JP2000139490A - Production of mannose and mannooligosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain mannose and a mannooligosaccharide not discoloring with time, and high in storage stability. SOLUTION: The mannose and a mannooligosaccharide are produced by the following process: copra meal pretreated with an organic solvent such as ethanol or hexane is subjected to a reaction with an enzyme such as mannanase or an acid such as sulfuric or hydrochloric acid to isolate mannose and mannooligosaccharide or the like followed by removing reaction residues and then subjecting the mannose and mannooligosaccharide to purification process such as treatment with active carbon or desalting concentration using an ion exchange resin, thereby obtaining the objective mannose and mannooligosaccharide not discoloring even after stored at room temperature or refrigeration temperatures.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for producing mannose and mannooligosaccharide, which is useful as an additive for preventing the transmission of harmful bacteria to feed, a food or a raw material for synthesizing pharmaceuticals such as mannitol.

[0002]

2. Description of the Related Art Mannose and mannooligosaccharides have been widely used as additives for preventing the transmission of harmful bacteria to feeds, used in foods, or used as raw materials for the synthesis of pharmaceuticals such as mannitol. The present inventors can produce mannose and mannooligosaccharide at low cost by directly acting on copramil an enzyme having the activity of releasing mannose and mannooligosaccharide by acting on copra meal, which is a residue obtained by pressing coconut oil. They have found that they can do so and have already proposed new production methods [see Japanese Patent Application No. 9-31863 (mannose production method) and Japanese Patent Application No. 9-178057 (mannobiose production method)]. In these manufacturing methods,
Copramyl, a raw material, is suspended in distilled water, and after an enzymatic decomposition reaction, the reaction product is subjected to a series of purification steps to obtain a colorless sugar solution of mannose and mannooligosaccharide.

However, when a colorless sugar solution of mannose and mannooligosaccharide obtained by the production methods described in Japanese Patent Application Nos. 9-31863 and 9-178057 is stored at room temperature or refrigerated temperature, There was a problem that it was colored. For this reason, for example, when this sugar solution is added to food, the original color of the food is impaired over time. Therefore, in order to produce mannose and mannooligosaccharide on an industrial scale, it was necessary to prevent this coloring.

[0004]

SUMMARY OF THE INVENTION In view of such circumstances, the present invention has been made to provide mannose and mannooligosaccharides which are not colored over time and have high storage stability.

[0005]

That is, the present invention relates to a method for producing mannose or mannooligosaccharide, which comprises subjecting copra meal pretreated with an organic solvent to a sugar-forming reaction using an enzyme or an acid.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The copra meal used in the present invention is a pulverized residue obtained by extracting coconut oil from copra, a coconut oil raw material, obtained by drying the core meat of coconut fruit. It contains about 25% by weight of copramannan. In the present invention, copra meal of any origin or production method can be used as long as it is produced in the usual coconut oil production process.

As the organic solvent used for the pretreatment of copramyl, methanol, ethanol, butanol, propanol and hexane are preferable, and in the case of food use, ethanol and hexane are preferable. The amount of the organic solvent used in the pretreatment is preferably 1 to 10 times, more preferably 2 to 5 times the amount of copra meal in terms of weight. After adding the organic solvent to the copra meal, the organic solvent is uniformly dispersed and left for 5 minutes to 1 hour, preferably 10 minutes to 30 minutes. The temperature at which the organic solvent is added is in the range of room temperature (5
-40 ° C). Separation of pretreated copra meal and organic solvent includes sedimentation, centrifugation, and filtration.
Steps such as press filtration and pressing can be used.
Since the remaining organic solvent hinders the next sugar generation reaction,
The pretreated copra meal is washed with water such as distilled water. The amount of distilled water used is preferably 1 to 6 times, preferably 2 to 4 times the weight of copra meal in terms of weight.

[0008] The method of decomposing copramyl to produce sugar is not particularly limited as long as copramil is decomposed to release sugar, and is decomposed with an acid such as sulfuric acid or hydrochloric acid. And a method of decomposing with an enzyme.

Examples of the enzyme that degrades copramyl include mannan degrading enzymes such as mannanase (mannanase) and mannosidase. The origin of the mannan-degrading enzyme is Bacillus subtilis [Bacillu
subtilis)], a filamentous fungus [Aspergillus
Asclegilus aculea
tus), Aspergillus awamori (A. awam)
ori), Aspergillus niger (A.
r), Aspergillus usamii (A. usami)
i), Humicola insolence
nsolens), Trichoderma harzianum (Tr
ichoderma harzianum, Trichoderma koningi (T. koningi), Trichoderma longibrachiatum (T. longibrachum)
iatum), Trichoderma viride (T. vir)
de), Basidiomycete [Corticium (Corticiu)
m), Pycnopors cocchineus
us coccineus)], and an enzyme derived from Aspergillus is preferable. Among them, mannanase derived from Aspergillus niger is particularly preferred.

