JP2000041624A - Dried food of cabbage juice and cooking of fish utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress the production of a mutagenic substance by dipping a fish in a solution containing a freeze-dried food of a cabbage juice and then carrying out a roasting treatment. SOLUTION: This dried food of a cabbage juice is obtained by subjecting ingredients of a cabbage to gel filtration with Sephadex G-25 (R) and fractionating the ingredients into F-II (fraction Nos. 33-75) and F-III (fraction Nos. 76-120) and then freeze-drying the fractions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to cabbage juice and cabbage juice fraction dried foods that suppress the occurrence of cancer and a method of cooking fish using the same.

[0002]

2. Description of the Related Art Human cancer is mainly caused by environmental factors,
In particular, it has been 20 years since it was reported that food consumption was very large. Meanwhile, methods for detecting carcinogens and mutagens from the environment have been studied using bacteria and animal cultured cells. Among them, as a method using bacteria, an excellent method for detecting a mutation was developed by Ames et al. And many studies have shown that there is a very high correlation between mutagenicity and carcinogenicity. At the same time, the foods we ingest directly include not only mutagenic substances caused by contamination during storage and additive compounds, but also mutagenic substances generated during processing and cooking of foods, and the safety of foods has been clarified. A problem has been raised from the aspect of carcinogenicity.

Under such circumstances, many researchers have repeatedly carried out epidemiological studies on cancer, animal experiments, and mutagenic tests. And, according to a report from the U.S. Academy that aggregates these data, vegetable ingredients reduce the incidence of all cancers in each area. In Japan, Kamiyama also showed significant differences in vitamin C, vitamin A, and nutrients other than iron between the two villages as a result of an epidemiological study of dietary habits and the age of death from gastric cancer in A village in Akita prefecture and B town in Iwate prefecture. Although there were no such cases, it was characteristic that the frequency of consumption of vegetables was high in town B, and it was concluded that high consumption of vegetables overall would lead to a reduction in stomach cancer mortality. Numerous other studies have reported the benefits of vegetable consumption for cancer. Apart from these studies, it has been reported that various substances that suppress mutagenic activity exist in vegetable foods such as vegetables and fruits.

[0004] On the other hand, even if a food ingredient has no mutagenic activity itself, it may be strongly carcinogenic or mutagenic due to the cooking, processing and storage of the food, and furthermore due to the interaction of the food ingredients. It has also been known that a substance showing the following may be produced. Examples thereof include nitrosamines produced by the reaction of nitrous acid and amines, and benz (a) pyrene and various heterocyclic amines produced when cooking protein foods, and their carcinogenicity has also been confirmed. ing. Under such circumstances, active research has been conducted on the mutagenicity-suppressing effects of many plant foods, triggered by the research by Kada et al.

[0005]

SUMMARY OF THE INVENTION The present inventors have found that the incidence of cancer (especially liver cancer and uterine cancer) in Nagasaki Prefecture is higher than in other prefectures, and that the annual trend is gradually increasing. Focusing on this, we thought that there might be area-specific factors. Nagasaki Prefecture is one of the leading fishing prefectures, and inevitably has more opportunities to eat grilled fish, which may lead to higher intake of Benz (a) pyrene and heterocyclic amine. On the other hand, fish is an excellent food as a protein food in terms of nutrition, and D-3 of n-3 series of highly unsaturated fatty acids.
It contains a large amount of various active ingredients such as HA, EPA, and taurine. Regardless of whether the high incidence of cancer in Nagasaki is related to the consumption of grilled fish, it is necessary to disseminate the fish diet in a safer manner.

Accordingly, the present inventors have focused on the mutagenicity-suppressing effect of vegetable foods, and have proposed a method of suppressing the generation of mutagenic substances by devising cooking and processing of grilled fish using the same, or a method of suppressing the generation of mutagenic substances. The inventors have found a method for suppressing the activity and completed the present invention.

[0007]

That is, as a means for solving the above problems, the dried food of cabbage juice according to claim 1 of the present invention is obtained by filtering and extracting the components of the cabbage and drying.

[0008] In the dried food of cabbage juice according to claim 2, the components of the cabbage are extracted by filtration and freeze-dried.

[0009] In the dried food of cabbage juice according to claim 3, the components of the cabbage are subjected to gel filtration to be separated into a high-molecular fraction and a low-molecular fraction, and the low-molecular fraction is freeze-dried.

[0010] In the dried food of cabbage juice according to claim 4, the components of the cabbage are subjected to gel filtration using Sephadex G-25, and F-II (fraction Nos. 33 to 7).
5) and F-III (fraction Nos. 76 to 12)
The fractionated solution of 0) was freeze-dried.

According to a fifth aspect of the present invention, there is provided a method of cooking a fish, wherein the fish is immersed in a solution or a seasoning liquid containing the substance according to the first, second, third or fourth aspect and a component thereof, followed by baking.

According to a sixth aspect of the present invention, in the method for cooking fish, the fish is immersed in at least one of cabbage juice, radish juice, ginger juice, lemon juice, and ascorbic acid solution, followed by baking. .

[0013]

Embodiments of the present invention will be described below with reference to the drawings. First, a method for detecting mutagenic activity from grilled fish will be described. As a detection method thereof, a modified method of the Ames method utilizing a mutation of a bacterium which has been recognized to be effective for the detection of mutagenic activity is employed. S that causes a frameshift mutation
almonella typhimurium TA9
Salmonell causing a mutation of base substitution type 8
Using atyphimurium TA100, it was tested whether mutagenic activity was detected in grilled fish by normal cooking. The fish used as the experimental material were cultured yellowtail, which was available throughout the year and had little variation in composition. As a result, when TA100 was used, S-9 Mix (which was added to activate the metabolism of mutagens) was added to the microsomal fraction of rat liver homogenate even if the baking treatment was performed to a carbonized state. Mutagenic activity was not detected irrespective of the presence or absence of various coenzymes). On the other hand, when TA98 is used, baking treatment at 260 ° C. in the presence of S-9 Mix (the surface temperature of the meat piece is 260 ° C.)
To the extent possible, baking treatment to the extent that ordinary cooking is possible). From this, it became clear that metabolic activation by S-9 Mix is necessary for normal grilled fish, and that a mutagen causing a frame-shift type mutation is generated. These properties are attributed to the heterocyclic amines Trp-P-1, Trp-P-2, Glu which have already been shown to form during the burning of the amino acids tryptophan, glutamic acid and protein during baking. −P
-1 and Glu-P-2, IQ and MeIQ. Therefore, it was found that these chemical mutagens were produced in grilled fish.

