JP2642774B2 - Food shelf life improver - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial applications]

The present invention relates to a shelf life improver for food. More specifically, the present invention relates to a food shelf life improver containing a chitosan hydrolyzate having a specific molecular weight as an active ingredient.

[Prior art]

In recent years, interest in food additives has increased, and in particular, synthetic preservatives, synthetic germicides, synthetic colorants, and the like are not only strictly regulated and restricted in use but also tend to be avoided by general consumers. Of these, synthetic preservatives such as sorbic acid, benzoic acid, and dehydroacetic acid are often used as food preservatives for the purpose of improving the shelf life of food, but general consumers do not trust the safety of synthetic preservatives. Due to the strong feeling, the amount used in food has leveled off. Attention has been paid to natural antibacterial agents in such a situation. But,
The logic that it is safe because it is natural is no longer valid, and it is required to confirm the safety of natural antimicrobial agents individually. Under such circumstances, chitosan has attracted attention for its safety and efficacy. Xatone is a basic homopolysaccharide in which 2-amino-2-deoxy-D-glucose (glucosamine) is linearly linked to β- (1 → 4), and is industrially usually used in crustaceans such as crabs and shrimps. Obtained by deacetylating the natural chitin contained in E. coli. For the safety of chitosan, Arai et al. (Tokai Water Research Bulletin, 56 , 89 (1968)) and M. Sugano et al. (Nutritino R
eports International, 18 , 531 (1978), The American
Journal of Clinical Nutrition, 33 , 787 (1980)). Chitosan is known to have various physiological activities and functions. Among them, regarding the antibacterial properties, some antibacterial properties against mold (CRAllan et al., Experimental Mycology,
3 , 285 (1979), DFKendra et al., Experimental Mycolog
y, 8 , 276 (1984), PSStoessel et al., Phytopathologisch
e Zeitschrift, 111 , 82 (1984)) and antibacterial properties against bacteria (Chemistry and Biology, 21 , 635, (1983), Polymer Digest, February, 14, (1985)). However, chitosan obtained by deacetylation of natural chitin is a polymer, and exhibits a very high viscosity when put into solution. In addition, chitosan has astringency, protein coagulation and the property of dissolving in water only under acidic conditions.
The disadvantage is that the foods that can be used are limited. Therefore, there has been a proposal in which a polymerized chitosan or oligo obtained by chemically or enzymatically dividing chitosan is expected to be effective as a food preservative. For example, there is a report that chitosan lightly degraded by chitosanase showed stronger antibacterial activity against bacteria than untreated chitosan (Uchida, Food Chemical, No. 2, 22 (1988), Tokuho 1-56755). . However, in this report, the extent of chitosan degradation was
The ratio is shown as the ratio of the amount of reducing sugars produced to the amount of original chitosan, and no information is provided on what molecular weight chitosan is produced. Reports that clearly indicate the chitosan molecular weight include molecular weights between 10,000 and 50,000
JP-A-63-169975, etc., including food preservatives (JP-A-1-128775) containing chitosan hydrolyzate as an active ingredient, and JP-A-1-291799, etc. regarding oligosaccharides. However, the antibacterial properties of oligosaccharides are known to be extremely weak, as described in the above-mentioned report by Uchida, and thus lack practicality.

[Problems to be solved by the invention]

When trying to use chitosan for improving the shelf life of foods, it is necessary to consider not only the antibacterial properties but also the improvement of the disadvantages of chitosan such as astringency and protein cohesion. However, there are few examples in which the relationship between the molecular weight of chitosan and the suitability as a food shelf life improver has been comprehensively studied in detail from the viewpoints of antibacterial properties, taste, cohesion, solubility and the like. The present invention provides a food shelf life improver containing chitosan as an active ingredient, which has improved defects such as taste, cohesion, and solubility so that it can be used in a wide range of foods while maintaining the antibacterial properties of chitosan. It was made for the purpose.

