JPH02231065A - Preservative for food - Google Patents

Abstract

PURPOSE:To obtain the title preservative having safety and sufficient antiseptic power, comprising a hydrolyzate having specific molecular weight prepared by hydrolyzing a chitosan having specific degree of deacetylation as an active ingredient. CONSTITUTION:The aimed preservative comprising a hydrolyzate having 5,000-50,000 molecular weight prepared by hydrolyzing a chitosan having 86-96% degree of deacetylation as an active ingredient.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a food preservative, and more particularly to a food preservative containing a chitosan decomposition product having a specific molecular weight and a specific degree of deacetylation as an active ingredient. .

[Prior art and problems to be solved by the invention] In recent years, the safety of food additives has become important, especially benzoic acid. Sorbic acid. Restrictions on the use of synthetic preservatives and fungicides such as dehydroacetic acid, paraoxybenzoic acid ester, and probionic acid are becoming stricter. In addition, these synthetic preservatives,
Consumers' concerns about disinfectants are increasing year by year, and their use in food products tends to be avoided.

Therefore, chitosan is starting to attract attention as a natural preservative that has antiseptic properties and is safe.

Chitosan is a deacetylated chitin contained in the cell walls of crustaceans such as shrimp and crabs and microorganisms. In particular, crustaceans such as shrimp and crabs have been eaten since ancient times.
It is highly safe. Much knowledge has already been obtained regarding the antibacterial properties of chitosan and its decomposed products, such as "Food Chemical" February 1988 issue, pages 22-29, JP-A-62-30103, JP-A-62-83877.
No. 62-198604, No. 63-9
This is reported in Publication No. 8395, Publication No. 63-251072, etc. Although chitosan or its decomposition products have excellent antibacterial properties, when they are used as food preservatives, chitosan or its decomposition products added for preservative purposes may be mixed into food due to the aggregation effect of chitosan or its decomposition products. It has the disadvantage that it causes aggregation reactions with proteins and other ingredients in the liquid, which causes turbidity especially in liquid foods that require clarity, reducing the value of the product and limiting its use. [Means for Solving the Problems] Therefore, the present inventors conducted extensive studies to solve the fatal drawback of turbidity caused by aggregation, and found that chitosan with a specific molecular weight and a specific degree of deacetylation was hydrated. The chitosan decomposition product obtained by decomposition has strong preservative power,
The present invention was completed based on the discovery that the agglomeration effect per unit of preservative power is extremely low, and even in liquid foods that require clarity, there is little turbidity, and the value of the product does not decrease.

That is, the present invention uses chitosan with a molecular weight of 5,000 to 50,000 obtained by hydrolyzing chitosan with a degree of deacetylation of 86 to 96%.
The purpose of the present invention is to provide a food preservative containing a hydrolyzate of as an active ingredient.

It is already known from "Food Chemical J, February 1988 issue, pages 22-29, that the antibacterial activity per unit weight of chitosan or chitosan decomposition product with increased degree of deacetylation increases, but the present inventors From the viewpoint that the aggregation effect of chitosan decomposition products reduces the commercial value,
While investigating the relationship between antibacterial activity and flocculating activity, they discovered that within the range of deacetylation of the present invention, the flocculating activity per unit of preservative activity was extremely low, which was completely unexamined and had not even been studied in the past. This is a new fact.

The present invention, which was completed based on this fact, has solved the problem of the aggregation effect of conventional chitosan decomposition products, and has made it possible to expand the range of applications of chitosan decomposition products. The chitosan used in the present invention is made from chitin, which is known as a component of the outer skin skeletal tissues of lower animals such as crustaceans, insects, and shellfish, and as a cell wall component of fungi. However, in the present invention, chitosan with a hexatylation degree of 86 to 96% is used.

Further, in the present invention, a decomposition product obtained by hydrolyzing the above-mentioned chitosan raw material and having a molecular weight of 5,000 to 50,000 is used as an active ingredient. Two methods can be used to decompose the above chitosan raw material: acid hydrolysis and enzyme hydrolysis. (2) Acid hydrolysis method The method described in Japanese Patent Application No. 62-284192 can be used, and an outline thereof will be described below.

