JPH01291799A - Production of chitosan-oligosaccharide and use thereof - Google Patents

Abstract

PURPOSE:To easily produce a feed preservative, etc., at a low cost, by decomposing a chitosan solution with an enzymatic agent containing pectinase. CONSTITUTION:A chitosan solution (A) having a pH of 3-5 is produced by adding 0.3-6 pts.wt. of acetic acid to 1 pt.wt. of chitosan. The solution A is added with 0.005-0.05 pts.wt. of an enzymatic agent (B) composed mainly of pectinase originated from Aspergillus niger, etc., and subjected to enzymatic decomposition at 40-60 deg.C for >=1hr to obtain a decomposed chitosan solution (C). The solution C is added with an organic solvent such as acetone together with NaOH, etc., and adjusted to a pH of >=6.5 to produce precipitate (D). A chitosan oligosaccharide (E) is produced by subjecting the component D to centrifugal separation, washing with water and drying. The component E is added, as necessary, to a food in an amount of 0.005-0.5W/W% (based on the food) as a food preservative.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing chitosan oligosaccharide by enzymatically decomposing chitosan, and a food preservative comprising the obtained chitosan oligosaccharide.

[Prior art and problems to be solved by the invention] Chitosan is a deacetylated chitin contained in the cell walls of crustaceans such as shrimp and crabs and microorganisms, and has recently been used as a water treatment agent and chelating agent. It has started to be used for various purposes such as industrial use; medicinal use such as anti-cholesterol effects, artificial skin, and immunostimulant; agricultural use such as germination promoting effect and anti-fungal effect; cosmetic use due to its moisturizing effect, and use in the food field. .

However, chitosan is a polymeric substance, and when it is made into a solution, it is extremely viscous. For example, chitosan 2-3% (w/v
) In solution, it appears gel-like and chitosan cannot be dissolved in high concentrations. Therefore, as a method for reducing the molecular weight of chitosan, hydrolysis using hydrochloric acid (S, T,
Horowitz et al., Journal of American
Chemical Society.

79, pp. 5046-5049 (1957)), oxidative decomposition method using nitrous acid (F, Yaku et al., Cellulose Chemistry and Technology, Vol. 11).

421-430 (1977)) and the oxidative decomposition method using chlorine (Shigehiro Hirano et al. 2 Japanese Society of Agricultural Chemistry, Abstracts of the 1980 Conference, p. 330), but the chemicals used There are some drawbacks, such as safety issues, complicated handling, and the need for a large amount of energy.

In addition, reports regarding chitosanase, a chitosan-degrading enzyme, include Bacillus sp.
Chitosanase produced by Penicillium islandicum (Penicillium p.)
Chitosanase produced by S. randicul 11) and chitosanase produced by Streptomyces (Stret tow ces) are known.

However, these chitosanases have problems with productivity such as low activity and require advanced purification techniques, and are not commercially available. Papain is a chitosan decomposition method using a commercially available enzyme.

Use of cellulases and acidic proteases (JP-A-63)
-63388), but the amount of each enzyme used relative to chitosan is large, and there are problems in terms of cost, and a fully satisfactory method has not yet been obtained.

[Means to solve the problem]

Therefore, as a result of intensive research to solve the above problems, the present inventors were able to produce chitosan oligotang by enzymatically decomposing a chitosan solution with an enzyme agent whose main component is pectinase. The present inventors have discovered that a food preservative without astringency can be obtained using chitosan oligosaccharide, and have completed the present invention.

That is, the present invention provides a method for producing chitosan oligosaccharides, which comprises enzymatically decomposing a chitosan solution with an enzyme agent containing pectinase as a main component to produce chitosan oligosaccharides, and a food preservative made from the chitosan oligosaccharides. It is something to do.

The chitosan used in the present invention is derived from crustaceans and insects.

Chitin, which is known as a component of the integumentary skeletal tissue of lower animals such as shellfish and a component of the cell wall of fungi, is used as a raw material and is obtained by deacetylating it using conventional methods such as alkali treatment.

Chitosan dissolves only under acidic conditions, so an acid is used to prepare the chitosan solution.The acid used at this time can be either an organic acid or an inorganic acid, but in consideration of adding it to food, acetic acid is used. It is desirable to use acids that are commonly used for food, such as lactic acid, citric acid, and malic acid.