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, the above object was achieved by enzymatically decomposing chitosan and using a chitosan fraction having a specific molecular weight distribution. They have found that this can be achieved and have completed the present invention. That is, the shelf life improver for foods of the present invention contains chitosan as a main component, which is obtained by decomposing chitosan with a chitosanase produced by a microorganism belonging to the genus Verticillium (Verticillium sp.) And having a molecular weight of 6,000 to 10,000. The chitosan hydrolyzate is used as an active ingredient. The raw material chitosan used in the present invention is not particularly limited in its molecular weight, whether it is a high-molecular-weight chitosan obtained by deacetylation of natural chitosan by an alkali treatment or the like or a chitosan obtained by chemically or enzymatically decomposing it to some extent. However, chitosan of any molecular weight can be used as a raw material. Colloidal chitosan in which chitosan is dispersed and suspended in water under basic or neutral conditions can also be used as a raw material. Both chitosan having a high degree of deacetylation (95 to 100%) and chitosan generally having a low degree of deacetylation (65 to 95%) can be used as raw materials. The molecular weight reduction of chitosan can be achieved both chemically and enzymatically. However, in order to obtain a chitosan hydrolyzate having a relatively narrow molecular weight range, in the present invention, Verticillium (Verticillium) is used.
illum sp.) using a chitosenase produced by a microorganism belonging to the group. In particular, Verticillium AF9-
The chitosenase produced by the V-156 strain (Microcosms No. 11377) and its mutants can be preferably used. A method for producing this enzyme has already been patented by the same applicant as the present application (Japanese Patent Application No. 2-81531). In order to obtain a chitosan-decomposed product mainly composed of low molecular weight chitosan having a molecular weight of 6,000 to 10,000, it is necessary to set a reaction condition by performing a preliminary test for each enzyme to be used. For detailed examination of conditions, an analysis method of molecular weight distribution by gel filtration is very effective. The combination of the reaction pH and temperature can be freely selected within the range in which the enzyme to be used acts, but if the chitosanase produced by the Verticillium genus AF9-V-156 strain or a mutant thereof is used, if the pH is 3.0 or more, Since the reaction can be carried out efficiently, the enzyme reaction can be carried out without adding alkali simply by adjusting the amount of acid added during chitosan dissolution and maintaining the solution pH after dissolution at 3.0 or more. The reaction temperature can be selected in the range of 30-80 ° C., which is usually the action temperature of chitosanase, but since the reaction at high temperature promotes the browning of chitosan,
To avoid browning as much as possible, a reaction at a low temperature is desirable. In the case of chitosanase produced by Verticillium AF9-V-156 strain, the enzyme reaction can be efficiently performed at 30 to 37 ° C. Thereafter, while analyzing the molecular weight distribution, the enzyme concentration and the reaction time may be adjusted to set conditions for obtaining chitosan having a target molecular weight. The food shelf life improver according to the present invention maintains antibacterial properties, improves the astringency and protein aggregation properties of conventional chitosan, and has solubility near neutrality, so that it can be applied to a wide range of foods. Can be. For example, processed cereals such as bread, noodles and cooked rice, processed vegetables and fruits such as pickles and jams, dairy products such as cheese, butter, margarine, meat products such as meat, ham, bacon, sausage, kamaboko, chikuwa, Processed fishery products such as tsukudani, buns, yokan, cakes and other sweets, seasonings such as miso, soy sauce, noodle soup, sauces and other processed foods such as tofu, soy milk, cooked beans and other processed foods, and fresh fish It can also be used for fresh foods such as fruits and vegetables and prepared foods.