The acid used for acid decomposition of chitosan is acetic acid, considering that it will be added to food. It is desirable to use lactic acid, citric acid, malic acid, or hydrochloric acid, which are commonly used for human consumption.

When using acetic acid, lactic acid, citric acid, or malic acid, the decomposition conditions are 1 at an acid concentration of 0.5 to 2.0χ (w/w).
Hydrolysis treatment may be carried out at 00 to 121°C for 5 to 180 minutes. In addition, when using hydrochloric acid, the concentration of hydrochloric acid is 0.05
~2. Hydrolysis treatment can be carried out at 100 to 121"C for 3 to 180 minutes at ON temperature. If the hydrolysis conditions are severe, use a component with a molecular weight of 5000 to 5000, which is a particularly effective component.
Both components of 0 are reduced in molecular weight and antibacterial activity is reduced. Conversely, if the decomposition conditions are gentle, the molecule 515 0 0 0 0
The polymer fraction with a molecular weight of 5000 to 500 increases.
The yield of the 00 fraction will decrease.

■Hydrolysis by enzymes JP-A-62-83877, JP-A-63-63
As described in Japanese Patent Application No. 388 and Japanese Patent Application No. 63-121852, chitosanase, papain. Hydrolysis methods using commercially available enzymes such as cellulase, acid protease, and vectinase can be used.

Since chitosan is soluble in acidic solutions, the enzymatic reaction needs to be carried out in the pH range of 3 to 5, so an enzyme with high chitosan degrading activity at low pH must be selected.

Since reaction temperature, reaction time, and enzyme concentration vary depending on each commercially available enzyme agent, the most effective chitosan decomposition product molecules 15
0 0 0 to 500,000 cars may be selected. For example, when using a commercially available bectinase preparation, acetic acid, lactic acid. Add 0.001 parts by weight or more of an enzyme preparation to 1 part by weight of chitosan in a chitosan solution prepared with citric acid, malic acid, or hydrochloric acid to a pt+ of 3 to 5, and decompose at 40 to 60°C for 1 to 24 hours. Bye.

In the present invention, the molecular weight was measured by a gel filtration method.

The molecular weight obtained in this way is 5,000 to 50,000.
A chitosan decomposition product having a degree of deacetylation of 86 to 96% may be used as a weakly acidic solution by adding an alkali in consideration of addition to foods.

Furthermore, the obtained chitosan decomposition product may be used after selectively separating only the active ingredients using means such as a dialysis membrane, an ultrafiltration membrane, or gel filtration.

When adding the above chitosan decomposition product to food as a food preservative, it can be used in paste or powder form in addition to liquid form. It can also be processed into shapes that are easy to add to foods, such as granules. The amount added to food is 0.00 as solid chitosan.
0 0 0 5 ~1. 5 (w/v), preferably 0.5 (w/v). 0 2 5 ~ 0. 5 (w/v).
The decomposed chitosan product of the present invention having a molecular weight of 5,000 to 50,000 and a degree of acetylation of 86 to 96% has significantly less flocculating action per antibacterial power than chitosan decomposed products outside this range, and especially has a flocculating action. It has superior characteristics compared to previously known preservatives that use chitosan decomposition products, which do not specify molecular weight or degree of deacetylation, in that they can be used in products whose use was previously limited. Food preservatives can be obtained. [Example] The present invention will be specifically explained below with reference to Examples, but the present invention is not limited thereto. The degree of deacetylation of chitosan used in the present invention is
It was measured by the following method. The sample chitosan was 0.5χ (
(v/v) 0.5x (I1/v) was dissolved in acetic acid solution, 1.0g of it was placed in a 150-mm Erlenmeyer flask, 3olII1 of distilled water was added, and the mixture was thoroughly mixed. Next, 1 to 2 drops of a 0.1% methylene blue solution was added, stirred with a magnetic stirrer, and then titrated with a 1/200 N-potassium polyvinyl sulfate (Wako Pure II) solution. The end point was defined as the point at which the blue color of the liquid turned to bluish-purple and white precipitate began to float. Next, based on the titration value, the degree of deacetylation was determined using the following formula. Degree of deacetylation (χ) = 16. IX titration number (xi!)
XE/100 (E represents the degree of esterification of polyvinyl sulfate.potassium.) Experimental example 1 (1) Preparation of chitosan decomposition product Commercially available chitin (manufactured by Kanto Kagaku Co., Ltd.) was
) to a caustic soda solution at a concentration of 5χ (w/w), decomposed by heating at 100°C for 2.5 hours, thoroughly washed with water, and freeze-dried to obtain chitosan with a degree of deacetylation of 88%. I got it.

