JP4232850B1 - Removal of paralytic shellfish poison components - Google Patents

Abstract

【Task】
To provide a safe and effective detoxification method for paralytic shellfish poisons from aquatic organisms as raw materials for food and feed, especially filtered edible aquatic organisms such as bivalves.
[Solution]
By feeding fresh shellfish with a feed containing a protein or peptide containing cysteine or cystine as a constituent amino acid in total of 1.5 to 40% by weight (dry weight), preferably a keratin-based feather meal, Detoxify paralytic shellfish poison component with sulfate ester group at 11th position. In addition, a paralytic shellfish venom component having no sulfate ester group at the 11-position, in a polyphenol solution, preferably a solution containing tannic acid, ellagic acid, chlorogenic acid, coumarin, catechin, gallic acid, propyl gallate or pyrogallol, Alternatively, it is removed by degrading with a polyphenol-containing food such as apple, tomato, tea, wine or grape juice.
[Selection figure] None

Description

  The present invention relates to a method for removing a paralytic shellfish poison component, a feed additive used therefor, and a feed. More specifically, a method for detoxifying a paralytic shellfish poison component having a sulfate ester group at the 11th position such as a goniotoxin group from fresh shellfish, a feed additive and feed used therefor, and a sulfate ester at the 11th position such as a saxitoxin group The present invention relates to a method for removing a paralytic shellfish poison component having no group.

  Shellfish is an excellent food resource that is high in protein, low in fat, and rich in trace elements such as zinc, which tends to be lacking in modern people. Filter feeders such as bivalves take phytoplankton directly as feed, but some phytoplankton produce natural poisons such as paralytic shellfish poisons.

  Paralytic shellfish poisons (PSP) is a highly lethal neurotoxin possessed by dinoflagellates such as Alexandrium tamarense. When algae that cause PSP occur in the ocean, filtered food aquatic organisms such as bivalves become highly poisonous and accumulate PSP causing human food poisoning. The sea area where PSP occurs is expanding worldwide, and in countries where bivalves are edible, the toxicity of shellfish is regularly monitored to prevent the outflow of poisoned shellfish to the market. In Japan, this shellfish poison monitoring works effectively, and it can be said that there is almost no poisoning caused by shellfish on the market. However, for a long time after the disappearance of the causal algae, bivalves often remain highly toxic, and the damage to the fishery economy due to shipping restrictions has become serious.

  Many studies have been conducted on the taxonomy of causative algae of PSP, physiological ecology, and the chemical and pharmacological properties of the causative components of PSP. To date, more than 20 PSP components have been separated and their structures confirmed (see Figure 1). These are derivatives of saxitoxin (STX), which has two guanidinium groups and a hydrated ketone on the tricyclic skeleton of the characteristic reduced purine. Ingredients included. These PSP ingredients lose little toxicity in normal cooking, and some ingredients change to more toxic ingredients when heated. This means that it is difficult to remove PSP from shellfish during food processing.

  Until now, the following researches have been carried out for the purpose of solving PSP problems. The first is to eliminate or control the toxic plankton that is the causative algae. However, it is currently difficult to control the occurrence of causal algae, which is a natural phenomenon, while preventing negative effects on the ecosystem. Second, it controls the generation of causal algae. A variety of researches have been conducted on the diffusion of artificial causal algae due to ballast water used for balancing cargo ships and the transplantation of cultured bivalve larvae. The third is to prevent the poisoning of shellfish by predicting the occurrence of causative plankton. The fourth is to develop a method that can detect PSP easily and quickly. The mouse test method currently used is a simple and reliable method for detecting PSP, but it has low detection sensitivity and has various problems associated with animal tests. To solve this problem, various research groups are developing a simple PSP analysis method that replaces the mouse test method. And fifth, the development of a method for effectively detoxifying poisoned shellfish.

The development of safe and effective early detoxification methods for shellfish is the most effective means for solving problems. As mentioned earlier, PSP is difficult to remove by cooking. However, bivalves can recover phytoplankton generated as a result of eutrophication as a high-quality protein source, so they are extremely valuable as food resources, and in some countries, consumer needs for fresh shellfish are also high. . In addition, bivalves often have large variations in toxicity even if they are of the same species collected at the same time in the same sea area. Non-Patent Document 1 reports that the PSP of shellfish is reduced by breeding poisoned bivalves in seawater blown with ozone. This method has been reexamined by many researchers, but there are many problems such as the fact that it takes a considerably long time until detoxification or a remarkable effect is not recognized, and it has hardly been carried out at present. For the development of such technology, it is indispensable to elucidate the mechanism of PSP metabolism in the living body such as shellfish. However, there are few such studies, and now the attempt to decontaminate shellfish is completely clogged.
Blogoslawski ME, Stewart WJ, 1979: Ozone detoxification of paralytic shellfish poison in the softshell clam (Mya Arenaria), Toxicon, 17, 650-654

  An object of the present invention is to provide a safe and effective method for removing paralytic shellfish poisons from aquatic organisms as food and feed materials.

