonding position of carbon was different from that of chitosan. Therefore, in order to clarify this, the binding position of each of the product of the present invention and the chitosan sample obtained in Example 3 was analyzed by 1H-NMR. For measurement of 1H-NMR, JEOL RESONANCE ECA-500 manufactured by JEOL Ltd. was used, and the solvent used was 1% heavy acetic acid in heavy water and 0.1% TSP-d4 as a chemical shift reference substance. This was measured by dissolving 1 wt% of each sample. The measurement temperature was 50 ° C.

The results of 1H-NMR spectra of the product of the present invention and the chitosan preparation are shown in FIGS. In chitosan, one H-1 signal at C-1 position derived from β-1 → 4 bond was detected at 4.85 ppm, and in the present invention, signals considered to be H at C-1 position were 5.1 ppm and 2 at 4.8 ppm. This was detected. Since there are two types of binding and the 1H integration ratio is 1: 1, it became clear that there are the same number of two types of binding.

The result of the spectrum by 13 C-NMR of the heavy water + 1% biacetic acid solution of this invention goods is shown in FIG. There are 12 C sites, and two signals belonging to C-1 are detected in the vicinity of 100 ppm, indicating that there are two types of sugar binding.

FIG. 17 shows the result of two-dimensional measurement (COSY, HMQC, HMBC, HMQC-TOCSY) of the present invention. In HMBC, the correlation between C and H separated by 2 to 3 bonds can be observed. The correlation of ₃ J _CH across O was confirmed. H at C-3 and C at C-1 '(1H: 4.1ppm and 13C: 99.4ppm), H at C-1' and C at C-3 (1H: 5.1ppm and 13C: 80.6ppm) ) Indicates β1 → 3 binding. H at C-4 'and C at C-1 (1H: 4.0ppm and 13C: 100.5ppm), H at C-1 and C at C-4' (1H: 4.8pm and 13C: 78.8ppm ) Indicates β1 → 4 binding. From this, the two types of binding are β1 → 3 and β1 → 4, and there are 12 C sites, so it is concluded that β1 → 3 and β1 → 4 are alternately linked. did.

Example 11 Molecular Weight Measurement An ultra-concentrated solution obtained by passing 300 ml of a culture supernatant obtained under the same culture conditions as in Example 1 through a filter for filtration (Diaflow Membrane XM50 manufactured by Amicon) was added with 2% sodium hydroxide. After concentration by precipitation by salting out with ammonium sulfate while adjusting the pH to 8 to 10 with a solution, this was dissolved in water, dialyzed under running water and freeze-dried to obtain a 0.2% solution in 1% acetic acid solution. HPLC was measured under the same conditions as in 3. In addition, amino sugar pullulan (Shodex STANDARD P-82) consisting of five kinds of kit products having different molecular weights was also measured as a standard sample for molecular weight test, and the molecular weight was estimated by comparing the retention times. At this time, the chitosan preparation was also measured and estimated in the same manner.

The results are shown in FIG. The running water dialysate was lyophilized without further fractionation, but no high molecular weight products other than the product of the present invention were detected and only the product of the present invention was selectively produced. The molecular weight was confirmed to be equivalent to 344,000 of the standard sample pullulan. The chitosan preparation was also eluted with approximately the same retention time.

Example 12 Viscosity
A 0.05, 0.1, 0.25, and 0.5% aqueous solution of the aminopolysaccharide obtained in Example 3 was prepared, and a chitosan preparation (Chitosan 7B Lot. NoTB25-M: manufactured by Funakoshi Co., Ltd.) was 0.05 with respect to 1% acetic acid. 0.1, 0.25, and 0.5% concentrations were prepared, and the viscosities at the respective concentrations were measured at 25 ° C. using an SV type viscometer (SV-10) manufactured by A & D Corporation. FIG. 19 shows the result.

The solution using the conventional chitosan preparation showed high viscosity as the concentration increased, whereas the increase in viscosity of the product of the present invention was extremely gradual. It was no exaggeration to say that it has almost no viscosity.

Examples 13-1 to 13-10, Comparative Examples 13-1 to 13-10 Purification of waste liquid 1 ml of 1% aqueous solution of each metal shown in Table 5 was prepared, and to this, 2 of the chitosan-like material obtained in Example 4 was prepared. The presence or absence of precipitation was qualitatively observed when 100 μl of a 1% solution (1% acetic acid) of conventional chitosan (chitosan 7B) was added as a comparative example. The results are shown in Table 4.
-: No precipitation formation +: Very small amount of precipitation formation ++: Gradual precipitation formation +++: Instantaneous precipitation formation In the case of chitosan 7B as a comparative example, a precipitation forming action was observed only for ferrous sulfate. On the other hand, the chitosan-like product of the present invention exhibited a precipitation forming action in the range of + to +++ in Examples 13-1 to 13-5, and had a higher precipitation forming ability and purification action than the conventional chitosan 7B. It was shown to have

Example 14 This strain was cultured for 5 days under the same conditions as in Example 1 except that sucrose was used as the purified carbon source using zinc sulfate heptahydrate. After completion of the culture, the mixture was centrifuged to obtain 65 ml of supernatant (pH 7.2). To this, 3 to 4 ml of a 1% aqueous solution of zinc sulfate heptahydrate was added and stirred. Simultaneously with stirring, a precipitate formed and gradually settled. When the precipitate was obtained by centrifugation, 10 ml of water was added and washed with stirring. This was repeated twice and then suspended again in 10 ml of water. To this was added 5 ml of strongly acidic ion exchange resin Dowex 50 [H], and the mixture was shaken and stirred. The supernatant was aspirated with a pipette and transferred to another container. The resin part was washed with water, and after standing, the supernatant part was sucked up with a pipette, transferred to the same container, and combined into one. Lyophilized to obtain 0.083 g. Yield 0.13%.

  The present invention can be expected to be widely applied to foods, health foods, supplements, food additives, cosmetics, toiletries, chemicals, pharmaceuticals, waste liquid treatment, and the like.

Claims (12)

An aminopolysaccharide having a structure in which glucosamine is bound in a structure in which β1 → 3 bonds and β1 → 4 bonds are alternately linked. Infrared absorption ^{spectrum: 891 ± 10 cm -1, 3300} ± 100cm -1, 2900 ± 50cm -1, 3300 ± 100cm -1, peak to 1550~1650cm ^-1 - amino polysaccharide according to claim 1 having a click. The aminopolysaccharide according to claim 2, wherein the elemental analysis value (% by weight) is as follows.
C: 38-40
H: 6-9
N: 6-9
O: 38-40  The aminopolysaccharide according to claim 2 or 3, which has a molecular weight of 300,000 to 500,000. The aminopolysaccharide according to any one of claims 1 to 4, produced by a Peyronellaea calorpreferens NITE P-01575 strain or a natural or artificial variant thereof. An aqueous solution containing the aminopolysaccharide according to any one of claims 1 to 5 and having a viscosity of 5 cP or less. A 15% by weight or less aqueous solution containing the aminopolysaccharide according to any one of claims 1 to 5. A waste liquid purifying agent comprising the aminopolysaccharide according to any one of claims 1 to 5. A purification method comprising purifying the waste liquid purifying agent according to claim 8 by a coagulation precipitation method. The foodstuff containing the amino polysaccharide in any one of Claims 1-5. The feed containing the amino polysaccharide in any one of Claims 1-5. The pharmaceutical containing the amino polysaccharide in any one of Claims 1-5.