A PROCESS FOR RECOVERING CHITIN FROM MATERIALS IN WHICH CHITIN OCCURS TOGETHER WITH OR CONNECTED TO PROTEINACEOUS SUBSTANCES

Abstract: A process according to which chitin and possibly astaxanthin are recovered from chitin sources, particularly shrimp and krill shells, by demineralization with an acid and removal of protein using the enzymatic activity of fish viscera. The chitin-decomposing enzymatic activity of the fish viscera is suppressed by treatment of the viscera with acid at pH 1.2 to 2.5, preferably 1.5 to 2.5, prior to or during the removal of protein. The recovering may be effected in the presence of an oil, preferably from cod liver, extracting the astaxanthin.

Description: A process for recovering chitin from materials in which chitin occurs together with or connected to proteinace- ous substances. Chitin is a nitrogen-containing polymer carbo¬ hydrate occurring widespread in nature, in particular in shells of insects, crustaceans and molluscs, but also in certain fungi. D Chitin and chitosan (the latter may be obtained by deacetylation of chitin using strong bases) are used in various industries, e.g. as viscosity-increasing agents, gelforming agents, film-forming agents, ion exchangers and flocculants and for heavy metal removal, 10 as suture materials and as wound healing agents. In the most important sources of chitin, e.g. the shells of shrimps, krill and crabs, the chitin occurs together with and partly connected to proteinaceous sub¬ stances. Said shells further contain a substantial 15 amount of mineral substances, in particular calcium car¬ bonate. Moreover, many sources of chitin comprise the dyestuff astaxantin and derivatives thereof which are very valuable because they can be used in fodder for 2.0 salmon or related fish. Several attempts have been made to develop methods of the production of chitin or chitosan using the shells of marine crustaceans as starting material. Such shells are at present regarded as being the most 25 important source of chitin, and the method according to the present invention is in the following described in connection with the processing thereof, in particular shells of shrimps and krill, but the process is general¬ ly applicable to all chitin sources from which protein 30 has to be removed. A common feature of the prior art methods is that the lime contained in the shells is removed by treatment with an acid, whereas it has been suggested removing the protein by treatment with a strong solution of sodium hydroxide or with enzymatic agents (pepsine from hog or fish) . It has further been suggested dissolving the pro¬ tein by proteolytic bacteria. The removal of protein by means of sodium hydro¬ xide or other strong bases has the disadvantage that a substantial part of the astaxanthin contained in the shells is lost and, moreover, a part of the chitin is hydrolyzed into chitosan which means that the obtained product is not pure. The use of proteolytic bacteria involves a com- plicated procedure, and the removal of protein using enzymatic agents has not resulted in an efficient dissolving of the protein and has given a rather poor yield of astaxanthin. A substantially higher yield of astaxanthin has been obtained by treating shrimp shells with silage pre¬ pared by treating cod viscera without lever, with formic acid and propionic acid to obtain pH 4.1-4.5 in the mix¬ ture, whereby pH was kept between 2.5 and 3.0 during the treatment of the shrimp shells. Said treatment resulted in an efficient dissolving of the protein, but simulta¬ neously a substantial part of the chitin was decomposed, for which reason the chitin yield was unsatisfactory. To separate the astaxanthin this has been ex¬ tracted with an oil, e.g. soybean oil. It has now been found that by treating fish viscera, preferably comminuted fish viscera, with an acid to achieve a pH between 1.2 and 2.5, the enzymatic activity of the viscera is effected in such a way that the chitin-decomposing activity is substantially sup- pressed, whereas the proteolytic activity only decreases to a minor extent. It has further been found that said suppressing of the activity of chitin-d composing enzy¬ mes is almost irreversible if the viscera are kept at a low pH for a prolonged period as is the case when they are ensiled. This means that with a silage of fish vis¬ cera of a pH between 1.5 and 2.5 a substantial increase of pH up to 4 is permissible during the treatment of the shrimp and krill shells and thereby an efficient proteo- lysis may be obtained without substantial decomposition of the chitin. However, if fresh fish viscera is used instead of silage the proteolysis would be performed at a pH of 1.2 to 2.5. Thus, the invention deals with a process for recovering chitin from materials in which chitin is pre¬ sent together with or connected to proteinaceous sub¬ stances by demineralizing by means of an acid and remo¬ val of protein by exploiting the proteolytic acticity of fish viscera, which process is characterized in that a) an aqueous suspension is produced comprising the optionally minced chitin-containing mate¬ rial and the fish viscera which have possibly been pre-ensiled, ensuring that the fish viscera or the silage prepared therefrom act on the shells at a pH of 1.2 to 2.5, preferab¬ ly 1.5 to 2.5, or the viscera have previously been ensiled at such a pH, b) the suspension is heated at a temperature be¬ tween 25 and 50°C for a period between a few hours and four days, preferably ^ to 3 days, and c) the suspension is separated to obtain at least (i) an aqueous phase containing dissolved hy- drolyzed protein, and (ii) a sludge fraction containing the chitin substantially without proteins and mineral substances. Between step b) and step c) it is preferred to carry out a partial neutralization and a heating of the suspension to inactivate the enzymes and microorganisms therein and to facilitate the subsequent separation. Particularly in cases where fish viscera or silage thereof are not available in sufficient amount, it is advantageous to omit to carry out such an enzyme activating heating and to return part of the aqueous phase separated in step c) to step a). This permits the exploitation of the remaining proteolytic activity in the aqueous phase and the need for fish viscera or fish viscera silage is reduced. Another way of reducing said need is to incor¬ porate proteolytically active material derived from the animals constituting the chitin source into the suspen¬ sion prepared in step a) . By the extraction of chitin from shrimp shells it is possible to remove the raw "shrimp heads" comprising the shrimp viscera in which proteolytic activation occurs. Correspondingly, by the processing of krill all parts of the animals in raw com¬ minuted form can be a constituent of the suspension pre¬ pared in step a) . The protease contained in the commi¬ nuted krill thus contributes in the liberation the chi¬ tin shells from proteins and the yield of protein hydro- lysate of the process is enhanced by the materials derived from the meat parts of the krill. Said separation at step c) of the process is pre¬ ferably effected by using a decanter combined with a centrifugation. The sludge fraction obtained by centri- fugation and containing the chitin is washed repeatedly. To improve the efficiency of the washing opera¬ tions the chitin is pressed after each washing. Espe¬ cially when sulfuric acid has been used for deminerali¬ zation of the shells the pressing is important since it helps to remove the only slightly soluble calcium sul- fate formed by the reaction of sulfuric acid with calci¬ um compounds in the shells. The purified product is dVied to obtain a stable product. In a following process or as an alternative to said drying the chitin may be further treated, i.e. by heating with a strong base to achieve chitosan. If the chitin-containing material used as start¬ ing material contains astaxanthin, this may be extracted by an oil, preferably in connection with a proteolytic treatment, and if so, the separation in step c) yields an oil fraction containing most of the astaxanthin from the starting material in addition to the aqueous frac¬ tion containing protein hydrolyzate and the sludge fraction containing the chitin. Said oil fraction is usually marketable without previous concentration of the astaxanthin therein. In a preferred embodiment the astaxanthin is extracted using a marine oil provided by adding fish lever or other oil-containing parts of fish from which the oil is released as a result of the proteolytic ac¬ tivity of viscera, said oil being isolated as a separate fraction in step c) as explained above. Alternatively, an oil such as soybean oil may be added, but to reduce costs it is often more advantageous to include fish lever, such as cod lever, or other oil- containing parts of fish when preparing the silage used in the process according to the invention, or to add cod lever or the like when preparing the suspension in step a). In both cases the proteolytic enzymes of the fish viscera cause such a decomposition of the oil-containing tissue that the oil is liberated and afterwards separ¬ ated in step c) . The aqueous phase obtained by the separation has a substantial content of protein hydrolysate and after an optional neutralization it may be used as a component in animal fodder, preferably in dried condition. From the above it appears that an embodiment of the process is characterized in that the suspension in step a) is prepared using a silage previously produced from fish viscera and an inorganic acid and having a pH of 1.2 to 2.5, preferably 1.5 to 2.5. The fish viscera silage may also comprise a minor amount of organic acid to suppress growth of microorganisms. When such a pre¬ viously manufactured silage is used, the pH of the sus- pension prepared in step a) may be higher than allowed when fresh viscera is used, but pH should not be higher than 4. In another embodiment of the process the suspen¬ sion is prepared in step a) by using fresh fish viscera and adjusting the pH of the suspension to 1.2 to 2.5, preferably 1.5 to 2.5. When shrimp shells are used as a source of chitin the shells are collected in a shrimp peeling plant and are usually pressed to a solids content of 50%. Due to the high deterioration rate the shrimp shells will usually be preserved by adding a strong inorganic acid to obtain a pH-value less than 3, and thereby a simul¬ taneous demineralization of the shells takes place. If the starting material is raw shrimp shells, the preser- vation is preferably effected at pH 1.2 to 2.5. Since the enzymatic activity of fish viscera and silage prepared therefrom varies substantially depending inter alia on the species of fish and the feed condition thereof as well as on the season and the age and storage temperature of the silage, it is not possible to indi- cate exact limits for the ratio of chitin source to fish viscera or silage to be used. However, when the chitin source is shrimp shells, said ratio is generally between 1:1 and 1:10, typically from 1:2 to 1:5, the amount of shrimp shells being calculated as a compacted material having about 50% solids. Since particularly the astaxanthin is very liable to oxidation, care is usually taken to ensure that anti- oxidants be present during the process, said antioxi- dants being preferably introduced as early in the pro¬ cess as possible, viz. during the possible preceding silaging or preservation of the chitin source with acid and by the possible preceding processing of fish viscera to silage. When using fresh starting materials the anti- oxidants are added during the preparation of the suspen¬ sion in step a) . As antioxidants, conventional compounds ■ may be used, e.g. butylhydroxyanisol. To obtain a high purity of the chitin obtained as sludge fraction at the separation it might be suitable to use a fish viscera silage previously freed from sludge, when preparing the suspension in step a) . The acid used in the possible preceding silaging or preservation of e.g. shrimp or krill shells and for obtaining the desired pH of the suspension, is hydro- chloric acid " or sulfuric acid or any other strong acid. The process according to the invention is further illustrated by means of the following embodiment example. E X A M P L E 6 batches of silage are prepared by using approx. 500 kg liver and viscera from cod, 15 1, 50%, (w/w) sulfuric acid and 5 g butylhydroxyanisol (as antioxi- dant) for each batch. The sulfuric acid is added while the liver and viscera are subjected to the action of a submerged mixer and blender in a 1000 1 tub. Mixing and blending is continued for 15 minutes whereupon the mix¬ ture is by pumping transferred to a tank wherein it is combined with the other five batches prepared in the same way. The pH of the mixture is approx. 2.0. The silage thus prepared is kept in the tank for two days during which it is intermittently agitated by means of a recycling pump. To remove par iculate material and sludge the silage is passed through a centrifugal decanter and pumped to a 5 m 3 spherical tank. At this stage the liver and viscera silage (approx. 3000 kg) has the following approximate com¬ position: Oil 20% by weight Non-fat solids 11% by weight - Protein 8% by weight Ash 2% by weight Shells from a shrimp shelling machine have the following approximate composition (after drainage of water) . Solids 20% by weight Protein 5% by weight Chitin 5% by weight Fat 0,5% by weight Ash 6,5% by weight Astaxanthin 50 ppm. Batches of each 250 kg shrimp shells are stirred with 600 liter water in a 1000 liter tub and 11 liter sulfuric acid (96% w/w) and 1 g butylhydroxyanisol is added. pH approx. 2.0. When a sufficient amount of shrimp shells has been silaged in this way they are pumped to a screw press to produce 1500 kg press cake having approx. 40% solids. Said press cake has the following composition: Solids 40 % by weight Protein 15 % by weight Chitin 15 % by weight Fat 1,5% by weight Ash 6 % by weight Astaxanthin 150 ppm. A substantial part of the calcium sulphate formed by reaction between the sulfuric acid and the calcium carbonate in the shells, is removed together with the press water. This press water may be used for treating a further amount of shrimp shells to reduce consumption of sulfuric acid and to avoid discharging of the acid liquid to the environment. The obtained press cake of acid treated shrimp shells is introduced into the spherical tank holding the liver and viscera ensilage into which the shells are suspended. The resulting suspension has a viscosity of 2-3000 cps. The pH is approx. 2. The mixture is heated at 35°C by means of a heat¬ ing coil through which water at 45°C is circulated. The mixture is agitated by pumping. After having been heated at 35°C and agitated for two days the suspension is heated to 80°C by means of steam and is subsequently pumped to the screw press in which oil and an aqueous solution of hydrolysed protein are separated from the raw chitin. The liquid phase is treated in a decanter centrifuge to remove sludge and is thereafter separated by centrifugation into an oil fraction and a fraction of aqueous protein hydrolysate. The oil contains 50-60% of the astaxanthin origi¬ nally present in the shrimp shells, corresponding to approx. 200 ppm astaxanthin in the oil. The oil may be used as such for the preparation of fodder for salmon and related fish in which a reddish meat colour is desired. The protein hydrolysate is evaporated and dried to obtain a product suitable as addition to fodder for cattle and poultry. The press cake which comprises the chitin of the shrimp shells is suspended in 2000 liter water at 80°C and the suspension is agitated by means of a pump for 15 minutes. The suspension is pumped to the press and the liquid phase obtained thereby is by means of a decanter and a centrifuge separated to recover a further amount of astaxanthin containing oil. The aqueous phase from said separation is discharged. The press cake of chitin is once more suspended in 2000 liter water at 80°C and 20 kg sodium carbonate is added. The suspension is agitated and the chitin recovered again by means of the screw press. In a similar way the chitin is rinsed once more using water without sodium carbonate and subsequently the rinsing is repeated adding 10 liter hydrochloric acid (30% w/w) . The obtained cake of chitin is finally washed with 2000 liter pure water, also at 80°C, and pressed again. The resulting cake of chitin is dried using hot air to obtain 225 kg chitin having the following data: Colour: white with a pale reddish tint Protein <0,5% by weight Oil <0,1% by weight Ash <0,5% by weight. When a product of extremely low ash content is desired a partially demineralized water is used in the washing process described above.

Claims: PATENT CLAIMS 1. A process for recovering chitin from materials wherein chitin is present together with or connected to proteinaceous substances by demineralization with an acid and removal of protein by utilizing the proteolytic effect of fish viscera, characterized in that a) an aqueous suspension is produced comprising the optionally minced chitin-containing mate¬ rial and the fish viscera which have possibly been pre-ensiled, ensuring that the fish viscera or the silage prepared therefrom act on the shells at a pH of 1.2 to 2.5, preferab¬ ly 1.5 to 2.5, or the viscera have previously been ensiled at such a pH, b) the suspension is heated at a temperature be¬ tween 25 and 50°C for a period between a few hours and four days, preferably to 3 days, and c) the suspension is separated to obtain at least (i) an aqueous phase containing dissolved hy- drolyzed protein, and (ii) a sludge fraction containing the chitin substantially without proteins and mineral substances. 2. A process according to claim 1, by which a chitin-containing material is used comprising astaxan¬ thin, characterized in that astaxanthin is extracted using a marine oil which is provided by using fish liver or other oil-containing parts of fish from which the oil is liberated by means of the proteolytic activity of the viscera, said oil being isolated as a separate frac¬ tion in step c) . 3. A process according to claim 1 or 3, charac¬ terized in that the suspension in step a) is produced using a silage prepared from fish viscera and inorganic acid and having a pH of 1.2 to 2.5, preferably 1.5 to 2.5, the pH of the suspension being adjusted to not more than 4. 4. A process according to claim 1 or 2, charac- terized in that the suspension is prepared in step a) by using fresh fish viscera and adjusting the pH thereof to 1.2 to 2.5, preferably 1.5 to 2.0. 5. A process according to any of the preceding claims, characterized in that the chitin source is shrimp shells whereby the ratio of said shells, calcu¬ lated as compacted shrimp shells having approx. 50% solids, to fish viscera or silage thereof, amounts to from 10:1 to 1:2. 6. A process according to any of the preceding claims, characterized in that an antioxidant is applied to the suspension, said antioxidant being preferably added during the possible silaging of the chitin source and/or of the fish viscera. 7. A process according to any of the preceding claims, characterized in that a partial neutralization and heating of the suspension at 80 to 85°C to inact¬ ivate enzymes therein and to decrease the viscosity, is performed between step b) and step c). 8. A process according to claim 3, characterized in that the silage of fish viscera is previously freed from sludge. 9. A process according to any of the preceding claims, characterized in that the source of chitin is raw shrimp shells preserved with an acid to pH 1.2 to 2.5. 10. A process according to claims 1 to 6 and 8 to 9, characterized in that part of the aqueous phase separated in step c) is returned to step a) . 11. A process as claimed in any of the preceding claims, characterized in that in the preparation of the suspension in step a) protease-containing parts of the animals serving as the chitin source are included.

Inventors: JOENSEN JON OLAVUR, JOENSEN, JON, OLAVUR
Applicants: MATCON RADGIVENDE ING FIRMA, MATCON RAODGIVENDE INGENIOERFIRMA A/S
Application year: 1986
Main classification: C08B37
IPC codes: C08B37, C08F12, C09B61, C12P19