WO2020049313A1

WO2020049313A1 - Process and product thereof

Info

Publication number: WO2020049313A1
Application number: PCT/GB2019/052489
Authority: WO
Inventors: David John Ritchie
Original assignee: 3F Bio Ltd
Priority date: 2018-09-06
Filing date: 2019-09-06
Publication date: 2020-03-12
Also published as: GB201814543D0

Abstract

There is described a process for increasing the nutrient content of a foodstuff, the process comprising: (i) performing a first fermentation on a first feedstock using a nutrient-producing microorganism to produce a nutritionally enhanced mixture comprising a nutrient and the nutrient- producing microorganism; (ii) performing a second fermentation on a second feedstock to produce an alcohol and a residue, the residue comprising a foodstuff; and (iii) isolating the foodstuff from the alcohol and residue mixture; wherein the nutritionally enhanced mixture is introduced into at least one of stage (ii) and stage (iii) of the process, the foodstuff thereby being nutritionally enhanced. The nutrient-producing microorganism may be inactivated (optionally thermally inactivated) after it is introduced into at least one of stage (ii) and stage (iii) of the process, and the nutrient may be an amino acid. There is also described a foodstuff obtainable, obtained or directly obtained from the process.

Description

Process and Product Thereof

Field of Invention

The present invention relates to a process for increasing the nutrient content of a foodstuff. In particular, the present invention relates to a process for increasing the nutrient content of a foodstuff, such as distiller’s grains, produced from an ethanol biorefinery.

Background

The term“biorefinery” refers to a facility that converts biomass to produce fuels and valuable by-products.

Typical by-products from a biorefinery are distiller’s grains, in particular Dried Distiller’s Grains with Solubles (DDGS). DDGS is a co-product of the ethanol biorefinery process, and can be used as an animal feed ingredient.

A typical ethanol biorefinery uses starch rich grain feedstock, such as wheat or maize, to produce ethanol as a primary product with carbon dioxide and distiller’s grains generated as co-products. The pellet form DDGS produced as a result of this process is rich in protein, fat and fibre and is, therefore, widely used as an animal feed ingredient. However, DDGS is deficient in essential amino acids (EAAs) such as L-lysine and L-tryptophan, which are required to improve the nutritional value of the feedstuff and optimise growth performance of the animals.

Therefore, DDGS is often supplemented with EAAs, such as L-lysine, to improve the nutritional quality of the animal feed. Typically, EAAs are produced via aerobic fermentation from a carbohydrate feedstock. The crude fermentation broth produced from the aerobic fermentation reaction must be processed through numerous sequential steps, e.g.,

centrifugation, purification, concentration, crystallisation and drying. The processing steps are required to separate EAA yielding microorganisms from the broth to ensure that the supplement is safe to add to the animal feed. The crystallised, solid EAA supplement, e.g., L-lysine

monohydrochloride, is then added to the DDGS to improve the nutritional quality of the feedstuff. These EAA additives require additional storage, transportation and handling in comparison to the use of DDGS alone. Additional training is also necessary to provide handlers with the knowledge to prepare suitable feed formulations using the solid EAA supplements. Therefore, it is expensive to produce and uneconomical to provide such solid EAA supplements for addition to DDGS.

WO 95/23129 describes a process for producing L-lysine salts in granular form. This document describes a process that involves a number of purification steps, e.g., recovery of L-!ysine from the fermentation broth by ion-exchange separation, adding a salt forming agent to the L-lysine to produce a non-stoichiometric salt of L-lysine in a solution or a slurry, introducing the solution or slurry to a granulator and recovering the L- lysine HCI product from the granulator. The L-lysine HCI granules are then separated into two or more size fractions and the lysine salt granules with the desired particle size range are selected for use as an animal feed supplement. US5431933 describes a process for the production of an animal feed supplement containing a high proportion of at least one amino acid.

Specifically, US5431933 describes the preparation of an amino acid animal feed supplement based on fermentation broth that still contains most of the contents of the fermentation broth excluding at least part of the biomass. Although this supplement includes most of the contents of the fermentation broth, a portion of the biomass and proteins are removed by mechanical separation techniques such as particular filtration and separation (e.g., centrifugation and decanting) to remove undissolved components and high molecular weight substances. The fermentation broth is then freeze dried to create the final product.