[0010] These enzymes are produced in the culture supernatant or cells of the above strains, but any fraction containing these enzymes may be used in the present invention. Further, if necessary, fractions containing these enzymes can be purified or partially purified by a conventional method and used. Alternatively, a commercially available enzyme may be used. Also, commercially available hemicellulases such as cellulase, xylanase, pectinase and galactanase may degrade copramyl.

The amount of the enzyme that acts on copra meal is not particularly limited, and may be any amount that releases carbohydrates.
Although it depends on the specific activity of the enzyme, for example, in the case of mannase, the amount is desirably 0.001 to 10% by weight and preferably 0.05 to 5% by weight based on the starting material copramil. More preferably, it is particularly preferably 0.01 to 2.5% by weight.

In the case of decomposition with an enzyme, for example, copra meal pretreated with an organic solvent may be suspended in an aqueous medium, the enzyme may be added thereto, and the copra meal may be decomposed with stirring. The condition for causing the enzyme to act on copramil is not particularly limited as long as it is a condition used for a normal enzyme reaction, and may be appropriately selected depending on the optimum working condition of the enzyme to be used and other factors. The reaction temperature is preferably a temperature at which the enzyme is not deactivated, and a temperature at which microorganisms are unlikely to grow in order to prevent spoilage. Specifically, 20 ° C to 90 ° C, preferably 40 ° C to
80 ° C, more preferably 50 ° C to 75 ° C. It is needless to say that the pH of the reaction solution is preferably pH 2 to 9, preferably pH 2.5 to 8, and more preferably pH 3 to 6, in which the reaction is desirably carried out under the optimal conditions of the enzyme. The reaction time depends on the amounts of copramil and enzyme to be used, but it is usually preferable to set the reaction time between 3 hours and 48 hours.

The sugar can also be released by reacting an acid such as sulfuric acid or hydrochloric acid with copra meal pretreated with an organic solvent. The concentration of the acid used is preferably 20 to 90% by volume, preferably 50 to 85% by volume, and more preferably 60 to 80% by volume in the case of sulfuric acid. The hydrolysis temperature is preferably from 80 to 121 ° C. When an enzyme or an acid is acted on under the above conditions, copramyl is decomposed, and monosaccharides such as mannose, glucose, and galactose and oligosaccharides such as mannooligosaccharides are released.

After removing the slag from the decomposition reaction of copra meal pretreated with an organic solvent, the residue is subjected to purification steps such as carbonation, activated carbon treatment, desalting with an ion exchange resin, and concentration to give a colorless sugar solution. Can be obtained. The colorless sugar solution obtained according to the present invention does not color even when stored at room temperature or refrigerated temperature.

[0015]

Next, the present invention will be described in detail with reference to examples. Example 1 After mixing 2 kg of copra meal (10% fat, 7.2% moisture) with 7 kg of special grade ethanol manufactured by Ishizu Pharmaceutical Co., Ltd.
The mixture was allowed to stand at room temperature for 10 minutes, and filtered with a membrane (product number Y110) made of Yabuta Kikai Co., Ltd. made of polyester and having an air permeability of 10 cc / cm ² to collect copra meal. Again, washing was performed in the same manner using 4 kg of distilled water. After suspending this copra meal in 20 L of distilled water, 10 g of cellulosin GM5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer 10,000 units / g) was added, and the mixture was stirred at 60 ° C. for 12 hours. Reacted.
After completion of the reaction, 20 L of a solution containing mannose was obtained. The sugar in this solution was analyzed by high performance liquid column chromatography. Using Aminex HPX-87P manufactured by Bio-Rad as an analytical column, a column temperature of 85 ° C,
Elution was performed with distilled water at a flow rate of 0.6 mL / min. The sugar was detected using a differential refractometer, and the mannose content was determined from the quantitative value of the standard product. As a result, this solution 2
0L contained 0.34 kg of mannose.

