WO2019211408A1 - Procédé enzymatique - Google Patents

Procédé enzymatique Download PDF

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Publication number
WO2019211408A1
WO2019211408A1 PCT/EP2019/061311 EP2019061311W WO2019211408A1 WO 2019211408 A1 WO2019211408 A1 WO 2019211408A1 EP 2019061311 W EP2019061311 W EP 2019061311W WO 2019211408 A1 WO2019211408 A1 WO 2019211408A1
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Prior art keywords
tma
oxidase
aquatic biological
product
biological material
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PCT/EP2019/061311
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English (en)
Inventor
Kjartan Sandnes
Stuart West
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Biomega Group AS
Biocatalysts Limited
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Priority to EP19723725.8A priority Critical patent/EP3788160A1/fr
Publication of WO2019211408A1 publication Critical patent/WO2019211408A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds

Definitions

  • the present invention concerns methods of modulating the fishy odour and/or taste of aquatic biological materials.
  • the seafood industry processes a large proportion of its catch, generating material comprising, for example, heads, tails, shells, scales, bones, and/or skin.
  • a large proportion of such material has traditionally been considered to be waste, as has so-called‘by-catch’.
  • such material can be processed into seafood products, such as fish oils, fishmeal and/or seafood hydrolysates.
  • Hydrolysates can represent a nutritious protein source for human or animal consumption and certain hydrolysates have been reported to have beneficial properties.
  • a key problem associated with aquatic biological materials, particularly marine biological materials, is that the odour and/or taste is typically“fishy” which can affect the attractiveness of such products to consumers.
  • autolysis After death of an aquatic organism, organic molecules are broken down into smaller molecules through the process of autolytic hydrolysis (autolysis). While autolysis proceeds, microbial decomposition begins. Microorganisms present on the external surfaces of aquatic animals are easily transferred to the flesh, for example after gutting. Such microorganisms can degrade the organism and induce the development of an unpleasant,“fishy” odour.
  • Trimethylamine is a colourless tertiary amine with a strong‘fishy’ odour. TMA, and its breakdown products dimethylamine and (mono)methylamine, contribute significantly to any fishy odour of aquatic biological material such as seafood material.
  • TMA can arise, for example, from the reduction of trimethylamine-N-oxide (TMAO) to TMA, and/or from the metabolism of other compounds, particularly choline.
  • TMAO trimethylamine-N-oxide
  • TMAO is abundant in the tissues of a variety of aquatic species, particularly in saltwater species such as saltwater fish.
  • TMAO is a common and compatible osmolyte in muscle tissues of marine organisms that is often credited with counteracting protein-destabilizing forces. TMAO is therefore typically highest in saltwater fish and gets less concentrated in fish living in water with lower salt contents.
  • TMAO is also commonly found in a variety of marine microorganisms, where it can serve as an important substrate in anaerobic metabolism.
  • Certain microorganisms such as the bacterium Shewanella putrefaciens, convert TMAO into trimethylamine (TMA).
  • Certain microorganisms are also capable of generating TMA from the metabolism of other compounds, for example choline.
  • the inventors have established that a trimethylamine oxidase may be used to modulate the fishy odour of aquatic biological material.
  • the present invention provides a method of modulating the odour of an aquatic biological material, the method comprising the use of a trimethylamine oxidase.
  • the fishy odour of an aquatic biological material may be reduced using such a method.
  • a method of modulating the taste of an aquatic biological material comprising the use of a trimethylamine oxidase.
  • the fishy taste of an aquatic biological material may be reduced using such a method.
  • TMA trimethylamine
  • TMA is a methylated amine which may be /V-demethylated into dimethylamine (DMA) and formaldehyde, e.g. by a trimethylamine dehydrogenase.
  • DMA may be /V-demethylated into methylamine (MA) and formaldehyde, e.g. by a DMA dehydrogenase; and MA may be /V-demethylated into ammonia and formaldehyde (FA), e.g. by an MA dehydrogenase.
  • TMA breakdown products DMA, MA, ammonia and FA may thus all be considered to be“TMA breakdown products”. Without wishing to be bound by theory, it is believed that reducing the level of TMA may reduce the amount of TMA available for the production of such TMA breakdown products.
  • TMA and/or one or more TMA breakdown products which may be selected from DMA, MA, ammonia and/or FA, of an aquatic biological material, the method comprising the use of a trimethylamine oxidase.
  • a method of modulating the odour and/or taste of an aquatic biological material through modulating the levels of TMA and/or one or more TMA breakdown products comprising the use of a trimethylamine oxidase.
  • TMA oxidase By“use of a TMA oxidase” is meant or includes contacting an aquatic biological material with a TMA oxidase.
  • the step of "contacting" the aquatic biological material with a TMA oxidase means or includes that a TMA oxidase is added to, or mixed with, or otherwise brought into contact with, the aquatic biological material.
  • the method of the invention generally involves a step of adding a TMA oxidase to the aquatic biological material.
  • the TMA oxidase may therefore be referred to as "exogenous" in that it did not form part of the aquatic biological material, although it is not excluded that the aquatic biological material contains small amounts of any endogenous TMA oxidases.
  • addition of a TMA oxidase to the material, or contact of the aquatic biological material with a TMA oxidase includes an incubation step for a suitable period of time and under suitable conditions, e.g. at a suitable temperature and/or pH, for example under conditions such that TMA levels, and/or odour and/or taste in the aquatic biological material are modulated (e.g. reduced).
  • a suitable incubation period is preferably at least 10, 15, 20, 25, 30, 40, or 50 minutes or at least 1 , 2, 3, 4, or 5 hours.
  • the incubation period is preferably no longer than 48, 40, 35, 30, 24, 20, 15, 10 or 6 hours. It may be, e.g. 0.5 to 5, 4, 3, 2, or 1 hours, e.g. about 1-2 hours.
  • a suitable incubation temperature will depend at least in part on the optimum temperature of the TMA oxidase used, so it may, e.g. be from 1-50°C. It may, e.g., be at least 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30°C and/or no more than 70, 60, 55, 50, 45, 44 or 35°C. Depending at least in part on the temperature optimum of the TMA oxidase, it may, e.g., be around 30-45°C, 30-37°C, 20-35°C, or 4-15°C.
  • the incubation temperature for the TMA oxidase of SEQ ID NO: 1 is preferably at or at least 20°C or 25°C, more preferably at or at least 30 or 35°C, most preferably at or around 40°C, for example at or around 37°C, or at or around 45°C, or between 30°C and 45°C or 30°C and 50°C, and generally should not be above 50°C or 55°C.
  • the method may involve adjusting the temperature to be as set out above.
  • a suitable pH will depend at least in part on the optimum pH of the TMA oxidase used, so it may, e.g. be from 3-1 1 , preferably 4-10, 4-8, 5-9, or 5.5-8.5, e.g. 6.5-8.5, 6-7, 6.5-8, 6.5-7.5, 7-7.5, 6.5-8.5, 7-8, or about 6, 7, or 8.
  • the incubation pH for the TMA oxidase of SEQ ID NO: 1 can be from 6.0 to 8.0 or 8.5 and is preferably at or about 8.5, 8.0, 7.5, 7.0, 6.5, or 6.0.
  • the method may involve adjusting the pH to be as set out above, e.g. using a suitable buffer.
  • Suitable amounts or concentrations of TMA oxidases to be used in the methods of the present invention can readily be determined by a person skilled in the art, for example so that the desired modulation of TMA levels, odour and/or taste of an aquatic biological material is achieved.
  • the amount or concentration will generally vary depending on the activity of the TMA oxidase used and the other conditions of the assay (for example temperature, pH, etc).
  • Exemplary concentrations of TMA oxidases might be 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w of enzyme, or concentrations of less than 2%, for example as low as 0.002% and up to 1.73%, for example at or around 0.04%, 0.26%, 0.44% 0.88% or 1.73% can be used.
  • levels of enzyme such as up to or about 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg or 100 mg of enzyme/kg raw material, e.g. raw amount of aquatic organisms, e.g. per kg of fish, can be used. Exemplary and preferred levels can be found in the Examples.
  • TMA oxidases are NADPH dependent, so a suitable level of NADPH should be present during the incubation step.
  • the NADPH may be endogenous and/or exogenous.
  • the method may involve adding sufficient NADPH, e.g. exogenous NADPH, to the material.
  • exogenous NADPH levels are often sufficient, for example when hydrolysates are used, as shown in the experimental Examples. Thus, in preferred embodiments, it is not required to add any exogenous NADPH.
  • TMA oxidases use a flavin-containing prosthetic group for activity, typically flavin adenine dinucleotide (FAD), which is typically tightly bound to the enzyme, forming a complex.
  • FAD flavin adenine dinucleotide
  • the method may involve adding FAD, e.g. exogenous FAD, to a TMA oxidase known or suspected not to contain sufficient FAD.
  • Addition of FAD may be carried out prior to, simultaneously with and/or after contacting the TMA oxidase with the aquatic biological material, preferably prior to.