  The present inventor has a mechanism whereby PSP components such as goniotoxin (GTX) group having a sulfate ester group at the 11-position react with thiol compounds such as glutathione (GSH) and mercaptoethanol (ME) and are reduced to STX group. In addition, we tried to detoxify GTX group from poisoned scallops by using cysteine and cystine, an oxidative polymer of cysteine, in the poisoned shellfish. Since cysteine is easily soluble in water, it is difficult to incorporate it as a bait in filter-feeding shellfish. As described above, filter edible organisms such as bivalves take up toxic plankton as food, and therefore, most of the poison is generally concentrated in the midgut gland, that is, the liver pancreas. The method of injecting cysteine into shellfish is not only considered to have a significant effect on the physiological state of shellfish, but it is also time consuming and expects a significant detoxification effect from the midgut gland where the poison is concentrated Can not. In contrast, cystine is relatively stable and difficult to dissolve in water, and can be easily taken up by shellfish as food. Cystine is rapidly reduced to cysteine in the body, and is expected to degrade the GTX group. It was. However, administration of cystine often causes moribund shellfish and is not appropriate as a detoxification method for marine products where freshness is important.

  The GTX group and C-toxin group with sulfate ester at the 11th position are the main PSP components found in the Alexandrium genus Dinoflagellate that occurs in the coastal area of the North Pacific Ocean and the shellfish that are poisoned as a result. Depending on the shellfish type and tissue, it may contain many saxitoxin (STX) groups that are reduced to the 11th position. The STX group is the main PSP component of the dinoflagellate Pyrodinium bahamense var. Compressum that occurs in tropical and subtropical waters. In these waters, shellfish poison the STX group as the main component, reflecting the toxic composition of the causative algae. To do. The STX group does not react with thiols and cannot be removed using this method.

  As a result of investigations, the present inventor found that a paralytic shellfish poison having a sulfate ester group at the 11-position feeds shellfish with a feed containing a protein or peptide containing cysteine or cystine as a constituent amino acid. It has been found that it can be remarkably detoxified, and that this method does not cause shellfish mortality. Furthermore, the present inventors have found that the paralytic shellfish poisons of the saxitoxin (STX) group having no sulfate ester group at the 11-position can be decomposed by polyphenol, thereby completing the present invention.

That is, the present invention is as follows.
(1) A sulfate group at the 11th position from fresh shellfish characterized by using a protein or peptide containing cysteine or cystine as a constituent amino acid and the total amount of cysteine and cystine being 1.5 to 40% by weight in dry weight To detoxify paralytic shellfish poison components.
(2) The method according to (1) above, wherein the protein is keratin.
(3) The method according to (2) above, which is performed by feeding fresh shellfish with a feed containing the protein or peptide.
(4) The method according to (3) above, wherein the protein is keratin.
(5) The method according to (4) above, wherein the feed containing keratin is feather meal.
(6) Paralytic shellfish poison component having sulfate ester group at position 11 is goniotoxin 1, goniotoxin 2, goniotoxin 3, goniotoxin 4, decarbamoyl goniotoxin 1, decarbamoyl goniotoxin 2, decarbamoyl goniotoxin 3. The method according to any one of (1) to (5) above, which is one or more of decarbamoylgoniotoxin 4, C1, C2, C3, or C4.
(7) The method according to any one of (1) to (5) above, wherein the fresh shellfish is a filter-feeding aquatic organism.
(8) Paralysis with a sulfate group at the 11th position, characterized by containing a protein or peptide containing cysteine or cystine as a constituent amino acid and the total amount of cysteine and cystine being 1.5 to 40% by weight in dry weight A feed additive for fresh shellfish for the detoxification of sex shellfish poison components.
(9) Paralysis having a sulfate group at the 11th position, characterized in that it contains a protein or peptide containing cysteine or cystine as a constituent amino acid and the total amount of cysteine and cystine being 1.5 to 40% by weight in dry weight Fresh shellfish feed for detoxification of sexual shellfish poison components.
(10) The feed according to (9) above, which is a feather meal.
(11) Paralytic shellfish poison component having sulfate ester group at position 11 is goniotoxin 1, goniotoxin 2, goniotoxin 3, goniotoxin 4, decarbamoyl goniotoxin 1, decarbamoyl goniotoxin 2, decarbamoyl goniotoxin 3. The feed according to (9) or (10) above, which is one or more of decarbamoylgoniotoxin 4, C1, C2, C3, or C4.
(12) The feed according to any one of (9) to (11) above, wherein the fresh shellfish is a filter-feeding aquatic organism.
(13) A method for removing a paralytic shellfish poison component having no sulfate ester group at the 11th position, which comprises decomposing a paralytic shellfish toxin component having no sulfate ester group at the 11th position in a polyphenol solution. .
(14) The paralytic shellfish venom component having no sulfate ester group at the 11-position is any of saxitoxin, decarbamoyl saxitoxin, neosaxitoxin, decarbamoyl neosaxitoxin, goniotoxin 5 (B1), or goniotoxin 6 (B2) The method according to (13) above, which is one or more.
(15) The method according to (13) or (14) above, wherein the polyphenol solution is a solution containing one or more polyphenol compounds, or a polyphenol-containing food.
(16) The method according to (15) above, wherein the polyphenol compound contained in the solution is tannic acid, ellagic acid, chlorogenic acid, coumarin, catechin, gallic acid, propyl gallate or pyrogallol.
(17) The method according to (15) above, wherein the polyphenol-containing food is apple, tomato, tea, wine or grape juice.