Next, in order to suppress the mutagenicity of the grilled fish or to seek a cooking / processing method for suppressing the generation of mutagenic substances, it was confirmed that the mutagenicity of heterocyclic amine was suppressed. The immersion experiment of grilled fish was performed using the vegetable juice which was done. Cabbage, as vegetable juice,
Fish was immersed in these juices for 20 minutes before or after baking (260 ° C. baking) using radish and ginger. The former treatment is called “post-immersion baking treatment”, and the latter treatment is called “pre-immersion baking treatment”. As a result, the mutagenicity-suppressing effect was observed in all of the juices in both treatments. From the above experiments, it was confirmed that the mutagenicity of the grilled fish could be suppressed in a state close to the actual cooking, but the result was more remarkable in the “post-immersion grilling treatment” (FIGS. 6, 8 to 8). 1
0). Although the mutagenic effect of many plant foods and various compounds has been reported so far, they are all mutagenic inhibitory effects of mutagenic substances except for a few experiments. Research on controlling the production of raw materials is very late. "Post-immersion baking treatment" found in this study
The strong mutagenic effect of the low molecular weight fraction of cabbage juice in the experiment is thought to be due to the suppression of mutagen production,
It was a very interesting result.

On the other hand, regarding the mutagenic effect of food, a series of studies by Kada et al. Found that various mutagenic inhibitors existed in foods and other natural products. It has been proposed to classify mutagenic inhibitors into three groups as shown in FIG. 1 in terms of their action.
The first group is factors that suppress the production of mutagens, and an example of this group is vitamin C (ascorbic acid). Carcinogenic nitrosamines are generated by the reaction between nitrous acid and amines in food, but vitamin C reductively decomposes nitrous acid and inhibits this nitrosation reaction. It is presumed that a similar effect also exists in vitamins E and A, which are antioxidants. In animal experiments, proline was administered, and it was confirmed that the production of N-nitrosoproline in urine was suppressed by plant phenols such as ascorbic acid, caffeic acid, and ferulic acid. It is presumed to have the function of suppressing in vivo. Therefore, in addition to ascorbic acid, food ingredients such as cysteine, vitamin A, and vitamin E having antioxidant ability are said to have the above-mentioned action. From the above, it is said that it is possible to suppress the production of mutagens by properly controlling the conditions of food processing and cooking.

The second group is called mutagen inactivator (factor) des-mutagen. This directly affects the mutagens or metabolically activated mutagens (degradation,
It is a factor that inhibits the reaction to inactivate by chemical modification reaction or the like, or to convert a mutagen into a metabolically active substance. The first factor in this group to be discovered is the mutagen 1,4-dinitro-2, which is formed from nitrous acid and sorbic acid.
This is a reaction in which vitamin C acts on methylpyrrole under mild conditions to reduce and inactivate it. In addition, the effect of vegetable peroxidase on heterocyclic amines and a macromolecule inactivating factor (lignin-like substance) that adsorbs and removes mutagens have been found in burdock and carrots. It is also presumed that fatty acids form complexes with benz (a) pyrene and heterocyclic amines to suppress them. In addition, it is speculated that antioxidants have an inhibitory effect in this group. In general, antioxidants suppress the peroxidation of unsaturated fatty acids, so they fall into the broad category of mutagen-inhibiting factors in that sense. It is speculated that antioxidants scavenge free radicals involved in the process of metabolic activation.

The third group includes anti-mutagenic agents,
It is a factor called bio-antimutagen. Mutagens damage DNA, which is immobilized and mutated in cells. It is a factor that affects the process of immobilization in cells and reduces the frequency of mutation. It is thought that there are a plurality of patterns in the mechanism (DNA correction / repair mechanism). Research in this field is a newer research field than the former groups 1 and 2, and in recent years its screening method has been established and several factors have been found. However, at present, the complete mechanism of DNA correction and repair is not clear, and detailed studies are required to prove that it is a mutagenic inhibitor belonging to this group.

As described above, the three mechanisms of the mutagenic suppression action have been described. The present inventors have found that cabbage, radish,
Ginger juice was found to have a mutagenicity-suppressing effect by both "pre-immersion grilling" and "post-immersion grilling" of the grilled fish. Or, it was presumed that there were factors of group 3. Therefore, in order to clarify the mutagenic inhibitory factors of grilled fish in vegetable juice including these points, cabbage juice was converted to Se.
phadex G-100 and Sephadex G
Gel filtration at -25, and the mutagenicity inhibitory effect of each fraction was baked using "blinding before baking" and "baking after baking".
I checked in. First, a high-molecular fraction and a low-molecular fraction were separated into two using Sephadex G-100, and the inhibitory effect of each fraction was examined. As a result, the polymer fraction has a remarkably strong “pre-immersion baking” effect, and the low-molecular fraction has both the “pre-immersion baking” and “post-immersion baking” effects. Was strong. Therefore, SePhadexG-
The low-molecular fraction is fractionated into 3 by 25, and using each fraction,
An experiment similar to the above was performed. As a result, the high-molecular fraction exhibits the same strong “pre-immersion baking” effect as described above, and the low-molecular fraction in which glucose or ascorbic acid elutes has both the “pre-immersion baking” and “post-immersion baking” effects. The effect was confirmed, and it was confirmed that the fraction eluted slightly later than the low-molecular-weight fraction had a strong effect of “post-immersion baking treatment”. From these results, it was found that at least three different mutagenic inhibitors existed in cabbage juice.

Until now, the mutagenic effect of vegetable juice on heterocyclic amines has been studied by studying the effect of chemical mutagens by acting on all chemical mutagens. No study has examined the mutagenicity of the baked and then baked products. Since this study started with suppressing the mutagenicity of grilled fish by devising cooking and processing, we tried a method of pre-treatment with vegetable juice as a method of cooking and found that the effect was remarkable. It was suggested that the effect was due to low molecular weight substances in vegetable juice. However, since the above studies are the results of experiments with grilled fish, it is necessary to prove the phenomenon with a more biochemical method.