In particular, when considering the use of the obtained chitosan oligosaccharide as a food preservative, acetic acid is desirable because the acid itself is known to have strong antibacterial activity. Vinegar is well known as a food containing acetic acid, and is most preferred from the standpoint of flavor and antibacterial properties. In addition, other preservatives and seasonings having a preservative effect may be used in combination. Specifically, sodium acetate, the vinegar-containing preservative described in Japanese Patent No. 959222, commercially available vinegar, and the like can be used.

Next, as the enzyme agent containing pectinase (pectin-degrading enzyme) as the main component used in the present invention, generally commercially available enzyme agents are sufficient, but the optimum pH is 3 to 5, and chitosan can be degraded by acetic acid. It is preferable to use one that allows the conditions for adjusting the optimum pH during solubilization and enzymatic decomposition to be applied as is. In addition, pectinases produced by Aspergillus niger (MP Kyu-sho ■U top 1 anus) and Rhizopus sp. is preferable.

The conditions for enzymatic decomposition of a chitosan solution using an enzyme agent whose main component is pectinase are as follows: 1 part by weight of chitosan and 0.00% acetic acid.
p [ containing 1 part by weight or more, preferably 0.3 to 6 parts by weight
0.001 part by weight or more of an enzyme agent containing pectinase as a main component, preferably 0.005 to 5 parts by weight, to a chitosan solution of +3 to 5.
Add 0.05 parts by weight and perform enzymatic decomposition at 40 to 60°C for 1 hour or more to obtain a chitosan decomposition solution. Next, the resulting chitosan decomposition solution is adjusted to pH 6.5 or higher using an organic solvent such as acetone, isopropanol, or sodium hydroxide to form a precipitate, and then filtered.

Chitosan oligosaccharide can be obtained by separating the precipitate by centrifugation, washing with water, and drying.

When using the chitosan oligosaccharide thus obtained as a food preservative, the chitosan oligosaccharide obtained by heating the chitosan decomposition solution to inactivate the enzyme, the chitosan oligosaccharide separated and purified from the decomposition solution, or the chitosan oligosaccharide separated from the decomposition solution and purified. Any one obtained by dissolving in an acid may be used. The amount added is 0.005 to 0.5% (w/w), preferably 0.01 to 0.2% (w/w) as chitosan oligosaccharide. If the amount added is small, no antibacterial effect can be expected, and if the amount added is too large, an astringent taste will develop, so care must be taken. The chitosan oligosaccharide to be added can be used in liquid, powder, or solid forms such as granules, and can be added by dissolving, kneading, or spraying.

Spraying, dipping, etc. are optional, and the shape and method that are easy to add to food may be selected as appropriate.

The present invention is a method for obtaining chitosan oligosaccharides by enzymatic hydrolysis of chitosan using an enzyme agent containing pectinase as a main component. By using acetic acid or vinegar from the solubilization stage to the final stage of decomposition, (a) the acetic acid or vinegar from the solubilization process remains even in the enzymatic decomposition stage, so it can be decomposed as it is or in an hourly preparation process. be able to. (a) Since an enzyme agent having enzymatic activity under acetic acid is used, sufficient enzymatic decomposition can be carried out. (1) Since acetic acid or vinegar, which have strong preservative properties, are present in the decomposition solution that is hydrolyzed by an enzyme agent whose main component is pectinase, it is possible to preserve the chitosan oligosaccharide solution for a long period of time after completion of decomposition. Thing (1)
Furthermore, various effects can be obtained, such as the ability to obtain a preservative with remarkable preservative power in which the preservative power of chitosan oligosaccharide and the preservative power of acetic acid or vinegar are synergistically enhanced after completion of decomposition.

〔Example〕

The present invention will be explained below with reference to Examples, but the present invention is not limited thereto.

Example 1 2.0 g of chitosan was dissolved in 100 d of 1% acetic acid aqueous solution. Next, 0.04 g of each enzyme shown in Table 1 was added.
The reaction was allowed to proceed for 6 hours with slow stirring in a 5°C constant temperature water bath. After the reaction was completed, the mixture was kept in boiling water for 5 minutes to inactivate the enzyme and obtain a chitosan oligosaccharide solution. The obtained chitosan oligosaccharide solution was subjected to a modified Shales method (A
gricultural Biological Che
mistry, vol. 35, pp. 1154-1156, 19
71), absorption was observed at 420 r+m, confirming that chitosan oligosaccharides were produced. Furthermore, the degree of decomposition, antibacterial activity, and astringency of each obtained chitosan oligosaccharide solution were measured.