  According to the present invention, it is possible to safely and effectively remove a paralytic shellfish poison that has not had an effective removal method so far. For the goniotoxin group and C-toxin group, which are shellfish poisons caused by dinoflagellates occurring in the North Pacific coastal waters, a protein or peptide containing abundant cysteine or cystine as a constituent amino acid is added to the feed, or as a feed By feeding fresh shellfish, it is possible to detoxify while keeping the freshness of the shellfish. Therefore, poisoning due to shellfish poison can be eliminated, and damage to the fisheries economy due to shipping restrictions caused by the generation of shellfish poison can be reduced. In addition, 11-reduced shellfish poisons such as saxitoxin group caused by dinoflagellate mainly in tropical and subtropical waters can be eliminated by polyphenols contained in plants and removed during shellfish processing. It enables the development of effective treatments for shellfish poisoning that had no clues.

  As shown in FIG. 1, the paralytic shellfish poisons targeted by the present invention include an 11-position sulfate ester type having a sulfate ester group at the 11-position, and an 11-position reduced form having no sulfate ester group at the 11-position. is there. The 11-position sulfate ester type includes goniotoxin 1 (GTX 1), goniotoxin 2 (GTX 2), goniotoxin 3 (GTX 3), goniotoxin 4 (GTX 4), decarbamoylgoniotoxin 1 (dcGTX 1), Goniotoxin (GTX) groups such as decarbamoylgoniotoxin 2 (dcGTX 2), decarbamoyl goniotoxin 3 (dcGTX 3), decarbamoyl goniotoxin 4 (dcGTX 4), and C such as C1, C2, C3, or C4 A toxin (C-toxin) group can be illustrated. These PSPs are the main components of PSP produced by the Alexandrium genus Dinoflagellates that occur in temperate and boreal waters.

  Examples of the 11-position reduced form include saxitoxin, decarbamoyl saxitoxin, neosaxitoxin, decarbamoyl neosaxitoxin, goniotoxin 5 (B1), and goniotoxin 6 (B2). These components are the main PSP components of the dinoflagellate Pyrodinium bahamense var. Compressum that occurs in tropical and subtropical waters.

  In the present invention, a protein or peptide containing 1.5 to 40% by weight (as dry weight) of cysteine or cystine in total is used for detoxification of the 11-position sulfate type PSP. Such a protein or peptide can be used without particular limitation as long as it contains cysteine or cystine in a total of 1.5 to 40% by weight (as dry weight), but preferably cysteine or cystine is added in total. And containing 2 to 40% by weight, particularly preferably 2 to 30% by weight. Of these, keratin containing about 8 to 20% by weight of cysteine and cystine is preferably used. As keratin, one purified from any raw material containing keratin can be used, but a material containing keratin protein as a main component may be used as it is. For example, the feather meal which has keratin protein as a main component and is used as an inexpensive feed material can be used. Feather meal is a livestock feed that reuses chicken wings that are produced as waste when shipping chickens on a poultry farm, and its main component is keratin protein rich in cysteine. When the feather meal is taken into the shell, it is digested and cysteine is released, acting on the shellfish poison of the goniotoxin group and degrading. Feather meal is an extremely inexpensive feed ingredient, and is useful not only as a feed or additive for PSP detoxification but also as a feed ingredient for filtered edible aquatic organisms such as bivalves. Other examples of proteins rich in cysteine or cystine include metallothionein.

  In order to administer a protein or peptide containing cysteine or cystine as a constituent amino acid to fresh shellfish, it is added to the feed or fed as a feed. The dose of protein or peptide varies depending on the time when the shellfish are collected, the content of shellfish poison, and the ratio of cysteine or cystine contained in the protein or peptide. The amount of the protein or peptide may be administered to the shellfish for about 1 mg to 100 mg per day, preferably 10 mg to 50 mg, for about 1 day to 7 days, preferably 1 day to 3 days. When using the feather meal as a feed for detoxification, the amount of the feather meal is about 0.04 g to 4 g per day, preferably about 0.5 to 1.5 g for about 1 to 6 days per 100 g of edible portion weight shellfish. Preferably, it may be given for 1 to 3 days.

