WO2018134415A1 - Composition and method of production thereof - Google Patents

Composition and method of production thereof Download PDF

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Publication number
WO2018134415A1
WO2018134415A1 PCT/EP2018/051481 EP2018051481W WO2018134415A1 WO 2018134415 A1 WO2018134415 A1 WO 2018134415A1 EP 2018051481 W EP2018051481 W EP 2018051481W WO 2018134415 A1 WO2018134415 A1 WO 2018134415A1
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WIPO (PCT)
Prior art keywords
food composition
microbial biomass
composition according
protein
dry cell
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PCT/EP2018/051481
Other languages
French (fr)
Inventor
Tom BRUDENELL-BRUCE
Bruno Sommer Ferreira
Original Assignee
Megatech Research Gmbh
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Application filed by Megatech Research Gmbh filed Critical Megatech Research Gmbh
Priority to EP18702173.8A priority Critical patent/EP3570690A1/en
Publication of WO2018134415A1 publication Critical patent/WO2018134415A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]

Definitions

  • the invention relates to improved food compositions comprising microbial biomass.
  • the present invention also relates to a method for growing a microbial biomass.
  • Omega-3 fatty acids are of significant commercial interest due to their recognition as important dietary compounds that can prevent arteriosclerosis and coronary heart disease, alleviate inflammatory conditions and retard the growth of tumour cells. They are also very important for brain health and cognitive development and it has been suggested they may reduce delinquency and mediate autism in specific cases. These fatty acids are not made by the fish de novo but are derived from their diet.
  • EAAs essential amino acids
  • Microbial based feeds offer advantages compared to terrestrial animal and plant based feeds due to continuous harvesting, low water and energy requirements, cost- effectiveness and protein rich constituency.
  • Microorganisms can provide a natural, sustainable supply of feeds comprising proteins, carbohydrates, oils and vitamins. Some microorganisms comprise a rich source of natural omega-3 fatty acids and provide a better omega-3/omega-6 ratio than soy and corn based feeds and derived vegetable oils.
  • Thraustochytrids are microorganisms of the order Thraustochytriales. Thraustochytrids include the genera Schizochytrium and Thraustochytrium which have been recognised as a potential source of omega-3 fatty acids as discussed in US 5130242.
  • Omega-3 fatty acid producing microorganisms can be grown and the oils produced used in food formulations.
  • exposure of fatty acids to air, for example during processing, extraction or product storage, can result in a loss of activity, decomposition and oxidation.
  • Decomposition of fatty acids can produce high levels of free fatty acids (FFA) which are highly undesirable, and generally speaking, no more than 3% FFA content is permitted in foodstuffs and feedstocks.
  • FFA free fatty acids
  • contact with light and certain metals can also accelerate decomposition and oxidation of fatty acids.
  • any decomposition products of the fatty acids can result in an undesirable taste and these products have the potential to be carcinogenic.
  • Antioxidants are often added to omega-3 oils to slow down oxidation of the oil.
  • antioxidants are expensive and can substantially increase the cost of the oil. If higher levels of FFAs are present in the oil, then the oil will need to be refined which is not only costly but also results in up to 10% of the oil being lost.
  • oils may be protected by encapsulation prior to incorporation into food products as detailed in WO2007150047. However, encapsulation procedures can be complex and expensive.
  • the present invention is based, in part, on studies undertaken by the inventors in which they show that surprisingly, it is possible to develop microbial biomass having particular contents of protein, oil and/or DHA which therefore make such biomass suitable for use in food compositions, particularly in feed formulations for fish, for example.
  • a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
  • the microbial biomass of the present invention comprises 15-95 dry cell wt% protein.
  • the microbial biomass comprises around 15-65 dry cell wt%, 25- 65 dry cell wt%, 30-65 dry cell wt%, 34-65 dry cell wt%, 45-65 dry cell wt% or 55-65 dry cell wt% protein.
  • the microbial biomass comprises around 30-95 dry cell wt%, 50-95 dry cell wt%, or 65-95 dry cell wt% protein. Most preferably the microbial biomass comprises around 75-95dry cell wt% protein.
  • the microbial biomass may comprise essential amino acids and non-essential amino acids.
  • the microbial biomass of the present invention may comprise 0.5-75 dry cell wt% oil. Therefore, in such embodiments, the microbial biomass may comprise:
  • the microbial biomass of the present invention may comprise 7-75 dry cell wt% oil. Therefore, in such embodiments, the microbial biomass may comprise:
  • the microbial biomass comprises around 0.5-65 dry cell wt%, 0.5-55 dry cell wt% or 0.5-45 dry cell wt% oil. Most preferably, the microbial biomass comprises around 0.5-25 dry cell wt% oil.