Therefore, the “pre-immersion baking treatment” of the polymer fraction
To demonstrate the effect of the high molecular weight fraction on the activity of the mutagen produced by baking, the effect of the heterocyclic amine Trp-P produced by baking of protein or amino acid tryptophan was proved. −
1. Using Trp-P-2 and IQ, it was examined which fraction of cabbage juice strongly inhibited its mutagenic activity. The results were almost the same as those obtained with grilled fish. That is, Trp-P-1, T
It has a strong mutagenic activity-suppressing effect on rp-P-2 and IQ, and then a low-molecular-weight fraction (F-II) containing eluted glucose and the like has a weaker but considerable inhibitory effect than the FI fraction. Was done. However, no inhibitory effect on the activity of these chemical mutagens was observed in the low-molecular fraction (F-III) eluted last.

Next, the strong mutagenic effect of the high molecular fraction on heterocyclic amines was attributed to the peroxidase of cabbage juice, indicating that the enzyme activity of commercially available horseradish peroxidase and its mutagenic activity were suppressed. (Activity) and the effects of heat treatment (100 ° C.) on their activities were clarified from the results of examination.
In addition, as a result of examining the effect of heat treatment on the enzyme activity and the mutagenic activity inhibitory effect (activity) of peroxidase, it was found that the mutagenic activity inhibitory effect is not essentially related to the enzyme activity, but rather has heme iron. It was presumed to be the effect of the structure itself. This indicates that the mutagenic activity inhibitory effect was maintained at a considerably high level even when peroxidase was inactivated by heating, and that other heme proteins (hemoglobin, catalase, and cytochrome C) also had a weak inhibitory effect. It also suggests that the protease hydrolyzate of hemoglobin has a weak inhibitory effect, and that when the hydrolyzate is subjected to gel filtration, the mutagenicity of the heme-rich fraction is high. Was.
Therefore, conventionally known des-mutagen is used.
It was revealed that the action of peroxidase was not limited to its enzymatic action.

Next, the mutagenic activity inhibitory factor of the fraction (F-II fraction (low molecular fraction eluted after the high molecular fraction)) from which the glucose and fructose in the cabbage juice are eluted ("baking before immersion") To estimate the "treatment" effect), the T of glucose, fructose, sucrose and L-acorbic acid apparently contained in this fraction is estimated.
The mutagenic activity inhibitory effect on rp-P-1 and IQ was examined. As a result, all three types of sugars showed an inhibitory effect on Trp-P-1, and glucose showed a relatively strong inhibitory effect, particularly. Glucose showed only a weak inhibitory effect on IQ. Although not shown in the results, L-ascorbic acid showed a considerable inhibitory effect on Trp-P-1, but did not show any inhibitory effect on IQ.

From these results, it was presumed that the effect of the "pre-immersion baking treatment" of the F-II fraction was affected by the sugar and ascorbic acid described above. There is no report on the reaction between the sugar and the heterocyclic amine, and glucose and fructose are considered to be acting by their reducing properties, but the inhibitory effect of sucrose cannot be explained by the reducing power. We believe that detailed research on the chemical reaction between these compounds and heterocyclic amines will be necessary in the future.

Next, the main purpose of the present invention was to demonstrate the mutagenicity-suppressing effect of the low-molecular-weight fraction, which was confirmed to have a strong "post-immersion baking treatment" effect in a grilled fish experiment, using a more chemical experimental system.
The experiment was conducted by replacing the grilled fish with tryptophan. That is,
350 ° C after adding cabbage juice fraction to tryptophan
For 30 minutes, and the mutagenic activity was measured to examine the effect of each fraction on the mutagen production. As a result, the strongest inhibitory effect was exhibited by low molecular weight (F) eluting later than glucose or fructose.
-III-1) It was a fraction. Next, the inhibitory effect of the fraction (F-II fraction) from which glucose or fructose eluted was strong, and the inhibitory effect of the polymer fraction was very weak. This result was in good agreement with the result for grilled fish. based on the above results,
It was strongly suggested that cabbage juice contained low-molecular substances having different elution positions due to at least two types of gel filtration that suppress generation of mutagen. That is, it was mentioned earlier that mutagenic inhibitors are divided into three groups (FIG. 1), but cabbage juice belongs to the first group in addition to peroxidase which is said to belong to the second group. ,
It was found that low molecular weight substances, which are thought to suppress the formation of heterocyclic amines, were present in two different fractions of the gel filtration by Sephadex G-25. Although there are many studies on mutagenic substances in plant foods, there are very few studies on substances that suppress the production of mutagen (factors belonging to the first group).

As an example of the first group, the example of ascorbic acid which inhibits the nitrosation reaction between nitrous acid and amines has been described. At present, no research has been conducted on substances that suppress the generation of clickamine. In this study, in order to identify the substance, the inhibitory effect of a few compounds contained in the low-molecular fraction of cabbage juice (F-II fraction, fraction in which glucose and fructose elute) was compared with tryptophan. In addition to the baking treatment (corresponding to the "post-immersion baking" experiment) was investigated. As a result, it was confirmed that glucose and fructose had an inhibitory effect.
This result is considered to be in common with the effect of suppressing sucrose in the “post-immersion baking treatment” experiment of grilled fish. However, no inhibitory effect was observed for L-ascorbic acid. Therefore, it was presumed that the inhibitory effect of the post-immersion baking treatment of the fraction from which glucose and the like eluted was due to these saccharides.

As described above, the mutagenic effect of cabbage juice fraction on the mutagen of grilled fish was examined. As a result, in order to reduce the mutagenicity of grilled fish as much as possible, "baking after immersion" is effective, and even if there is an effect of individual differences in cabbage, judging from the results of several experiments, It was presumed that grilled fish having almost no mutagenicity could be obtained by the treatment. Of course, in actual cooking and processing, since both "pre-immersion baking treatment" and "post-immersion baking treatment" can be used, it is considered that the effect can be further enhanced by cooking using both treatment methods. .