WG20G7141111 describes a process for producing animal feed additives based on fermentation broth having a high content of L-lysine. In particular, the fermentation broths are obtained by fermentation using Coryneforme bacteria, such as Corynebacterium glutamicum, that contains selected mutations. After completion of the fermentation reaction, W02007141111 teaches lowering the pH of the mixture by the addition of sulphuric acid and optionally adding ammonium sulfate, which results in a broth comprising sulphides and L-lysine. The mixture is then concentrated, dried and granulated for use as a feed additive.

US 2011/269185 describes a method for increasing the value output of a fermentation reaction that yields a first product, intended for

commercialisation, such as ethanol, and a fermentation residual, which can be used as animal feed. Specifically, US 2011/269185 describes using genetically modified microorganisms that, in a fermentation process, yield a fermentation residual comprising a nutrient, such as an essential amino acid, in a greater concentration than that of unmodified

corresponding microorganisms when used in the fermentation process.

However, the animal feed supplements described require complex processing steps, which makes them expensive to produce. They also require additional storage, handling and transportation, which increases the cost of the animal feed ingredient considerably and reduces the overall profit achieved from the DDGS co-product revenue stream in the ethanol biorefinery industry.

Furthermore, the use of genetically modified microorganisms in the anaerobic fermentation reaction of the ethanol biorefinery increases the cost and complexity of the process.

Therefore, it is an object of the present invention to provide an efficient, cost effective process for obtaining a nutritionally enhanced fermentation residue from an ethanol biorefinery without the need for any modifications to the existing ethanol biorefinery process.

It is a further object of the invention to mitigate at least some of the disadvantages of the prior art. Further objects of the invention will be apparent from reading the following.

Disclosure of Invention

According to the first aspect of the invention, there is provided a process for increasing the nutrient content of a foodstuff, the process comprising:

(i) performing a first fermentation to produce a nutrient, the first

fermentation comprising the steps of:

a. providing a first feedstock;

b. introducing the first feedstock to a first vessel;

c. introducing a nutrient-producing microorganism to the first vessel; and

d. fermenting the first feedstock to produce a nutritionally

enhanced mixture comprising the nutrient and the nutrient- producing microorganism;

(ii) performing a second fermentation to produce an alcohol, the second fermentation comprising the steps of: a. providing a second feedstock;

b. introducing the second feedstock to a second vessel; and c. fermenting the second feedstock to produce a mixture

comprising an alcohol and a residue, the residue comprising a foodstuff; and

(iii) isolating the foodstuff from the alcohol and residue mixture;

wherein the nutritionally enhanced mixture is introduced into at least one of stage (ii) and stage (iii) of the process, and wherein the foodstuff is thereby nutritionally enhanced.

By the term“foodstuff” is meant a substance intended for consumption by humans and/or animals as food.

By the term“nutritionally enhanced” is meant a substance with an increased nutrient content over its ordinary nutrient content.

The nutrient-producing microorganism may be inactivated after it is introduced into at least one of stage (ii) and stage (iii) of the process.

Optionally, the nutrient-producing microorganism may be thermally inactivated after it is introduced into at least one of stage (ii) and stage (iii) of the process.

By the term“inactivated” is meant the destruction of activity of the nutrient- producing microorganism.

By the term“thermally inactivated” is meant the destruction of activity of the nutrient-producing microorganism by the action of heat.

The thermal inactivation may occur during the process without an additional heat treatment step. Optionally, the thermal inactivation may occur passively during the process. By the term“passively” is meant without being initiated by an additional step, i.e. , the thermal inactivation occurs during the process without an additional heat treatment step.

Optionally, the thermal inactivation may occur as a consequence of the process.

The nutrient-producing microorganism may be thermally inactivated by at least one of stage (ii) and stage (iii) of the process, wherein at least one of stage (ii) and stage (iii) of the process comprises heating to a temperature that thermally inactivates the microorganism. As the process does not require an additional heat treatment step to thermally inactivate the nutrient-producing microorganism, no modifications to stage (ii) and stage (iii) of the process (e.g., in an existing ethanol biorefinery) are required. Therefore, this reduces the cost and complexity of the process.

The first feedstock may comprise a carbohydrate suitable for producing a nutrient. Optionally, the second feedstock may comprise a carbohydrate suitable for producing an alcohol. Optionally, the carbohydrate may be a sugar. Optionally, the carbohydrate may be glucose or a source thereof.