The results are shown in Table 1. The degree of disassembly is Tokyo Keiki B.
The viscosity at 20°C was measured using a BL adapter with a type viscometer. The antibacterial activity was determined using Saccharomyces cerevisiae as the test bacterium.
visiae) IFOO203 in YM medium (yeast extract 3g, IE budding kiss 3g peptone 5g, glucose L
og to 12 with distilled water, pH 5,5) at 30°
2d of the solution pre-cultured for 24 hours in C was mixed with YM agar medium (the above YM medium + agar 1.5% (iv.
/v)) A plate was prepared by adding 100 ml of the chitosan oligosaccharide solution, and 30μ2 of the chitosan oligosaccharide solution was appropriately diluted according to a conventional method.The presence or absence of halo formation was examined after 24 hours at 30"C using the cup method. The minimum concentration is M
It was made into an IC. For the astringency, the chitosan oligosaccharide solution was diluted 10 times with water, and the strength of the astringency was determined by sensory evaluation. In addition, as a control, the same operation as above was performed except that no enzyme was added.

As is clear from Table 1, it was found that the chitosan oligosaccharide obtained using an enzyme agent whose main component is pectinase, especially pectinase produced by Aspergillus niger, is superior.

Next, we investigated acids that dissolve chitosan.

After dissolving 2.0 g of chitosan in a 1% aqueous solution of the acids shown in Table 2, the pH was adjusted to 4.0 with sodium hydroxide. There Sumiteam AP2 (manufactured by Shin Nippon Chemical Industry ■) 0.0
5 g was added thereto, and the reaction was allowed to proceed for 6 hours with slow stirring in a constant temperature water bath at 45°C. After the reaction was completed, the mixture was kept in boiling water for 5 minutes to inactivate the enzyme and obtain a chitosan oligosaccharide solution. The degree of decomposition and antibacterial activity of each obtained chitosan oligosaccharide solution were measured by the same method as described above. The results are shown in Table 2.

As is clear from Table 2, acetic acid was found to be preferable when solubilizing chitosan and enzymatically decomposing it.

Next, we examined the amount of enzyme added. Chitosan 2. O
Dissolve g in 100 ml of 1% acetic acid aqueous solution,
P2 (manufactured by Shin Nippon Chemical Co., Ltd.) at 0.2 for chitosan.
5 to 4% (w/w) was added, and the mixture was reacted for 6 hours with slow stirring in a constant temperature water bath at 50°C. After the reaction was completed, the mixture was kept in boiling water for 5 minutes to inactivate the enzyme and obtain a chitosan oligosaccharide solution.

The viscosity of the obtained chitosan oligosaccharide solution was measured by the same method as described above, and the results are shown in FIG. As is clear from Figure 1, the viscosity of 1460 cp when no enzyme is added is reduced by the addition of enzyme, and the amount of enzyme added is approximately 1% (w) of the substrate.
/w) remained almost constant, indicating that chitosan was decomposed.

Next, we examined the enzyme reaction time. chitosan 2.0
Dissolve g in 100 ml of 1% acetic acid aqueous solution, and add Sumiteem AP2 (manufactured by Shin Nippon Chemical Co., Ltd.) to 2% chitosan.
(w/w) and reacted for 1 to 18 hours with slow stirring in a constant temperature water bath at 50°C. After the reaction was completed, the mixture was kept in boiling water for 5 minutes to inactivate the enzyme and obtain a chitosan oligosaccharide solution. The viscosity of the obtained chitosan oligosaccharide solution was measured by the same method as described above, and the results are shown in FIG.

As is clear from FIG. 2, it was found that the decomposition of chitosan became almost constant in the enzymatic reaction after about 6 hours.

Example 2 5.0 g of chitosan was added to commercially available vinegar (acetic acid acidity 4.2% (w/
v)) Dissolved in 100 ttti. Next, 0.1 g of Sumiteam AP2 (manufactured by Shin Nihon Kagaku Kogyo ■) was added, and the mixture was reacted for 6 hours with slow stirring in a constant temperature water bath at 50°C. After the reaction was completed, the mixture was kept in boiling water for 5 minutes to inactivate the enzyme and obtain a chitosan oligosaccharide solution (A).