[0027]

[Examples] There have been many studies on natural mutagenic activity inhibitors (also called anti-mutagens) against chemical mutagens generated when meat or fish are baked. Very few studies have been conducted on changes in mutagenic activity or suppression of mutagen production under conditions close to the standard.
In particular, there is no study on the latter phenomenon of suppressing the production of mutagens. It is well known that the baking of proteinaceous foods, such as meat and fish, produces various heterocyclic amines, which are carcinogens with mutagenic activity. There are many experiments in which a mutagenic substance is separated and purified from charred charcoal by performing extremely extreme baking treatment, which is slightly different from the actual cooking conditions. Therefore, in this chapter, The effect of heating conditions (baking conditions) on the expression of mutagenic activity in grilled fish was examined, and mutagenic activity was detected even by grilling close to ordinary cooking using meat pieces of cultured yellowtail cut to a certain size. I was sure that. Then, 2. The effects of (1) vegetable juice, fruit juice, and ascorbic acid solution, and (2) the effect of seasoning solution on the generation of mutagen and the activity of the generated mutagen in grilled yellowtail were examined by two treatment methods. One is a method in which a piece of yellowtail is immersed in the above-mentioned various solutions and then baked, and the other is a method in which the piece of meat is immersed after baking.

In the present embodiment, the former treatment is called "post-immersion baking treatment", and the latter treatment is called "pre-immersion baking treatment".
Extracts (mutagenic activity measurement sample solutions) were prepared from the meat pieces treated by both treatment methods, and the mutagenic activity was measured. As a result, it was confirmed that the mutagenic activity of burnt yellowtail was remarkably reduced by both of the above-mentioned treatment methods using the liquids (1) and (2). That is, it was suggested that the mutagenicity of grilled fish could be reduced by devising the cooking process, and the results were summarized.

Experimental Materials and Experimental Methods Preparation of sample liquid for measuring mutagenic activity (baked yellowtail extract) As a sample fish, cultured yellowtail (body length about 65 cm) with little change in the amount of body fat per year was used as an experimental material. The back muscle was cut with a sharp scalpel and a 2 x 5 x 0.5 cm piece of meat (weight 5
g) It was used for the baking treatment experiment. Put the meat pieces of this cultured yellowtail into (1) vegetable juice (cabbage, radish, ginger),
Fruit juice (lemon), 0.1% L-ascorbic acid solution,
(2) Seasoning liquid (50% sucrose, soy sauce, 50%
20ml each of 1: 1 mixture of sucrose and soy sauce)
After being immersed at room temperature for 20 minutes, it was baked. Such a case where the baking treatment is performed after immersion in a specific solution is referred to as a “post-immersion baking treatment” experiment. On the other hand, when the cultured yellowtail meat pieces are baked and then immersed in 20 ml of each liquid for 20 minutes,
This is referred to as a “pre-immersion baking treatment” experiment. A small amount of dimethyl sulfoxide was added to the samples subjected to the “post-immersion baking treatment” and “pre-immersion baking treatment”, and the mixture was ground with a homogenizer for 5 minutes, and transferred to a weighing bottle using a total of 15 ml of dimethyl sulfoxide.

After leaving it at 37 ° C. for 24 hours,
man filter paper No. 2, and the mixture was subjected to suction filtration to obtain an extract of grilled fish. This was used as a sample for measuring mutagenic activity. The baking process is performed by bonding the tip of a thermocouple sensor to the center of the surface of the meat piece and using a roaster (NF-730 manufactured by National).
Mold). The conditions were set at 200 ° C. and 260 ° C., which are generally levels of eating, and at 400 ° C. and 600 ° C. where carbonization occurs, and heating was performed until this temperature was reached. Each piece of meat was subjected to the above-described baking treatment, and the roaster was cooled to room temperature and used each time. In the control test, in the case of the “baking after immersion” experiment, the meat pieces were immersed in distilled water at room temperature for 20 minutes and then baked (in this case,
The result was almost the same even if the baking treatment was simply performed.) In the case of the “pre-soaking baking treatment” experiment, the baking treatment with the roaster was followed by immersion in 20 ml of distilled water for 20 minutes. Vegetable juices and the like were prepared by using 200 g of fresh vegetables or three lemons, washing with water and then thoroughly wiping the water, and collecting them with a juicer and a lemon squeezer, respectively. The soy sauce used was Choco Koikuchi (manufactured by Choko Shoyu Co., Ltd., Honjozo).

2. Measurement of mutagenic activity Mutagenic activity was measured by a modification of the Ames method. 1) Strain to be used and its preculture The mutagenic activity was detected by detecting a frame-shift type mutant, Salmonella typhimurium TA9.
8 and a base substitution mutant Salmonella t
yphimurium TA100 was used. This was pre-cultured and used. For pre-culture, 4 g of nutrient broth (manufactured by Difco) and 2.5 g of sodium chloride were dissolved in 500 ml of distilled water, and 7.5 ml of each solution was dispensed into an L-shaped test tube with a silicon stopper, and autoclaved for 12 hours.
The solution was sterilized at 1 ° C. for 20 minutes and stored refrigerated until use. Then, the above L-shaped test tube was inoculated with 15 μl of the bacterial suspension which had been previously dispensed at −80 ° C. and stored frozen, and this was inoculated at 37 ° C.
For 16 hours with shaking.

2) Medium (i) Minimum glucose agar medium: (a) vogel
Bonner minimal medium (magnesium sulfate heptahydrate 0.
2 g, citric acid monohydrate 2.0 g, anhydrous dipotassium phosphate 10.0 g, ammonium hydrogen phosphate sodium 4
Dissolve 3.5 g of water salt in 100 ml of distilled water), (b) Di
fco Agar Powder 5 g distilled water 800
(c) 20 g of D-glucose was dissolved in 10 ml of distilled water.
Dissolve in 0 ml. The above (a), (b), and (c) were sterilized in an autoclave and then mixed.
A minimum glucose agar plate was prepared using 20 ml per m). (Ii) Soft agar: 0.7% Difco Aga
r Powder-0.6% sodium chloride solution was sterilized in an autoclave, and 1/20 volume of 0.5 mM L-histidine and 0.5 mM
After adding the M biotin solution, keep at 45 ° C until use,
The experiment used 2 ml per plate.