  In the present invention, the polyphenol used for the decomposition of the 11-position reduced PSP (STX group) is one or two polyphenol compounds such as tannic acid, ellagic acid, chlorogenic acid, coumarin, catechin, gallic acid, propyl gallate, and pyrogallol. It may be a solution containing seeds or more, or a food containing polyphenols such as apple, tomato, tea, wine, grape juice. The tea may be any kind such as green tea, brown rice tea, bancha, black tea, strawberry tea, oolong tea. Degradation of STX group shellfish toxins is thought to be due to oxidative degradation of the STX group by polyphenol radicals, and polyphenols having high redox potential and strong oxidizing power can be preferably used. In particular, it is desirable to have a redox potential of 150 mV or more, particularly 200 mV or more when dissolved in an aqueous solution at a concentration of 0.1% by weight under neutral and 25 ° C conditions.

In order to degrade STX group shellfish poisons using polyphenols, heating or boiling in a neutral aqueous solution containing polyphenols is preferred in terms of increasing the degradation rate, but degradation under physiological conditions is also not possible. Is possible . The concentration of polyphenol in the solution varies greatly depending on the concentration of the STX group contained and other coexisting components and its concentration, but is preferably 0.001% to 1%, particularly 0.02% to 0.2% in terms of the weight ratio in the solution. preferable. This method using a polyphenol solution can be applied, for example, to detoxification during the processing of shellfish, and is considered to be effective as a countermeasure in the early stage of poisoning.

  The STX group is the main PSP component of Pyrodinium bahamense ver. Compressum that occurs in tropical and subtropical regions. To date, in tropical and subtropical countries, there have been many cases of death from poisoning due to paralytic shellfish poisons caused by STX. In these countries, cheap and nutritious shellfish soup is used as baby food, and infants can become seriously addicted even if they consume a very small amount of PSP compared to adults. It has become. However, there are currently no effective treatments for paralytic shellfish poisoning, and there are only symptomatic treatments such as gastric lavage and artificial respiration in medical institutions. The present invention not only improves the food safety of shellfish but also establishes an effective treatment method for food poisoning caused by paralytic shellfish poison.

Feather meal feeding test for scallops
(sample)
The following tests were conducted after acclimatizing 75 toxic scallop shellfish collected at the Ofunato Bay Shimizu fixed point on October 10, 2006, in 25 liters each in a 500-liter acrylic test tank, preliminarily raised in running water for 1 day. It was used for. The total content of cysteine and cystine in the used feather meal was just over 3% by weight.

(Reagents)
Feather meal was donated from Amatsutake headquarters (Ofunato, Iwate Prefecture), liquid nitrogen was added and pulverized in a mortar, and particles of 125 μm or less were collected and stored frozen at −80 ° C. until use. DIC diet (Dainippon Ink Chemical Co., Ltd.), a commercially available bivalve artificial sample, was also stored at low temperatures until it was used for testing.

(Feather meal administration to scallops)
500 L of seawater filtered through a 0.5 μm filter was placed in the acrylic water tank, and 25 g of feather meal was suspended therein, and the water temperature and turbidity were measured while stirring by aeration. A cushion cushion containing the above 25 scallop raw shellfish was hung in this aquarium and bred in a still water while being aerated for 24 hours. Thereafter, the water tank was switched to running seawater, and further bred for 2 days without feeding. After breeding for a total of 3 days, the shell height, shell length, total weight, and stripped weight were measured for each individual.

  In addition to the above test, 25 scallop mussels were fed with 25 g of DIC diet instead of feather meal and reared in the same manner as a control group (control). Another 25 individuals that had been preliminarily raised were taken as they were, and the shell height, shell length, total weight, and stripped weight were measured for each individual and designated as the initial group. The strips of the test group, the control group and the initial group were frozen individually and stored in a freezer at -80 ° C. until extraction.

(Analysis of PSP components in scallops)
The test area, control area, and initial area were each homogenized by stripping 5 individuals, and the homogenate was heat-extracted with dilute hydrochloric acid according to the method described in the Food Sanitation Inspection Guidelines (2005). Analytical quantification was performed by the mouse test method (Sommer H., Meyer KF, 1973: Paralytic shellfish poisoning, Arch. Pathol., 24, 560-598). Part of the extract was filtered with an ultracentrifugation kit (Ultrafree-MC NMWL5000, Millipore), and the PSP components in the filtrate were separated by HPLC fluorescence method (Oshima Y, Post-column derivatization HPLC methods for paralytic shellfish poisons. In: GM Hallegraeff, DM Anderson, SD Cambella, HO Enevodsen, eds. Manual on Harmful Marine Microlgae, Paris: UNESCO, pp. 81-94, 1995).

