JPS6131095A - Mucilaginous polysaccharide and preparation thereof - Google Patents

Abstract

PURPOSE:To prepare a novel mucilaginous polysaccharide, by cultivating Spirulina subsalsa in preparing the novel mucilaginous polysaccharide, and extracting the aimed substance from the cultivated algal body. CONSTITUTION:Spirulina sabsalsa is cultivated to separate the algal body, which is then extracted with a lower alcohol to extract and remove dyestuff in the algal body. The algal body is then extracted with hot water containing sodium hydrogencarbonate and sodium chloride, and a precipitant is added to the extract solution to precipitate the aimed mucilaginous polysaccharide. The resultant mucilaginous polysaccharide contains rhamnose, fucose, xylose, galactose, glucose, mannose, uronic acid and N-acetylated saccharide as constituent components.

Description

[Detailed description of the invention] (Industrial usage Wf) This invention is based on the algae Spirulina subsalf (8
The present invention relates to a novel mucilage polysaccharide produced by P. pjrulina subsalwa and a method for producing the same. In this specification, this sticky polysaccharide is referred to as spirulinan (spiru-1
1nan).

(Prior Art) It is well known that algae, especially multicellular red algae, brown algae, and green algae, produce sticky polysaccharides such as agar, carrageenan, alginic acid, and fucoidan, but microalgae, especially blue-green algae, It is not known that it produces mucilage polysaccharides.

(Problems to be Solved by the Invention) An object of the present invention is to provide svirulinan, a novel sticky polysaccharide produced by Spirulina sapsarsa, a type of microalgae, and a method for producing the same.

(Means for Solving the Problems) (1) Production Algae A typical example of algae used in the production of spirulinan of this invention is Spirulina subsalsa, but any algae capable of producing spirulinan can be used. Either can be used in this invention.

Representative algae include Spirulina, Sapsarsa, Onirst, Variety, and Spiru-1ina.
tubsa19a 0erst, var, class
ior), which was isolated in pure form from hot spring water in the Imperial Valley, California, USA. When this algae is cultured under the following conditions, it grows by elongating its filamentous algal bodies and reaches %i+ on the liquid surface. It has a more compact spiral filament than Spirulina platens.

(2) Cultivation When culturing Spirulina sapsalsa in carrying out the present invention, the medium composition and culture conditions are not particularly limited as long as the Spirulina sapsalsa of the present invention can grow. For example, as a medium, NaHCO3, K2HPO
4, NaNO3, NaC6°Mf804. CaCl2
．． A medium containing FeSO4 and other trace amounts of inorganic salts can be used.

When culturing, it is necessary to irradiate light.1. Natural light (sunlight) f: can be used as this light, but in order to efficiently culture algae, it is preferable to use an artificial light source such as a fluorescent lamp. The culture temperature is preferably in the range of 37°C to 40°C, but the culture can also be carried out at 40°C to 45°C.

When cultured under such conditions, the Spirulina subsalsa of the present invention secretes polysaccharides outside the algae in the form of villi.

(3) Collection of svirulinan In order to collect svirulinan from the culture obtained as described above, it is preferable to first separate and harvest the algae together with the spirulinan secreted outside the algae.

For this algae separation, commonly used separation means such as centrifugation, F
Using hendo is f! Ru. Next, it is preferable to remove the pigments in the algal bodies by heating with a lower alcohol such as methanol, ethanol, acetone, or ether, or by heating with these sequentially to extract and separate the pigments in the algal bodies. Next, the algae after this decolorization treatment is heated at high temperature with water or an aqueous solution containing a basic agent such as sodium carbonate, for example, an aqueous solution containing 0.1% sodium bicarbonate and 0.1rX sodium chloride. , for example at about 90°C, for example for 1 hour N
Extract over jK. This extraction operation yields an aqueous solution containing spirulinan. Spirulinan is precipitated by adding a suitable precipitant to this aqueous solution.

As a precipitant, for example, cetyltrimethylammonium bromide (Cetabron) can be used in a concentration of 2 to 1. Also, instead of this method, ethanol,
For example, spirulinan can be precipitated by adding about 3 times the volume of the spirulinan-containing aqueous solution.

The precipitate thus obtained is separated by a conventional method such as filtration or centrifugation. Crude spirulinan can be obtained by drying this, but in order to obtain even purer spirulinan, it is preferable to wash this precipitate with ethanol, ether, etc. to remove impurity gold, and then dry it.

In this way, white spiranan is obtained with a yield of 10-55%.