3) Preparation of S-9 Mix S-9 (manufactured by Oriental Yeast Co., Ltd.) is added for activating metabolism of mutagen, and 9000 g of supernatant of rat liver homogenate (S-Mix) is added. 9 fractions). Generally, it is said that carcinogens are metabolized directly in animals to become carcinogens, and are often activated as ultimate carcinogens, and many microorganisms lack this metabolic pathway. Therefore, mammalian liver homogenates have been used to activate metabolism in a mutation detection experiment system using microorganisms. In this experiment, according to a modification of the Ames method, a compound obtained by adding the following compound to the S-9 fraction prepared from rat liver was designated as S-9 Mix. 0.1 ml was added to 1 ml of the S-9 fraction of rat liver homogenate.
0.2 ml of 4 M magnesium chloride, 0.2 ml of 1.65 M potassium chloride, 0.05 m of 1 M glucose-6 phosphate
1, 0.1M NADPH 0.4ml, 0.1M NADH
0.4 ml, 0.25 M sodium phosphate buffer (pH
7.4) 4 ml was added, and the total amount was adjusted to 10 ml with distilled water. s-9Mix was prepared each time for each test, and used after being sterilized by filtration.

4) Detection of mutagenic activity Based on a modified method of Ames, typhimuriu
m The number of revertants to histidine non-auxotrophy induced in TA98 and TA100 was used as an index. A mutagenic activity measurement sample solution (baked yellowtail extract) and 0.1 ml of a test solution diluted with sterile water (2 to 20-fold dilution) are placed in a sterilized small test tube, and S-9 Mix or 0.
0.5m 1M sodium phosphate buffer (pH 7.4)
1 and 0.1 ml of the pre-cultured bacterial suspension were added and adjusted to a total volume of 1.0 ml with sterile water. This is 37
After shaking and keeping at 20 ° C. for 20 minutes, 2.0 ml of soft agar stored at 45 ° C. was added and layered on a minimum glucose agar plate prepared in advance. This is 37
After culturing at 48 ° C for 48 hours, the number of histidine non-requiring revertant colonies on the plate (expressed as the number of His + revertants or revertants) was counted. In addition, the mutagenicity (activity) suppression effect was calculated | required by the following formula. Mutagenic (active) inhibitory effect (%) = [(AB) / A] ×
100A is the number of revertant colonies in the control test, that is, the number of revertant colonies in the extract prepared from a sample immersed in distilled water before or after baking the meat pieces. B is the number of revertant colonies of the extract prepared from the sample in which the meat pieces were immersed in a liquid other than distilled water. Note that A and B are numerical values obtained by subtracting the number of spontaneously mutated colonies (using sterilized distilled water in place of the baked yellowtail extract). The above experiments were performed using three plates for each experiment, and the average value was calculated.

1. Mutagenic activity of roasted yellowtail extract The temperature, the time required to reach the temperature and the state of roasted yellowtail when roasting a piece of yellowtail meat with a roaster are shown in FIG.
As shown in FIG. In addition, the mutagenic activity of cultured yellowtail under various baking treatment conditions. typhimurium T
Fig. 3 shows the mutagenic activity against A98 (S-9Mix added).
It was shown to. The horizontal axis indicates the number of μl of the grilled yellowtail extract per plate, and the vertical axis indicates the number of revertant colonies per plate. No mutagenic activity was observed in raw cultured yellowtail that was not subjected to baking treatment. In the case of baking at 200 ° C., which gives the most ideal baking state (provides a light brown baking state) that is generally eaten, no increase in the number of revertant colonies is observed even when the amount of the extracted sample solution is increased.
No mutagenic activity was observed. However, in the case of a baking treatment at 260 ° C. (a baking state in which browns appear around fish meat, a state often observed during home cooking processing; also referred to as intermediate baking in the present embodiment), an increase in the amount of extracted sample solution Accordingly, the number of revertant colonies increased, and mutagenic activity was clearly detected. In the baking treatment at 400 ° C. and 600 ° C. (fully carbonized state in which the inside of the meat piece was carbonized), about twice and about 6 times the mutagenic activity was observed in the baking treatment at 260 ° C., respectively. However, when S-9Mix was not added, mutagenic activity was not observed even after baking at 600 ° C. (FIG. 4). In addition, S.I. typhimuri
When um TA100 was used, almost no mutagenic activity was detected by baking at 600 ° C. regardless of whether S-9Mix was added or not (FIG. 5).

2. Effect of vegetable juice on mutagenicity of grilled yellowtail
Influence of fruit juice, L-ascorbic acid and several seasonings According to the experiment in the preceding section, S-9 was obtained in grilled yellowtail at 260 ° C or higher.
Mix in the presence of Mix. typhimurium TA98
When was used, mutation was clearly caused, and it was confirmed that mutagenic activity was detected. In the presence of S-9Mix, Using the reverse mutation of T. typhimurium TA98, the effect of vegetable juice and seasoning liquid on the mutagenicity of grilled yellowtail at 260 ° C. was examined.

1) Influence of vegetable juice, fruit juice, L-ascorbic acid solution (i) Influence of cabbage juice The mutagenicity of the intermediate-baked cultured yellowtail, which was baked at 260 ° C, was immersed in cabbage juice and baked. Both samples of “baking after immersion” and “baking before immersion” baked before immersion in cabbage juice were suppressed by cabbage juice (FIG. 6). The mutagenic (active) activity inhibitory effect was particularly remarkable in the case of "baking treatment after immersion", and was 90% or more. On the other hand, in the case of the “pre-immersion baking treatment”, it was about 60%. The mutagenicity (activity) suppression effect was calculated by extracting 50 μl of the extracted sample solution in which the number of revertant colonies was high.
/ Plate and 100 μl / plate, and the average value was used. Although not shown in the figure, the baking treatment at 200 ° C. did not show the mutagenic activity as in the case of the raw fish, so that the influence of the cabbage juice immersion treatment was not recognized. In the carbonized cultured yellowtail subjected to the baking treatment at 400 ° C., a weak suppression effect was observed only in the sample of “post-immersion baking treatment”. In the case of the fully carbonized state subjected to baking treatment at 600 ° C., strong mutagenic activity was detected, and no suppression effect by cabbage juice was observed. In addition, the same detection experiment was performed on the samples that were subjected to “baking after immersion” and “baking before immersion” using distilled water. Since no test was observed, only the result of the specimen which was baked at 260 ° C. without performing the immersion treatment with distilled water as a control test of the 260 ° C. baking treatment was shown.

Next, in order to estimate whether or not the causal juice mutagenic (active) inhibitory factor observed in the above-mentioned baking treatment at 260 ° C. is easily denatured by heating, for example, due to a protein or the like, it was examined for 20 minutes. Using the cabbage juice heated and boiled, a sample “baked after immersion” was prepared, and the effect of the heat treatment of the cabbage juice was examined. As a result, even when immersed in heated cabbage juice, mutagenicity was suppressed, and no difference from the suppression effect of fresh cabbage juice was observed (FIG. 7).

(Ii) Influence of radish juice, ginger juice, lemon juice and L-ascorbic acid The sap of radish, ginger, lemon, etc. and mutagenicity of cultured yellowtail after 260 ° C. baking treatment and L-ascorbic acid in 0. The effect of the 1% solution (concentration similar to the vitamin C content in lemon juice) was examined by both experiments of "baking after immersion" and "baking before immersion". as a result,
In each case, the mutagenic (active) activity suppressing effect was observed (FIGS. 8 to 11). Mutants calculated from the average values of 50 μl / plate and 100 μl / plate of “radish after immersion” and “pre-immersion baked” samples of radish juice, ginger juice, lemon juice and 0.1% L-ascorbic acid solution The activity-suppressing effects were 95% and 72% for radish juice, 92% and 72% for ginger juice, 80% and 48% for lemon juice, and 91% for 0.1% L-ascorbic acid solution, respectively. And 82%, indicating that the effect of suppressing mutagenicity was greater in the “post-immersion baking treatment” than in the “pre-immersion baking treatment”.

2) Influence of seasoning liquid (i) Influence of "post-immersion baking treatment" TA98 (in the presence of S-9 Mix) when aquacultured yellowtail was immersed in several types of seasoning liquids and then baked at 260 ° C.
FIG. 12 shows the mutagenic activity against. As the seasoning liquid, a 50% sucrose solution, soy sauce, and a mixed solution (1: 1) of 50% sucrose solution and soy sauce were used. As a result, 5
At the average value of 0 μl / plate and 100 μl / plate, the mutagenic activity was about 74% in soy sauce immersion, about 69% in sucrose solution immersion, and sucrose and soy sauce mixed solution compared to the control test. About 56% was suppressed, and the effect of the immersion treatment was recognized by any of the seasoning liquid treatments.

(Ii) Influence of "Baking treatment before immersion" FIG. 13 shows the mutagenic activity of cultured yellowtail after baking at 260 ° C. in various seasonings. 50 as before
In the mean of μl / plate and 100 μl / plate,
The soaking effect was about 90% in soy sauce immersion, about 84% in sucrose solution immersion, and about 81% in sucrose and soy sauce mixed solution immersion. It was confirmed that these seasonings had an effect of significantly suppressing the mutagenic activity of the mutagen produced in the grilled fish.

Differences in mutagenic activity due to differences in the baking temperature of cultured yellowtail, and the effects of vegetable juice, fruit juice, ascorbic acid and seasoning on the production of mutagenic substances in yellowtail and the activity of the mutagen produced in yellowtail The effect of S. t
Yphimurium TA98 and TA100 were examined by a modified method of Ames. At the same time, S-
The effects of adding and not adding 9Mix were examined.

First, the applicability of the sample solution (mutagenicity measurement sample solution) extracted from grilled yellowtail to the Ames method will be discussed. The Ames method is a method using a histidine auxotrophic strain and detecting a reversion to histidine non-auxotrophy. However, the Ames method employs a method in which a certain amount of histidine is added to a medium. On the other hand, it is considered that the free histidine in fish meat is naturally contained in the sample solution for measuring mutagenic activity (baked yellowtail extract) in this experiment. Therefore, it is thought that histidine in this sample solution affects the results of the Ames method,
This experiment was not a comparison of the mutagenic activity of grilled yellowtail meat pieces with other fish species or other foods, but an experiment in which the histidine content in the sample was compared under the same conditions. It was judged that it hardly affected the judgment result. In the Ames method, it is said that the number of spontaneously mutated colonies increases as the amount of histidine in the extracted sample solution increases. This means that in experiments that examine the suppression effect as in this experiment, it can be a factor that moves the suppression effect in the negative (negative) direction, but cannot be a factor that moves the suppression effect in the positive (both) direction. Means Furthermore, as shown in FIGS. 3 to 5, no tendency was observed such that the number of revertant colonies in the extract of the raw fish increased with an increase in the extract per plate. From these results, it is clear that the amount of free histidine in fish meat hardly affects the measurement of mutagenic activity within the scope of this experiment.

In the case of the baking treatment at 200 ° C. (the most ideally baked state as a cooking process), the mutagenic activity against TA98 and TA100 is the same as that of the cultured yellowtail which is not subjected to the baking treatment. None was recognized, with or without presence. In the case of the baking treatment at 260 ° C., at which the meat pieces start to burn, the number of revertant colonies clearly increases with TA98 (S-9Mix added) with the increase in the extract sample solution used for the measurement. Extracted sample liquid 50
The average value of μl and 100 μl / plate was about 10 times that of the raw fish. Furthermore, the mutagenic activity increased with an increase in the baking temperature, and the number of revertant colonies increased by about 20 times and about 60 times that of the raw fish by baking at 400 ° C. and 600 ° C. (charging). . Note that S
600 ° C for TA98 when -9 Mix is not added
No mutagenicity was observed in the sample which was baked.
In addition, no mutagenic activity of burnt yellowtail was detected for TAl00 regardless of the presence or absence of S-9Mix.

As described above, the fact that S-9 Mix was necessary to cause reversion and that no mutagenic activity was detected in TA100, which causes a base substitution type mutation, indicate that during the baking treatment, The mutagen produced in the above requires metabolic activation for the expression of the mutagenesis, and suggests that it is a substance causing a frameshift type mutation, not a base substitution type. ing. From the above results, the mutagenic activity of grilled fish was S-9M
It became clear that detection was possible by using TA98 in the presence of ix. In addition, it has been reported that the heat-decomposed product of the protein requires metabolic activation by S-9 Mix and shows strong activity on the frame-shift type TA98, and this experiment was consistent with these reports. Also,
The relationship between the baked state of the fish and the mutagenic activity was such that the baked state could be an actual cooking process, that is, the baked state was such that brown appeared on the meat surface (baking treatment at 260 ° C in this experiment).
It was revealed that a substance having mutagenic activity was produced.

Based on the above results, the following experiment was conducted using TA98 and adding 0.5 ml of S-9 Mix to examine the mutagenic activity of grilled fish. Trp-P-1 and Tr generated when tryptophan of protein or amino acid is heated at high temperature
The mutagenic activity inhibitory effect of vegetable extracts on chemical mutagens such as p-P-2 has already been reported,
Here, using grilled yellowtail meat pieces, making grilled fish close to the actual cooking process, in the process, immersing the meat pieces with vegetable juice, L-ascorbic acid and several seasonings, and performing mutagenicity of grilled fish The effect of those immersion treatments on the water content was investigated.

The mutagenicity of the intermediate grilled cultured yellowtail which had been baked at 260 ° C. was suppressed by cabbage juice in both the “post-immersion baking” and “pre-immersion baking” treatment methods. The average mutagenicity of 50 μl and 100 μl of the extracted sample solution per plate was about 90% for “post-immersion baking” and “pre-immersion baking”.
Was about 60%, and the former treatment method was more effective. In addition, in an experiment of “baking after immersion” in which the mutagenicity suppressing effect was high, the effect of heating and boiling (20 minutes) cabbage juice, which is an immersion liquid, was examined. As a result, a result showing the same mutagenicity suppression effect as that of raw cabbage juice was obtained. This suggested that cabbage juice contained a heat-resistant substance as a mutagenic inhibitor in the “post-dipping baking treatment”.

The suppression effect of radish juice, ginger juice, and L-ascorbic acid solution on the mutagenicity of cultured yellowtail at 260 ° C. is similar to that of cabbage juice by “post-immersion baking treatment” and “pre-immersion baking treatment”. Accepted by both treatments. The mutagenicity suppression effect of both treatment methods was 5
Looking at the average values of 0 μl and 100 μl, 95% and 72% were immersed in radish juice, 92% and 72% were immersed in ginger juice, and 80% and 4% were immersed in lemon juice.
8%, 0.1% ascorbic acid solution (substantially equivalent to the vitamin C content in cabbage juice) immersed in 91% and 82%, and the suppression effect in "baking after immersion" rather than "baking before immersion" it was high. It is completely unknown whether the mutagenic inhibitory factor in both treatment methods for each vegetable juice and the like is due to the same substance, but until now, Trp-P-1 and Trp-P produced when the protein is baked. -2
It has been reported that various vegetable juices have a mutagenic activity-suppressing effect on and against several other chemical mutagens.

However, when these reports are applied to the present experiment, there is no report corresponding to the “pre-immersion baking treatment” experiment, and no report corresponding to the “post-immersion baking treatment” experiment. Among the many studies corresponding to "pre-immersion baking treatment", only a few studies have revealed the effect of suppressing the mutagenic activity of specific components of plant foods. There are some reports of the effects of peroxidase in green and yellow vegetables, the effects of burdock lignin-like polymer compounds, the effects of plant fibers adsorbing mutagens, and certain components in red algae. In addition, effects of vitamin E, vitamin C (L-ascorbic acid) and the like are estimated as substances involved in the antioxidant action in the living body. No studies have been proven.
Inferring from the results of these previously reported studies, peroxidase is considered to be a substance that has the effect of suppressing vegetable juice in the “pre-immersion baking treatment” experiment in this experiment.

As another factor, in this experiment, L-ascorbic acid alone showed this effect. This suggested that one of the inhibitory substances in the vegetable juice was L-ascorbic acid. However, a strong inhibitory effect was observed even in ginger juice with a low vitamin C content (about 1/20 of cabbage in the food composition table). In the case of "pre-soaking treatment" of lemon juice with a high vitamin C content The effect of ascorbic acid is not considered to be great, because the effect of suppressing ascorbic acid is not so high. In addition, as described above, adsorption to fiber is also reported as one of the mutagenicity-suppressing effects of plant foods, but the results of this experiment are those using juices of vegetables and fruits. Therefore, it was presumed that there was no influence of at least the insoluble fiber substance. On the other hand, as described above, the results of this experiment revealed that the vegetable juice contained a substance having an effect of “baking after immersion”. That is, it was suggested that a substance that suppresses the production of mutagenic substances during the baking treatment was present in vegetable juice.

Next, the effect of the seasoning liquid on the mutagenicity of grilled yellowtail was examined for sugar and soy sauce, which are widely used for grilling and teriyaki. In the experiment, sucrose was used instead of sugar. A 50% sucrose solution, soy sauce, and a mixture of soy sauce and 50% sucrose solution (1: 1) were used. The effect was recognized. In addition, the effect of suppressing mutagenicity was also observed when "baking treatment after immersion" was performed. However, unlike the case of vegetable juice and lemon juice, the effect of "baking before immersion" was slightly stronger than that of "baking after immersion". One of the effects of sucrose and soy sauce is the product of the Maillard reaction (aminocarbonyl reaction). In this regard, it has been reported that melanoidin produced by an aminocarbonyl reaction has a mutagenic activity inhibitory effect on heterocyclic amine produced by baking fish meat. Therefore, it is considered that the inhibitory effect of the seasoning liquid confirmed in this experiment is influenced by melanoidin. However, on the other hand, Hanaoka
According to the research report, the brown pigment of melanoidin also has an effect of inhibiting the growth of certain bacteria. In addition, there is a report on the effect of 2-methyl or 2,5-dimethylpyrazine generated by the Maillard reaction on IQ generated when beef is baked, which is the reverse effect of enhancing the mutagenic activity of IQ. It is unknown whether Maillard reactants are involved in the immersion effect of sucrose and soy sauce confirmed in this experiment, but sucrose and soy sauce are used in the process of grilling and cooking fish meat. It was suggested that the mutagenic activity of grilled fish could be suppressed to some extent.