(result)
In the control group fed with DIC diet, two individuals died on the third day of breeding. In the test plot fed with feather meal, no moribund individuals were found. The toxicity of the initial plot calculated by HPLC is 6.58 ± 1.08MU / g, which is higher than the shipping regulation value of 4MU / g, and the initial plot is 4.94 ± 1.44MU / g (n = 5) in the control plot. There was almost no change. In the test plot fed with feather meal, toxicity was markedly reduced to 3.72 ± 0.57 MU / g, which was below the regulation value for shipping (Figure 2). Also in the mouse test, only the test section was below the shipping regulation value. Fig. 3 shows the toxic content of each test section divided into 11-sulfo type (11-sulfo) and 11-reduced type (11-reduced). In contrast to the initial group, the 11-sulfo-type PSP component was not significantly reduced in the control group, whereas it was confirmed that this was greatly reduced in the test group administered with feather meal. From the above results, it was confirmed that the 11α-sulfo-type PSP component in the poisoned scallop raw shellfish can be remarkably removed by administration of feather meal. Feather meal is digested in the body of scallops, and it is thought that abundant cysteine is released and 11-sulfo type components are decomposed.

(Reference example)
Detoxification test of poisoned scallops by feeding cystine 11 scallops (edible weight 108.5 + 18.3 g) were collected from the test tub of Ofunato Bay Shimizu fixed point, and two tanks containing 40L of filtered seawater with a water temperature of 6.5 ° C Four individuals were placed in each. 10g DIC diet (Dainippon Chemical Co., Ltd.) was added to one tank, and 10g DIC diet and 5g L (-)-cystine (Wako Pure Chemicals, 031-05295) were added to the other tank. . After raising for 2 days with aeration, the seawater was changed and the animals were raised for 2 days without feeding. Remove the midgut gland from each scallop and follow the method described in the Food Sanitation Inspection Guidelines (Japan Food Hygiene Association, 2005 edition, RIKEN, pp. 673-680). An extract was prepared, and the paralytic shellfish toxin components contained in the HPLC fluorescence method (Oshima, 1995) were analyzed and quantified. Separately, midgut glands were taken out from 3 individuals at the start of the test, extracted and analyzed in the same manner.

  Figures 4 and 5 show the toxicity and toxic content of each scallop midgut gland (initial group) at the start of the test, the control group fed with DIC diet alone, and the test group fed with DIC diet and cystine. Results are shown. When only DIC diet was fed, there was almost no change in the toxicity of the initial gut and midgut gland, whereas in the test plot fed cystine, there was a marked decrease in toxicity compared to the initial plot and the control plot. It was. In particular, it was confirmed that the poison component having a sulfate ester at the 11α position (11α-sulfo type component) was greatly reduced in the test section.

  11β-sulfo-type toxic components such as C2, GTX4, GTX3 are the main toxic components produced by the genus Alexandrium genus Dinoflagellate, but they gradually isomerize in neutral aqueous solutions or shellfish bodies, and C1 , GTX1, GTX4, and other 11α-sulfo type toxic components, and finally it is known that the molar ratio of 11β type: 11α type becomes an equilibrium mixture of about 1: 3. On the other hand, it has been clarified that various thiol compounds react directly with 11α-sulfo-type components to form a conjugate via the thiol sulfur atom at the 11-position. Thiol compounds that have an amino group near the SH group, such as cysteine, also react in vitro with 11α-sulfo-type toxic components, but the resulting conjugate has been confirmed to be extremely unstable and spontaneously degraded. Yes. The result of this test is thought to be due to the fact that cystine is reduced to cysteine in the shell, which acts on the 11α-sulfo-type toxic component contained in the midgut gland in a high concentration and decomposes it. It is done.

(Reference example)
Elimination effect of saxitoxin group PSP by polyphenol In this experimental example, it is shown that saxitoxin (hereinafter referred to as STX), a PSP component reduced at the 11th position, is eliminated by polyphenol and polyphenol-containing foods.
(Samples and reagents)
STX is extracted from the midgut gland of the toxic scallops of Ofunato Bay with dilute hydrochloric acid and is subjected to conventional chromatography using activated carbon, Bio-Gel P-2 and Bio-Rex 70 column chromatography (Shimizu, 2000, Chemistry and mechanism of action, in: Seafood and Freshwater Toxins, Botana, LM ed., Marcel Decker, NY, pp. 151-172).