Next, this sugar solution containing mannose was pressed with a hydraulic press machine KS-2 type manufactured by Komagata Machinery Co., Ltd. to recover 18 L of sugar solution. Calcium hydroxide manufactured by Ishizu Pharmaceutical Co., Ltd. was added to this solution until the pH reached 7.2. In addition, 20 g of carbofin (trade name activated carbon activated by wood, chemical activation) manufactured by Takeda Pharmaceutical Co., Ltd.
Stirred at room temperature (25 ° C) for 20 minutes. This treatment liquid was filtered using a membrane made of Yabuta Machinery Co., Ltd. made of polypropylene and having a gas permeability of 0.5 cc / cm ² (part number Y2) under a pressure of 5 kg / m ² to obtain a fine insoluble fraction. The powdered activated carbon to which the oil was adsorbed was removed to obtain 17 L of a clear sugar solution containing mannose. This solution was treated with an anion exchange resin (Dowex SAR, OH manufactured by Muromachi Chemical Co., Ltd.).
^- type, bed Volume - No 1L), cation exchange resin (Muromachi Chemical Co., Ltd. of Dowex HCRW2, H ⁺
The solution was passed through a mold, a bed volume (1 L) and activated carbon (Diahop S80 manufactured by Mitsubishi Chemical Corporation) in this order, and a solution containing mannose was recovered. The collected solution was concentrated by an evaporator until it became Brix 70, and a sugar solution containing mannose was obtained. This sugar solution contained 0.34 kg of mannose. A indicating the coloring of this sugar solution
bs420 (absorbance at 420 nm) was 0.05. Abs when this sugar solution was left at 20 ° C. for 30 days
420 was 0.06, and no coloring was observed.

Example 2 After mixing 2 kg of copra meal and 10 kg of special grade hexane manufactured by Ishizu Pharmaceutical Co., Ltd., the mixture was allowed to stand at room temperature for 20 minutes, and then squeezed using a hydraulic press KS-2 to remove copra meal. Collected. Again, the same washing was performed using 5 kg of distilled water. After suspending this copra meal in 15 L of distilled water, Sumiteam ACH (Cellulase manufactured by Shin Nippon Chemical Co., Ltd., titer 50,000 units /
g) was added and the mixture was reacted under stirring at 55 ° C. for 24 hours. After completion of the reaction, a solution 1 containing mannose
3 L were obtained. The sugar in this solution was analyzed in the same manner as in Example 1. As a result, 13 L of this solution contained 0.37 kg of mannobiose.

Next, this sugar solution containing mannobiose is
The same purification process as in Example 1 was performed to obtain a colorless sugar solution.
This sugar solution contained 0.25 kg of mannobiose. Abs420 of this sugar solution was 0.07. Abs420 when this sugar solution was left at 20 ° C. for 30 days
Was 0.09, and no coloring was observed.

Example 3 After mixing 2 kg of copra meal and 9 kg of special grade hexane, the mixture was allowed to stand at room temperature for 20 minutes, and then a hydraulic press KS-
Copra meal was recovered by pressing with a mold.
Again, washing was performed in the same manner using 4 kg of distilled water. This copra meal was suspended in 5 L of 60% sulfuric acid (manufactured by Ishizu Pharmaceutical Co., Ltd.), and reacted at 100 ° C. for 8 hours with stirring. After completion of the reaction, the mixture was neutralized to pH 7.0 with sodium hydroxide to obtain 5 L of a solution containing mannose. The sugar in this solution was analyzed in the same manner as in Example 1. As a result, 2 L of this solution contained 0.61 kg of mannose. Then, this sugar solution containing mannose was subjected to the same purification step as in Example 1 to obtain a colorless sugar solution. This sugar solution contained 0.43 kg of mannose. Abs420 of this sugar solution was 0.02. Abs420 when this sugar solution was allowed to stand at 20 ° C. for 30 days was 0.05, and no coloring was observed.

Comparative Example A mannose sugar solution was produced in the same manner as in Example 1. However, copra meal was washed twice with 6 kg of distilled water. As a result, a colorless sugar solution containing 0.35 kg of mannose was obtained. Abs420 of the sugar solution was 0.04. Abs420 when this sugar solution was left at 20 ° C. for 30 days
Was 0.53 and coloring was observed.

[0021]

According to the method of the present invention, it is possible to obtain mannose and mannooligosaccharide which are not colored over time and have high storage stability.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Takuma Yano 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Central Research Laboratory F-term (reference) 4B064 AF02 AF04 CA21 CB07 CE09 DA01 DA10 DA11 4C057 AA06 BB02 BB04

Claims (3)

[Claims] 1. A method for producing mannose or mannooligosaccharide, comprising subjecting copra meal pretreated with an organic solvent to a sugar-forming reaction using an enzyme or an acid. 2. The method according to claim 1, wherein the organic solvent is ethanol or hexane. 3. Mannose or mannooligosaccharide obtained by the method according to claim 1 or 2.