GB2428682A - A process for the production of oil and protein from fish waste - Google Patents

Abstract

A process for the production of oil and protein from fish waste comprises the steps of mixing fish, fish waste or fish material together with a low pH preserving/stabilising solution consisting of both aqueous-soluble and lipid-soluble antioxidants and a lipase inhibitor, comminuting, subjection to a controlled autolytic proteolysis by endogenous proteolytic enzymes, heating to denature the proteolytic enzymes, phase separation and centrifugation to separate out an oil containing omega-3 polyunsaturated fatty acids and protein. The pH of the solution may be between 3-4.5 by the incorporation of a mineral acid or lower monocarboxylic acid. The lipase inhibitor may be green tea catechin flavanols. The aqueous-soluble and lipid-soluble antioxidants may comprise a synergistic combination of synthetic or natural agents exhibiting a range of established or putative antioxidant mechanisms including oxygen scavenging, metal ion sequestering and free radical quenching. Also disclosed are products produced by the process afore described.

Description

HIGH QUALITY OMEGA-3 OILS AND PROTEINS FROM FISH WASTE The invention

herein described relates to a method whereby fish, fish offal or fish or shellfish waste or fisheries and aquaculture processing by-product material of any kind is first stabilised and protected by the addition of a preserving/stabilising solution containing a multi-component antioxidant system and a lipase inhibitor, comminuted, subjected to controlled autolytic proteolysis by endogenous enzymes under mild and optimised reaction conditions in a reaction solution and then immediately separated by centrifugation into two stable product phases, a lipid stream and an aqueous protein stream. For the first time both omega-3 long chain polyunsaturated fatty acid rich oils and proteins and protein fractions and\or derivatives can be produced in a stable and high-quality form by a low-cost, non-polluting, natural bioprocess. Both the omega-3 oils and the partially hydrolysed proteins produced are suitable for a wide range of food, pharmaceutical, nutritional supplement, dietetic, nutraceutical, functional food and skincare applications in humans and animals and are safer, of lower cost and of better nutritional quality than fishmeal and fish oil products currently being produced by malodorous, energy-intensive processes.

It is recognised throughout the world that supplying fish for human consumption is inefficient and wasteful in terms of utilising the biomass harvested. Depending upon the fish species, its established end-use, methods of processing and cultural practices, only about 40-50% of the catch is channelled into human nutrition, either by direct consumption or indirectly as an animal feed, for example in the production of poultry, pigs, and ruminants. The material not used and discarded comprises bycatch and trash-fish harvested inadvertently, and processing waste (viscera, off-cuts, heads, tails, skin, and frames) from both fisheries and aquaculture. The present invention offers a simple, safe, robust process for converting all kinds of fish waste into two value-added product streams; high-quality proteins and health-enhancing oils rich in the nutritionally essential omega-3 long chain polyunsaturated fatty acids and its development has been driven by: 1. Environmental concerns about the hazards associated with fish waste: microbiological spoilage; contamination and disease; vermin and pests; and, decaying material and noxious fumes spoiling amenity, leisure and tourism value of beach and shore areas. Punitive legislation is imposing fines and forcing producers and processors to incur ever-escalating haulage and disposal costs.

2. Demand for fish and all fish derivatives are increasing rapidly and global fish reserves and catches are dwindling. This demands more effective utilisation of existing resources.

3. l'he realisation of the nutritional benefits of fish protein, at present dominantly in the form of fishmeal, in animal productivity has created enormous demands in the aquaculture and animal production industries.

4. The increasing awareness of the significant health benefits of omega-3 long chain polyunsaturated fatty acids and their use in preventing and treating human disease and in promoting animal well being, production and fertility.

The established methods of converting fish waste and industrial' fish species, i.e. those deemed unsuitable for direct human consumption, such as menhaden, sand eel and capelin, into fishmeal protein and as a byproduct, fish oil, are expensive in terms of capital investment, energyintensive to operate, emit objectionable odours and produce low-quality products. There are two other drawbacks associated with channelling fish waste into fishmeal production. Fish viscera because of its soft texture, high oil content and high chemical and biological reactivity cannot readily be processed in fishmeal factories and in any case, fishmeal is not consumed by humans and is used mostly in pig and poultry feeds and in aquaculture production.

The two general methods of fishmeal production are by high-temperature cooking and pressing and by rendering at elevated temperatures. The oil by-product produced can often be oxidised and thermally denatured and needs remedial treatment by a sequence of energy intensive chemical and physical techniques, including degumming, alkalisation/neutralisation, bleaching and/or molecular distillation before it is safe for use in animal feeds. Oxidised chemical species from the residual oil in the fishmeal, including highly reactive free radicals, interact with the proteins resulting in denaturing and in the generation of malodorous volatiles. Another process for dealing with fish waste termed, silaging', has been introduced commercially in Scandinavia. This involves ensiling material in a mineral or organic acid for indeterminate and long periods of time at ambient temperatures and allowing the lipid content to be released from the degrading fish tissue. A supernatant oily scum is formed, which is periodically collected by decanting or skimming. The degraded protein is used as a low-grade, feed material on fur farms or as fertiliser. The oil produced is highly oxidised and of low quality and if not discarded is burned as a partial replacement for boiler fuel.

The emphasis of these existing processes is on producing protein products, with the oil component considered as an inconvenience or a low-value byproduct. In contradistinction, the present process was developed specifically to optimise the extraction of fish oils, to conserve their properties by preventing oxidation and lipolysis, to protect their endogenous antioxidants and vitamins, and to produce stable products rich in omega-3 long chain polyunsaturated fatty acids. This is seen in very low Acid Values, Peroxide Values, Anisidine Values, Totox Values, Oxidative Stability Index values and in high triglyceride, vitamin E, caroteniods, other biologically active components and omega-3 levels. In protecting and stabilising the oil and in quantitatively and rapidly separating it from the partially hydrolysed protein produced, the process also produces higher quality protein products than the conventional processes. Contact with the destructive free radicals generated from the polyunsaturated fatty acids is avoided and the protein products have low Total Volatile Nitrogen values, low histamine levels, low levels of biogenic amines, and have an acceptable malty odour. Additiorafly they have high digestibility, contain all the essential amino acids, conserved teyJs of tryptophan, asparagine, and glutamine, and are highly resistant to microbiological attack and have a prolonged shelf-life.

The use of the two solutions in the present invention is central in controlling the complex series of post-mortem biochemical and microbiological reactions and in ensuring the quality of the lipid and protein products. The chemical nature and low pH of the preserving/stabilising solution prevents microbiological growth and the added antioxidants and lipase inhibiting agents prevent oxidative attack and lipase degradation of the lipids, respectively. The nature of the reaction solution ensures that for the first time the endogenous enzymes are regulated, so that the pH-mediated proteolytic activity responsible for tissue breakdown is enhanced, whilst the lipase activity, which attacks the native triglyceride structure of the oil and reduces its quality, is inhibited.

The preferred material for the present invention is fresh viscera and offcuts from recently processed fish from marine fisheries and freshwater or aquaculture sources.

The waste or offal material can be any commercial pelagic, demersal or shellfish species including herring, mackerel, sardine, anchovy, pilchard, tuna, cod, haddock, plaice, salmon, perch, bream, trout, tilapia, carp, squid, shrimp, scampi, prawn, crab.

Fresh whole fish of underutilised or trash' or industrial species can be successfully processed including saithe, sand-eel, sprats, capelin, and menhaden as well as whole fish of edible species such as herring, mackerel, sardines, anchovy and pilchards.

The present process exploits the endogenous proteolytic enzymes present in fish tissue and optimises their behaviour and activity by controlling the fish tissue particle size, the ratio of enzyme to substrate (fish tissue) concentrations, and the pH, temperature, and time of the reaction. The enhanced proteolytic activity achieved rapidly breaks down the tissue structure and releases the oil from the fish tissues. The released oil is protected from the destructive effects of lipase activity by the selection of the reaction conditions and the presence of the lipase inhibiting agents, for example, the catechin flavanols from green tea extract, in the preserving/stabilising solution. These conditions, primarily pH and temperature, whilst optimal for proteolysis, reduce lipase activity. Moreover, in the process the oil phase is rapidly separated from the aqueous protein phase so that contact with the lipase enzymes is reduced, the unwanted generation of free fatty acids is minimised and the native triglyceride structure of the oil is conserved. The antioxidants used prevent the oxidative attack on the double bonds of the unsaturated fatty acids, and particularly the omega-3 long chain polyunsaturated fatty acids, present in the fish oil The process is essentially a five-stage operation, preserving/stabilising, comminution, proteolytic reaction and oil release, phase separation and evaporation and is described as follows: After slaughter and bleeding andlor filleting, the fresh material straight from the degutting line, processing plant, harvesting facility, fishing vessel, slaughter house or abattoir is transferred directly into a receiving tank containing the preserving/stabilising solution as described in detail below and fitted with a slowly rotating paddle stirrer. The preserving/stabilising solution, generally containing the same components and proportions as the reaction solution used in the reaction stage, is made up at ambient temperature. The preserving/stabilising solution is used at half the weight ratio with respect to the incoming fish mass as reaction solution used in the autolytic proteolysis reaction stage. After the batch of fish material has been received in the tank and been in contact with the preserving/stabilising solution for a short time the tank contents is transferred for comminution. Fresh material, which cannot be handled immediately by the present process can be chopped into large chunks and stored in the preserving/stabilising solution contained in a light-proof, air-tight vessel for comminution and processing at a later date. Similarly frozen material can be chipped and thawed in the preserving/stabilising solution prior to comminution.

Comminution stimulates proteolyitc activity, which is then further accelerated by temperature and pH during the reaction stage. Large pieces of bone, shell and extraneous material such as polystyrene and wood are separated on a screen arrangement before the comminution system and discarded. Similarly, metal fragments are removed by an in-line magnet prior to comminution. The fish material is comminuted by an in-line pump/macerator or series of macerators or similar comminution devices known by those skilled in the art, so that a particle size of less than 2 cm, more preferably less than 1 cm, and most preferably less than 2 mm is achieved. Oversize pieces of tissue are retained on a screen positioned at the exit of the comminution system. The screen is fitted with a device to scrape its surface the oversize material is recycled back to the maceration system for repeat comminution.

The comminuted material, which has the consistency of a porridge, is passed into a jacketed and temperature controlled reaction tank with an appropriate agitation system and mixed with a volume of water to give a ratio of I part fish mass to between 0.25 and 2.0 parts water depending on the nature of the material used. Gut material containing higher amounts of proteolytic enzymes will generally receive greater dilution. The acid added to regulate the pH, stimulates the proteolytic enzymes and prevents microbiological spoilage can be a mineral acid, an organic acid or a mixture of both. The simple monocarboxylic acids or their derivatives are preferred and formic acid is the most preferred for the present invention. Formic acid is preferred because its low molecular weight and hydrophobicity allow it to penetrate tissue fragments and to cross cell membranes. The use of formic acid ensures that the required pH is achieved homogeneously and rapidly throughout the tissue particles and the whole reaction milieu. The same attributes of formic acid contribute to its established antibacterial and antifungal activity.

Formic acid (as 85% w/w) is added to the reaction medium at the rate to give a proportion of between 1-6% by weight, and most preferably between 2-4% by weight and mixed thoroughly and continuously throughout the process to ensure homogeneity.

The range of pH required can be between 2 and 5, more preferably between 3 and 4.5, and most preferably in the range 3.3 to 4.0.

A multi-component antioxidant system is added to protect the oil against oxidation particularly as it is released from the fish tissue and also to protect the protein, polypeptides and amino acids in the aqueous phase against attack from the highly reactive free radicals generated from lipid oxidation. Thus, both lipid soluble and water soluble antioxidants are added before comminution in the preserving/stabilising solution and further amounts can be added during the maceration process. The antioxidant agents used are selected from a wide range of substances, both natural and synthetic and perform the following roles: 1. Oxygen scavenging - substances such as ascorbic acid (vitamin C), ascorbyl palmitate, ascorbyl stearate, erythorbic acid, sodium erythorbate, etc., react with various forms of oxygen (singlet oxygen, hydroxyl radicals, superoxide) and hence reduce the amount of dissolved oxygen available during the oil extraction process 2. Metal ion sequestering - citric acid, ethylenediaminetetraacetic acid, and phosphoric acid derivatives chelate pro-oxidant metal ions such as iron and copper present in the fish tissue and thus prevent them from promoting oxidative deterioration.

3. Free radical quenching - synthetic phenolic substances such as butylated hydroxtoluene, butylated hydroxyanisole, tert-butly hydroxyquinine, and propyl gallate and natural phenolic compounds such as vitamin E, rosemaric acid, flavonoids in general, and flavanols in particular, react with lipid free radicals and hence reduce autoxidation in the oil. This activity is known as primary antioxidant activity reflecting its importance in protecting lipids.

Various combinations of these or other antioxidant agents can be added, but the preferred system utilises the protective effect of the three classes of antioxidant. Both ascorbic acid and ascorbyl palmitate are used at concentrations of between 20 and 1000 ppm, and preferably between 100 and 300 ppm. The former is water soluble, but is also known to be active at the aqueous/lipid interface, and the latter is the lipid soluble form.

Thus, there is effective oxygen scavenging activity from immediately after degutting or slaughter when material is contacted with the preserving/stabilising solution prior to comminution, and throughout the reaction stage. The same is true for the sequestering of pro-oxidant metal ions, where citric acid can be added at the rate of between 20 and 1000 ppm and preferably at about 50-250 ppm; a proportion of the citric acid will be solubilised in the lipid phase, especially as the reaction mixture is heated and as the oil is released from the fish tissues. The primary antioxidant preferred is vitamin E and particularly a combination of natural vitamin E and alpha-tocopheryl acetate each added at the rate of between 50 and 300 ppm. The former is moderately lipid soluble and the latter is slightly soluble in water with temperature increasing the relative solubility of both. In aqueous acid media the latter dissociates to generate active tocopherol at the aqueous/lipid interface. Another important water-soluble antioxidant mixture is added to the stabilisation/protection solution. Tea catechin fiavanols in the form of a powdered green tea extract are very effective at addition rates of between 20 and 1000 ppm. Concentrated forms and individual catechin flavanols can also be used, with epigallocatechin gallate at addition rates of between 20 and 200 ppm being particularly potent. Moreover, as described previously the catechin flavanols in the green tea extract added at these levels also inhibit lipase activity and help to conserve the native triglyceride structure of the oil.

The reaction mixture is heated by passing through a heat exchanger or by an internal or external heating coil or jacket containing a heating medium or any other heating means known to those skilled in the art. The reaction temperature can be between 25 and 50 C, and preferably between 35 and 45 C. The reaction mixture is held at the reaction temperature for between 0.5 and 8 hours and preferably between I and 4 hours depending upon the fish material used, its origin and the proportion of viscera., off-cuts, bones etc. In general viscera and material containing a high proportion of visceral tissue requires shorter incubation periods. The reaction mixture is agitated continuously throughout the reaction process so that enzyme and substrate are in constant contact and so that temperature and pH are homogeneous. The type of agitation can be any stirring system which gives gentle slow mixing which ensures thorough contact between the phases, whilst avoiding turbulence, shearing and possible emulsification.

It has been found that the pH and temperature regimen used in the present process favours the activity of particular enzymes at the expense of others to the benefit of the quality of the products produced. The endogenous protease enzymes optimised are pepsin from the viscera and digestive tract and the catheptic enzymes from the lysosomes of muscle cells. Trypsin and chymotrypsin are rendered largely inactive under the conditions selected. Similarly the activity of lipase enzymes has been found to be suppressed by the pH and temperatures used and by the presence of green tea extract, thus protecting the native triglyceride structure of the oil released during the process from hydrolysis to free fatty acids.

Once the controlled autolytic reactions are complete and the reaction substrate is transformed into a liquefied, viscous slurry, it is transferred by any suitable pumping system via a heat exchange unit at 6090 C to a phase separating arrangement. It is preferred to heat the reaction mass to between 75 and 90 C to denature the endogenous enzymes, to halt the proteolytic breakdown and to ensure good phase separation. The slurry, because of its sensitivity to temperature should be brought to the required temperature level as rapidly as possible. Similarly temperatures higher than 90 C are, in general, to be avoided so as to prevent possible destruction of the highly labile double bonds of the polyunsaturated species present in the oil. Material known to contain oil with only a small proportion of polyunsaturated fatty acids can be, however, subjected to higher temperatures to improve separation throughput.

The choice OI separation system should be such that quantitatively complete separation is achieved so that the oil phase contains only traces of moisture and no protein and the aqueous protein phase contains very small amounts of oil. Those skilled in the art can readily devise continuous centrifugation systems or the like to achieve this required degree of separation of the two phases. This complete separation enhances the quality and stability of both product phases since it has been found that even minor contamination of one phase with the other leads to oxidative deterioration. In some cases it is necessary to introduce a second centrifugal separation to ensure the removal of small proteinaceous particles, silicaceous grains and other suspended solids from the oil phase. Such a clarifying or polishing operation can also be conveniently carried out by a clarifying centrifuge or by filtration. Similarly, the aqueous protein stream may be treated by a further dc- oiling operation by a clarifying centrifuge, by filtration or by any process known to those skilled in the art.

The protein stream can be converted into a number of products depending upon type of solids, dry matter content and degree of hydrolysis. The product directly after separation from the oil phase can contain between 10 and 20% solids and this can be used directly in the animal feeds industry for incorporation into liquid feed systems.

This stream can be further concentrated to 40-50% dry matter content and used in a wide variety of animal, aquaculture feeds and pet foods. The product can also then be spray-dried by any of the industry standard techniques now available commercially to produce a smell-free, protein dense, free-flowing powder product with a dry matter of up to 95%. This product can be used in a wide variety of human nutrition products and/or nutritional supplements and functional foods. It is readily incorporated into soups, sauces, relishes and into dry and baked goods such as pasta, bread, health bars, biscuits, etc. Another process route can be followed for the protein products stream after the oil removal stage, which allows the production of highly hydrolysed products. The aqueous protein can be kept at 30-40 C for the time required to achieve the selected level of protein breakdown to peptides and amino acids. Residual enzyme activity and low acid pH drive this hydrolysis, whilst the absence of oil, the presence of the water soluble components of the antioxidant system and the acidic medium prevent both microbiological spoilage and oxidative deterioration during the prolonged reaction period. Additional exogenous proteolytic enzymes can also be added to accelerate the hydrolysis and/or to take it towards stoichiometric completeion. It has been found that endogenous enzymes in the form of macerated fish viscera can be added to achieve this and also commercially available proteases can be used. The amino acid products resulting from these processes can be used as nitrogenous fertilisers, or after purification, as growth media in fermentation and other biotechnological processes.

The high-quality, stabilised oil produced from the present controlled autolytic proteolysis process is in a form suitable to undergo a variety of refining and/or further processing operations to make it more suitable for specialist pharmaceutical, nutritional, chemical, or industrial applications. For example, the oil can readily be winterised to reduce the saturated fatty acid content and subjected to an adsorption bleaching regime to remove traces of possible contaminants, such as pesticide residues, polycyclic aromatic hydrocarbons, dioxins/dibenzofurans, polychlorinated biphenyls, heavy metals, etc. There are a range of high specification materials, such as activated- carbon, silicas, benthonite clays, etc., which have been specifically developed to remove such contaminants. The oil can then be deodorised by a short-path distillation technique and used in pharmaceutical, skin-care or nutritional supplement preparations.

The oil can also be processed for specific industrial purposes by those skilled in the art, for example by sulphating (or suiphiting) to produce a tanning liquor or by emulsifying and incorporating additives to produce a high-temperature drilling fluid.

Another aspect of the process allows another product to be produced. When the aqueous protein phase is permitted to settle in a vessel immediately after phase separation, a bottom layer or stickwater' fraction is formed. This dilute solution can be concentrated by any of several techniques available and then dried in an evaporator to give a fish solubles' product containing water soluble components valuable as palatates and used as flavours or attractants in relishes, fish feeds, lures, etc. In another embodiment of the process, when the production site is not immediately adjacent to the source of raw material and fresh material cannot be fed directly into the process, material can be stored for later use. This is achieved by using the preserving/stabilising solution as previously described. It has been found that fish material, including fish viscera, is effectively protected from both microbiological spoilage and oxidative attack for as long 5 to 20 days depending upon the ambient temperature, the species of fish, the type of waste and its previous processing history.

Such a pre-treatment allows the rest of the process to be carried out at a location remote from the site of production of fish or fish waste, where transportation would take a significant time. It also allows small producers of waste fish or fish processing waste to accumulate quantities of material in collection tanks over several days for regular collection on a milk round' basis. Moreover, it permits the use of gutting waste produced at sea and also the use of trash' fish species. Fishing vessels can simply store the by-catch, trash' fish, and/or gutting waste, etc., in tanks containing the preserving/stabilising solution until they return to port, where the material can be pumped into the processing plant or road tanker for transport to the plant.

In another embodiment of the process, several of the processing stages can be combined. For example, the stabilisation/protection, comminution and reaction/oil release stages can take place in one vessel appropriately fitted with a submerged macerating pump and a re- circulation system. Such a system and many possible variations on it conceived by those skilled in the art can be operated in either batch or continuous production mode.

Some non-limiting examples are given to illustrate the features of the invention

Example 1

Species & Source: whole herring from market, about 1 day from landing.

Conditions: 1. 10 kg fish crudely chopped into 3 cm pieces and divided into two 5 kg batches for further treatment.

2. Treatment A: 150 g formic acid (of 85% w/w strength) was added to 5 kg of the chopped fish material in addition to 200 ppm of the antioxidant ethoxyquinone and stored in an enclosed glass vessel (1OL) at ambient temperature (18-22 C) in a light- proof cupboard until the material had largely liquefied and no further quantities of oil were appearing on the surface. This was achieved in 8 days.

3. Both the supernatant oil phase and the aqueous protein phase were collected by decanting and stored in 50 ml brown glass jars for analytical sampling.

4. Treatment B: 5 kg of the chopped fish material was mixed with 500 kg of water in an enclosed glass fermentation vessel (1OL) fitted with a slowly rotating paddle stirrer. Formic acid (85% w/w) was added to give a 3% w/w solution and the contents were gently stirred for 30 minutes at ambient temperature (1 8-22 C). The pH over this period came down from 6. 0 and stabilised at 3.8.

5. An antioxidant system consisting of ascorbyl palmitate, natural vitamin E (as mixed tocopherols) and lecithin was added in the proportions 25%, 5% and 70%, respectively. The total amount of antioxidant added gave a concentration of 200 ppm 6. Steps 4. and 5. completed the preparation and use of the preserving/stabilising solution and the fish pieces in the solution were repeatedly maccrated in a modified laboratory blender operated with the cutting blades at slowest speed until a porridge consistency was achieved with particle sizes in the approximate range 1.70-3.35 mm. This was judged by subjecting sub-samples to sieve analysis until the fish tissue particles passed through 5 mesh and were retained on 10 mesh (BS 410).

7. A 3 kg sample of the thick gruel was transferred to an enclosed glass fermentation vessel (10 L) fitted with a slowly rotating paddle stirrer and 1 L of water was added. The reaction solution was added consisting of formic acid (85% w/w) to

give a 3% solution and the antioxidant system as described in 5. above at the rate of 100 ppm.

8. The vessel was submerged in a water bath thermostated at 38 C and the proteolytic reaction was allowed to continue at pH 3.5 and 38 C until liquefaction was complete and the two phases had formed and separated (3 h).

9. The material was immediately transferred to a bench centrifuge operating at 5,000 rpm and the two phases were quantitatively separated and the two phases collected and stored in a series of 50 ml brown glass jars for analytical purposes.

10. Samples of the aqueous products from both treatments were carefully dried in a laboratory vacuum oven until dry powders were obtained.

Results: Proximate Analysis Start Material Protein Phase cx A Protein Phase ex B Water% 67 70 73 Oil% 12 8 2 Protein% 17 15 19 Ash% 3 5 3 Dry Protein Powder Treatment A Treatment B Protein (N x 6.25), % 54 78 Total Volatile Nitrogen, mg/I OOg 150 75 Histamine, ppm 810 50 Oil Analysis Treatment A Treatment B Colour Brown Yellow Odour Strong rancid fish smell Slight fresh fish smell Acid Value, mg KOHIg 14.9 3.9 Peroxide value, mEq 02/kg 17.9 4.3 Fatty Acid Composition*: Eicosahexaenoic acid, % 11.7 13.8 Docosahexaenoic acid, % 7.6 8.9 Total Omega-3 22.0 26.2 * Determined as fatty acid methy' esters by gas chromatography and expressed in terms of peak height/area percentages.

Treatment A simulated the silaging method and it can be seen clearly that the present invention results in much better protein and oil quality. With the new process in Treatment B there has been much less protein degradation and much lower levels of degradation products as indicated by the low TVN.

Under the conditions used in the new process the quality parameters of the oil produced are also much better than the results obtained from the silaging method. The low Peroxide Value demonstrates that the oil has been protected against oxidation and as a consequence the levels of the nutritionally important omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid and docosahexaenoic acid, have been conserved. The Acid Value is also much lower and indicates that lipase activity has been reduced by the processing conditions and the integrity of the triglyceride structure has been maintained.

Example 2

Tuna viscera was obtained from a canning factory within 2-3 hours of processing and deep frozen for transportation. 20kg of the frozen material was chipped into large pieces and 10 L of water at ambient temperature was added and continuously stirred to aid thawing. To this mixture, 750 g of formic acid (85% w/w) was immediately added to give a concentration of 2.5%. An antioxidant system consisting of citric acid, ethoxyquin and ascorbic acid at rates of 100 ppm, 200 ppm and 100 ppm, respectively was also added and the fish tissue was allowed to thaw completely at ambient temperature. The formic acid and three-component antioxidant system comprised the preserving/stabilising solution. The following treatments were then applied: 1. All the fish viscera chunks were crudely chopped into 2.5-5 cm chunks and four 4 kg batches of pieces were collected for further treatment. The melt water including the preserving/stabilising solution was drained into a separate container 2. Treatment A: 1L of a 12.5% formic acid (85% w/w) was added to 4 kg of the chopped fish material to give a 2.5% concentration of the acid and stored in an enclosed glass vessel (1 OL) at ambient temperature (20-26 C). In accordance with the silage method the vessel was stored until the material had largely liquefied and no further quantities of supernatant appeared (6 days).

3. Both the supernatant oil phase and the aqueous protein phase were collected by decanting and stored in 50 ml brown glass jars for analytical sampling.

4. Treatment B: 4 kg of the chopped fish material was repeatedly maccrated in a modified lab blender operated with the cutting blades at slowest speed until a porridge consistency was achieved. During this operation 2 L of the preserving\stabilising solution retained in I above was added.

5. The thick gruel was mixed slowly with 2 L of a 7.5 % formic acid (85% w/w) solution in an enclosed glass fermentation vessel (1 OL) fitted with a slowly rotating paddle stirrer so that the correct (2.5%) concentration of formic acid and the correct substrate to enzyme ratio be maintained..

6. To complete the reaction solution an antioxidant system consisting of ascorbyl palmitate, tocopheryl acetate and a lecithin material (ammonium phosphatides) was added in the proportions: 50 ppm, 50 ppm, and 100 ppm.

7. The vessel was submerged in a water bath thermostated at 40 C and the proteolytic reaction was allowed to continue at p11 3.5 and 40 C until liquefaction was complete and the two phases had formed and separated (3 h).

8. The material was transferred to a bench centrifuge operating at 5,000 rpm and the two phases were collected and stored in a series of 50 ml glass jars for analytical purposes.

9. Treatment C was identical to Treatment B except that 150 ppm of a powder containing green tea catechin flavanols was added in addition to the antioxidant system at 6.

10. Samples of the aqueous products from the three treatments were carefully dried in a laboratory vacuum oven until dry powders were obtained.

Amino Acid Composition (mg/I OOg) of Protein Stream Treatment C Treatment A Ala 1008 1070 Arg 779 665 Asp 1403 1265 Cys 108 130 Glu 2165 1958 Gly 982 1124 His 366 306 lie 886 832 Leu 1466 1437 Lys 301 247 Met 431 265 Phe 700 635 Pro 715 714 Ser 537 583 Tau 482 351 Trp 196 194 Thr 680 855 Tyr 289 158 Val 892 872 Dry Protein Powder Treatment A Treatment B Treatment C Protein (N x 6.25), % 58 77 82 Total Volatile Nitrogen, mg/lOOg 175 100 35 Histamine, ppm 550 50 10 Tuna Oil Analysis _____________ FattyAcid A B C 14:0 MA 5.3 41 52 PA 16.8 16.6 15.1 0.3 0.4 08 SA 32 2.3 3.0 Sum saturated 25.6 234 24 1 161w7 4.5 34 3.0 181w9 128 12.1 12.6 181w7 3.6 32 4.0 20:lwll 1.5 1.8 1 5 20:1w9 08 0.5 0.7 22:1w9 1 9 1.4 2.1 22:lwll 04 04 0.4 24:1 11 07 06 Sum mono-unsaturated 26 2 23 5 24.9 18:2w6 LA 3.0 3.2 2.8 2w6 0.4 0.4 0.3 4w6 AA 0.5 0.6 0.5 224w6 1.5 1.6 2.4 225w6 03 0.4 0.3 Sum w6-PUFAS 57 62 63 16:3w3/16:4w3 0.5 07 0.9 183w3 ALA 1.2 1.4 1 4 18:4w3 1.5 2.2 20 204w3 0.5 1 0 0.7 5w3 EPA 4.6 5.8 6.2 22 5w3 3.3 4 1 3.9 22.6w3 DHA 20.7 24.7 25.9 Sum w3-PUFAS 38.4 42.9 40 0 AcidValue,mgKOHg1 13.9 44 07 Peroxide Value mEq 02 kg 1 29 6 11.1 43 Anisidine Value 71 21 13 Totox Value 130 43 21

Example 3

A series of trials were conducted on trout processing waste from a fillet producing factory. The waste, combining all waste streams, gut, off-cut and frames, was immediately collected, chopped into 3-6 cm fragments and sprayed with fresh water so that one part of water was added to two parts of waste. Formic acid (85% wlw) was added to form a 2.5% solution and ethoxyquin and phosphoric acid were added to complete the preserving/stabilising solution at rates of 200 ppm and 75 ppm, respectively. The material was stored in a lidded, light-proof container for 12 hours.

The following treatments were carried out on the material.

Trial I - Treatment Variable: Antioxidant System.

The material was maccrated by passing through a maccrating pump and various antioxidant systems were added in the reaction solution: Treatment A: A 2.5% HCOOH (85% w/w) solution was added to an equal volume of the comminuted fish mass and vitamin E (as mixed natural tocopherols) was added at the rate of 200 ppm. The controlled autolytic reaction was carried out in a stirred vessel at 40 C for a period of 3 hours. The phases were separated by centrifugation and samples of both the aqueous protein product and the fish oil were kept and prepared for analysis.

Treatment B: The same as A in all respects except that additional antioxidants were added to the reaction solution: aqueous soluble ascorbic acid at S0ppm plus lipid soluble ascorbyl palmitate at 50 ppm.

Treatment C: The same as B in all respects except that additional antioxidants were added to the reaction solution: flavanols (cx green tea extract) at 150 ppm Results Analysis of Trout Oil _________________ _________________ Treatment Acid value Peroxide Anisidine value value A 5. 5 6.1 18 B 3.1 1.1 6 C 0.2 0.7 I Treatment C, containing the synergistic mixture of antioxidants and lipase inhibitor, was extremely effective in preventing the generation of free fatty acids (i.e. low A.V.) and both primary and secondary oxidation products (as seen in low P.V. and An.V., respectively).

Trial 2 - Treatment Variable: Time between Dc-Gutting and Processing.

The process was carried out as in Trial I with the following variations: Treatment 1: As Treatment C in Trial 1 except that trout waste was retained for 3 hours before the addition of the preserving/stabilising solution and then subsequent processing.

Treatment 2: As Treatment C in Trial I except that the preserving/stabilising solution was added and the material was retained for 2 days before processing.

Treatment 3: As Treatment C in Trial 1 except that the preservative/antioxidant was added to the fresh material and then it was retained for 4 days before processing.

Results Analysis of Trout Oil _________________________________ Treatment Acid value Peroxide Anisidine value value 1 11.8 32.3 47 2 1.4 2.8 12 3 1.2 3.2 14 It is clearly necessary to add the preserving/stabilising solution to the waste material as soon as possible after it has been produced at the fillet manufacturing plant. The use of the potent antioxidantllipase inhibitor system allows much more flexibility in collection and transportation between the time of degutting and processing by the controlled autolytic proteolysis process.

Fish oil must have an AV below 4, a PV below 10 and an AnV below 20 to allow free sale on the international commodity market and using the multicomponent antioxidant system will ensure this.

Trial 3 - Treatment Variable: Reaction Temperature Treatment 15: As C in Trial I except that after comminution the slurry was kept at 15 C until all the oil was judged to be released, tiisue breakdown was achieved liquefaction was complete - this took 36 h. Treatment 25: As Treatment 15 except that the reaction temperature was maintained at C until completion - this took 12 h. Treatment 35: As Treatment 15 except that the reaction temperature was maintained at C until completion - this took 4 h. Treatment 45: As Treatment 15 except the reaction temperature was kept at 45 C until completion - this took 2 h. Results Trout Oil Analysis Treatment Acid value Peroxide Anisidine value value 4.6 20.1 32 2.3 8.4 14 1.1 0.9 9 0.9 1.9 5 Using temperature to accelerate the autolytic proteolysis reactions under the controlled conditions used results in better quality oil than using time to effect the tissue breakdown and oil release. From an industrial perspective reducing the reaction time will also allow more throughput capacity in a production plant.

Example 4

Salmon processing waste from a commercial fillet production factory, consisting of whole viscera (the pluck'), heads and tails and other offcuts, and the frames, were collected straight from the automated processing lines and mixed with fresh water in the ratio of 2 parts salmon waste to 1 part water. Formic acid (85% wlw) was added so that a 2. 5% concentration was obtained. The following additives were used at the rates specified to complete the preserving/stabilising medium: lipid soluble antioxidants, ascorbyl palmitate I O0ppm, ammonium phosphatides 1 OOppm, tocopheryl acetate 50 ppm; and the aqueous soluble compounds, 1 O0ppm epigallocatechin gallate, citric acid 100 ppm.

The salmon waste was stored for 24 hours before further processing.

The material was passed through a chopper pump, which produced tissue fragments of approximately 2-4 cm and then through a maceration pump, which further comminuted the material to produce a porridge-like slurry. This slurry was transferred into a lidded reactor vessel with a stirrer and an external jacket for hot water circulation. An equal volume of fresh water was added. To make up the reaction solution formic acid (85%) was added at the rate of about 2-3 % to titrate the solution to give a pH of 3.3-3.6. The same antioxidant system as used in the preserving/stabilising solution was used at the same concentrations.

The contents of the reactor were stirred at slow speed and kept at 42-45 C for 2.5 hours.

The material was then heated to 85 C over a short period in the reactor and passed into a continuous decanting centrifuge to separate the two phases. The oil was collected directly and the aqueous protein phase was collected via a screen, which removed the small amount of undigested particles.

Samples of the oil and aqueous protein products were kept in brown glass, stoppered bottles for analysis. Additionally samples of the aqueous protein were dried in an oven to about 50% dry matter content and subsamples of this liquid concentrate were further dried to about 90% dry matter content in a laboratory spray drier.

Results.

The protein products had high protein content and retained very low levels of oil Proximate Analysis Liquid Protein Concentrate Protein Powder Water, % 55 7 Protein (N X 6.25), % 34 80 Oil,% 1 2 Ash,% 7 8 The protein powder had an acceptable malty odour and had a cooked fish taste when examined by a taste panel. It also had excellent quality parameters; high digestibility and very low volatiles, i.e. aldehydes, ketones, alcohols, ammonia, trimethylamine, etc., and very low levels of histamine.

Dry Protein Powder Appearance CreanifLight Brown Odour Malty Protein (N x 6.25), % 80 Digestibility (Pepsin), % 97 Total Volatile Nitrogen, mg/lOOg 48 Histamine, ppm 21 The protein contained all the essential amino acids and the particularly labile species were conserved, such as tryptophan and glutamine were conserved.

Amino Acid Composition of Protein, gIlOOg Lysine: 6.90 Histidine: 1.50 Methionine: 3.10 Aspartic acid: 5.20 Asparagine: 1.09 Cysteine: 0.73 Phenylalanine: 4.90 Tryptophan: 1.55 Glycine: 12.90 Threonine: 4.00 Glutamicacid: 14.36 Glutamine: 0.95 Taurine: 1.23 Isoleucine: 2.95 Proline: 6.10 Valine: 4.10 Serine: 5.57 Alanine: 7.26 The powder with a pH of 3.3 was very resistant to microbiological spoilage.

Powder after storage for 3 months: Microbiological Analysis CFU / g Total mesophilic aerobes: < 10 000 Total coliforms: 10 Enterobacteria: 10 Suiphite reducing anaerobes: 10 Staphylococcus aureus: 10 Salmonella: absence I 25 g.

The process has protected the oil from oxidation and other chemical degradation and the oil produced has all the characteristics of an excellent oil for human and animal nutritional and pharmaceutical purposes. This is seen in the low Totox Value, which combines the primary and secondary oxidation products and the very low Acid Value.