(4) Separation of λ-spirulinan and κ-spirulinan The spirulinan obtained as described above can be divided into two fractions based on its behavior towards potassium chloride. That is, the above-mentioned spirulinan is dissolved in hot distilled water to a concentration of 01, potassium chloride is added thereto, the concentration is set to α3M, and the solution is cooled on ice. Next, it is divided into an insoluble fraction and a soluble fraction. The insoluble fraction is washed with 80% ethanol to remove chlorine, then washed with ethanol, acetone and ether, and dried to separate the κ-type mucilage polysaccharide which is insoluble in an aqueous potassium chloride solution. This is called ni-spirulinan.

On the other hand, a precipitate is generated by adding 2.5 times the volume of 100% ethanol to the soluble fraction, and this precipitate is separated,
By washing with ethanol, acetone and ether and drying, a λ-type sticky polysaccharide soluble in a potassium chloride water bath is obtained. This is called λ-spirulinan.

In this specification, when simply referred to as spirulinan, the term ``spirulinan'' refers to a mixture of λ-spirurinan and E-spirulinan, when it refers to each of λ-spirulinan and E-spirulinan, and when it refers to these collectively. There are cases.

(5) Scientific properties of spirulinan ■ Elemental analysis and ash content The results of the elemental analysis and measurement of ash content are shown in Table 1 below.

Table 1 ■ Detection reaction The results of various detection reactions are shown in Table 2 below.

As is clear from Table 2, Yaphe's resorcinol reaction did not show an obvious positive result, and the spirulinane of this invention does not contain 3,6-anhydrogalactose, or even if it does, its t is extremely small. , clearly different from carrageenan.

This is consistent with the optical results obtained by hydrolyzing spirulinan t-1N with hydrochloric acid for 2 hours and examining its constituent components by paper chromatography.

Mata, the carbazole-sulfuric acid reaction which is a qualitative test of uronic acid, and the qualitative test of N-acetyhexosamine 1%! Morgan-Blson
All reactions were positive, and spirulinan was detected as uron.
and N-acetylhexosamine.

Furthermore, since the Dodgi+on reaction was positive, it was confirmed that subirulinan contains a sulfate group.

(2) IR spectrum The IFL-spectrum is shown in FIG. In this diagram,
Near 172551, absorption derived from the aldehyde group or carbon group of carboxylic acid is observed.

The presence of the C═O group in the carbonic acid indicates that κ-spirulinane and λ-spirulinane in the form of potassium salts
It is also estimated by the appearance of nearby absorption. 1660m
Absorption of acetamide near m-' and 1530tan-1,
Absorption derived from the methyl group is seen near 1450m-" and 1400cIM-1. Absorption derived from the sulfuric ester (S-0) group is seen at 1240ffl-1, and C-0-8 at 820m-1 and 840cIM-1. Absorption derived from the group is seen, indicating the presence of sulfate ester.The absorption at 8203-' indicates the presence of equatorially bound 6-sulfate, and the absorption at 840 crs-' indicates the presence of axially bound 4-sulfate. The absorption at 940 m-'', indicating the presence of 3.6-anhydrogalactose, was slight.

The above IFL spectrum results agree well with the detection results described above.

■ The constituent sugar spirulinan is treated with trimethyl sylate (trimetll).
When the hydrolyzate was analyzed by gas chromatography, the results shown in the following Tx5 table were obtained.

As is clear from Table 3 below, spirulinan contains a small amount of tomorhamnose, fucose, xylose, galactose, glucose, and himannose, and is expected to contain other sugars as well.

The presence of galactose and glutenose was also confirmed by test # using each oxidase.

■ Gel-forming property The spirulinan of this invention has K4. Gels are formed in the presence of Nl-[, +, or Ca+.

The mucilage polysaccharide with the above properties is Spirulina prana 7
Cis (5pirulina platen9is) K
It is a novel sticky polysaccharide, as its existence has not been confirmed in either blue-green algae or other blue-green algae.

(Effects of the Invention) The present invention provides spirulinan, a novel sticky polysaccharide.

This spirulinan is produced by Spirulina sapsarsa, a microalgae that can be artificially cultured, and therefore can be advantageously produced industrially.

Moreover, this spirulinan is tasteless and odorless.

In addition, it has the property of forming a gel with +, N) 14+, and Ca++, so it is used as a raw material for foods, medicines, and other industrial industries.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Production of subirrinan A medium having the following composition was prepared.