Next, the chitosan obtained above was added to a 12 (w/w) acetic acid solution at a ratio of 7x (1/li), and a pectinase enzyme preparation (manufactured by Shin Nihon Kagaku Kogyo Co., Ltd.) was added at a ratio of 0.04x.
(w/w) and then reacted for 12 hours while heating at 45-65°C. After the reaction was completed, the temperature was maintained at 80°C for 5 minutes to inactivate the enzyme, and then the pH was adjusted with caustic soda.
Adjust to 4.5 and add water to make chitosan enzymatically decomposed product 5χ
A (w/w) solution was obtained.

(2) Molecular weight fractionation of the chitosan decomposition product obtained in (1), using a 3χ (mono/gastric) solution of the chitosan decomposition product diluted with deionized water as a sample, gel filtration method using the following method. Molecular weight fractionation was performed using Gel filtration packing material Sephadex G-75 (PHALMAC
Made by IA FINE CHEMICALS. Molecular weight 100
0 to 50,000) by a conventional method, and then placed in a glass column with an inner diameter of 16 m + and a height of 100 cm.
Pack the chitosan decomposition product obtained in (I) to 0.
Developed with 05N acetate buffer (pH 5.5),
The effluent was collected in 10 d portions. Dextran MW500000 (PHALMACIA F
Made by INECHEMICALS), text run m pupil 9
400 (manufactured by SIGMA) and chitopentaose hydrochloride (manufactured by Seikagaku Corporation) were used. Note that sugar elution was measured by the phenol-sulfuric acid method.

(3) Evaluation of antibacterial activity of molecular weight fractions The antibacterial activity of each molecular weight fraction obtained in (2) was evaluated using the following method. Medium (glucose 100 g, ammonium chloride 0.2 g, monopotassium phosphate 2.0 g,
Magnesium sulfate 1.0g, calcium chloride 0.2g,
Dissolve 0.2 g of sodium chloride in 1 l of distilled water and adjust the pH to 4.
．． In order to keep the chitosan decomposition product content of each molecular weight fraction obtained in (2) constant, the total nitrogen content of the sample was measured, and the content was adjusted to 3.0 × 10-Sχ ( w/
w) and added each of the 5 relatives to make the total amount 10 relatives, and then added the test bacteria Candida mycoderma IF00.
164 to 4. The cells were inoculated at OX10Scell/Id and cultured with shaking at 30°C. 24 hours after the start of culture, the growth of the inoculated bacteria was determined from the presence or absence of turbidity of the culture solution. The results are shown in FIG. In addition, + in the figure indicates that the culture solution is turbid, 2 indicates that the culture solution is slightly turbid, and 1 indicates that the culture solution is not turbid.

As is clear from Figure 1, molecules 15000~5
Both parts of 0000 were found to have antibacterial properties. Experimental Example 2 (1) Preparation of chitosan with different degrees of deacetylation After adding commercially available chitin (manufactured by Kanto Kagaku Co., Ltd.) to a 50χ (w/w) caustic soda solution to a concentration of 5χ (w/w), 1
After thermal decomposition at 0 0'C and thorough washing with water,
In the freeze-drying method, the thermal decomposition time is 0.
5. 0.75, 1.0. 1.5. 2.0, 2
．． 5.3.0, 3.5. 4.0. 4.5 hours 1
The degree of deacetylation was 65χ, 71χ, 75χ, 80χ, 86χ. 882.9
0%. 94χ, 96χ. Ten types of chitosan with different degrees of deacetylation of 99χ were obtained.