  • the addition of FAD can act to increase the activity of the TMA oxidase enzyme, so this can be a preferred step in some methods, especially where an increase in TMA oxidase activity is required or desired, for example because the natural oxidase activity of the enzyme is low, or because low concentrations of TMA oxidase are present.
  • FAD FAD
  • a TMA oxidase reaction as described herein has been shown to increase the rate of reaction, for example by increasing enzyme activity e.g. by around 4 to 10 fold.
  • the experimental Examples herein show that the addition of FAD is not necessary for methods of the invention to be carried out.
  • no exogenous FAD is added.
  • Also provided is a method of processing an aquatic biological material e.g. a method of making or refining an aquatic biological product, said method comprising the use of a trimethylamine oxidase.
  • an aquatic biological material e.g. a refined aquatic biological product, having a desired TMA level, a desired odour and/or a desired taste.
  • an aquatic biological material e.g. a refined aquatic biological product, having a TMA level, e.g. a desired TMA level, a desired odour, and/or a desired taste.
  • a TMA level e.g. a desired TMA level, a desired odour, and/or a desired taste.
  • This may, e.g., be obtainable or obtained via a method provided herein.
  • an aquatic biological material e.g. a refined aquatic biological product, obtainable or obtained via a method provided herein.
  • Preferred products thus have a reduced TMA level, a reduced fishy odour, and/or a reduced fishy taste, for example when or if compared to an untreated product (a product not treated with TMA oxidase) or a product which has not been subjected to the methods of the invention.
  • Preferably said products have a weakly or non-fishy odour and/or taste, or a neutral odour and/or taste.
  • Exemplary levels of TMA in such products are described elsewhere herein.
  • Such products may thus further comprise a TMA oxidase (e.g.
  • an active TMA oxidase as described herein, for example any TMA oxidase suitable for use in the methods of the invention, for example a TMA oxidase of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, or 17, or a variant thereof, as defined elsewhere herein.
  • hydrolysates as described elsewhere herein.
  • Such hydrolysates (or other products) preferably contain at least 60% dry matter.
  • Such hydrolysates (or other products) optionally contain other suitable components such as stabilisers and/or preservatives, examples of which are described elsewhere herein.
  • Stabilisers and/or preservatives are particularly useful when hydrolysates (or other products) have less than 60% dry matter. Such products may also be subjected to filtration, e.g.
  • Such products may take any suitable form.
  • preferred products are dried products (products with water removed), e.g. freeze dried or spray dried products, or powders, which can conveniently be stored and can then for example be reconstituted in liquid form, e.g using an aqueous solvent such as water.
  • such products may be liquid products.
  • composition comprising such a product, which may, e.g. be a food or food ingredient, animal feed or feed ingredient, supplement, or nutraceutical.
  • a product which may, e.g. be a food or food ingredient, animal feed or feed ingredient, supplement, or nutraceutical.
  • nutraceutical denotes a product that may provide health benefits in addition to nutritional benefits.
  • supplier includes protein powders for sports nutrition.
  • modulating the odour is meant that the strength and/or quality of the odour is changed. In particular, this may include decreasing the strength of the odour and/or improving the quality of the odour.
  • the modulation is preferably a reduction, so the provided methods are preferably methods of reducing the fishy odour.
  • Aquatic biological products with a weak and/or higher quality odour e.g. a reduced fishy odour, or a weak, or non-fishy, or neutral odour, are typically preferred by consumers, so the method may be considered to be a method of improving the odour of an aquatic biological product.
  • modulating the taste is meant that the strength and/or quality of the taste is changed. In particular, this may include reducing the strength of the taste and/or improving the quality of the taste.
  • the modulation is preferably a reduction of the fishy taste.
  • Aquatic biological products with a reduced fishy taste, or a weak, or non-fishy, or neutral taste, are typically preferred by consumers, so the method may be considered to be a method of improving the taste of an aquatic biological product.
  • the modulation of odour and/or taste may be assessed, e.g., through organoleptic evaluation, which may be carried out by an individual assessor or a panel of assessors.
  • the assessor may optionally be trained in organoleptic evaluation.
  • Organoleptic evaluation may involve sniffing (or smelling) and/or tasting, as appropriate, one or more test materials and optionally comparing the odour and/or taste to one or more control materials.
  • the evaluation may be blind (where the assessor does not know the identity of the test materials) or double-blind (where any personnel supervising the evaluation do not know the identity of the test materials).
  • the organoleptic evaluation may, e.g., be a smell test to evaluate whether and/or to what extent a material has a“fishy” odour, for example such tests may confirm that a material has a fishy, or non-fishy, or neutral odour.
  • a suitable test is described in Example 1.
  • Organoleptic evaluation may involve the scoring of the odour and/or taste on a simple binary scale (e.g. fishy versus non-fishy, or moderately/highly fishy versus mildly/non- fishy), or on a more detailed scale.
  • a suitable scale may, for example be 1-2: intensely (or strong) fishy smell/taste respectively; 3: moderately fishy smell/taste respectively; 4-5:
  • the scoring may combine odour and taste, or one or both of these may be scored independently. With regard to the scoring of odour, the scoring may combine strength and quality of odour, as set out above, or these features may be scored individually.
  • organoleptic evaluation may involve the scoring of the strength of the odour on a simple binary scale or on a more detailed scale.
  • a suitable scale may, for example be 1-2: strong odour, 3: moderate odour, 4-5: weak or non-detectable odour, although more detailed scales may be used, if desired.
  • organoleptic evaluation may involve the scoring of the quality of the odour on a simple binary scale or on a more detailed scale.
  • a suitable scale may, for example be 1-2: poor quality odour, 3: acceptable quality odour, 4-5: good quality odour, although more detailed scales may be used, if desired.
  • odour is meant an odour that is not an intensely or strong fishy smell, preferably an odour that is at most moderately fishy, more preferably an odour that is at most weakly fishy, most preferably an odour that is non-fishy or neutral.
  • the odour may be neutral, or predominantly non-fishy.
  • the desired odour may be a“reduced fishy odour”,“weakly fishy” odour or“non-fishy” odour or“neutral” odour. Appropriate tests would be well known in the art.
  • the desired odour may be assessed via organoleptic evaluation as set out above.
  • “desired” taste is meant a taste that is not an intensely or strong fishy taste, preferably a taste that is at most moderately fishy, more preferably a taste that is at most weakly fishy, most preferably a taste that is non-fishy or neutral.
  • the taste may be neutral, or predominantly non-fishy.
  • the desired taste may be a“reduced fishy taste”, “weakly fishy” taste or“non-fishy” taste or“neutral” taste. Appropriate tests would be well known in the art.
  • desired taste may be assessed via organoleptic evaluation as set out above.
  • “Reducing” or“reduced” fishy odour and/or taste means that the intensity of the fishy odour and/or taste is weakened or even eliminated. Thus, the fishy odour and/or taste may be improved or even eliminated. “Reducing” or“reduced” fishy odour and/or taste may be assessed via organoleptic evaluation as set out above.“Reduced”,“reducing” and the like may be assessed by comparison to one or more appropriate reference(s) and/or one or more appropriate control(s).
  • an appropriate reference or control may, e.g. be a material that has not (yet) been treated with a TMA oxidase, or which has been treated under different conditions.
  • a control may be a material known to have an intense, moderate or weak fishy odour and/or taste, as appropriate, or a material known not to have a fishy odour and/or taste.
  • a "reduction" in the fishy odour and/or taste or a “reduced” fishy odour and/or taste includes any measurable decrease of the fishy odour and/or taste when the fishy odour and/or taste is compared with a control or reference.
  • a material may, e.g., be considered to be“weakly fishy or non-fishy” if in an organoleptic evaluation at least 30%, preferably at least 40, 50, 55, 60, or 65%, most preferably at least 70, 75, or 80% of assessors rate the material to be“weakly fishy or non- fishy” with regard to odour and/or taste.
  • The“modulation” or“modulating” of the TMA level is preferably the reduction of the TMA level, e.g. reduction to a desired level.
  • By“desired TMA level” is meant that the concentration of TMA is reduced or low or non-detectable (e.g. ⁇ 1 mg/100g or ⁇ 1 mg N/100g TMA).
  • a "reduction" in the TMA level or a “reduced” TMA level includes any measurable decrease of the TMA level when the TMA level is compared with an appropriate control or reference.
  • a reference may, e.g., be a material that has not (yet) been treated with a TMA oxidase, or which has been treated under different conditions.
  • a control may be a material known or suspected to have a high, moderate or low TMA level, as appropriate, or a material known to contain a specific TMA level or known not to contain any TMA.
  • the control TMA level may, e.g., be a discrete figure or a range.
  • TMA levels are“reduced” or“low” can be expressed in relative or absolute terms.
  • the TMA level is reduced or low when it is below a predetermined threshold, i.e. a cut-off, when compared to the control or reference.
  • a material is considered to have a reduced or low TMA level if it falls below the cut-off.
  • a reduced TMA level may be at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0,
  • cut-off for "reduced" level can be defined as least 1.2, 1.4, 1.6,
  • a reduced or low TMA level compared to a relevant control or reference level can be assessed as a percentage, e.g. as a percentage of the control or reference level or value.
  • a reduced or low TMA level may be a reduction of at least 20%, 25%, 30%, 35% or 40%, compared to a control or reference level or value.
  • Appropriate control or reference levels are described elsewhere herein, for example can be the level of TMA in a corresponding sample which is untreated with TMA oxidase.
  • a reduced or low TMA level is preferably below 50, 40, 30, 20, 15, or 10 ppm, more preferably below 9, 8, 7, 6, 5, or 4 ppm, even more preferably below 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 ppm.
  • a reduced or low TMA level is preferably a TMA level of less than 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg N/100g.
  • a reduced or low TMA level is preferably a TMA level of less than 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg/100g.
  • the combined level of TMA and DMA, or TMA, DMA, and MA, and optionally FA is below 50, 40, 30, 20, 15, or 10 ppm, more preferably below 9, 8, 7, 6, 5, or 4 ppm, even more preferably below 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ppm.
  • a reduced or low combined level of TMA and DMA, or TMA, DMA, and MA, and optionally FA is preferably a level of less than 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg N/100g.
  • a reduced or low combined level of TMA and DMA, or TMA, DMA, and MA, and optionally FA is preferably a level of less than 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg/100g.
  • TMA and/or one or more TMA breakdown products can be used. Preferred methods are as described in the experimental Examples, and include Conway and Byrne's micro-diffusion method (Conway, E. I., and A. Byrne (1933), Biochem. J. 27:419-429) and adaptations thereof.
  • TMA and/or one or more TMA breakdown products may be assessed qualitatively and/or quantitatively.
  • Qualitative assessment may be carried out via
  • Quantitative assessment may be carried out, for example, via photospectrometric methods and/or gas chromatography.
  • photospectrometric methods for example, via photospectrometric methods and/or gas chromatography.
  • photospectrometry may be used to analyse NADP+ levels in a sample, which provides a measure of how much NADPH has been converted into NADP+, which has a direct correlation with the level of TMA that has been converted into TMAO (see the reaction set out below).
  • TMA trichloroacetic acid
  • a perchloric acid extract of biomass is extracted with monoethylamine in combination with tert. -butyl methyl ether, or alternatively with n- propylamine in combination with isooctane.
  • the amines can then be separated by gas chromatography, using either tert.-butyl methyl ether or isooctane as solvent.
  • a micro-diffusion method of determining the concentration of ammonia in a fluid is disclosed in Conway and Byrne, 1933, Biochemical Journal, 27, 2: 419, incorporated by reference herein in its entirety.
  • the method can be adapted, e.g. to determine the total concentration of volatile nitrogen (TVN) in a sample.
  • TVN volatile nitrogen
  • TMA oxidases are flavoprotein enzymes capable of converting TMA into A/,A/,A/-trimethylamine A/-oxide (TMAO).
  • the human TMA oxidase is also known as flavin-containing monooxygenase (FM03).
  • FM03 flavin-containing monooxygenase
  • TMA oxidases from other species, including bacteria, are also referred to as FM03 enzymes.
  • TMA oxidases catalyse the following reaction:
  • TMA oxidase activity may be assayed by assessing changes in TMA levels, e.g. via any of the qualitative and/or quantitative assessments described above. Changes in TMAO levels may also be assessed, for example increased TMAO levels can be indicative of TMA oxidase activity.
  • TMA oxidases have many important physiological functions in eukaryotes. For example, inhibition of FM03 expression in mice using antisense oligonucleotides has been shown to attenuate atherosclerosis, while human individuals lacking functional FM03 exhibit trimethylaminuria with the TMA causing a fishy odour in the urine and the breath.
  • trimethylamine oxidases are known in the art.
  • a trimethylamine oxidase is disclosed in Choi et al. (A novel flavin-containing monooxygenase from
  • Any suitable trimethylamine oxidase may be used in any of the methods provided herein, e.g. a TMA oxidase having an amino acid sequence of SEQ ID NO: 1 or an active fragment thereof, or a variant thereof having at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity SEQ ID NO: 1. Any variant sequences should retain TMA oxidase activity, in particular the ability to convert TMA to TMAO.
  • Any variant sequences preferably have at least 50%, at least 60% or at least 70%, most preferably at least 80, 85, 90, 95, 98, or 99%, or 100%, of the ability of the TMA oxidase of SEQ ID NO: 1 to convert TMA into TMAO.
  • TMA oxidases/FM03 enzymes have sequences in the enzyme class grouping EC 1.14.13.148 and thus any of these sequences can also be used.
  • the present inventors have identified a number of novel TMA oxidase enzymes for use in the present invention. These enzymes are discussed below and any of these can be used in the methods of the present invention. These enzymes and enzyme sequences (at both the amino acid and nucleic acid level, including expression vectors comprising such nucleic acids and host cells expressing such nucleic acids) form yet further aspects of the invention.
  • TMA oxidase enzymes or polypeptides of, or for use in, the present invention are generally isolated or recombinant enzymes or polypeptides.
  • the present invention provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:3 or an active fragment thereof, or an amino acid sequence (variant sequence) having at least 66, 67, 68, 69, 70, 75, 80, 85, 90, 91 , 92, 93,
  • Preferred such polypeptides comprise an amino acid sequence of SEQ ID NO:3, 7,
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:7 or an active fragment thereof, or a sequence (variant sequence) having at least 95, 96, 97, 98, or 99% identity thereto.
  • a polypeptide e.g. an isolated polypeptide
  • TMA oxidase enzyme trimethylamine oxidase activity
  • Preferred such polypeptides comprise an amino acid sequence of SEQ ID NO:7, 9, or 11 , or an active fragment thereof.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:9 or an active fragment thereof, or a sequence (variant sequence) having at least 95, 96, 97, 98, or 99% identity thereto.
  • a polypeptide e.g. an isolated polypeptide
  • TMA oxidase enzyme trimethylamine oxidase activity
  • Preferred such polypeptides comprise an amino acid sequence of SEQ ID NO:7, 9, or 11 , or an active fragment thereof.
  • the present invention provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO:3 or an active fragment thereof, or a sequence (variant sequence) having at least 66, 67, 68, 69, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO:3 is derived from Pelagibacterales bacteria.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO:5 or an active fragment thereof, or a sequence (variant sequence) having at least 77, 78, 79, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO:5 is derived from alpha-proteobacterium HIMB59.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO:7 or an active fragment thereof, or a sequence (variant sequence) having at least 94, 95, 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO:7 is derived from Candidatus pelagibacter ubique.
  • the present invention further provides a polypeptide (e.g.
  • SEQ ID NO:9 is derived from Candidatus pelagibacter ubique.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO:1 1 or an active fragment thereof, or a sequence (variant sequence) having at least 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO:1 1 is derived from Candidatus pelagibacter ubique.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO: 13 or an active fragment thereof, or a sequence (variant sequence) having at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO: 13 is derived from Candidatus pelagibacter ubique.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO: 15 or an active fragment thereof, or a sequence (variant sequence) having at least 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • a polypeptide e.g. an isolated polypeptide
  • TMA oxidase enzyme trimethylamine oxidase activity
  • SEQ ID NO:15 is derived from Candidatus pelagibacter TMED239.
  • the present invention further provides a polypeptide (e.g. an isolated polypeptide) with trimethylamine oxidase activity (a TMA oxidase enzyme) said polypeptide comprising an amino acid sequence of SEQ ID NO: 17 or an active fragment thereof, or a sequence (variant sequence) having at least 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. It is believed that SEQ ID NO: 17 is derived from Soonwoa buanensis.
  • a yet further aspect provides a composition
  • a composition comprising: i) a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), and ii) an aquatic biological material.
  • TMA oxidase enzyme or polypeptide may be used as described elsewhere herein, for example a TMA oxidase comprising an amino acid sequence of SEQ ID NO:1 , 3, 5, 7, 9, 1 1 , 13, 15, or 17, or an active fragment thereof, or a sequence having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • a yet further aspect of the invention provides a composition comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:7 or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and
  • composition comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:9 or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and
  • a yet further aspect provides a composition comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:1 1 or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and
  • a yet further aspect provides a composition comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:3, 5, 13, 15 or 17, or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and
  • Any aquatic biological material as defined elsewhere herein can be used in such compositions, for example a hydrolysate.
  • a yet further aspect of the invention provides a dry product or composition, e.g. a dry powder product or composition, e.g. a freeze dried or spray dried product or composition, comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:7 or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • Preferred polypeptides for use in such dry products or compositions comprise an amino acid sequence of SEQ ID NO:3, 5, 7, 9, 11 , 13, or 15, or an active fragment thereof.
  • Alternative dry products or compositions e.g. dry powder products or compositions of the invention may comprise a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:3, 5, 7, 9, 1 1 , 13, 15, or 17, or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • TMA oxidase enzyme trimethylamine oxidase activity
  • variant sequence having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95,
  • a yet further aspect provides a liquid product or composition comprising:
  • a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:7 or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • Preferred polypeptides for use in such liquid products or compositions comprise an amino acid sequence of SEQ ID NO:3, 5, 7, 9, 11 , 13, or 15 or an active fragment thereof.
  • Alternative liquid products or compositions of the invention may comprise a polypeptide with trimethylamine oxidase activity (a TMA oxidase enzyme), said polypeptide comprising an amino acid sequence of SEQ ID NO:3, 5, 7, 9, 11 , 13, 15, or 17, or an active fragment thereof, or a sequence (variant sequence) having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • TMA oxidase enzyme trimethylamine oxidase activity
  • variant sequence having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%
  • Such liquid products or compositions would conveniently be aqueous products or compositions, for example containing water.
  • the TMA oxidase polypeptides can conveniently be provided, for example dissolved or solubilised in aqueous compositions.
  • Preferably such liquid products or compositions would be stabilised liquid or aqueous compositions, for example containing stabilisers and/or preservatives, or other appropriate components to for example maintain the activity and functionality of the TMA oxidase enzyme.
  • Preferred stabilisers and/or preservatives include antioxidants, antimicrobial agents and chelating agents. More preferred stabilisers and/or preservatives include: alcohols, for example ethanol; polyols, for example polyvinyl alcohol, glycerol, sorbitol, mannitol and xylitol; cellulose and derivatives thereof, for example methyl cellulose, ethyl cellulose and hydroxypropyl cellulose; benzoates, for example sodium benzoate and potassium benzoate; parabens, for example methyl-paraben and propyl-paraben; and other compounds such as ascorbic acid, polyethylene glycol, sodium chloride, sodium bisulfite, sodium sulphite, sodium citrate, citric acid, EDTA, benzalkonium chloride, chlorobutanol, cysteine, methionine, phenol, vitamin A, vitamin C and vitamin E.
  • alcohols for example ethanol
  • polyols for example polyvinyl
  • Preferred stabilisers and/or preservatives also include potassium sorbate, sucrose, magnesium sulphate, ammonium sulphate, sodium chloride, calcium chloride, potassium phosphate, sodium phosphate, potassium chloride, sodium sulphate, zinc sulphate, corn starch, dextrose, sorbitol, lactose, mannitol, malt extract, maltose, maltodextrin, sucrose, trehalose and any combination of the above.
  • compositions of the invention may optionally comprise a mineral mixture, for example minerals originating from the raw material used as the aquatic biological material which is processed or treated with a trimethylamine oxidase in accordance with the present invention.
  • compositions or products for example dry products or compositions or liquid products or compositions such as described above, but comprising nucleic acid molecules encoding such TMA oxidase polypeptides, are also provided.
  • Any variant sequences or fragments of TMA oxidase enzymes as described herein should retain TMA oxidase activity, in particular the ability to convert TMA to TMAO.
  • Any variant sequences preferably have at least 50%, at least 60% or at least 70%, most preferably at least 80, 85, 90, 95, 98, or 99%, or 100%, of the ability of the relevant, e.g. parent or original, TMA oxidase to convert TMA into TMAO.
  • TMA oxidase isolated from or corresponding to a TMA oxidase enzyme isolated from Pelagibacterales bacteria, for example Candidatus pelagibacter ubique or Candidatus pelagibacter TMED239.
  • Alternative sources include alpha-proteobacterium HIMB59 or Soonwoa buanensis.
  • the invention also provides nucleic acid molecules encoding the TMA oxidases of and for use in the invention, and active fragments thereof.
  • Nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15 and 17 are disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, respectively (see Table 1 ), and the nucleic acids of or for use in the invention may comprise these nucleotide sequences, or variants thereof having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
  • Degeneracy of the genetic code means that each of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 are each only one of many possible nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15 and 17, respectively. Accordingly, the invention extends to nucleic acid molecules and uses of nucleic acid molecules comprising nucleotide sequences which are degenerate versions of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18.
  • the nucleic acid molecules of or for use in the invention may be nucleic acid vectors, e.g. cloning vectors or expression vectors.
  • Preferred vectors are plasmids compatible with bacterial and/or yeast cells.
  • the TMA oxidases of the invention may be isolated from a natural source, e.g. isolated from extracts of the organisms described elsewhere herein, or produced recombinantly in a host cell and isolated and purified therefrom.
  • the TMA oxidases of the invention may therefore be recombinant enzymes, in particular isolated recombinant enzymes.
  • the TMA oxidase is produced by
  • a host cell that is not, or not from, an organism which is the same as that in which the TMA oxidase is found naturally, i.e. a heterologous host cell.
  • a cell-free expression system can be used for production of the TMA oxidase.
  • a method for the isolation and purification of a TMA oxidase or an active fragment thereof as described herein represents a further aspect of the present invention.
  • the invention provides such a method, said method comprising culturing cells in which the TMA oxidase is expressed and subsequently separating the TMA oxidase from said cells and/or the media in which said cells have been cultured.
  • the method comprises expressing said TMA oxidase in a suitable heterologous host cell (e.g. E. coli), and subsequently separating the TMA oxidase from said host cells and/or the media in which said cells have been cultured.
  • TMA oxidase can be achieved by incorporating into a suitable host cell an expression vector encoding said TMA oxidase, e.g. an expression vector comprising a nucleic acid molecule encoding any of the amino acid sequences of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15 and 17, for instance a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, respectively (see Table 1 ).
  • Host cells comprising these expression vectors and nucleic acid molecules are encompassed by the invention.
  • the TMA oxidase enzyme may be separated, or isolated, from the host cells/culture media using any of the purification techniques for protein known in the art and widely described in the literature or any combination thereof.
  • Homology may be assessed by any convenient method. However, for determining the degree of homology (e.g. identity) between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994).
  • the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1 , so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment.
  • BLOSUM 62 scoring matrix Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992
  • gap opening penalty 10
  • gap extension penalty 10
  • Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl.
  • sequences according to the present invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, adjoin, France).
  • Variants of the abovementioned SEQ ID NO:s include amino acid sequences in which one or more amino acids of said SEQ ID NO:s have undergone conservative substitution.
  • substitutions are silent substitutions and modifications in that the modified forms of the TMA oxidases of, or for use in, the invention have the same or essentially the same enzymatic characteristics as the unmodified forms.
  • a “conservative amino acid substitution”, as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g. lysine, arginine, histidine
  • acidic side chains e.g. aspartic
  • the "aquatic biological material” may include any aquatic organisms, including animals, plants and/or microbes, such as fish, shellfish, echinoderms, algae and/or bacteria. It may be a seafood material, i.e. comprise or consist of fish and/or shellfish material.
  • the aquatic biological material may be saltwater, estuarine and/or freshwater material. Saltwater biological material is preferred, e.g. a marine biological material, which may, e.g., comprise or consist of pelagic and/or mesopelagic biological material. It may be a marine seafood material.
  • Shellfish includes crustaceans and molluscs.
  • Crustaceans include crab, lobster, crayfish, krill, shrimp and prawns.
  • Preferred fish are marine or saltwater fish, such as Anchovy, Carp, Catfish, Cod, Eel, Haddock, Halibut, Herring, Mackerel, Salmon, Eel, Whiting, Sardine, Scad, Snapper, Tilapia, Trout, Tuna, and/or Lantern fish, preferably salmon.
  • the aquatic biological material may comprise or consist of whole aquatic biological organisms, such as fish, shellfish and/or algae. Alternatively and/or in addition it may comprise or consist of one or more parts of such aquatic biological organisms.
  • the parts may comprise or consist of, e.g., flesh, heads, tails, shells, scales, bones, viscera, and/or skin from fish and/or shellfish.
  • the aquatic biological material may comprise or consist of marine organisms that are considered to be by-catch and/or one or more seafood parts that are typically considered to be waste material, for example, heads, tails, shells, scales, bones, viscera, and/or skin from fish and/or shellfish.
  • Fish frames, particularly salmon frames, are especially preferred.
  • the aquatic biological material may comprise or consist of one or more seafood parts that are typically considered to be particularly suited for human consumption without further processing, e.g. fish fillet or edible seaweed.
  • the aquatic biological material may be processed, e.g. subjected to one or more processing steps.
  • the methods of the present invention may thus involve or comprise one or more processing steps. Alternatively viewed, such methods can be referred to as methods of processing an aquatic biological material. Appropriate processing steps would be well known to a person skilled in the art.
  • Refining steps may generate, concentrate and/or purify the component(s) of interest, which may, e.g. be selected from (i) proteins, polypeptides, oligopeptides and/or amino acids; and/or (ii) lipids, e.g. oils.
  • the method of processing an aquatic biological material may therefore comprise one or more refining steps, in which case it may be referred to as a method of refining an aquatic biological material.
  • the processing and/or refining steps may include mechanical, chemical, enzymatic and/or heat treatment steps.
  • the refining steps may, e.g., include hydrolysis, centrifugation, filtration, and/or chromatography.
  • Chromatography may, e.g., include ion- exchange chromatography and/or high performance liquid chromatography (HPLC), e.g. reverse-phase HPLC.
  • Filtration may, e.g., include gel filtration, Ultrafiltration (UF) and/or nanofiltration (NF).
  • the material may, e.g., be filtered through a filter having a specific pore size, yielding a permeate which should only contain molecules no larger than the pore size.
  • a pore size of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 kDa or less may be suitable, although sometimes filters with larger pore sizes can be used in the methods, e.g. filters with pore sizes up to 100 kDa, e.g. pore sizes of about or up to 15, 20, 30, 40, 50, or 100 kDa may be used.
  • Preferred methods may involve a step of nanofiltration, for example using filters with a pore size of about or up to 10 nm, e.g. 1 to 10 nm.
  • Preferred methods may involve a step of nanofiltration and the use of a hydrolysate as an aquatic biological material.
  • Preferred methods may involve a step of nanofiltration with a cutoff of at or around 200 Da, as provided in the Examples.
  • the method of processing/refining an aquatic biological material may, e.g., comprise one or more of the following steps:
  • the aquatic biological material may, e.g. be the starting material in an aquatic biological material processing method, e.g. it may be what has traditionally been considered to be“waste” material as discussed above. It may, e.g., be an intermediate in an aquatic biological material processing method, e.g. it may be an intermediate in a process of making or refining a product, such as a hydrolysate. It may, e.g., be the product of an aquatic biological material processing method, e.g. it may be a hydrolysate.
  • the product of an aquatic biological material processing method may be referred to as an“aquatic biological product” and where such a method included one or more refining steps, the product may be referred to as a“refined” product.
  • The“refined” product may, for example, comprise or consist of an oil, emulsion, fishmeal, silage, hydrolysate, or a product derived from any of these through further refinement, e.g. a composition predominantly comprising or predominantly consisting of oligopeptides, which are preferably oligopeptides of 2-5 amino acids.
  • oligopeptides which are preferably oligopeptides of 2-5 amino acids.
  • “predominantly” is meant at least 60%, preferably at least 70, 80, 85, or 90% (w/w of dry matter).
  • the aquatic biological material may, e.g., be a silage of an aquatic biological material.
  • Silage is a liquid product made from aquatic organisms that are liquefied by the action of enzymes in the presence of added acid.
  • the enzymes present in the acidic medium break down proteins into smaller soluble units while the acid helps to speed up their activity and prevent bacterial spoilage.
  • the method of processing an aquatic biological material may therefore include a step of making a silage.
  • the aquatic biological material may, e.g., be a fishmeal.
  • Fishmeal is typically made by heat treatment, pressing, drying, and grinding whole fish and/or fish parts.
  • the method of processing an aquatic biological material may therefore include a step of making a fishmeal.
  • the aquatic biological material may, e.g., be a hydrolysate of an aquatic biological material, e.g. a seafood hydrolysate, such as a salmon or cod hydrolysate.
  • Hydrolysates are typically made by hydrolysis, e.g. with enzymes, e.g. proteases.
  • the method of processing an aquatic biological material may therefore include a hydrolysis step, e.g. include or be a method of making a hydrolysate of an aquatic biological material, e.g. a seafood hydrolysate.
  • hydrolysate is meant the reaction product obtained subsequent to hydrolysis of organic components, e.g. protein components, of a starting aquatic biological material.
  • a peptide is a molecule that is formed by linking at least two amino acids via an amide bond, also called a peptide bond.
  • a peptide comprises or consists of at least 2 amino acids.
  • Peptides consisting of two amino acids are called dipeptides, peptides consisting of three amino acids tripeptides and so on.
  • Peptides consisting of between 2 and 20 amino acids may be referred to as oligopeptides, and peptides having less than about 50 amino acids are typically referred to as polypeptides.
  • protein is typically used to refer to large polypeptides or complexes of polypeptides.
  • the hydrolysate may therefore comprise or predominantly consist of proteins, polypeptides, peptides and/or free amino acids, with peptides and/or free amino acids being preferred.
  • the material may, e.g., contain substantially no polypeptides/proteins having a sequence of more than 50 contiguous amino acids.
  • hydrolysate may optionally also be present in the hydrolysate, or the hydrolysate may be substantially free of carbohydrates and/or lipids.
  • the step of contacting the aquatic biological material with a TMA oxidase may, e.g., be carried out on a refined aquatic biological product, or it may, e.g. be part of a method of refining an aquatic biological material.
  • the step of contacting the aquatic biological material with a TMA oxidase may, e.g. be carried out on an aquatic biological material/product that is sterile or at least substantially sterile and/or free or at least substantially free of active proteases, e.g. active endogenous or exogenous proteases.
  • the aquatic biological material/product that is sterile or at least substantially sterile and/or free or at least substantially free of active proteases is at least substantially free ofactive endogenous enzymes.
  • “endogenous” is used to refer to any enzymes that are naturally present in the aquatic biological material, i.e. have not been deliberately added.
  • the TMA oxidase may be considered to be“exogenous”, so it is not covered by the expression“substantially no endogenous enzymes”.
  • “at least substantially free of endogenous enzymes” is meant that substantially any endogenous enzymes, if present, are inactive, e.g. have been heat-inactivated.
  • the material may, e.g., contain substantially no polypeptides/proteins having a sequence of more than 50 contiguous amino acids.
  • the aquatic biological material may therefore be, or have been, subjected to one or more treatments to render the aquatic biological material at least substantially sterile and/or at least substantially free of active proteases, preferably any endogenous or exogenous enzymes, such as exogenous proteases used for hydrolysis.
  • a suitable treatment may, e.g., involve heating to a suitable temperature, e.g. at or at least 60, 65, 70, 75, 80, 85, 90, 95,
  • enzymes e.g. proteases, and/or microbes.
  • an 85°C to about 90°C temperature may be maintained for about 10 or 15 minutes to inactivate the target enzyme(s), e.g. protease(s).
  • Appropriate methods would be well known to a person skilled in the art.
  • a suitable treatment may, e.g., involve drying the aquatic biological material, e.g. via spray or freeze drying.
  • a suitable treatment may, e.g., involve chemical inactivation.
  • inactivating is meant rendering the target enzyme(s), e.g. protease(s), at least about 90% inactive, preferably at least about 95% inactive, and more preferably at least about 99% inactive.
  • one or more acids or bases may be added to the reaction mixture to adjust the pH so as to substantially inactivate the one or more target enzymes.
  • the target enzymes are inactivated when the reaction mixture has a pH of less than about 4 or greater than about 9.
  • the present invention provides a method of making an aquatic biological product, e.g. a refined aquatic biological product, having a desired TMA level, desired odour and/or desired taste, wherein said method comprises:
  • step (b) a method for modulating TMA levels, odour and/or taste of an aquatic biological material as described herein, wherein step (b) is carried out prior to, during and/or after step (a)(i) and/or step (a)(ii).
  • said aquatic biological material or product has a reduced TMA level, a reduced fishy odour, and/or a reduced fishy taste, more preferably a weakly or non-fishy odour and/or taste.
  • TMA levels e.g.
  • said aquatic biological material or product may have a TMA level of less than 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg N/100g.
  • said aquatic biological material or product may have a TMA level of less than 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg/100g.
  • said material or product may have a TMA level, or a combined level of TMA, dimethylamine (DMA) and/or methylamine (MA), of less than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ppm.
  • DMA dimethylamine
  • MA methylamine
  • a TMA oxidase may be used at any appropriate stage of any of the methods of processing, e.g. refining, an aquatic biological material disclosed herein.
  • the method of reducing the TMA levels, fishy odour and/or fishy taste may be incorporated into such a method of processing/refining an aquatic biological material at any appropriate stage.
  • the biological material may be contacted with a TMA oxidase before, during and/or after any of the processing/refining steps, e.g. any of the steps discussed herein.
  • the TMA oxidase may be contacted with the material prior to, during and/or after hydrolysis thereof.
  • TMA oxidase After one or more refining steps, e.g. after a hydrolysis step has been completed, is preferred. Contacting the material with a TMA oxidase after any step of inactivating enzymes, e.g. inactivating enzymes (e.g. exogenous enzymes) used for hydrolysis, e.g. after protease inactivation, see for example Figure 7, and/or substantially sterilizing the material, is also preferred.
  • inactivating enzymes e.g. exogenous enzymes
  • Hydrolysis is a process in which an organic molecule is split into two parts by the addition of a molecule of water. Hydrolysis reactions are typically chemically or enzymatically catalysed. Chemical hydrolysis may be acid-catalysed or base-catalysed (alkaline hydrolysis, e.g. using NaOH), whereas enzymatic hydrolysis is catalysed by an enzyme.
  • hydrolysis may be chemical and/or enzymatic, enzymatic hydrolysis being preferred.
  • Enzymatic hydrolysis of proteins is catalysed by a protease, which catalyses hydrolysis of peptide bonds in proteins/polypeptides, so enzymatic hydrolysis requires the presence of a protease.
  • Hydrolysis of a protein may also be referred to as "proteolysis”.
  • protease an enzyme which catalyses the hydrolytic breakdown of proteins/polypeptides into polypeptides, oligopeptides and/or free amino acids.
  • Proteases which may be used to prepare a hydrolysate may be any type of protease including endoproteases and exoproteases.
  • endoprotease refers to an enzyme that hydrolyses internal peptide bonds in oligopeptide or polypeptide chains. Suitable
  • endoproteases include Serine proteases, Threonine proteases, Cysteine proteases, Aspartate proteases, Metalloproteases and Glutamic acid proteases, Serine proteases being preferred.
  • suitable serine endoproteases include pepsin, trypsin, chymotrypsin and elastase.
  • the protease may, for example, be selected from subtilisin (Alcalase ® ), bacillolysin (Neutrase ® ), (each of which can be obtained from Novozymes of Denmark), Pescalase ® (Gist-brocades of the Netherlands) Promo 31 ® (Biocatalysts Ltd.
  • Promod P439L Biocatalysts Ltd. of Wales
  • mixtures thereof e.g. a mixture of subtilisin and bacillolysin (Protamex ® ).
  • Endopeptidase and exopeptidase mixtures may be used, e.g. Flavourzyme (Novozymes of Denmark).
  • Aquatic biological material hydrolysates are typically generated by the hydrolysis of the organic molecules of aquatic biological material.
  • the various components of the reaction mixture for example, the raw material comprising the molecules to be hydrolysed, water, and an appropriate enzyme
  • the various components of the reaction mixture are typically mixed together under appropriate reaction conditions.
  • proteins obtained from aquatic biological sources can be processed by hydrolysis (e.g. base-catalysed hydrolysis) to obtain a wide array of peptides all the way down to single amino acids - depending on the enzymes used and the processing conditions.
  • the method of processing an aquatic biological material may therefore include mixing an aquatic biological material with an appropriate hydrolytic enzyme (e.g. a protease) and optionally water and/or further components and incubating this mixture under appropriate reaction conditions for a suitable length of time.
  • an appropriate hydrolytic enzyme e.g. a protease
  • the process may include dilution of the appropriate aquatic biological material, such as aquatic biological material processing waste material, in water and addition of a hydrolytic enzyme (e.g. a protease), allowing enzymatic hydrolysis of the aquatic biological material at a temperature at which the hydrolytic enzyme has good activity, typically 20-65° C, for a suitable length of time, such as 1 or 2 hours. Base-catalysed hydrolysis can conveniently be used for this step.
  • the incubation may be followed by heat inactivation of the enzyme by heating, e.g. to around 80-90° C for an appropriate time period, e.g. 10-20 minutes.
  • any solids may then be separated from the aqueous phase, the aqueous phase containing the protein hydrolysate.
  • Fats which typically originate from cell membranes, may optionally be removed, for example by cooling the crude hydrolysate to cause fats to solidify, followed by removal of the solidified fats by
  • the method of processing an aquatic biological material may, e.g. include method steps and/or involve the use of an apparatus as disclosed in any one of WO2017/158188, WO 2016/041896 and/or WO 2004/049818, each incorporated by reference herein in its entirety. Exemplary and preferred methods of preparing hydrolysates are also described in the attached Examples.
  • a hydrolysis step which may comprise incubating the aquatic biological material with a protease (e.g. an exogenous protease); and subsequently (b) an inactivation step which may comprise inactivating the protease (e.g. by heating as described elsewhere herein, e.g. by heating at a high temperature, e.g. at around 90° C); and optionally (c) a separation step (e.g. by centrifugation) which may comprise separating the product of step (b) into at least one substantially liquid component and at least one substantially solid-containing component.
  • the aquatic biological material may be incubated with a TMA oxidase prior to, during and/or after any of these steps.
  • the TMA oxidase is used after step (b), e.g. after step (b) but before step (c).
  • Preferred methods also involve the steps of filtration, e.g. nanofiltration which can for example be used to remove unwanted components.
  • Preferred methods also involve a drying step, e.g. spray drying, to form a powder or other dry product.
  • hydrolysis area that provides hydrolysis of aquatic biological material by reacting a reaction mixture comprising said aquatic biological material and at least a protease, present in said area, wherein the reaction mixture contains both solids and liquid, and wherein upon hydrolysis, said reaction mixture further comprises hydrolysis product;
  • an inactivation area that receives reaction mixture from the hydrolysis area and substantially inactivates the protease present in the reaction mixture
  • a separation area that receives at least a portion of the reaction mixture from the inactivation area and is capable of separating it into two or more components, including at least one substantially liquid component which comprises water-soluble protein, polypeptides and/or peptides.
  • the hydrolysis step and/or the incubation with a TMA oxidase may, e.g., be carried out in a rotating drum, e.g. a rotating drum as disclosed in WO2017/158188.
  • the method may involve the use of a rotating drum apparatus for the mixing and processing of materials, the rotating drum apparatus comprising: a rotating drum arranged with the length of the drum and the axis of rotation of the drum extending along the horizontal; an inlet at a first point on the drum for receiving materials (e.g.
  • a screw within the drum for mixing the materials whilst conveying them lengthwise along the drum, wherein the screw includes a helical blade extending along the length of the drum with the outer edge of the helical blade being fixed to the inner surface of the drum such that material can be conveyed and mixed in separated volumes between each turn of the screw blade; an outlet at a second point along the drum for discharge of materials after mixing and/or processing; and a plurality mixing devices for promoting mixing of the material in each of the separated volumes of material as the material is conveyed along the screw, wherein the plurality of mixing devices are spaced apart along the blade of the screw, and wherein there is at least one mixing device for each turn of the screw blade.
  • the hydrolysis step and/or the incubation with a TMA oxidase may, e.g., be carried out in a turbulence-generating pipe, e.g. a turbulence- generating pipe as disclosed in WO2016/041896.
  • the method may comprise passing a reaction mixture through a first enzymatic processing area comprising a turbulence- generating pipe, the turbulence-generating pipe having a repeatedly changing centre-line and/or a repeatedly changing cross-section, the turbulence generated by the turbulence- generating pipe being used to mix the reaction mixture and to prevent sedimentation of particles as the mixture is flowing through the turbulence generating pipe, wherein the reaction mixture is subjected to turbulence within the enzymatic processing area, e.g. for a reaction time of 15 minutes or more.
  • the turbulence-generating pipe may be a corrugated pipe.
  • the method may involve the use of an enzymatic processing plant arranged for such a method.
  • the aquatic biological material may optionally be ground through dyes, e.g. 6 mm dyes, preferably prior to a hydrolysis step and/or prior to incubation with a TMA oxidase.
  • the method may include a second or further hydrolysis step, which may, e.g., involve the use of enzymes designed to optimize taste and reduce bitterness.
  • a suitable example is Flavourzyme (Novozymes) which is an exopeptidase/endopeptidase complex specially designed to optimize taste and reduce bitterness.
  • a hydrolysate may be diluted to contain, e.g., about 10 % dry matter, of which protein should be the major part (preferably approx. 90 %).
  • the hydrolysate should preferably contain virtually no lipids.
  • the reaction time may e.g. be about 20 minutes and the reaction temperature about 55°C.
  • the enzyme concentration may, e.g., be about 0,1 % (d.w) of raw material (w.w).
  • the method may comprise hydrolysis of an aquatic biological material, inactivating any proteases in the resulting hydrolysate, and incubating the hydrolysate with a TMA oxidase.
  • the method may comprise hydrolyzing an aquatic biological material, inactivating any proteases in the resulting hydrolysate, incubating the hydrolysate with a TMA oxidase, and then carrying out a second or further hydrolysis step.
  • a first and second hydrolysis steps, and optionally any protease inactivation steps may be carried out prior to a step of incubation with a TMA oxidase.
  • the methods provided herein may include a step of inactivating a protease.
  • the methods provided herein may include a step of inactivating a TMA oxidase after a suitable incubation step, for example after it has been used to convert TMA in the sample.
  • Any of the methods provided herein, or relevant steps thereof, may, e.g., be run as a continuous process or as a batch process.
  • the aquatic biological material comprises or consists of fish (whole or parts thereof) and a hydrolysis reaction is carried out with a protease concentration of about 0.1 % (d.w) of raw material (w.w) excluding added water, a reaction temperature of about 45- 75 ° C, e.g. about 60 ° C or about 45° C, at an appropriate pH, e.g. about 7.5, and a reaction time of about 15-120 minutes, e.g. about 45 minutes or 60 minutes; the material is subjected to a heat-inactivation step, after which a TMA oxidase is then contacted with the material.
  • An exemplary protocol is outlined in Figure 7 and in the Examples.
  • prior to in the context of a process step is meant that the step which is “prior to” a specific step chronologically precedes said step, so the prior step may be immediately prior to said specific step, i.e. without any further intermediate steps, but one or more intermediate steps may also be present.
  • “prior to” includes in one embodiment “immediately prior to”.
  • immediately prior to is meant that no intermediate steps are present between the two steps referred to and “immediately” does not in any way mean that there may not be a temporal space between the two steps. Indeed, at any stage the aquatic biological material may be stored prior to performance of the next step. Storage for short periods of time, e.g. a few minutes or hours, may conveniently be done at room temperature, e.g. 20-40° C. However, to minimise the risk of contamination and growth of
  • storage may preferably be at about or below 4° C or at freezing
  • Storage may involve drying, if this is the case, then after storage the material should be reconstituted with a suitable solvent, preferably water, particularly deionised water to reverse the effect of the drying.
  • a suitable solvent preferably water, particularly deionised water to reverse the effect of the drying.
  • step which is “after” a specific step chronologically follows said specific step, so the subsequent step may be immediately after said specific step, i.e. without any further intermediate steps, but one or more intermediate steps may also be present.
  • step includes in one embodiment “immediately after”.
  • intermediately after is meant that no intermediate steps are present between the two steps referred to and “immediately” does not in any way mean that there may not be a temporal space between the two steps.
  • a trimethylamine oxidase to modulate the TMA levels, odour and/or taste of an aquatic biological material.
  • a TMA oxidase in any of the methods described herein, in a method of processing an aquatic biological material, e.g. a method of making a refined aquatic biological product.
  • these terms include the term“consists of” or“consists essentially of”, or other equivalent terms.
  • the term "increase" (or equivalent terms) as described herein includes any measurable increase when compared with an appropriate control.
  • Appropriate controls would readily be identified by a person skilled in the art, examples of which are described elsewhere herein and include non-treated samples or samples subjected to a different treatment, or might include a level of a particular parameter in the same individual sample measured at an earlier time point (e.g. comparison with a "baseline” level in that sample).
  • the increase will be significant, for example statistically significant, for example with a probability value of ⁇ 0.05, when compared to an appropriate control level or value.
  • Table 1 shows the sequences of TMA oxidases
  • Figure 1 is a graph showing the percentage of individuals who reported weakly or non-fishy smell (organoleptic evaluation score of 4-5) in salmon hydrolysate samples after incubation with different concentrations of the TMA oxidase at 37 °C for 1 hr.
  • Figure 2 is a graph showing the percentage of individuals who reported weakly or non-fishy smell (organoleptic evaluation score of 4-5) in salmon hydrolysate samples after incubation with different concentrations of the TMA oxidase at 37 °C for 1 hr, 7 hrs and 24 hrs.
  • Figure 3 is a graph showing the percentage of individuals who reported weakly or non-fishy smell (organoleptic evaluation score of 4-5) in salmon hydrolysate samples after incubation with different concentrations of the TMA oxidase at 25 °C for 1 hr, 7 hrs and 24 hrs.
  • Figure 4 is a graph showing the enzymatic activity of the TMA oxidase over a range of temperatures at pH 7.5. Enzymatic activity is shown in enzyme units per gram (u/g).
  • Figure 5 is a graph showing the enzymatic activity of the TMA oxidase over a range of pH values at 40 °C. Enzymatic activity is shown in enzyme units per gram (u/g).
  • Figure 6 is a graph showing the enzymatic activity of the TMA oxidase under three different sets of conditions. Enzymatic activity is shown in enzyme units per gram (u/g) (see Example 4).
  • Figure 7 is a flow chart showing the protocol of Example 5.
  • Figure 8 is a set of graphs showing results of Example 5, i.e. the different levels of TMA, TMAO and total volatile nitrogen in cod fillet hydrolysates following treatment with different concentrations of TMA oxidase.
  • Figure 9 is a graph showing results of Example 6, i.e. a reduction in TMA level in salmon hydrolysates following treatment with TMA oxidase for 1 hr at 45 °C compared to control.
  • Figure 10 is a flow chart showing the protocol of Example 7.
  • Figure 11 is a set of graphs showing results of Example 7, i.e. the Total Volatile N and TMA levels of the L60, spray-dried peptides and permeate fractions generated from the protocol of Example 7.
  • TMA oxidase enzyme A positive effect of the TMA oxidase enzyme on fish smell was observed, even after only 1 hour of incubation at 37°C, as shown in Figure 1. There was a dose response, with the higher concentrations of TMA oxidase enzyme (10 or 5% enzyme) having a more pronounced effect, compared to low (1.25%) and no TMA oxidase enzyme. After just 1 hour, 79% of people surveyed reported the sample with 10% TMA oxidase enzyme having a slight/ no fish smell, compared to only 21% reporting the samples with no enzyme having no/ a slight fish smell.
  • TMA oxidase enzyme on fish smell did not vary significantly at 37°C over the 24 hour time course examined, as shown in Figure 2. After 24 hours 71% of people surveyed reported the sample with 10% TMA oxidase having a slight/no fish smell compared to 21% reporting the samples with no enzyme having no/ a slight fish smell.
  • TMA oxidase enzyme The effect of TMA oxidase enzyme on fish smell improved over time on incubation at 25 °C over the 24 hour time course examined, as shown in Figure 3.
  • 36% of people surveyed reported the sample with 10% TMA oxidase enzyme having no/ a low fish smell, compared to 7% reporting the samples with no TMA oxidase enzyme having no/ a slight fish smell.
  • 79% of people surveyed reported the sample with 10% TMA oxidase enzyme having no/ a low fish smell, compared to 21% reporting the samples with no TMA oxidase enzyme having no/ a slight fish smell.
  • exogenous NADPH was also investigated by incubating salmon samples at 37 °C with (a) TMA oxidase plus NADPH; (b) TMA oxidase without added NADPH; or (c) no enzyme. Both (a) and (b) showed a reduction in fishy smell compared to the control, indicating that the addition of exogenous NADPH may not necessarily be required.
  • the concentration of NADP+ is measured by following its formation from NADPH by recording the change in absorbance at 340nm.
  • the rate of decrease in the A340 is directly proportional to the TMA oxidase activity in the sample.
  • Water should be reagent grade unless otherwise specified.
  • TMA T rimethylamine
  • Vt total reaction volume (3.0ml
  • Vs sample volume (0.1 ml)
  • the standard NADPH assay was carried out with the incubation temperature varied between 25 ° C and 60 ° C at 5 ° C intervals. For these tests the 50mM phosphate buffer at pH7.5 was used throughout and a reaction time of 1 minute.
  • the starting absorbance was always in the range 0.64-0.61 and enzyme blanks showed no breakdown of the substrate without the enzyme present.
  • the optimal temperature is about 40 ° C (see Figure 4).
  • the temperature for these assays was maintained at 40 ° C.
  • the pH rage measured was 3-9. Enzyme blanks were carried out at all pHs and showed to be significant at several points. Buffering was achieved using citrate buffers within the range of pH3-5.5, phosphate buffers within the range 6-8 and borate buffers within the rage 8-9.
  • the TMA oxidase gene of SEQ ID NO: 2 was synthesised and sub-cloned into the pET-24a plasmid (NovagenTM)to generate an expression vector, denoted ST003-MSK1-pET24a.
  • the expression vector was transformed into E.coli BL21 (DE3) to generate a host strain, denoted ST003-MSK1 strain.
  • the strain was grown in the Overnight ExpressTM Autoinduction medium (NovagenTM) with the flasks incubated at 30 ° C, 300rpm. At harvest, the cultures were centrifuged and the supernatants discarded. The cell pellets were lysed using chemical lysis and centrifuged. The soluble fractions were pooled together and freeze dried. All subsequent tests were carried out with the freeze-dried powders.
  • pET-24a plasmid, BL21 (DE3) and Overnight ExpressTM Autoinduction medium are commercially available, e.g. from Merck Chemicals Ltd. (Boulevard Industrial Road, Padge Road, Beeston, Nottingham, East Midlands NG9 2JR United Kingdom)
  • Example 2 assay measures the concentration of NADP+ by following its formation from NADPH by recording the drop in absorbance at 340nm.
  • the rate of decrease in absorbance at 340nm is directly proportional to the TMA oxidase activity in the sample.
  • TMA oxidase uses NADPH as a cofactor to convert TMA into TMAO, see reaction below. In this way we can measure the conversion of TMA into TMAO.
  • TMA oxidase SEQ ID NO: 1
  • the protocol for the experiments is set out in Figure 7.
  • the protocol involves (a) a hydrolysis step comprising incubation of the cod fillet with a protease (Promod P439L from Biocatalysts Ltd. of Wales) under the following conditions for 1 hour in order to generate a hydrolysate:
  • protease solution/ cod fillet ratio 660 ppm (600 mg protease solution/ kg cod fillet)
  • an inactivation step comprising inactivation of the protease by incubation of the hydrolysate at 90°C for 10 min
  • a TMA removal step comprising incubation of the hydrolysate with a concentration of TMA oxidase at 45°C for 1 hr; and (d) centrifugation of the reaction product.
  • TMA oxidase dose 2 20 mg TMA oxidase/ kg cod fillet
  • Table 2 Exemplary amounts of the components that could be used in a single run are shown in Table 2:
  • the aqueous fraction containing TMA and TMAO was separated by a centrifugation step. Thereafter, the aqueous fraction was freeze-dried (water removed). The chemical analyses were performed on the freeze-dried samples.
  • TMA oxidase treatment Treatment with the highest TMA oxidase dose (20 mg/ kg raw material) reduced TMA from 363 to 263, ca -28%.
  • concentration of TMAO increased after TMA oxidase treatment, high dose, from 244 to 349 (43%).
  • TMA oxidase treatment on cod raw material is successful to substantially reduce the level of TMA. It can be noted that TMA levels in salmon are low as compared to levels found in cod.
  • Salmon raw material was incubated with protease (Promod 439L from Biocatalysts Ltd. of Wales) for 1 hr to generate a salmon hydrolysate.
  • protease Promod 439L from Biocatalysts Ltd. of Wales
  • the salmon hydrolysate was then heated at high temperature (>90°C) to inactivate the protease.
  • the resulting hydrolysates sample (named L60) was then used in the procedure below.
  • TMA oxidase Treatment with TMA oxidase reduced TMA level from 20 to 13 mg N/ 100g (-35%). TMAO levels would have been expected to increase in the TMA oxidase treated samples. However this could not be detected due to the insufficient sensitivity of the detection method.
  • L60 (salmon hydrolysate with protease inactivated by heating) was prepared as shown in Example 6. The protocol is illustrated in Figure 10.
  • the sample was filtered using a 100 kDa filter (Atech Innovations GmbH) ceramic membrane.
  • the 150 L permeate was subject to nanofiltration using a piperazin-amide spiral membrane.
  • the cutoff in the nanofilter is in the range of 200 Daltons.
  • the volatile nitrogen compounds are extracted from the test material with an acidic deproteinization reagent, trichloroacetic acid.
  • Method for determination of TVN Base is added to increase the pH of the sample above pH 12, thereby releasing the TVN compounds from the sample. The TVN compounds diffuse into a boric acid solution. The boric acid solution is back-titrated to the original pH with low concentration sulfuric acid.
  • TMA levels Formalin is added to bind ammonia. Base is added to increase the pH of the sample above pH 12, thereby releasing the TMA from the sample. The TMA diffuses into a boric acid solution. The boric acid solution is back-titrated to the original pH with low concentration sulfuric acid.
  • Titanium trichloride is added to reduce TMAO to TMA.
  • Formalin is added to bind ammonia.
  • Base is added to increase the pH of the sample above pH 12, thereby releasing the TMA from the sample.
  • the TMA diffuses into a boric acid solution.
  • the boric acid solution is back-titrated to the original pH with low
  • Table 5 below provides the concentrations of various components in L60 (the hydrolysate), the spray-dried peptides and the permeate.
  • the Total Volatile N and TMA levels of Table 5 are illustrated graphically in Figure 1 1.
  • Table 6 below provides the molecular weight distribution for the proteins in L60, the spray- dried peptides and the permeate.
  • TMA oxidase The amount of TMA reduced from 7 (1 1 on dry matter) in L60 to ⁇ 1 mg N/100g (i.e. below detection level) in spray-dried peptides by TMA oxidase (FM03) treatment. Nanofiltration removed peptides ⁇ 200 Dalton (80% of peptides in permeate ⁇ 200 Da).
  • L60 material was diluted in water to make material at the appropriate percent solids, (5%, 24% and 37.5%). Diluted material was titrated to the required pH if needed using 5 M KOH.
  • Diluted material was mixed for approximately 15 minutes at the specified temperature in a water bath.
  • liquid NADPH was added to the samples before the addition of the enzyme.
  • Enzyme was added to the diluted material to the desired final concentration. Samples were incubated with mixing in the water bath for the specified time and at the specified temperature. Samples were heated to 70°C for 10 minutes to deactivate the enzyme. Samples were frozen, and then either analysed in liquid form (tests 1 (Tables 7a and 7b) and 2 (Table 8)), or freeze dried and then analysed (test 3 (Table 8)).
  • TMA oxidase used was that of SEQ ID NO: 1.
  • TMA and TMAO levels were determined by the methods of Example 7.
  • Tables 7a and 7b show the results from test 1.
  • Table 8 shows the results from test 2.
  • Table 9 shows the results from test 3.
  • This Example was to look at the parameters in which the enzyme can be used to reduce the TMA levels in salmon hydrolysate.
  • Enzyme concentration was tested from a dose of 0.002 % to 1.73 % based on 5 % solids in the substrate and all resulted in a decrease in TMA.
  • Percent solids in the salmon hydrolysate were looked at from a range of 5% to 37.5% and all saw a reduction in TMA levels.
  • Incubation times were investigated between 1 and 18 hours and all showed a reduction in the levels of TMA.
  • a pH range of 6.1-8 was tested and all pH values showed a reduction in the levels of TMA.
  • Temperatures of 40°C and 43°C were shown to result in a decrease in TMA levels using different conditions. NADPH is not necessary for reduction in levels of TMA using the enzyme.
  • the material from tests 1 and 2 was sent for analysis as liquids and test 3 was freeze dried before sending for analysis. This explains the increased initial figure of TMA in the powdered (
  • TMA oxidases 8 novel TMA oxidases (flavin containing monooxygenase 3s; FM03s) were discovered using the MetXtraTM platform (Biocatalysts Ltd. of Wales). These 8 TMA oxidases have been produced at small scale and tested using the TMA oxidase assay (as set out in Example 2) for their action on converting trimethylamine (TMA) into trimethylamine-N-oxide (TMAO) at different temperatures and pH values.
  • MetXtraTM platform 8 novel TMA oxidases (flavin containing monooxygenase 3s; FM03s) were discovered using the MetXtraTM platform (Biocatalysts Ltd. of Wales). These 8 TMA oxidases have been produced at small scale and tested using the TMA oxidase assay (as set out in Example 2) for their action on converting trimethylamine (TMA) into trimethylamine-N-oxide (TMAO) at different temperatures and pH values.
  • Biocatalysts Ltd have worked with The European Bioinformatics Institute (EMBL-EBI) to develop a novel, unique software platform for data analysis called MetXtraTM, launched at Food Ingredients Europe 2017 in Frankfurt, Germany.
  • MetXtraTM is a system for identifying completely novel enzymes rapidly from large metagenomic DNA sequence libraries. This allows food industry researchers, among others, to discover unique enzymes quickly and rationally for specific applications.
  • Biocatalysts Ltd coupled their proprietary metagenomic libraries with open datasets in EBI Metagenomics (https://www.ebi.ac.uk/metagenomics/), a public resource for metagenomics data developed at EMBL-EBI. Mining this data resulted in a collection of over 11 1 million unique protein sequences, which function in diverse environmental niches (e.g. hot/cold, acid/alkaline, saline).
  • EMBL-EBI a publicly funded intergovernmental research institute and data provider - developed a new technology for searching and analysing very large datasets automatically.
  • the new proprietary platform, MetXtraTM enables screening of metagenomic libraries for enzymes in minutes, rather than days. This platform was used to identify the new TMA oxidases as described herein.
  • MetXtraTM filters results through bespoke algorithms. This rational selection process ultimately gives fast, low-cost access to enzymes with a higher probability of being commercially successful.
  • Example 9A The pH profiles were then prepared for each specific enzyme using the optimal temperature for that enzyme as determined in Example 9A.
  • the pH profiles for the 8 novel TMA oxidases are shown in Table 11 below, with the highest activities shown in bold and italics.
  • TMA oxidase activity is defined as the amount of enzyme that catalyses the oxidation of one micromole of NADPH per minute at pH 7.5 and 40 ° C. All 8 enzymes are thus able to catalyse the reaction of converting TMA into TMAO.

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Abstract

L'invention concerne un procédé de modulation des taux de triméthylamine (TMA), de l'odeur et/ou du goût d'un matériau biologique aquatique, le procédé consistant à mettre en contact le matériau biologique aquatique avec une triméthylamine oxydase. L'invention concerne également de nouvelles TMA oxydases destinées à être utilisées dans le procédé de la présente invention, et des produits biologiques aquatiques produits par le procédé de la présente invention.
PCT/EP2019/061311 2018-05-02 2019-05-02 Procédé enzymatique WO2019211408A1 (fr)

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WO2021087631A1 (fr) * 2019-11-08 2021-05-14 Latorre Salas Hector Rodrigo Composition biotechnologique pour éviter l'augmentation de composés volatils d'azote et réduire la viscosité dans le concentré d'eau de colle durant la production de farine et d'huile de poisson

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