All of the polyphenol preparations used Wako Pure Chemical and Sigima chemical and biochemical reagents. Foods containing polyphenols, ie, green tea, brown rice tea, bancha, black tea, strawberry tea, oolong tea, wine (4 types of red wine), grape juice (3 types), cocoa, tomato juice, and apples are all purchased on the market. did.
(STX elimination test with polyphenol preparation)
Take 10 mg each of tannic acid, ellagic acid, chlorogenic acid, coumarin, catechin, gallic acid, propyl gallate and pyrogallol in separate test tubes, add 10 mL of 50 mM sodium phosphate buffer (pH 7.4) to each, Dissolved or suspended. To these solutions, STX was added to a final concentration of 10 μM and warmed in a boiling bath for 5 minutes. Thereafter, STX in the filtrate obtained by treatment with an ultracentrifugation kit (Ultrafree-MC, NMWL5,000, Millipore) was analyzed by HPLC fluorescence method (Oshima, mentioned above). What was added and mixed in 50 mM sodium phosphate buffer (pH 7.4) so as to have a final concentration of 10 μM was treated as initial, and this was boiled in a boiling bath for 5 minutes in the same manner as a control and analyzed.
(STX elimination test with polyphenol-containing food)
1 g of green tea, brown rice tea, bancha, black tea, strawberry tea and oolong tea leaves and 1 g of cocoa were placed in separate beakers, and 100 mL of 50 mM sodium phosphate buffer (pH 7.4) was added thereto. This was heated in a boiling bath for 5 minutes and filtered through a filter paper to obtain a neutral solution. The four types of red wine (red wine-1,2,3,4), tomato juice and grape juice (3 types) were all adjusted to pH 7.4 by adding sodium carbonate. The apple was grated with the skin, neutralized with sodium carbonate, and an equal amount of water was added to make a suspension. STX was added to each solution or suspension to a final concentration of 10 μM and warmed in a boiling bath for 5 minutes. Thereafter, STX in the filtrate obtained by treatment with an ultracentrifugation kit (Ultrafree-MC, NMWL5,000, Millipore) was analyzed by HPLC fluorescence method (Oshima, mentioned above). What was added and mixed in 50 mM sodium phosphate buffer (pH 7.4) so as to have a final concentration of 10 μM was treated as initial, and this was boiled in a boiling bath for 5 minutes in the same manner as a control and analyzed.
(result)
FIG. 6 shows the results of heating STX in neutral aqueous solutions of various polyphenol preparations. In a solution to which no polyphenol was added, 8.6 μM STX remained after boiling (CTRL in the figure) with respect to the initial 10 μM. In contrast, STX was significantly reduced in the solution containing tannic acid, ellagic acid, chlorogenic acid, coumarin, catechin, gallic acid, propyl gallate and pyrogallol. In particular, catechin and tannic acid, which are the main polyphenols of tea leaves, and gallic acid and its constituents, propyl gallate and pyrogallol, which are constituents thereof, were confirmed to have particularly high STX scavenging action.

FIG. 7 shows the results of heating STX in a neutral solution of various polyphenol-containing foods. When heated for 5 minutes in neutral sodium phosphate buffer, 8.6 μM STX remained for the initial 10 μM. In contrast, when STX was heated in neutral solutions of red wine, grape juice, green tea, brown rice tea, bancha, black tea, strawberry tea and oolong tea, the amount of STX recovered was significantly reduced. Similar effects were observed in tomato juice, cocoa and apple . The bancha, black tea, and oolong tea solutions were found to have particularly high STX erasing activity.

(Reference example)
Using STX, neoSTX, GTX 5 (B1) and GTX 6 (B2) purified and isolated in the same manner as in Example 2, a decomposition experiment with tannic acid was performed.
GTX 5 standard solution (56.9 μM), GTX 6 standard solution (38.6 μM), STX standard solution (354 μM), and neoSTX standard solution (100 μM) contain 0.01% tannic acid so that the final concentration of each PSP is 1 μM. DMSO / 0.01M sodium phosphate (pH 7.4) was added and mixed. These mixtures were heated in a boiling bath for 5 minutes and cooled with ice, and the filtrate obtained by ultrafiltration was analyzed by HPLC fluorescence.

  For initials and controls, 0.01 M sodium phosphate (pH 7.4) with GTX 5, GTX 6, STX, neoSTX adjusted to a concentration of 1 μM and a solution that was heated in a boiling bath for 5 minutes and cooled to room temperature Each was prepared similarly and analyzed by HPLC fluorescence.

  As shown in FIG. 8, STX, neoSTX, GTX 5 and GTX6 were significantly reduced in the test group heated in a 0.01% tannic acid solution compared to the control. From the above results, it was revealed that the tannic acid solution has an effect of specifically eliminating STX, neoSTX, GTX 5 and GTX 6 in which the 11th position is reduced.

  In contrast, the GTX group (an equilibrium mixture of GTX1 and GTX 4 and an equilibrium mixture of GTX2 and GTX 3), which is an 11-sulfo-type PSP component (a paralytic shellfish poison component having a sulfate ester at the 11th position), is similarly 0.01 When heated in a neutral solution containing 1% tannic acid, no clear decrease was observed in these PSP components compared to before warming.

(Reference example)
Elimination of STX by polyphenol under physiological conditions In this example, STX purified and isolated in the same manner as in Example 2 was used. Spanish red wine (Olelia) was neutralized by adding 1 M sodium carbonate, and STX was added and mixed to a final concentration of 10 μM. Incubate this solution in a 37 ° C water bath, collect a portion of the solution over time (5, 10, 20, and 30 minutes after the start of the reaction), add an equal volume of 0.5M acetic acid and ultrafilter it. The filtrate was analyzed by HPLC fluorescence method (Oshima, 1995, mentioned above), and the remaining STX concentration was analyzed and quantified. A neutral aqueous solution containing 10 μM STX was similarly heated at 37 ° C., and analyzed and quantified over time in the same manner as a control.