【Example】

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1. In order to examine in detail the relationship between the molecular weight of chitosan and the suitability as a shelf life improver for food, chitosan was decomposed with chitosanase to prepare four decomposed products having different molecular weight distributions. Taste (astringency), protein aggregation, and solubility were compared. (1) Preparation of chitosan decomposition product: Low viscosity chitosan LLWP (trade name, manufactured by Kimitsu Chemical Industry Co., Ltd .;
800 ml of pure water was added to 50 g of the deacetylation degree (75-85%), and 50 ml of 10M acetic acid was added with stirring to dissolve chitosan.
After complete dissolution, the volume was adjusted to 1,000 ml to give a 5% chitosan solution. The pH of this solution was 4.2. Chitosanase produced by Verticillium sp. AF9-V-156 strain was added to this chitosan solution at a concentration of 10 mU / ml, followed by oxygen reaction at 30 ° C. After 2 hours (decomposition A), 6 hours (decomposition B), 18 hours (decomposition C) and 42 hours (decomposition D) after the start of the reaction, 200 ml of each sample was taken, and the enzyme was subjected to a heat treatment at 80 ° C. for 15 minutes. After inactivation, analysis of molecular weight distribution, antibacterial test,
A cohesion test and a solubility test were performed. (2) Analysis of molecular weight distribution of chitosan decomposition product: The molecular weight distribution was analyzed by a gel filtration method. That is,
1 ml of the chitosan hydrolyzate solution diluted to 0.2% was subjected to gel filtration using a column (15 mm × 44 mm) packed with a gel filtration carrier Sephacryl S-200 (manufactured by Pharmacia), and the molecular weight distribution was estimated. As molecular weight markers, molecular weight 510,000,7
1,000, 39,000, 9,400 dextran (Sigma), chitohexaose hydrochloride (molecular weight: about 1,200, manufactured by Seikagaku Corporation), and glucosamine hydrochloride were used. As a result, the decomposition product A mainly has a molecular weight of 40,000 or more, and the decomposition product B
Was widely distributed around the molecular weight of 10,000 to 40,000, the decomposition product C was distributed around the molecular weight of 6,000 to 10,000, and the decomposition product D was distributed around the molecular weight of 2,000 to 3,000. (3) Measurement of reducing sugar amount: The reducing sugar amount of undecomposed chitosan (LLWP, 5% solution) and each chitosan hydrolyzate (AD, 5% solution) was determined by the p-HBAH method (M. Lever, Anal. Biochem., 47 , 273 (1972)). Glucosamine was used as a standard substance. As a result, the amount of each reducing sugar is undegraded chitosan: 0.427 mg / m
l, Decomposition A: 1.07 mg / ml, Decomposition B: 2.37 mg / ml, Decomposition C:
6.56 mg / ml and degradation product D: 12.5 mg / ml. (4) Antibacterial test: The antibacterial properties of each decomposed product obtained in the above (2) were tested by the following method. Commercial broth medium (Eiken Chemical Co., Ltd.)
Was dissolved in 1,000 ml of distilled water, the pH was adjusted to 6.0, and 4 ml of the solution was dispensed into test tubes and autoclaved. Using this test tube, to prepare a 2-fold dilution series of each degradation product, was added overnight BaiYoeki of E. coli K-12 strain was appropriately diluted to each tube such that the 10 ⁵ / ml The cells were cultured with shaking at 37 ° C for 72 hours. The minimum concentration at which no bacterial growth was observed was defined as the MIC (minimum inhibitory concentration) of each degradation product. The results are shown in Table 1. ○ When MIC is 125ppm or less ○, 250 ~ 500ppm △,
In the case of 500 ppm, it was indicated by x. (5) Taste test: Each decomposed product was made into a 0.5% solution, and each bitterness was tested by 10 panelists. The results are shown in Table 1.
When a feeling of astringency was not felt, it was indicated by 、, when feeling slightly, △, and when feeling considerably ×. (6) Protein agglutination test: Each of the decomposed products was added to the bouillon medium at a concentration of 0.5%, and the presence or absence of ori was examined. The results are shown in Table 1. ○ in the table
Indicates that no deposits are formed, △ indicates that there is slight deposits, and X indicates that there is deposits. (7) Solubility test: Each decomposed product is made into a 1% solution, and pH is adjusted with 1N sodium hydroxide.
It was increased to 7.0 and the presence or absence of precipitate formation was examined. The results are shown in Table 1.も の indicates that chitosan was dissolved without precipitation, and x indicates that chitosan was precipitated.
From the results of (4) to (6), the decomposition product C, that is, the molecular weight
Chitosan hydrolyzate mainly composed of low molecular weight chitosan of 6,000 to 10,000 has antibacterial properties, astringency and protein cohesion are improved, and further has solubility at pH 7.0, It turned out to be optimal for use in food. Example 2 The effect of adding the decomposed product of chitosan C obtained in Example 1 to food was tested with lightly pickled Chinese cabbage. 3.5% salt, chemical seasoning
Make a pickling solution with a composition of 0.4% and liquid seasoning 0.1%, and decompose chitosan C (5% as chitosan solid content)
0.2% (0.01% as chitosan solid content) and 1.0% (0.05% as chitosan solid content) were added, and
And Test Zone 2. The group to which the chitosan hydrolyzate was not added was used as a control group. Table 2 shows the results obtained by examining the degree of cloudiness of the pickling solution over time. In addition,-in the table
Indicates no turbidity of the pickling solution, + indicates slight turbidity of the pickling solution, +
+ Indicates that the pickling solution is fairly cloudy. In addition, the taste was evaluated by having 10 panelists sample, and it was evaluated that no astringency was felt in any of the test plots. From this result, it was found that the decomposition product C had an effect of improving the shelf life for several days or more according to the amount added without changing the taste of the pickles. Example 3 The effect of adding the chitosan hydrolyzate C prepared in Example 1 to custard cream was tested. Based on a basic composition of 20 g of egg yolk, 40 g of sugar, 10 g of flour, 10 g of corn starch, and 420 g of milk, the chitosan digest C is 0.02
% (Test zone 1) and 0.1% (test zone 2) to prepare a custard cream according to a conventional method. Each sample was stored at 20 ° C. and observed for changes in appearance. Table 3 shows the results. In the table,-indicates no change, + indicates slight change,
++ clearly indicates deterioration. As is clear from Table 3, the chitosan hydrolyzate C was able to significantly improve the shelf life of custard cream. Sensory tests were also conducted by 10 panelists at the same time.
In all respects, such as odor, there was no difference from the control plot, and no quality problems were recognized.

【The invention's effect】

The food shelf life improver of the present invention can be used without impairing the texture and appearance of the food, because the astringency and protein cohesion of conventional chitosan are improved while maintaining the antibacterial properties. Moreover, since it maintains solubility near neutrality, it can be used for many types of foods, and is very useful in the food industry.

Claims (2)

(57) [Claims] 1. The method of claim 1, wherein the chitosan is of the genus Verticil.
lium sp.), which has a molecular weight of 6,000 to 10,000 obtained by decomposing with a chitosanase produced by a microorganism belonging to a microorganism belonging to a low molecular weight chitosan as an active ingredient. . 2. The microorganism belonging to the genus Verticillium is Verticillium AF9-V-156 (No. 11377, a microbial bacterium).
The food shelf life improver according to claim 1, wherein: