Patent Application: US-89059497-A

Abstract:
this invention relates to a process for detoxifying shellfish contaminated with paralytic toxin . actually no industrial method has been described that is 100 % effective for detoxification of shellfish containing this type of toxins . decontamination is achieved through a process involving the chemical treatment of shellfish . this chemical treatment consists of preincubating and then cooking the shellfish in an alkaline ph solution . this treatment can be combined with one or more other procedures for the reduction of the products &# 39 ; final toxicity , these other procedures including , for example , partial shellfish detoxification by depuration , removal of the most toxic parts , and commercial cooking / canning of the shellfish . the process reduces total toxicity levels in shellfish to values below 80 μg of eq . stx / 100 g shellfish , independently of the toxin profile , or the initial toxicity present in the shellfish . this is the first known industrial process that guarantee the shellfish decontamination 100 % compatible with the international regulatory norms for human consumption .

Description:
in the present invention an effective procedure for the decontamination of paralytic toxin contaminated shellfish is described . such procedure consists in a chemical treatment of shellfish , combined or not with one or several other procedures for a reduction in the final toxicity of the product . the additional procedures can be selected from : partial shellfish detoxification by deputation , removal of the more toxic parts , and commercial cooling / canning . the shellfish can be fresh live , fresh dead , frozen , pre - cooked ( by water vapour treatment , autoclave , boiled , fried , etc . ), with or without shell , whole or chopped . this mollusc can be whatever species of monovalve such as snails , locos , abalons , limpets , etc . ; or any species of bivalves such as all kind of mussels , big mussels , chilean mussels , clams , oysters , scallops , tumbaos , culengues , navajas , navajuelas , machas , picorocos , etc . ; or any another species of shellfish . any other marine food product contaminated with psp toxins can also be included in this decontamination procedure . the alkaline treatment consists of submerging paralytic toxin contaminated shellfish in an alkaline ph solution and then submitting them to a thermal process . the alkaline solution can be obtained using any buffer that is able to regulate the ph to alkaline values . for example the following buffers can be used : carbonate / bicarbonate ; carbonate / sodium hydroxide ; sodium hydroxide ; h 2 po 4 − / hpo 4 2 − ; hpo 4 2 − / po 4 3 − ; citrate / sodium hydroxide ; barbital ; barbital / hcl ; barbital / sodium hydroxide ; borate ; borate / hcl ; borate / boric acid ; borate / sodium hydroxide ; aminoacid ; aminoacid / sodium hydroxide ; phosphate / borate ; phosphate / citrate ; citrate - phosphate - borate / hcl ; imidazole / hcl ; trizma / hcl ; tricine / hcl , among others . the concentration of this alkaline solution can vary from 1 mm up to 5 m , more specifically between 5 mm and 2 m , depending on the product to be treated and on the quantity of toxin to be destroyed . by alkaline ph is meant any ph that is equal to or above 6 . 0 . the more alkaline the ph the more efficient the paralytic toxin destruction . the maximum alkaline ph that it is possible to use will depend on the product being treated , the initial toxicity , and the capacity of the product to maintain an organoleptic characteristic acceptable for human consumption . the shellfish immersion time can vary from some seconds up to 2 days . in general , time is selected in order to permit the complete imbibition of the shellfish with the alkaline solution and its diffusion to the contaminated tissues . then , the time will depend on the shellfish size ; their form of presentation , that is , if molluscs are or not shucked , if the molluscs are whole or not , if they have been pre - cooked or not , etc . ; and if they are live or not . the live shellfish aspire the alkaline solution through the siphons and , in these conditions , it diffuse faster to the inside of the shellfish the thermal process is important for the paralytic toxin destruction . once molluscs are contacted and completely imbibed with the alkaline solution , the shellfish are thermally treated by boiling in the treatment solution , or by injection of hot water vapour directly in the imbibed shellfish , or by autoclave of the imbibed shellfish , among other possibilities . the time and the temperature of the treatment will depend on the shellfish being treated and on the amount of toxin that is necessary to destroy . in general , it can vary between 10 seconds to 5 hours . it is possible that not enough toxin is destroyed to bring the final level of toxicity down to the permitted levels . in this case it is possible to repeat the alkaline treatment as many times as necessary . using this process it is possible to destroy the toxins in the contaminated shellfish , to reach acceptable levels of toxicity for human consumption . during the cooking of the contaminated shellfish in the presence of the alkaline solution , besides destruction , an extraction of toxins from the shellfish occur . the supernatant liquid contains an important amount of toxin , initially present in the shellfish . this phenomenon also produces a migration of the toxins within the shellfish from more to less contaminated parts , for example , from digestive glands to adductor muscles and the foot . thus , in highly psp contaminated shellfish , it may be necessary to remove the more toxic parts prior to alkaline treatment , thus avoiding contamination by toxin diffusion the toxins extracted from the shellfish using the alkaline treatment , remain in the liquids in which shellfish are submerged . these can be removed , and / or concentrated using well - known methods and technique , or be destroyed totally through the alkaline treatment described in this invention , using , if necessary , more drastic conditions . to optimize the industrial decontamination procedure and utilize this procedure with raw materials containing high toxicity levels , the alkaline treatment can be associated with other already existent procedures for toxin decontamination . for example , in a same production line can exist , besides the alkaline treatment , one or several ( or all ) treatments that can include partial depuration of shellfish , removal of the more toxic parts and commercial cooking / canning . the production plant can be equipped with depuration pools . these pools can be filled with non - psp contaminated seawater and shellfish can be deposited there for variable times . the presence of non contaminated seawater will permit the reduction of the initial toxin load in the shellfish . the time of treatment can vary from hours up to weeks depending on the shellfish and on the initial toxicity . subsequently the shellfish are collected and subjected to the industrial decontamination process of this invention , and canning . great part of shellfish can be commercialized as a non whole product . thus , there can exist tongue or foot canned from diverse types of shellfish , like for example machas , clams , tumbaos , culengues , navajas , navajuelas , or adductor muscles from scallops . in this case these products have the siphons , rims , digestive glands , gills and gonads eliminated , constituting generally the most contaminated tissues . the residual toxicity of the adductor muscles and tongues or feet can be eliminated through the alkaline treatment . on the other hand , discards of the canning process of tongues or feet and adductor muscles , especially rims and in some cases gonads and siphons , can be decontaminated using the alkaline treatment and then used for the elaboration of other canned products , for example soups , shellfish paste , etc . because cooking / canning is a normal stage of the canning process for shellfish , the inventive treatment described also takes advantage of it for the decontamination of psp toxins . as noted , the canning of contaminated shellfish itself can reduce to a great extent the toxicity levels . therefore , if the alkaline treatment doesn &# 39 ; t reduce the total toxicity to permitted levels , the subsequent canning can often reduce them to safe levels . the ph of the final product must be regulated to control both the organoleptic characteristic and the reologic properties of the shellfish . this regulation is carried out easily when the treated products are destined for commercial canning . the ph can be controlled by the addition of a covering liquid that contains a buffer to the appropriate ph or an acid . this can be selected , for example , from acetic acid , citric acid , ascorbic acid , butyric acid , phosphoric acid , hydrochloric acid , glutamic acid , any aminoacid , ftalic acid , succinic acid , pyruvic acid , glyceric acid , malic acid , boric acid , acetic / acetate , h 3 po 4 / h 2 po 4 − , h 2 po 4 − hpo 4 2 − , polyphosphate , tripolyphosphate , edta , among others . within these compounds those that improve the organoleptic , reologic or any another properties of the final product &# 39 ; s acceptability , are preferred . for example phosphates and polyphosphates produce an increment in the water retention capacity of canned shellfish , ascorbic acid possesses antioxidant properties , etc . the possibility of utilizing other buffers as the covering liquid that give other interesting properties to the final product , is also possible . if the decontamination procedure by alkaline treatment destroys & gt ; 90 % of toxins , and the removal of the more toxic parts and cooking / canning decrease by 90 % and 80 % the shellfish toxins , respectively , then a process that involves all three stages of decontamination could theoretically diminish toxicity levels as high as 40 , 000 μg stx equivalent / 100 g . to permitted levels . however , these high levels of toxicity are not frequent , and usually the toxicity level in the psp - endemic zones oscillates between 80 and 10 , 000 μg stx equivalent / 100 g . thus this decontamination procedure is applicable to any kind of mollusc from psp - endemic zones . detoxification of mussels contaminated with paralytic toxin using an alkaline ph treatment mussels were collected from the xii region of chile , a zone that possess an endemic problem with an alexandrium catenella bloom . the samples were contaminated with paralytic toxin with a toxicity level of 6 , 800 μg stx eq ./ 100 g , measured by mouse bioassay , or 7 , 816 μg stx eq ./ 100 g , measured by hplc . the samples were submitted to the alkaline treatment for 24 hours after their catch . during their transit samples were kept refrigerated at 4 ° c . live mussels , whole and with shell , were submerged in 100 mm sodium bicarbonate solution ( ph 9 . 0 ) and were incubated 1 hour at room temperature . subsequently , the shellfish were washed with abundant water , submerged again in 100 mm sodium bicarbonate solution ( ph 9 . 