Prepare neutral aqueous solutions containing 0.1% by weight of tannic acid, gallic acid, propyl gallate or pyrogallol separately, add STX to the final concentration of 10 μM, mix and incubate at 37 ° C. As in the case of red wine, the remaining STX concentration was analyzed and quantified.
(result)
As shown in FIG. 9, when STX was incubated at 37 ° C. in neutralized red wine, the STX concentration decreased to about 30% of the initial after 5 minutes. Similar results were observed when STX was incubated in a neutral solution containing tannic acid, gallic acid, or propyl gallate (Figure 10). On the other hand, the STX erasing action of the solution added with pyrogallol was slightly inferior, and in the control without red wine or polyphenol, no significant decrease in STX was confirmed.

  When an adult ingests the 10 μM STX solution used in this example, serious poisoning occurs in the amount of about one cup. For food poisoning caused by paralytic shellfish poisons, no effective treatment has been established at present, and only symptomatic treatment such as gastric lavage and artificial respiration has been provided. From this example, at least in the early stage of poisoning, where most of the unabsorbed poison remains in the stomach, the administration of neutralized red wine or gallic acid solution may significantly reduce the symptoms of food poisoning caused by the STX group. It is considered expensive. The results of this example suggest that the use of these polyphenols or polyphenol-containing foods can effectively remove the STX group from shellfish or its soup at the cooking / processing stage.

(Reference example)
Influence of bioreductive agent on STX removal effect by polyphenol In Examples 2 to 4 above, it was confirmed that STX was erased when incubated in a polyphenol solution. Paralytic shellfish poisons such as STX are well adsorbed on activated carbon and gel filtration resins such as Sepahdex and Bio-Gel P-2 under neutral conditions. Similar to such a carrier, if STX is strongly adsorbed to polyphenol, it cannot be detected by HPLC analysis. On the other hand, polyphenols are known to function as quenchers for oxygen radicals (active oxygen) generated in the body of photosynthetic plants. Therefore, it is conceivable that the radical generated on the polyphenol by such a reaction directly decomposes STX. In this example, for the purpose of elucidating the STX elimination mechanism by polyphenol, the influence of an in vivo reducing agent on the STX removal effect by polyphenol was examined.

(Samples and reagents)
As the purified poison for STX, one obtained in the same manner as in Example 2 was used. The reagents used are as follows.

Polyphenol tannic acid: Wako, for chemical use, 203-06331
Bioreducing agent Aspartic acid: Wako, reagent grade, 013-04832
Glutamic acid: Wako, reagent grade, 070-00502
Glutathione: Wako, Wako Special, 071-02014
Cysteine hydrochloride: Wako, reagent grade, 033-05272
Ascorbic acid: Wako, reagent grade, 016-04805
(Method)
0.1% tannic acid was added to 0.1 phosphate buffer (pH 7.4) containing 1, 3 or 10 mM aspartic acid. In addition, STX was added to these solutions to a final concentration of 10 μM. These mixed liquids were boiled in a boiling bath for 5 minutes, and filtrates obtained by ultrafiltration using an ultracentrifugation kit (Ultrafree-MC NMWL5000, Millipore) were analyzed by HPLC fluorescence method (Oshima, supra). Moreover, it replaced with aspartic acid, performed the same operation using glutamic acid, glutathione, and cysteine, and analyzed by HPLC fluorescence method. For ascorbic acid, solutions having concentrations of 0.1, 0.3, 0.5, 1, 3, and 10 mM were prepared and the same experiment was performed. In addition to these, 0.1 M phosphate buffer (pH 7.4) with only a final concentration of 10 μM STX added to the initial, tannic acid added to a concentration of 0.1% and boiling in a boiling bath for 5 minutes This was used as a control. Each of the above solutions was analyzed by HPLC fluorescence method.

(result)
The results are shown in FIG. When STX was heated in a solution obtained by adding aspartic acid or glutamic acid to a tannic acid solution, most STX disappeared to the same extent as the control using a tannic acid solution not containing these amino acids. On the other hand, when glutathione or cytosine was added, the remaining STX concentration tended to increase as the concentration of these thiols increased. With ascorbic acid, the same phenomenon was confirmed even when a lower concentration was added.

  From the above results, it was found that glutamic acid and aspartic acid do not suppress the effect of removing STX by polyphenol (tannic acid), whereas glutathione and cysteine having an SH group suppress this effect. Similar effects were observed with ascorbic acid distributed in vivo as an antioxidant similar to thiol. Thiols and ascorbic acid eliminate radicals generated on polyphenol molecules. This means that STX is erased only when radicals are generated on the tannic acid molecule. That is, the elimination of STX by polyphenols such as tannic acid is considered to be due to polyphenols acting directly on STX and oxidizing them.

(Reference example)
Elimination of STX and redox potential in polyphenol solution The oxidation-reduction potential of neutral aqueous solution was measured for various polyphenols and compared with the STX elimination effect.