(2) Preparation of chitosan decomposition product by acid hydrolysis method 0.7
The 10 kinds of chitosans with different degrees of deacetylation obtained in (1) were added to a 5N hydrochloric acid solution so that each had a concentration of 7χ (w/w), and the mixture was hydrolyzed at 100°C for 3 hours.
The pH was adjusted to 4.5 with caustic soda, and water was added to obtain 52 (w/w) solutions A-J of chitosanic acid decomposition products having 10 different degrees of deacetylation shown below.

Doge Tsuchi Z Yoshibun Gosen Acetyl Voice 2) A
65 B 71 C 75 D 80 E 86 F 8B G 90 H 94 J 99 (3) Molecular weight fractionation of chitosan decomposition products From the 10 types of chitosan decomposition products with different degrees of deacetylation obtained in (2), Examples Using the same method as in (2) of 1, fractionate both fractions with a molecular weight of 5,000 to 50,000,
Ten types of chitosan decomposition products A' to J'' shown below were obtained.

Experimental Example 3 (1) Preparation of chitosan with different degrees of deacetylation Experimental Example 2
By the same method as in (1) above, 10 types of chitosan having different degrees of deacetylation were obtained.

(2) Preparation of chitosan decomposition product by enzymatic decomposition method 1χ(e
The 10 types of chitosan obtained in (1) with different degrees of deacetylation were added to the acetic acid solution (v/w) so that each chitosan was 7χ (18), and a pectinase enzyme preparation (manufactured by Shin Nippon Chemical Co., Ltd.) was added. ) to 0. 0 4 2 (in/w) Tonaichi''
j''E- 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 5000-50000 After adding 1 unit of additive, 50 After the reaction was completed, the temperature was maintained at 80°C for 5 minutes to inactivate the enzyme, and the pH was adjusted to 4.5 with caustic soda. 5% (w/w) solutions a-j of chitosanic acid decomposition products with different degrees of deacetylation were obtained.

Earth, Earth, Decoy, Five Stands, Acetylhi Agency (χ)a
65 b 71 c 75 d 80 e 86 f 8B g 90 h 94 i 96 j 99 (3) Molecular weight fractionation of chitosan decomposition products Performed from 10 types of chitosan decomposition products with different degrees of deacetylation obtained in (2) Using the same method as in Example 1 (2), fractions each having 15,000 to 50,000 molecules were fractionated to obtain 10 types of chitosan decomposition products a~j~ shown below.

Experimental Example 4 Chitosan decomposition products A゜~J'' obtained in Experimental Example 2 and chitosan obtained in Experimental Example 3 Regarding decomposition products a゛～j゛,
Antibacterial activity was compared using the following method.

Medium (glucose 100 g. ammonium chloride 0
．． 2g, monopotassium phosphate 2.0g, magnesium sulfate
jj''E-5000~50000 5000~50000 5000~50000 5000~soooo 5000~50000 5000~50000 sooo~50000 5000~50000 5000~50000 5000~50000 Torture 1I limit [1L Mul. og, 0.2 g of calcium chloride and 0.2 g of sodium chloride were dissolved in 1 liter of distilled water and adjusted to pH 4.5).
J' and chitosan decomposition products a~j obtained in Experimental Example 3
'The total nitrogen content was measured, and after adding chitosan decomposition products and distilled water to the predetermined concentrations shown in Table 1 to make the total amount 1O, Candida mycoderma IFO 0164 was added to 4.3 x 10 ' Cells/mN were inoculated and cultured at 30°C. 24 hours after the start of culture, the presence or absence of growth of the inoculated bacteria was examined.

In addition, the antibacterial power per unit total nitrogen is determined by molecular weight 5000-50.
000 and expressed as an antibacterial magnification when the antibacterial activity of a decomposed product of chitosan with a degree of acetylation of 65% is set to 1.

The results are shown in Table 1. In the table, + indicates growth of the inoculated bacteria, and 1 indicates no growth of the inoculated bacteria.