0 ) and boiled for 20 minutes . after cooling at room temperature , mussels were chucked by hand and washed with abundant water . analysis of these samples revealed that the toxicity was 550 μg stx eq ./ 100 g , measured by mouse bioassay , or 408 μg stx eq ./ 100 g . measured by hplc ( fig3 a ), reaching a reduction of 91 . 9 % ( bioassay ) or 94 . 8 % ( hplc ) in their initial toxicity . a second treatment of mussels with a fresh solution of bicarbonate , in the same conditions described previously , reduced the final toxicity to 69 μg stx eq ./ 100 g . measured by mouse bioassay , or 61 μg stx eq ./ 100 g , measured by hplc ( fig3 b ). the percentage of decontamination was 99 . 0 % ( bioassay ) or 99 . 9 % ( hplc ) relative to the initial toxicity . this second treatment with the alkaline solution reduced the toxicity levels to a safe level which is completely acceptable for human consumption . a single alkaline treatment of fresh chucked mussels reduced the initial toxicity by 96 . 8 % ( bioassay ) or 95 . 3 % ( hplc ). the better efficiency of this treatment could be due to the improvement in the alkaline solution accessibility to the contaminated tissues . the paralytic toxin profile changed drastically due to the alkaline treatment ( see fig3 c ). observe that gtxs toxins and the neosaxitoxin were principally destroyed . their proportion in treated shellfish decrease substantially relative to that of the initial raw material . on the other hand , saxitoxin was slowly destroyed , increasing their relative proportion in the treated products . the biotoxicologic assay was carried out according to the method of helrich ( 1990 ) and corresponds to the official method of the a . o . a . c ( association of official analytical chemists ). as the standard for mouse bioassay calibration , an international standard of stx , kindly donated by dr . s . hall , food and drug administration , washington , dc , was used . briefly , 100 g of shellfish , previously milled , were homogenized with 100 ml of hcl 0 . 1n , the ph was adjusted to & lt ; 4 . 0 , when necessary , and then boiled for 5 min . after cooling , final volume was adjusted to 200 ml and then filtered 1 ml of filtrate was injected intraperitoneally to a preweight mouse ( 15 - 21 grams ) and the death time was registered . using the sommer and meyer table ( sommer and meyer , 1937 ; helrich , 1980 ) and according to the death time , mouse units were calculated and corrected by the mouse weight . the μg stx eq ./ 100 g were calculated through the calibration with an international standard of stx . the hplc analysis was carried out basically as described by oshima et al . ( 1988 ). briefly , the acid extracts utilized for the mouse bioassay , were chromatografied in a sep pak c18 ™ column ( waters co ) and then deproteinized by ultrafiltration ( milipore ultrafree c3gc ™ membrane , exclusion 10 , 000 pm ). the eluted was injected into a hplc equipped with a rp8 column and subjected to an elution at 0 . 8 ml / min with buffer a ( 2 mm sodium 1 - heptanesulfonate in 10 mm ammonium phosphate ph 7 . 2 ) for the gtxs toxins and dcgtxs , and an elution with buffer b ( a : acetonitrile = 9 : 1 ), for the stx toxins , neo and dcstx . the detection was carried out with a post column derivatization with 0 . 4 ml / min of 7 mm peryodic acid in 50 mm sodium phosphate ph 9 . 0 , heating at 65 ° c . in a 10 meter teflon ™ tube ( 0 . 5 mm i . d . ), and 0 . 4 ml / min of 0 . 4 m acetic acid , and detection with fluorescence detector ( ex 330 nm ; em 390 nm ). this example describes an industrial decontamination procedure for culengue . this bivalve shellfish was collected in a zone endemically contaminated with paralytic toxin and then transported to a canning plant in order to carry out the experiment . three shellfish samples that possessed different toxicity levels were collected . the assay was carried out on processing the culengues in 3 batches of approximately 300 kilos each ( a batch per sample ). in order to carry out the analysis , 10 kilograms of randomly taken samples , were collected in order to do a representative analysis the whole sample was homogenized and aliquots were utilized for the psp determination by mouse bioassay and hplc . the industrial process used is detailed in the flow diagram described in the fig4 . the process is a continuous process for traditional shellfish canning in which variations were done in order to include the toxin decontamination steps . the results obtained in this process were summarized in the table 3 . with the 3 processes a considerable reduction in the culengues toxicity was achieved . the reduction in toxicity was always greater than 90 %. the final toxicity of the canned products permitted their commercialization and they were below the established limits for the prohibition of consumption . cembella , a . d . and shumway , s . e . 1995 . anatomical and spatio - temporal variations in psp toxin composition in natural populations of the surfclam spisula solidissima in the gulf of maine . in “ harmful marine algal blooms ” ( lassus , p ., arzul , g ., erard , e ., gentlen , p . and marcaillou , c . eds .) lavoisier , intercept ltd ., pp . 421 - 426 . cembella , a . d ., sullivan j . j ., boyer , g . l ., taylor , f . j . r . and anderson , r . j . 1987 . variations in paralytic shellfish toxin composition within the protogonyaulax tamarensis / catenella species complex ; 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