  • An oil content of less than around 25 dry cell wt% can reduce the likelihood of cells bursting and releasing oil during feed formulation processes, for example an extrusion process. The release of oil could cause problems with shear during extrusion.
  • a low ratio of oil to protein in the microbial biomass provides a food composition which can meet all of a consumer's protein requirements. This is especially advantageous in the provision of, for instance, fish feed for farmed fish. Generally the diets of wild fish contain far higher amounts of protein than those of farmed fish. This is because of the economic pressure on fish feed producers to produce more cost effective and balanced diets thus resulting in feed with an increased proportion of oil to protein to provide energy and as well as essential lipids.
  • the microbial biomass of the present invention can provide a cost-effective and complete source of protein in consumer's diets.
  • the oil comprises around 95-97% fatty acids. Therefore, the present invention also provides a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
  • the present invention also provides a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
  • the microbial biomass comprises 25-55% DHA as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 30-55% DHA as a percentage of total fatty acids, more preferably around 35-55% or 40-55% DHA as a percentage of total fatty acids.
  • farmed fish require preformed DHA and the essential amino acids in their diets for each phase of production and critically during early developmental stages.
  • the present inventors have found that it is possible to provide a microbial biomass with between 25-55% DHA as a percentage of total fatty acids, which makes the microbial biomass particularly suitable for use as a fish feed composition.
  • the microbial biomass may comprise or additionally comprise around 6-1 1 % docosapentaenoic acid (DPA) as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 8-10% DPA as a percentage of total fatty acids.
  • the microbial biomass may comprise or additionally comprise around 15-55% 16:0 fatty acids as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 25-50% 16:0 fatty acids, more preferably around 28-40% 16:0 fatty acids as a percentage of total fatty acids.
  • the microbial biomass may comprise cultures of Thraustochytrids, Thraustochytrium, Aurantiochytrium or Schizochytrium.
  • the microbial biomass comprises a culture of Schizochytrium mangrovei.
  • the present inventors have surprisingly found that it is possible to produce food compositions in accordance with the first aspect of the invention in particularly high yields when the microbial biomass comprises a culture of Schizochytrium mangrovei. This results in a highly economic product which can be used throughout the life cycle of fish, for example.
  • the microbial biomass is in the form of microbial whole cell biomass.
  • the consumption of intact, whole microbial cells and their post-consumption digestion protects oils within microbial cells from oxidation.
  • oxidised oils can be damaging to consumers.
  • cell wall residues can have positive effects on gut morphology and overall function in many fish species.
  • the microbial biomass may further comprise vitamins and minerals.
  • the microbial biomass may comprise vitamin E a-tocopherol, vitamin D, vitamin A and vitamin K.
  • the microbial biomass may further comprise iron, manganese, zinc, copper and selenium.
  • the food composition may further comprise starch, vitamins, minerals and binding agents, such as soybean meal.
  • the food composition may additionally comprise one or more essential amino acids selected from: methionine, arginine, threonine, tryptophan, histidine, isoleucine, lysine, leucine, valine and phenylalanine.
  • the quantities of the one or more essential amino acids provided in the composition will be in accordance with usual animal or fish food compositions and will largely be dependent upon the desired application.
  • the food composition may form part of the diet of fish, dogs, cats, chickens, other domestic animals and/or of humans.
  • the food composition forms the whole diet for fish, dogs, cats, chickens, other domestic animals and/or humans.
  • the food composition is for consumption by fish and in particular high value species such as salmon, trout, sea bass and seabreams.
  • the food composition may be adapted for fish to meet their essential fatty acids requirements and provide enhanced fatty acid (Omega-3) for the consumer.
  • the food composition is for consumption by sea bream, sea bass, salmonids, flounder, turbot, sole and rainbow trout at various production stages but especially from grow-out to harvest.
  • the food composition may be employed for consumption by crustaceans such as penaid shrimp and to provide enhanced HUFA (highly unsaturated fatty acids) in the flesh for species like tilapia as an added value niche market for this important fish species.
  • the oil and protein concentrations of the microbial biomass may be selected based on the age of the consumer. For example, rapidly growing fish and shellfish (juvenile to pre- harvest) require a higher protein to fat (oil) ratio in their diets typically 45-50% protein: 18- 20% oil. When fish such as salmon approach the end of their growth phase, they require relatively more fat (oil) (>25%) than protein (35%) in their diet. Suitably they also need a particular omega-3 fatty acid profile in their flesh for subsequent consumption.
  • the food composition is adapted for juvenile fish and shellfish and comprises: a) 7-25 dry cell wt% oil; and
  • the composition may preferably comprise: a) 45-50 dry cell wt% protein;
  • the food composition is adapted for adult fish and shellfish and comprises: a) 15-25 dry cell wt% oil; and
  • the composition may preferably comprise: a) around 25 dry cell wt% oil;
  • the food composition of the present invention may be in any suitable form, for example pellets, micro-pellets, granules, crumbles, powders, microcapsules or flakes.
  • the composition may also be in a liquid (such as a solution or suspension) or in a dried form.
  • the food composition may be in the form of a pellet.
  • the food composition may be extruded.
  • a method for growing a microbial biomass wherein the microbial biomass comprises:
  • nitrogen and carbon are fed to the microbial biomass during fermentation.
  • Nitrogen and carbon feed rates can of course be adjusted to change microbial cell growth rate and microbial biomass oil and protein content.
  • the present inventors have shown that increasing the feed rate of carbon (for example in the form of glucose or glycerol) during fermentation results in high cell growth rate and high oil content and conversely, increasing the feed rate of nitrogen (for example in the form of yeast extract, corn steep liquor (CSL) and/or (NhU ⁇ SCU) during fermentation favours cell growth and results in high yields of microbial biomass and a high protein content.
  • carbon for example in the form of glucose or glycerol
  • nitrogen for example in the form of yeast extract, corn steep liquor (CSL) and/or (NhU ⁇ SCU
  • the mass ratio of total carbon to nitrogen sources is less than around 5.
  • the mass ratio of total carbon to nitrogen sources is between around 3 and 5, preferably around 4.
  • the mass ratio of total carbon to nitrogen sources may be in the range of around 1 .8 to 4.2, around 1 .9 to 4.2, around 2.2 to 4.2, around 2.4 to 4.2, around 2.7 to 4.2, around 2.9 to 4.2, around 3.2 to 4.2, around 3.4 to 4.2, around 3.5 to 4.3 or around 3.8 to 4.2.
  • the mass ratio of total carbon to nitrogen sources may be in the range of around 1 .8 to 4.2, around 1 .8 to 3.8, around 1 .8 to 3.5, around 1.8 to 3.4, around 1 .8 to 3.2, around 1.8 to 2.9, around 1 .8 to 2.7, around 1 .8 to 2.4, around 1 .8 to 2.4, around 1.8 to 2.2, or around 1 .8 to 1 .9. Most preferably, the mass ratio of total carbon to nitrogen sources is between around 4.1 and 4.2.
  • the present inventors have surprisingly shown that by having a mass ratio of total carbon to nitrogen sources as described above, microbial biomass with high protein and high levels of DHA can be produced, making such biomass highly suited for use in feed compositions, particularly fish feed compositions.
  • the method of the present invention may be grown in aerobic fermentation conditions.
  • the oil to protein ratio is in the region of around 0.02 to 1 .24.
  • the oil to protein ratio may be in the region of around 0.03 to 1 .24, around 0.04 to 1 .24, around 0.06 to 1 .24, around 0.08 to 1 .24, around 0.1 1 to 1 .24, around 0.14 to 1 .24 or around 0.61 to 1 .24.
  • the oil to protein ratio may be in the region of around 0.02 to 0.61 , around 0.02 to 0.14, around 0.02 to 0.1 1 , around 0.02 to 0.08, around 0.02 to 0.06, around 0.02 to 0.04 or around 0.02 to 1 .03.
  • the oil to protein ratio is in the region of around 0.02 to 0.61.
  • the set-point of the dissolved oxygen concentration in the fermentation medium is controlled between around 20% and 30% of the saturation concentration.
  • the microbial biomass grown by the method of the present invention is in accordance with the microbial biomass of the first aspect of the present invention.
  • Figure shows the mass ratio of total carbon to nitrogen correlating with the oil to protein ratio of the product biomass produced in accordance with the invention.
  • Microbial cells believed to be Schizochytrium mangrovei, were initially grown on a culture plate before being transferred to a shaker flask and fermentation tank. Twelve trials were undertaken, each using different fermentation conditions, as shown in table 1 .
  • Trial 1 was a batch fermentation reaction, whereas trials 2-12 were fed-batch fermentations. The components of the additional feed used in trials 2-12 are shown in table 2.
  • the mass ratio of total carbon to nitrogen sources used in the twelve trials is shown 4.
  • the present inventors have surprisingly shown that high protein contents can be achieved in the resultant microbial biomass.
  • the present inventors have surprisingly shown that as shown in Figure 1 a mass ratio of total carbon to nitrogen sources correlates with the oil to protein ratio of the product biomass achieved.
  • the microbial biomass produced in the twelve trials was spray dried and the protein, oil, carbohydrate and moisture content determined.
  • Carbohydrate content was determined by Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances Anal. Chem. 28, 350-356. The results (along with analytical methods used) are shown in table 5.
  • the fatty acid composition (% total fatty acids) of total lipid from the spray dried biomass are shown in table 6.
  • DHA, DPA and 16:0 are highlighted in the table.
  • the limit of quantification for fatty acid analysis is 0.06%.
  • the relatively high proportions of DHA in the biomass demonstrate the suitability of microbial biomass to be used in a food composition which can supply health improving omega-3 fatty acids to consumers.

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Abstract

The invention relates to food compositions comprising microbial biomass, the microbial biomass comprising:(a) 15-95 dry cell wt% protein; and(b) 25-55% DHA as a percentage of total fatty acids. The present invention also relates to a method for growing microbial biomass by fermentation at 25-39°C, for less than 4 days resulting in a yield of 100-350 g/l.

Description

COMPOSITION AND METHOD OF PRODUCTION THEREOF
Technical Field of the Invention
The invention relates to improved food compositions comprising microbial biomass. The present invention also relates to a method for growing a microbial biomass. Background to the Invention
Fish are an important source of protein and omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) and docosahexaenoic acid (DHA) in human diets. Omega-3 fatty acids are of significant commercial interest due to their recognition as important dietary compounds that can prevent arteriosclerosis and coronary heart disease, alleviate inflammatory conditions and retard the growth of tumour cells. They are also very important for brain health and cognitive development and it has been suggested they may reduce delinquency and mediate autism in specific cases. These fatty acids are not made by the fish de novo but are derived from their diet. Additionally fish cannot synthesise 10 essential amino acids (EAAs) for growth and development: methionine, arginine, threonine, tryptophan, histidine, isoleucine, lysine, leucine, valine and phenylalanine- all of which are essential for a balanced protein intake. Therefore farmed fish require preformed DHA and the essential amino acids in their diets for each phase of production and critically during early developmental stages.
Fish meal and fish oils can provide farmed fish with all of the DHA and the essential amino acids necessary for optimum growth and development. However, global supplies of fish meal and fish oils are finite and expensive and many environmental and consumer acceptance concerns surround them. With increasing demand for fish food due to the expansion of world-wide aquaculture, there is increased pressure to find suitable alternative sources of protein, energy and essential fatty acids. Consequently, numerous plant byproducts, as well as specific rendered proteins and fats from terrestrial farmed animals have been investigated as alternative food sources. As such, soybean meal and corn gluten meals have been used as alternative protein sources for farmed fish. However, while vegetable protein sources are more cost effective than fishmeal they generally contain only 40% protein compared to 70% in fishmeal. Soy bean meal is also unable to provide all the essential amino acids needed by fish and therefore supplementation with synthetic amino acids is required for some fish species, especially carnivorous fish like salmon and trout. Also plant and animal based oils and fats do not contain DHA.
Animal by-products do not meet consumer acceptability in Europe and other regions and their use is quite limited outside the European Union.
Microbial based feeds offer advantages compared to terrestrial animal and plant based feeds due to continuous harvesting, low water and energy requirements, cost- effectiveness and protein rich constituency. Microorganisms can provide a natural, sustainable supply of feeds comprising proteins, carbohydrates, oils and vitamins. Some microorganisms comprise a rich source of natural omega-3 fatty acids and provide a better omega-3/omega-6 ratio than soy and corn based feeds and derived vegetable oils. Thraustochytrids are microorganisms of the order Thraustochytriales. Thraustochytrids include the genera Schizochytrium and Thraustochytrium which have been recognised as a potential source of omega-3 fatty acids as discussed in US 5130242.
Omega-3 fatty acid producing microorganisms can be grown and the oils produced used in food formulations. However exposure of fatty acids to air, for example during processing, extraction or product storage, can result in a loss of activity, decomposition and oxidation. Decomposition of fatty acids can produce high levels of free fatty acids (FFA) which are highly undesirable, and generally speaking, no more than 3% FFA content is permitted in foodstuffs and feedstocks. Furthermore, contact with light and certain metals can also accelerate decomposition and oxidation of fatty acids. If included in food products, any decomposition products of the fatty acids can result in an undesirable taste and these products have the potential to be carcinogenic. Antioxidants are often added to omega-3 oils to slow down oxidation of the oil. However, the use of antioxidants is expensive and can substantially increase the cost of the oil. If higher levels of FFAs are present in the oil, then the oil will need to be refined which is not only costly but also results in up to 10% of the oil being lost. Alternatively such oils may be protected by encapsulation prior to incorporation into food products as detailed in WO2007150047. However, encapsulation procedures can be complex and expensive.
It is therefore an object of the present invention to alleviate one or more of the aforementioned problems associated with current biomasses used for animal and fish feed. It is also an object of the present invention to provide an improved food composition which preferably comprises microbial biomass.
Summary of the Invention
The present invention is based, in part, on studies undertaken by the inventors in which they show that surprisingly, it is possible to develop microbial biomass having particular contents of protein, oil and/or DHA which therefore make such biomass suitable for use in food compositions, particularly in feed formulations for fish, for example.
In accordance with a first aspect of the present invention there is provided a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
(a) 15-95 dry cell wt% protein; and
(b) 25-55% DHA as a percentage of total fatty acids. The present inventors have surprisingly found that it is possible to develop microbial biomass having the abovementioned protein and DHA contents, which therefore make such biomass suitable for use in food compositions, particularly in fish food compositions.
The microbial biomass of the present invention comprises 15-95 dry cell wt% protein. In preferred embodiments, the microbial biomass comprises around 15-65 dry cell wt%, 25- 65 dry cell wt%, 30-65 dry cell wt%, 34-65 dry cell wt%, 45-65 dry cell wt% or 55-65 dry cell wt% protein. In additional preferred embodiments the microbial biomass comprises around 30-95 dry cell wt%, 50-95 dry cell wt%, or 65-95 dry cell wt% protein. Most preferably the microbial biomass comprises around 75-95dry cell wt% protein. The microbial biomass may comprise essential amino acids and non-essential amino acids.
In embodiments, the microbial biomass of the present invention may comprise 0.5-75 dry cell wt% oil. Therefore, in such embodiments, the microbial biomass may comprise:
(a) 15-95 dry cell wt% protein;
(b) 0.5-75 dry cell wt% oil; and
(c) 25-55% DHA as a percentage of total fatty acids.
In other embodiments, the microbial biomass of the present invention may comprise 7-75 dry cell wt% oil. Therefore, in such embodiments, the microbial biomass may comprise:
(a) 15-65 dry cell wt% protein;
(b) 7-75 dry cell wt% oil; and
(c) 25-55% DHA as a percentage of total fatty acids.
Preferably the microbial biomass comprises around 0.5-65 dry cell wt%, 0.5-55 dry cell wt% or 0.5-45 dry cell wt% oil. Most preferably, the microbial biomass comprises around 0.5-25 dry cell wt% oil.
Limiting the oil content of the microbial biomass results in easier downstream processing. An oil content of less than around 25 dry cell wt% can reduce the likelihood of cells bursting and releasing oil during feed formulation processes, for example an extrusion process. The release of oil could cause problems with shear during extrusion.
Advantageously a low ratio of oil to protein in the microbial biomass provides a food composition which can meet all of a consumer's protein requirements. This is especially advantageous in the provision of, for instance, fish feed for farmed fish. Generally the diets of wild fish contain far higher amounts of protein than those of farmed fish. This is because of the economic pressure on fish feed producers to produce more cost effective and balanced diets thus resulting in feed with an increased proportion of oil to protein to provide energy and as well as essential lipids.
Advantageously increasing protein levels in the microbial biomass has been found to reduce the cost of production. Therefore the microbial biomass of the present invention can provide a cost-effective and complete source of protein in consumer's diets.
In the compositions of the present invention, the oil comprises around 95-97% fatty acids. Therefore, the present invention also provides a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
(a) 15-95 dry cell wt% protein;
(b) 0.5-75 dry cell wt% oil; and
(c) 25-55% DHA as a percentage of total fatty acids; wherein the oil comprises around 95-97% fatty acids.
The present invention also provides a food composition comprising a microbial biomass, wherein the microbial biomass comprises:
(a) 15-65 dry cell wt% protein;
(b) 7-75 dry cell wt% oil; and
(c) 25-55% DHA as a percentage of total fatty acids; wherein the oil comprises around 95-97% fatty acids.
The microbial biomass comprises 25-55% DHA as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 30-55% DHA as a percentage of total fatty acids, more preferably around 35-55% or 40-55% DHA as a percentage of total fatty acids. As described above, farmed fish require preformed DHA and the essential amino acids in their diets for each phase of production and critically during early developmental stages. The present inventors have found that it is possible to provide a microbial biomass with between 25-55% DHA as a percentage of total fatty acids, which makes the microbial biomass particularly suitable for use as a fish feed composition.
The microbial biomass may comprise or additionally comprise around 6-1 1 % docosapentaenoic acid (DPA) as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 8-10% DPA as a percentage of total fatty acids. The microbial biomass may comprise or additionally comprise around 15-55% 16:0 fatty acids as a percentage of total fatty acids. More preferably, the microbial biomass comprises around 25-50% 16:0 fatty acids, more preferably around 28-40% 16:0 fatty acids as a percentage of total fatty acids.
The microbial biomass may comprise cultures of Thraustochytrids, Thraustochytrium, Aurantiochytrium or Schizochytrium. Preferably the microbial biomass comprises a culture of Schizochytrium mangrovei. The present inventors have surprisingly found that it is possible to produce food compositions in accordance with the first aspect of the invention in particularly high yields when the microbial biomass comprises a culture of Schizochytrium mangrovei. This results in a highly economic product which can be used throughout the life cycle of fish, for example.
In embodiments, the microbial biomass is in the form of microbial whole cell biomass. The consumption of intact, whole microbial cells and their post-consumption digestion protects oils within microbial cells from oxidation. As mentioned above, oxidised oils can be damaging to consumers. In addition, cell wall residues can have positive effects on gut morphology and overall function in many fish species. The microbial biomass may further comprise vitamins and minerals. The microbial biomass may comprise vitamin E a-tocopherol, vitamin D, vitamin A and vitamin K. The microbial biomass may further comprise iron, manganese, zinc, copper and selenium.
The food composition may further comprise starch, vitamins, minerals and binding agents, such as soybean meal.
The food composition may additionally comprise one or more essential amino acids selected from: methionine, arginine, threonine, tryptophan, histidine, isoleucine, lysine, leucine, valine and phenylalanine. The quantities of the one or more essential amino acids provided in the composition will be in accordance with usual animal or fish food compositions and will largely be dependent upon the desired application.
The food composition may form part of the diet of fish, dogs, cats, chickens, other domestic animals and/or of humans. Alternatively the food composition forms the whole diet for fish, dogs, cats, chickens, other domestic animals and/or humans. Preferably the food composition is for consumption by fish and in particular high value species such as salmon, trout, sea bass and seabreams. In such embodiments the food composition may be adapted for fish to meet their essential fatty acids requirements and provide enhanced fatty acid (Omega-3) for the consumer. Most preferred, the food composition is for consumption by sea bream, sea bass, salmonids, flounder, turbot, sole and rainbow trout at various production stages but especially from grow-out to harvest. Alternatively, the food composition may be employed for consumption by crustaceans such as penaid shrimp and to provide enhanced HUFA (highly unsaturated fatty acids) in the flesh for species like tilapia as an added value niche market for this important fish species.
The oil and protein concentrations of the microbial biomass may be selected based on the age of the consumer. For example, rapidly growing fish and shellfish (juvenile to pre- harvest) require a higher protein to fat (oil) ratio in their diets typically 45-50% protein: 18- 20% oil. When fish such as salmon approach the end of their growth phase, they require relatively more fat (oil) (>25%) than protein (35%) in their diet. Suitably they also need a particular omega-3 fatty acid profile in their flesh for subsequent consumption.
Therefore in embodiments of the present invention the food composition is adapted for juvenile fish and shellfish and comprises: a) 7-25 dry cell wt% oil; and
b) 35-65 dry cell wt% protein.
In embodiments in which the food composition is adapted for juvenile fish and shellfish, the composition may preferably comprise: a) 45-50 dry cell wt% protein; and
b) 18-20 dry cell wt% oil.
In alternative embodiments of the present invention, the food composition is adapted for adult fish and shellfish and comprises: a) 15-25 dry cell wt% oil; and
b) 30-40 dry cell wt% protein.
In embodiments in which the food composition is adapted for adult fish and shellfish, the composition may preferably comprise: a) around 25 dry cell wt% oil; and
b) around 35 dry cell wt% protein.
The food composition of the present invention may be in any suitable form, for example pellets, micro-pellets, granules, crumbles, powders, microcapsules or flakes. The composition may also be in a liquid (such as a solution or suspension) or in a dried form. In preferred embodiments of the invention the food composition may be in the form of a pellet.
In embodiments of the present invention the food composition may be extruded. In a further aspect of the invention there is provided a method for growing a microbial biomass, wherein the microbial biomass comprises:
(a) 15-95 dry cell wt% protein; and
(b) 25-55% DHA as a percentage of total fatty acids; wherein the microbial biomass is grown by fermentation for less than 4 days, preferably less than 2 days, at 25-39°C to produce a concentration of microbial biomass in the range of 100-350 g/l based on dry cell weight.
Preferably, nitrogen and carbon are fed to the microbial biomass during fermentation. Nitrogen and carbon feed rates can of course be adjusted to change microbial cell growth rate and microbial biomass oil and protein content.
The present inventors have shown that increasing the feed rate of carbon (for example in the form of glucose or glycerol) during fermentation results in high cell growth rate and high oil content and conversely, increasing the feed rate of nitrogen (for example in the form of yeast extract, corn steep liquor (CSL) and/or (NhU^SCU) during fermentation favours cell growth and results in high yields of microbial biomass and a high protein content.
In preferred embodiments of the invention, the mass ratio of total carbon to nitrogen sources is less than around 5. Preferably, the mass ratio of total carbon to nitrogen sources is between around 3 and 5, preferably around 4. The mass ratio of total carbon to nitrogen sources may be in the range of around 1 .8 to 4.2, around 1 .9 to 4.2, around 2.2 to 4.2, around 2.4 to 4.2, around 2.7 to 4.2, around 2.9 to 4.2, around 3.2 to 4.2, around 3.4 to 4.2, around 3.5 to 4.3 or around 3.8 to 4.2. The mass ratio of total carbon to nitrogen sources may be in the range of around 1 .8 to 4.2, around 1 .8 to 3.8, around 1 .8 to 3.5, around 1.8 to 3.4, around 1 .8 to 3.2, around 1.8 to 2.9, around 1 .8 to 2.7, around 1 .8 to 2.4, around 1 .8 to 2.4, around 1.8 to 2.2, or around 1 .8 to 1 .9. Most preferably, the mass ratio of total carbon to nitrogen sources is between around 4.1 and 4.2. The present inventors have surprisingly shown that by having a mass ratio of total carbon to nitrogen sources as described above, microbial biomass with high protein and high levels of DHA can be produced, making such biomass highly suited for use in feed compositions, particularly fish feed compositions. In embodiments, the method of the present invention may be grown in aerobic fermentation conditions.
In preferred embodiments of the invention, the oil to protein ratio is in the region of around 0.02 to 1 .24. The oil to protein ratio may be in the region of around 0.03 to 1 .24, around 0.04 to 1 .24, around 0.06 to 1 .24, around 0.08 to 1 .24, around 0.1 1 to 1 .24, around 0.14 to 1 .24 or around 0.61 to 1 .24. The oil to protein ratio may be in the region of around 0.02 to 0.61 , around 0.02 to 0.14, around 0.02 to 0.1 1 , around 0.02 to 0.08, around 0.02 to 0.06, around 0.02 to 0.04 or around 0.02 to 1 .03. Most preferably, the oil to protein ratio is in the region of around 0.02 to 0.61.
In embodiments, the set-point of the dissolved oxygen concentration in the fermentation medium is controlled between around 20% and 30% of the saturation concentration.
In embodiments, the microbial biomass grown by the method of the present invention is in accordance with the microbial biomass of the first aspect of the present invention.
It will be apparent to the skilled addressee that a number of the features of the compositions listed in respect of the various aspects of the invention will be interchangeable. Furthermore, additional steps may be incorporated such as the incorporation of the food composition or microbial biomass alone into foodstuffs. Detailed Description of the Invention
Embodiments of the present invention will now be described, by way of example only in which:
Figure shows the mass ratio of total carbon to nitrogen correlating with the oil to protein ratio of the product biomass produced in accordance with the invention.
Microbial cells, believed to be Schizochytrium mangrovei, were initially grown on a culture plate before being transferred to a shaker flask and fermentation tank. Twelve trials were undertaken, each using different fermentation conditions, as shown in table 1 . Trial 1 was a batch fermentation reaction, whereas trials 2-12 were fed-batch fermentations. The components of the additional feed used in trials 2-12 are shown in table 2.
The pH of some of the fermentation trials was controlled by adding a base. The amounts and base added are shown in table 3.