Experimental Example 5 Regarding the chitosan decomposition products A′ to J゜ obtained in Experimental Example 2 and the chitosan decomposition products a゜ to j゛ obtained in Experimental Example 3,
The flocculation effect was evaluated using the following method. Commercially available brewed soy sauce (manufactured by Kikkoman Co., Ltd.) at 10χ (w/
diluted with distilled water so that the total nitrogen content of the chitosan decomposed products A゜~J゜ obtained in Experimental Example 2 and the chitosan decomposed products a~j゜ obtained in Experimental Example 3 were diluted with distilled water so that the total nitrogen content was 0.0
Add the soy sauce adjusted to 04χ (w/v) to 2χ (ν/v), and after 5 minutes, the turbidity of the soy sauce solution was reduced to 660n.
The absorbance was measured at m. The results are shown in Table 2.

Experimental Example 6 Chitosan decomposed products B′ to J° obtained in Experimental Example 2 and the experiment were prepared to have antibacterial activity equivalent to that of chitosan decomposed products A° and a′ obtained in Experimental Examples 2 and 3. The chitosan decomposition products b゜~j゜ obtained in Example 3 were diluted according to the antibacterial activity per unit of chitosan decomposition product, and chitosan decomposition products B''~J'' and chitosan decomposition products b''~j'' were prepared. I got it.

For example, chitosan decomposition product B' has an antibacterial activity of 1.3 per unit of chitosan decomposition product, so chitosan decomposition product B' is 1.
．． It was diluted 3 times to obtain chitosan decomposition product B''. By adjusting each chitosan decomposition product in this way, chitosa becomes equivalent. For each chitosan decomposition product obtained, the same method as in Experimental Example 5 was carried out. The aggregation effect was evaluated using the following method.The results are shown in Table 3.

From the results of Experimental Examples 4 to 6, (1) the antibacterial activity improves as the degree of deacetylation increases, (2) the aggregation effect becomes stronger as the degree of deacetylation increases, (3)
) The degree of deacetylation is 86 to 96 for the aggregation effect per antibacterial power.
% was found to decrease.

What is shown in (1) is already known; for example, in Food Chemical, February 1988 issue, pages 22-29, it was reported that as the degree of deacetylation of chitosan increases, its antibacterial activity against Fusarium solani improves. has been done. However, until now, there has been no study on the relationship between the antibacterial activity and the aggregation effect on the molecular weight and degree of acetylation of decomposed chitosan products. , molecular weight is 5000-50000
Contains active ingredients and has a degree of deacetylation of 88-96%
They found that it decreased.

Example 1 Desalted and dehydrated cucumbers were soaked in a seasoning liquid with the composition shown below at a solid-liquid ratio of 1:1, and the chitosan decomposition products obtained in Experimental Examples 2 and 3 were added thereto, and the mixture was incubated at 30°C. A storage test was conducted for 2 days. The amount of chitosan decomposition products added was 1.6χ (w/w) for chitosan decomposition products A and a, and 1.6χ (w/w) for chitosan decomposition products B to J and b-j after dilution in the same manner as in Experimental Example 6. w/w).

120g 200g 1.4g Log 0.4g 0.4g 0.4g 24g 1.0g 20g 1.0g + Yield 1000g Take out a portion of the liquid immediately after pickling and prepare standard agar culture soy sauce amino acid solution lactic acid vinegar citric acid malic acid succinic acid Using sodium glutamate sodium succinate amino acid powder flavoring agent glycine base, the number of general viable bacteria was measured, and the mixture was soaked in the same manner as in Experimental Example 5, and the turbidity of the liquid was measured to evaluate the aggregation effect. In addition, a portion of the liquid after 2 days of storage was collected and the number of viable bacteria was measured using a standard agar medium. The results are shown in Table 4.