From the above results, in addition to the technique of “pre-dipping baking”, which has been used as a traditional eating habit, such as adding grated radish to grilled fish, eating ginger, and adding lemon juice, this experiment The mutagenicity suppression effect of the “post-immersion baking treatment” first revealed in
It can be applied to cooking methods such as "teriyaki" and shows that the mutagenicity of grilled fish can be further suppressed by devising the cooking method, and its utility is expected to spread. Also, T
Freeze-drying cabbage juice of cabbage having a mutagenic activity-suppressing effect and a mutagenic-product generation-suppressing effect caused by high-temperature overheating of fish and shellfish such as rp-p-1, Trp-p-2 and IQ By processing it as a freeze-dried food and using it easily in your daily diet,
It can help prevent cancer.

The embodiments and examples of the present invention have been described above. However, the specific configuration of the present invention is not limited to the present embodiments, and changes and the like in a range not departing from the gist of the invention are described. The present invention is also included in the present invention. For example, the method of filtering cabbage juice described in the above-described embodiment is not limited to gel filtration, and the present invention includes any other filtration method or centrifugation. The cabbage juice drying method according to the above embodiment is freeze-drying. However, the same operation and effect can be obtained when the cabbage juice is used as a liquid without drying or when another drying method is used.

[0054]

As described above, in the dried food of cabbage juice according to the first and second aspects, the filter extract component of cabbage suppresses the mutagenicity of grilled fish.

In the dried food of cabbage juice according to the third aspect, the low molecular fraction of the gel filtration component of cabbage effectively suppresses the generation of mutagen and the activity of the mutagen of grilled fish.

In the dried food of cabbage juice according to the fourth aspect, the F-II (fraction Nos. 33 to 75) fraction effectively suppresses the production of mutagenic substances and the activity of mutagenic substances in grilled fish. . Furthermore, the F-III (fraction Nos. 76 to 120) fraction most effectively suppresses the production of mutagen in grilled fish.

According to a fifth aspect of the present invention, there is provided a method for cooking grilled fish, wherein the fish is immersed in a low-molecular fraction of a filtered extract component of cabbage or a gel filtration component of cabbage, followed by baking treatment. Suppress.

In the method for cooking grilled fish according to claim 6, cabbage juice, radish juice, ginger juice, lemon juice,
A method of immersing the fish in at least one of the ascorbic acid solutions and then performing the baking treatment suppresses the mutagenicity of the baked fish.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing a mutagenic inactivation mechanism.

FIG. 2 is an explanatory diagram showing a required time during a baking process, an attained temperature of a meat surface, and a state of a piece of meat;

FIG. 3 is an explanatory diagram showing mutagenic activity of yellowtail under various baking treatment conditions.

FIG. 4 is an explanatory diagram showing the mutagenic activity of carbonized yellowtail.

FIG. 5 is an explanatory diagram showing the mutagenic activity of carbonized yellowtail.

FIG. 6 is an explanatory diagram showing the effect of immersion in raw cabbage juice on the mutagenicity of yellowtail (260 ° C.) yellowtail.

FIG. 7: Intermediate baking (26
It is explanatory drawing which shows the influence of heating cabbage juice immersion on the mutagenicity of yellowtail (0 degreeC).

FIG. 8 is an explanatory diagram showing the effect of radish juice immersion on the mutagenic activity of yellowtail (260 ° C.) yellowtail.

FIG. 9 is an explanatory diagram showing the effect of ginger juice immersion on the mutagenic activity of yellowtail (260 ° C.) yellowtail.

FIG. 10 is an explanatory diagram showing the effect of lemon juice immersion on the mutagenic activity of yellowtail (260 ° C.) yellowtail.

FIG. 11 is an explanatory diagram showing the effect of immersion in a 0.1% L-ascorbic acid solution on the mutagenic activity of yellowtail (260 ° C.) yellowtail.

FIG. 12: Intermediate baking (2)
It is explanatory drawing which shows the influence of heating cabbage juice immersion on the mutagenic activity of yellowtail (60 degreeC).

FIG. 13: Intermediate baking (2
It is explanatory drawing which shows the influence of various seasonings on mutagenic activity of yellowtail (60 degreeC).

FIG. 14 is an explanatory diagram showing a procedure for preparing cabbage juice.

FIG. 15 is an explanatory diagram showing a modification of the Ames method.

FIG. 16 is an explanatory diagram showing an elution pattern of gel filtration using Sephadex G-100.

FIG. 17 is an explanatory diagram showing an elution pattern of gel filtration using Sephadex G-25.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B018 LB05 LE06 MS09 MS11 MS14 4B041 LC10 LD10 LH01 LH08 LK05 LK19 LK36 LP05 LP07 LP09 LP16 4B042 AC08 AC10 AD08 AE03 AG30 AH01 AK04 AK11 AP04 AP07

Claims (6)

[Claims] 1. A dried food of cabbage juice, wherein components of the cabbage are extracted by filtration and dried. 2. A dried food of cabbage juice, wherein components of the cabbage are extracted by filtration and freeze-dried. 3. A dried food product of cabbage juice, wherein the components of the cabbage are subjected to gel filtration, separated into a high molecular fraction and a low molecular fraction, and the low molecular fraction is freeze-dried. 4. The component of cabbage is Sephadex G
Gel filtration through F-25 (fraction N
o. 33-75) and F-III (fraction no.
76-120), and dried and freeze-dried cabbage juice. 5. A method for cooking fish, comprising immersing the fish in a solution or seasoning solution containing the substance according to claim 1, 2, 3 or 4 and a baking treatment. 6. Cabbage juice, radish juice, ginger juice,
A method for cooking fish, comprising immersing the fish in at least one of a lemon juice solution and an ascorbic acid solution, followed by baking.