Table 1
Figure imgf000013_0001
Figure imgf000014_0001
Table 2
Figure imgf000014_0002
Figure imgf000015_0001
Figure imgf000016_0001
Table 3
Figure imgf000016_0002
Figure imgf000017_0001
The mass ratio of total carbon to nitrogen sources used in the twelve trials is shown 4.
Table 4
Figure imgf000019_0001
By modifying the mass ratio of total carbon to nitrogen sources, the present inventors have surprisingly shown that high protein contents can be achieved in the resultant microbial biomass. In particular, the present inventors have surprisingly shown that as shown in Figure 1 a mass ratio of total carbon to nitrogen sources correlates with the oil to protein ratio of the product biomass achieved.
The microbial biomass produced in the twelve trials was spray dried and the protein, oil, carbohydrate and moisture content determined. Carbohydrate content was determined by Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances Anal. Chem. 28, 350-356. The results (along with analytical methods used) are shown in table 5.
Table 5
Figure imgf000021_0001
The fatty acid composition (% total fatty acids) of total lipid from the spray dried biomass are shown in table 6. DHA, DPA and 16:0 are highlighted in the table. The limit of quantification for fatty acid analysis is 0.06%. The relatively high proportions of DHA in the biomass demonstrate the suitability of microbial biomass to be used in a food composition which can supply health improving omega-3 fatty acids to consumers.
Table 6
Figure imgf000023_0001
Analysis of the protein and oil of the dry microbial biomass, corrected for moisture content was also determined. The results (along with analytical methods used) are shown in table 7 below. The results show a high protein, high oil, and intermediate protein and oil of the microbial biomass for the trials.
Table 7
Figure imgf000024_0001
The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.

Claims

Claims
1 . A food composition comprising a microbial biomass, wherein the microbial biomass comprises:
(a) 15-95 dry cell wt% protein; and
(b) 25-55% DHA as a percentage of total fatty acids.
2. The food composition according to claim 1 wherein the microbial biomass comprises a biomass derived from one or more of the following: Thraustochytrids, Thraustochytrium, Schizochytrium or Aurantiochytrium.
3. The food composition according to either claim 1 or 2 wherein the microbial biomass is derived from Schizochytrium mangrovei or Aurantiochytrium sp.
4. The food composition according to any preceding claim wherein the microbial biomass comprises 30-95 dry cell wt% protein.
5. The food composition according to any preceding claim wherein the microbial biomass comprises 75-95 dry cell wt% protein.
6. The food composition according to any preceding claim wherein the microbial biomass further comprises 0.5-75 dry cell wt% oil.
7. The food composition according to any preceding claim wherein the microbial biomass further comprises 0.5-25 dry cell wt% oil.
8. The food composition according to claim 6 or 7 wherein the oil comprises around 95-97% fatty acids.
9. The food composition according to any preceding claim wherein the food composition is adapted for fish.
10. The food composition according to any preceding claim wherein the food composition is adapted for juvenile fish and shellfish and wherein the microbial biomass comprises: a) 7-25 dry cell wt% oil; and
b) 35-65 dry cell wt% protein.
1 1 . The food composition according to any one of claims 1 -9 wherein the food composition is adapted for adult fish and shellfish and wherein the microbial biomass comprises:
a) 15-25 dry cell wt% oil; and
b) 30-40 dry cell wt% protein.
12. The food composition according to any preceding claim wherein the food composition is in the form of one or more selected from: pellets, micro-pellets, granules, crumbles, powders, microcapsules or flakes.
13. The food composition according to any preceding claim wherein the microbial biomass is microbial whole cell biomass.
14. A method for growing microbial biomass wherein the microbial biomass comprises:
(a) 15-95 dry cell wt% protein; and
(b) 25-55% DHA as a percentage of total fatty acids; wherein the microbial biomass is grown by fermentation for less than 4 days, at 25- 39°C and the resulting concentration of microbial biomass is 100-350 g/l.
15. The method for producing a food composition according to claim 14 wherein nitrogen and carbon are fed to the microbial biomass during fermentation and wherein the feed rates of nitrogen and carbon can be adjusted to change microbial cells growth rates and microbial biomass oil and protein content.
16. The method for producing a food composition according to claim 14 or 15 wherein a mass ratio of total carbon to nitrogen sources is below 5.
17. The method for producing a food composition according to any one of claims 14-16 wherein a mass ratio of total carbon to nitrogen sources is between 3 and 5.
18. The method for producing a food composition according to any one of claims 14-17, wherein a mass ratio of total carbon to nitrogen sources is around 4.
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