Table 4: Number of forks immediately after pickling: 3.4 x 10' JO. 16 3. IX1031.12 2.7 X 10' 0.96 3.2X10' 0.95 3.3X10' 0.94 3. OX1030.79 2.9xlO" 0.78 2.7X10' 0.50 2.O OX10' 3. O X 10' 1. O x 10' 5. O x 10' 3. OXIO'1.8X10''' 1.I X 10' 8. I X 103 7.6 Enjoy the warm weather
l. 9X10″1.01 3.6X10
'b 2.5xl030.96 2.8
xlO'c 2.7X10" 0.93
4.8X103d 2.2X1030.
95 5.7X10"e 3.5X1
0' 0.81 7.9X10"f
1.5X10' 0.74 3.9X10'
g 2.6 x 10' 0.53
2.4 X 10'h 3.9X1030.8
0 6.2X10'i 3. IX10
30.85 1.9X10'j 2.
7 X 10' 1.20 3.3
Contains ~50,000 active ingredients and a degree of deacetylation of 8
Chitosan decomposition products E-1 and e-i with 6 to 96% have antibacterial properties equivalent to chitosan decomposition products A and a with a degree of deacetylation of 65%, despite the low solid content of chitosan decomposition products. In addition to showing its strength, it also significantly suppressed the turbidity of the pickling liquid, which reduces the commercial value of light pickles.

Example 2 The chitosan decomposition products obtained in Experimental Examples 2 and 3 were added to the yakiniku sauce having the composition shown below, and Satsuriromyces roxie AHU 3013 was added to each sample using IO''call/
The seeds were inoculated in equal amounts and a storage test was carried out at 30°C for 14 days. The amount of chitosan decomposition products added was 4χ (w/w) for chitosan decomposition products A and a, and 4X (w/w) for chitosan decomposition products B-J and b-j after dilution in the same manner as in Experimental Example 6. ).

Notare Soy Sauce Light Flavor Liquid Sugar Salt G1 & Tamic Acid Sodium Beef Extract Tomato Juice Sesame Oil 230g 260g 40g 25g 13g 40g 7g Notare Onion Extract 0.05g Garlic Extract 0.03g Ginger Extract 0.03g Capsicum Extract 0.03g + Yield In addition to evaluating the presence or absence of precipitate after adding 1l of chitosan decomposition product, a portion of the liquid after 14 days of storage was collected and added to yeast MY medium (N
aCjl! 5% content) was used to measure the number of general seedlings.

The results are shown in Table 5. In addition, the presence or absence of precipitation is shown in 1, with a significant amount of precipitation, 2, with a slight amount of precipitation, and 1, with no precipitation observed.

5th table of pictures and ships 10 + ++ + 10 + 10 + + ++ ++ + 1 + + ++ + 14 days after storage 5.8 x 10' 9.8 x 10' 2.3 x 103 4.6 X 10' 3.8 X 103 7.6 X 103 9. O X 103 1.8 X 103 6.6 X 10' 7.2X103 4.3X10' 3. I X 10' 8.8 X 103 3.7 X 103 1.2 X 10' 8.5 X 10' 6. I X 10' Table 5 (continued) Chitosan Preservation 1
4L solution after 4 days” Jishiki Reno “]W
:2B Lord's help λg −
6.2X103h
+5.9X10'i
+2.2X10'j
++5. IX10' *Blank...Test area in which water was added instead of chitosan decomposition product As is clear from Table 5, the molecule ffi50
Chitosan decomposition products E-1 and e~ containing 0 to 50,000 active ingredients and having a degree of deacetylation of 86 to 96%
Despite the low solid content of the chitosan decomposition product, i shows the same zero antibacterial activity as chitosan decomposition products A and a with a degree of deacetylation of 65%, and reduces the commercial value of yakiniku sauce. The precipitation caused by this process was also significantly suppressed.

〔Effect of the invention〕

The food preservative of the present invention is safe and has sufficient preservative power, and is useful in the food industry because it reduces the turbidity of foods due to the aggregation effect of decomposed products of chitosan, which is the main raw material. Furthermore, the food preservative of the present invention does not reduce its commercial value, so it can be used in a wide variety of foods.

[Brief explanation of the drawing]

FIG. 1 shows the results of the molecular weight fractionation of the chitosan decomposition product obtained in Experimental Example 1 (1) and the results of the antibacterial property test for each molecular weight fraction.

Claims (1)

[Claims] A food preservative whose active ingredient is a hydrolyzate with a molecular weight of 5,000 to 50,000 obtained by hydrolyzing chitosan with a degree of deacetylation of 86 to 96%.