10 kg of Chinese cabbage cut into appropriate size and 1 part of 10% salt solution
After adding 0 kg and salting for 18 hours using a 5 kg weight, the Chinese cabbage was taken out and lightly drained. 300g of this salted Chinese cabbage, 5kg of salt, 0 of sodium glutamate
．． 5 kg and chitosan oligosaccharide solution (A) in the amount shown in Table 3, and then added water to make 100 f Seasoning liquid 30
0d was added as a dipping solution, which was then placed in a plastic bag and sealed to obtain a prototype. In addition, as a control, a control product was prepared using a seasoning liquid having the same composition as above except that the chitosan oligosaccharide liquid (A) was not added.

20 samples each of the obtained prototype and control products were kept at room temperature (
The preservative effect was determined from the degree of turbidity of the soaking solution. In addition, 10 panelists tasted the 18th preserved product and evaluated its taste. The results are shown in Table 3.

Table 3 As is clear from Table 3, the amount of chitosan oligosaccharide (as solid content), which has a preservative effect and does not cause any taste problems, is 0.005% to 0.5% (W/ W) was found to be desirable.

Example 3 Beat 225 g of whole chicken egg liquid, add 50 d of stock, then add 5 d of soy sauce, 1 g of salt, 14 g of sugar, and the chitosan oligosaccharide solution (A) 3II1 obtained in Example 2. Add water to make a 40% test solution and mix. Next, heat a thin layer of oil in an omelet pan and add 1/4 of the above omelet ingredients.
Pour in the amount, and when the bottom part has hardened, roll it toward you.

Transfer the rolled item to the other side of the pot, reapply oil to the pot, pour in the ingredients as before, and when the bottom part has hardened, start rolling (= using the baked part as the core and rolling it towards you. After baking in the same way, rolling in one after another,
Cut it into the desired size to obtain a fried egg product. In addition, as a control, fried eggs were prepared in the same manner as above except that the chitosan oligosaccharide solution (A) was not added.

Storage tests and taste evaluations were conducted on the obtained products and control products. The results are shown in Table 1. The storage test shows the degree of spoilage when the product and control product are placed in a tray bag, wrapped in Saran wrap, and stored at 20°C. In addition, - in the table was not corrupted, and 2 players were corrupted. ++
indicates significant corruption. Taste evaluation was conducted by having 20 panelists taste the freshly made products.

The results are shown in Table 4.

As is clear from Table 4, it was found that the addition of the chitosan oligosaccharide solution improved the preservability of the fried eggs and also provided good taste.

Additionally, some panelists rated the product as having less darkening and better color tone than the control product.

Example 5 5.0 g of chitosan and 100 ml of 2% (-/I) lactic acid solution
Next, 0.2 g of Macerozyme 2S (I$3 manufactured by Yakult Honsha Co., Ltd.) dissolved in
The reaction was allowed to proceed with slow stirring for several hours. After the reaction is complete,
The enzyme was inactivated by keeping it in boiling water for 5 minutes to obtain a chitosan oligosaccharide solution (B). Using the obtained chitosan oligosaccharide solution (B), make pickled cucumbers with the following composition,
A storage test was conducted.

Note that the solid:liquid ratio of cucumber and pickling liquid was 1:1.

The results are shown in Table 5. In the table, - indicates no turbidity of the pickling solution, + indicates turbidity of the pickling solution, and 10+ indicates severe turbidity of the pickling solution.

As is clear from Table 5, the preservation effect was significantly improved by the addition of chitosan oligosaccharide.

Example 5 Sugars 50% (w/w), flavoring agent flavor liquid 20%
Add the test solution shown below to (w/w) and add water to 1
I made a mirin-style seasoning with 00%.

h-min”? A Chitosan oligosaccharide solution obtained in Example 2 (A)
LX (v/w) B Brewed vinegar (acetic acid acidity 5%)
2χ (v/w) C water Each of these products was sealed in a sachet and stored at 30°C to examine the presence or absence of foaming caused by yeast. In addition, taste evaluation was conducted in the same manner as in Example 1. The results are shown in Table 6.

Table 6 (=): Foaming not observed (+): Foaming present As is clear from Table 6, product A was found to have good preservability and good taste.

Example 6 400 g of peeled and cut potatoes, 100 g of pork
Stir-fry with 12 at of salad oil and add 37 ml of soy sauce. Alcohol 1
5d, Mirin 8d, sand T7!13.5g, salt 2.
5g, chitosan oligosaccharide solution (A) obtained in Example 2 1
0m, 5 parts of brewed vinegar (acetic acid acidity 10%) and 1 part of water
Add 75d and simmer until the liquid evaporates.
550 g of product Bl) was obtained.