Salmon Oil Analysis Colour Light red Odour Slight fresh fish Acid Value, mg KOH/g 1.12 Peroxide value, mEq 02/kg 3.2 Anisidine Value 10.7 Totox Value 18.5 Unsaponifiable Matter, % 2.1 Astaxanthin Content, ppm 20 Triglyceride Content, % 95.7 Fatty acid composition, %: Eicosapentaenoic acid 14.8 Docosapentaenoic acid 2.8 Docosahexaenoic acid 8.9 Total Omega3 29.5 The oil also has high resistance to oxidation during storage and using the Oxidative Stability Index instrument a value of 45 h was obtained. This is because the endogenous antioxidants such as astaxanthin have bee conserved by the process and also because of the presence of the exogenous antioxidants introduced in both the preserving/stabilising solutions.

Claims (36)

1. A process whereby fish, fish waste or fish material of any kind is mixed with a low- pH preserving/stabilising solution consisting of both aqueous-soluble and lipid- soluble antioxidants and a lipase inhibitor, comminuted to a pre- determined particle size, subjected to controlled autolytic proteolysis by endogenous proteolytic enzymes under specified and optimised conditions in a reaction solution, heated to denature the proteolytic enzymes and prepare the liquefied mass for phase separation and then immediately separated by centrifugation into two high-quality, stable products, oil containing omega-3 polyunsaturated fatty acids and protein.

2. A process according to claim I wherein the fish material is whole or damaged fish of any edible or industrial' pelagic, demersal, freshwater, or shellfish species harvested from the wild or produced by aquacultute, or any waste material resulting from processing any marine or freshwater species, including viscera, heads, tails, off-cuts and all other waste products and offal.

3. A process according to claim 1 wherein the ratio of the stabilising/protecting solution to fish mass used is between 0.25:1 and 2:1.

4. A process according to claim I wherein the pH of the stabilising/protecting solution is set at between 3 to 4.5 by the incorporation of a mineral acid or lower monocarboxylic acid.

5. A process according to claim 4 wherein the pH is in the range 3.3 to 4. 0.

6. A process according to claim 4 wherein the concentration of acid used in the preserving/stabilising solution is between 1-6%.

7. A process according to claim 4 wherein the aqueous-soluble and lipid soluble antioxidants used in the stabilising/preserving solution comprise a synergistic combination of synthetic or natural agents exhibiting a range of established or putative antioxidant mechanisms including oxygen scavenging, metal ion sequestering and free radical quenching.

8. A process according to claim 4 wherein a lipase inhibitor such as green tea catechin fiavanols are used singly or in combination in the preserving/stabilising solution to protect the structural integrity of the triglycerides present and to prevent the generation of free fatty acids.

9. A process according to claim 4 wherein the stabilising/preserving solution is added to fresh, frozen, or thawing tissue in a whole or comminuted state.

10. A process according to claim 1 wherein the fish mass is chopped and macerated so that it is comminuted to a particle size less than 2 cm, more preferably less than I cm and most preferably less than 2 mm.

11. A process according to claim 1 where the viscous comminuted fish slurry is subjected to controlled autolytic proteolysis in a reaction solution.

12. A process according to claim 11 wherein the viscous slurry produced is diluted with fresh water in the ratio of 1 part of fish mass slurry to between 0.25 to 2.0 parts of water so that the correct protein to substrate ratio is obtained.

13. A process according to claim 11 wherein the pH of the reaction solution is set at between 3 to 4.5 by the incorporation of a mineral acid or lower monocarboxylic acid.

14. A process according to claim 11 wherein the pH is in the range 3.3 to 4.0.

15. A process according to claim 11 wherein the concentration of acid used in the preserving/stabilising solution is between 1-6%.

16. A process according to claim 11 wherein the aqueous-soluble and lipid soluble antioxidants used in the stabilising/preserving solution comprise a synergistic combination of synthetic or natural agents exhibiting a range of established or putative antioxidant mechanisms including oxygen scavenging, metal ion sequestering and free radical quenching.

17. A process according to claim 11 wherein the reaction is maintained at a selected temperature within the range 25-50 C.

18. A process according to claim 11 wherein the reaction is allowed to proceed for a time of between 0.5 and 8 hours to allow tissue breakdown and liquefaction to occur.

19. A process according to claim 1 wherein the reaction mixture is heated to terminate enzyme activity.

20. A process according to claim I wherein the reaction mixture is heated to prepare the material for phase separation.

21. A process according to claim 1 wherein the temperature of the reaction mixture is elevated to a selected temperature within the range 60-90 C.

22. A process according to claim 1 wherein the aqueous protein phase and the oil phase are rapidly and quantitatively separated.

23. A process according to claims 1-22 which produces high quality fish oil and protein products.

24. All products and their derivatives produced by the process according to claims 1-22.

25. All products and their derivatives produced by the process according to claims 1-22 presented as liquids, powders, emulsions, suspensions, pellets, pills, tablets, capsules, parenterals, enterals, creams, lotions and any other delivery form.

26. Products produced by the process according to claims 1-22 used in the human or animal nutrition products of any kind.

27. Products produced by the process according to claims 1-22 used in human or animal food products.

28. Products produced by the process according to claims 1-22 used in feeds for aquaculture and in commercial preparations for use in aquaria and ponds.

29. Products produced by the process according to claims 1-22 used in baits or attractants used in fishing.

30. Products produced by the process according to claims 1-22 used in human or animal nutritional supplements.

31. Products produced by the process according to claims 1-22 used in human or animal specialist dietetic food products.

32. Products produced by the process according to claims 1-22 used in human or animal functional food products.

33. Products produced by the process according to claims 1-22 used in human or animal nutriceutical products.

34. Products produced by the process according to claims 1-22 used in human or animal pharmaceutical products.

35. Products produced by the process according to claims 1-22 used in medicated products.

36. Products produced by the process according to claims 1-22 used in skincare preparations.