K2HPO40,s: CaCl2Q,0'
; H2O1, Ot; NaNOs 2.5:
FeSO4Q,01:NaC1,1,0:ED
TA 00os; (*) ^5 solution H3SO52,86?: MnCl2.4H20j,
81f: Zn804.7H200,22: CuSO
4,5H20o, oa ; Na2MO4Q, 021:
Conc, H2SO41 drop: H2O1, Ot; Spirulina subsarsa was inoculated into this medium 100-
The cells were cultured at 37° C. to 40° C. under irradiation with a fluorescent lamp at 4000 lux and without aeration of carbon dioxide.

It was confirmed that mucilage polysaccharide was secreted outside the algae during the culture process. Under these conditions, this strain grew by elongating its filamentous body and floated to the surface of the liquid. It formed a more compact spiral filament compared to Spirulina platensis.

Progress of culture (increase in algal bodies and production of spirulinan) t-
Shown in Figure 512.

Wash the algae with water, add 80% methanol, 80π acetone%1
The dye was removed by boiling with 0.00% ethanol and ether. Collect the algae and 0.2
The dispirulinane was extracted by heating at 90° C. for 1 hour in an aqueous solution containing sodium chloride and 0.1% sodium bicarbonate and treated with F.

Subirurinan was precipitated by adding cetyltrimethylammonium bromide (-1!tribrone) to the subirurinan extract solution in a ratio of 2 to 1. The precipitate was washed with 80π ethanol, 100% ethanol, and ether, and dried. As a result, white subirrinan 44w#I was obtained.

Example 2 Production of Spirurinan Svirulinan was produced in the same manner as in Example 1, and
However, in the extraction of spirulinan, distilled water was used instead of water containing sodium chloride and sodium hydrogen carbonate. Almost the same results as in Example 1 were obtained.

Example 3 Production of spirulinan Spirulinan was produced in the same manner as in Example 1.

However, 3 times the volume of 100% ethanol was used instead of 2% cetyltrimethylammonium bromide in the step of precipitating spirulinan from the svirulinan extract. Almost the same results as in Example 1 were obtained.

Example 4 Separation of λ-Spirulinan and κ-Svirulinan Spirulinan Q prepared according to the method described in Example 1, 1 r'i, was heated to 90° C. with vigorous stirring.
．． It was dissolved in distilled water to a concentration of 1:1 and incubated in the oven. 0.1 volume of 3M potassium chloride solution was added to Solution F, the solution was mixed and cooled in a crushed ice bath for 1 hour. Centrifugation at 10.00 OrpmK for 1 hour at 50°C;
It was divided into an insoluble fraction and a soluble fraction.

The insoluble fraction was resuspended in α3M sodium chloride solution, centrifuged, the insoluble material was dechlorinated by washing with 80% ethanol, and washed with 100π ethanol, acetone and ether, dried and - Subirinan cL03 fft obtained fC,.

On the other hand, the soluble fraction was precipitated by adding 2.5 volumes of 100% ethanol, washed with 80% ethanol, 100% ethanol, acetone, and ether, and dried;
Seussville linane 0.069 f was obtained.

The ratio of λ-spirulinan to κ-svirulinan is approximately 69
．． 1=30.9.

[Brief explanation of drawings]

FIG. 1 shows the IR spectrum of spirulinan of the present invention, and the IIZ diagram shows the progress of culturing Spirulina subsalsa.

Claims (1)

[Claims] 1. At least rhamnose, fucose,
xylose, galactose, glucose, mannose,
Spirulina subsalsa contains uronic acid and N-acetyl sugar.
) is a mucilage polysaccharide produced by 2. The mucilage polysaccharide according to claim 1, comprising a λ-type mucilage polysaccharide soluble in an aqueous potassium chloride solution and a κ-type mucilage polysaccharide insoluble in an aqueous potassium chloride solution. 3. The mucilage polysaccharide according to claim 1, which is a λ-type mucilage polysaccharide soluble in an aqueous potassium chloride solution. 4. The mucilage polysaccharide according to claim 1, which is a κ-type mucilage polysaccharide insoluble in an aqueous potassium chloride solution. 5. At least rhamnose, fucose,
xylose, galactose, glucose, mannose,
In a method for producing a mucilage polysaccharide comprising uronic acid and N-acetyl sugar, after culturing Spirulina subsalsa capable of producing the mucilage polysaccharide, separating the cultured algae, and drying this if desired. It is characterized by removing the pigment by extraction with a lower alcohol, optionally extracting with hot water containing sodium bicarbonate and sodium chloride, and precipitating and recovering the viscous polysaccharide in this extract. How to do it. 6. The method according to claim 5, further comprising separating a λ-type mucilage polysaccharide soluble in an aqueous potassium chloride solution. 7. The method according to claim 5, further comprising separating a κ-type mucilage polysaccharide that is insoluble in an aqueous potassium chloride solution.