(Method)
Several drops of 12N hydrochloric acid were added to 10 μM STX, and the acidified solution was lyophilized. This was dissolved in 10 ml of water, passed through Sep-Pak C-18, and neutralized with 0.2 M phosphate buffer (pH 7.4). Take tannic acid, catechin, ellagic acid, chlorogenic acid, and coumarin used in Example 2 in separate beakers, add 1000 volumes of 0.1 M phosphate buffer (pH 7.4), and dissolve or suspend. did. Each 0.1 M phosphate buffer (pH 7.4) solution containing 1 mM of aspartic acid, glutamic acid, glutathione, cysteine, and ascorbic acid used in Example 3 was prepared. Furthermore, the liquid mixture which combined said tannic acid solution and ascorbic acid was produced. These neutral solutions were measured for their respective redox potentials (ORP values) using a redox potential measuring device (Eutech Instruments).
(result)
As shown in FIG. 12, the redox potential (ORP value) of a 1 mM neutral STX aqueous solution is 150 mV. On the other hand, the ORP values of 0.1% neutral aqueous solutions of various polyphenol samples were all higher than that of the STX solution. The ORP values of the catechin, tannic acid, coumarin, ellagic acid, and chlorogenic acid solutions were 261 mV, 258 mV, 237 mV, 207 mV, and 204 mV, respectively.

  As described above, the ORP values of catechin, tannic acid, etc. exhibiting a remarkable STX elimination action were all 200 mV or more. The ORP value of the neutral aqueous solution of ascorbic acid was 135 mV, whereas the ORP value of the mixed solution of tannic acid and ascorbic acid was 95 mV, which was lower than either one of the solutions. The above results indicate that the STX scavenging action by polyphenols depends on its redox potential.

  From the above, it is clear that the elimination of STX by polyphenol involves the radicals or oxidized form generated in polyphenol, that is, the degradation reaction due to oxidation occurs when STX disappears.

It is a figure which shows the structure of a typical paralytic shellfish poison component. It is a figure which shows the result of the feather meal feeding test with respect to a scallop. It is a figure which shows the poison content according to a composition in the feather meal feeding test with respect to a scallop. It is a figure which shows the toxicity of the poisoned scallop midgut gland by cystine feeding. It is a figure which shows the poison content according to composition of the poisoned scallop midgut gland by cystine feeding. It is a figure which shows the result of the STX removal experiment by various polyphenol preparations. It is a figure which shows the result of the STX removal experiment by various polyphenol containing foodstuffs. It is a figure which shows the result of the STX degradation test by tannic acid. It is a figure which shows the result of the STX decomposition | disassembly test by red wine. It is a figure which shows the result of the STX degradation test by tannic acid, gallic acid, propyl gallate, and pyrogallol. It is a figure which shows the influence of the in-vivo reducing agent on the STX removal effect by polyphenol. It is a figure which shows the oxidation-reduction potential (ORP value) in a neutral solution.

Claims (12)

A protein or peptide containing cysteine or cystine as a constituent amino acid and having a total amount of cysteine and cystine of 1.5 to 40% by weight in dry weight is administered to the fresh shellfish by sulfate to the 11th position. A method for detoxifying paralytic shellfish poison components having ester groups. The method of claim 1, wherein the protein is keratin. The method according to claim 1, wherein the method is performed by feeding fresh shellfish with a feed containing the protein or peptide. 4. The method of claim 3, wherein the protein is keratin. The method according to claim 4, wherein the feed containing keratin is a feather meal. The paralytic shellfish poison component having sulfate ester group at the 11th position is goniotoxin 1, goniotoxin 2, goniotoxin 3, goniotoxin 4, decarbamoyl goniotoxin 1, decarbamoyl goniotoxin 2, decarbamoyl goniotoxin 3, decarbamoyl goniotoxin 3, The method according to any one of claims 1 to 5, wherein one or more of carbamoylgoniotoxin 4, C1, C2, C3, or C4 is used. The method according to claim 1, wherein the fresh shellfish is a filter-feeding aquatic organism. Paralytic shellfish poison having a sulfate ester group at the 11-position, comprising a protein or peptide containing cysteine or cystine as a constituent amino acid and having a total amount of cysteine and cystine of 1.5 to 40% by dry weight Feed additive for fresh shellfish for detoxification of ingredients. Paralytic shellfish poison having a sulfate ester group at the 11-position, comprising a protein or peptide containing cysteine or cystine as a constituent amino acid and having a total amount of cysteine and cystine of 1.5 to 40% by dry weight Fresh shellfish feed for detoxification of ingredients. The feed according to claim 9, which is a feather meal. The paralytic shellfish poison component having sulfate ester group at the 11th position is goniotoxin 1, goniotoxin 2, goniotoxin 3, goniotoxin 4, decarbamoyl goniotoxin 1, decarbamoyl goniotoxin 2, decarbamoyl goniotoxin 3, decarbamoyl goniotoxin 3, The feed according to claim 9 or 10, which is one or more of carbamoylgoniotoxin 4, C1, C2, C3, or C4. The feed according to any one of claims 9 to 11, wherein the fresh shellfish is a filter-feeding aquatic organism.

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