In addition, as a control, meat potatoes (product B2) were made in the same manner as above, except that 15 μl of brewed vinegar (10% acetic acid acidity) and 115 ml of water were used without adding the chitosan oligosaccharide solution (A). Furthermore, chitosan oligosaccharide solution (A
) and brewed vinegar were not added and meat potatoes (control product) were made in the same manner as above except that 190 d of water was used.

The obtained products and control products were packed and subjected to a storage test at 25°C for a predetermined period of time, and the number of bacteria was measured. - The shipowner's bacterial count was measured every day by the pouring method using a standard agar medium according to the usual method, and when it reached 10' bacteria/g, it was judged as the date of spoilage. In addition, panelists were asked to sample the freshly made products and evaluate their taste.

The results are shown in Table 7.

As is clear from Table 7, product B1 suppressed spoilage caused by microorganisms and had good taste.

Example 7 30 g of salt was added to 1000 g of ground cod, and the mixture was thoroughly crushed, followed by 60 g of potato starch and 10 g of sand W.

15 m of vinegar solution obtained by mixing 25 relatives of the chitosan oligosaccharide solution (A) obtained in Example 2 with 11 brewed vinegar (10% acetic acidity) and 33.3 g of sodium hydroxide! After adding 160 d of water and thoroughly crushing and attaching a plate,
The mixture was placed in a steamer and steamed for 40 minutes. After the steaming was completed, the mixture was removed from the steaming process to obtain kamaboko (product CI). In addition, as a control, kamaboko (product C2) was made in the same manner as above except that no vinegar solution was added and 175% of water was used. ml
Kamaboko (control product) was made in the same manner as above except that .

The obtained products and control products were stored at 20°C for a predetermined period of time, and a storage test and taste evaluation were conducted in the same manner as in Example 2. The results are shown in Table 8.

As is clear from Table 8, product CI and product C2 are superior to the control product in terms of shelf life and taste, especially product C.
1 was found to be superior.

Example 8 After washing 1.5 kg of polished rice with water, 30 mf of the chitosan oligosaccharide solution (A) obtained in Example 2 and 2220 mk of water were added and the rice was cooked in a commercially available rice cooker.
It was possible to obtain cooked rice with good taste.

Example 9 The chitosan oligosaccharide solution (A) obtained in Example 2 was dropped into acetone to obtain a precipitate. This precipitate was separated, washed with water, and dried to obtain chitosan oligosaccharide.

Add 150 g of salt, 5 g of chitosan oligo F' obtained above and 1500 mA of water to 4 kg of wheat (wheat flour),
When udon noodles were made by mixing the mixture well and making noodles, it was possible to obtain udon noodles with excellent storage stability and good taste.

Example 10 300 kg of minced pork edible parts, 15 kg of starch, 3 kg of salt, and 5 kg of grated garlic.

Add appropriate amounts of chemical seasonings and spices, and add eggshell 30.0 to chitosan oligosaccharide i (A) 8 ffi obtained in Example 2 and brewed vinegar (acetic acid acidity 10'/, later) IN.
g.

14.0 g of sodium bicarbonate and 6 g of sodium hydroxide
．． 67g was added and all of the mixed liquid 4N was added and mixed well, and sausages were manufactured by filling casings according to the conventional method, heating and cooling, and it was possible to manufacture sausages with good taste and excellent storage stability. .

〔Effect of the invention〕

According to the present invention, chitosan oligosaccharides can be produced easily and at low cost. Furthermore, the chitosan oligosaccharides obtained by the present invention are useful as food preservatives and the like.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the amount of enzyme added and the degree of chitosan decomposition (viscosity) in Example 1, and FIG. 2 shows the relationship between the enzyme reaction time and the degree of chitosan decomposition (viscosity) in Example 1. Figure) Enzyme addition amount (% for 1 chitosan) Enzyme reaction time (hours)

Claims (2)

[Claims] (1) A method for producing chitosan oligosaccharides, which comprises producing chitosan oligosaccharides by enzymatically decomposing a chitosan solution with an enzyme agent containing pectinase as a main component. (2) A food preservative comprising chitosan oligosaccharide produced by the method according to claim 1.