By the term“sugar” is meant a poly-, oligo- or mono- saccharide. Suitable sugars include: starch, amylose, amylopectin, dextrins, maltose and glucose. At least one of the first feedstock and the second feedstock may be a biomass feedstock, optionally at least one of the first feedstock and the second feedstock may comprise a grain, optionally wherein the grain is a cereal grain, optionally at least one of wheat, maize, buckwheat, rye, barley, millet and rice.

The first fermentation may comprise the additional step of hydrolysing the first feedstock, optionally before introducing the first feedstock into the first vessel.

The second fermentation may comprise the additional step of hydrolysing the second feedstock, optionally before introducing the second feedstock into the second vessel. The nutritionally enhanced mixture comprising the nutrient and the nutrient-producing microorganism may be introduced into the second feedstock during the step of hydrolysing the second feedstock.

At least one of the first feedstock and the second feedstock may be hydrolysed, optionally the hydrolysed feedstock may be wheat

hydrolysate.

By the term“wheat hydrolysate” is meant the substance produced by hydrolysis of the wheat feedstock.

The hydrolysed first feedstock may comprise a solid phase and a liquid phase. Optionally, the solid phase and liquid phase may be separated, and substantially only the liquid phase of the hydrolysed first feedstock may be introduced to the first vessel. The second fermentation may comprise the steps of:

(i) introducing alcohol-producing fermentation conditions to the

second vessel; and

(ii) fermenting the second feedstock to produce a mixture comprising an alcohol and a residue, wherein the residue comprises the foodstuff.

Isolating the foodstuff from the alcohol and residue mixture may comprise the steps of:

(i) introducing the alcohol and residue mixture to a distillation

vessel; and

(ii) distilling the alcohol, wherein the distillation process is

configured to substantially separate the alcohol from the residue.

The nutritionally enhanced mixture may be introduced into the distillation vessel before the step of distilling the alcohol.

The residue comprising the foodstuff may comprise a solid phase and a liquid phase, the liquid phase comprising a nutrient and a solvent, optionally an aqueous solvent.

Isolating the foodstuff from the residue may further comprise the steps of: (i) separating the solid phase and the liquid phase;

(ii) evaporating at least part of the solvent from the liquid phase to increase the concentration of the nutrient in the liquid phase;

(iii) combining the concentrated liquid phase with the solid phase to provide a residue comprising the solid phase and the

concentrated liquid phase; and (iv) drying the residue comprising the solid phase and the

concentrated liquid phase to provide a foodstuff.

The nutritionally enhanced mixture may be introduced into the liquid phase before the step of evaporating at least part of the solvent from the liquid phase.

The foodstuff may be a feedstuff.

By the term“feedstuff” is meant a substance intended for consumption by animals as food.

The feedstuff may be selected from one or more of the group consisting of: distiller’s grains, wet distiller’s grains, wet distiller’s grains with solubles and dried distiller’s grains with solubles. Optionally, the feedstuff may be dried distiller’s grains with solubles.

The nutrient may be an amino acid, optionally an essential amino acid.

Optionally, the amino acid is selected from one or more of the group consisting of: lysine, methionine, arginine and leucine.

The essential amino acid may be selected from one or more of the group consisting of: lysine, methionine, threonine, tryptophan, valine, histidine, isoleucine, leucine and phenylalanine. Optionally, the essential amino acid is selected from one or more of the group consisting of: lysine, methionine and leucine. Optionally, the essential amino acid may be lysine. Optionally, the amino acid is arginine.

The first fermentation may be an aerobic fermentation and/or the second fermentation may be an anaerobic fermentation. The nutrient-producing microorganism may be an amino acid-producing microorganism. Optionally, the nutrient-producing microorganism may be an essential amino acid-producing microorganism.

The nutrient-producing microorganism may be selected from one or more of the group consisting of: Corynebacterium glutamicum, Bacillus megaterium, Bacillus brevis, Brevibacterium heali, Corynebacterium lilium, Candida biodini, Kluyveromyces lactis, Methylomonas sp., Providencia rettgeri, Pseudomonas sp., Saccharomyces cerevisiae, Brevibacterium flavum, Corynebacterium acetoacidophilum, Escherichia coli, Aerobacter aerogenes, Serratia marcescens, Aureobacterium flavescens,

Micrococcus glutamicus, Paracolobacterum coliforme, P. intermedium, Bacillus anthracis, and Bacillus cereus. Optionally, the nutrient-producing microorganism may be Corynebacterium glutamicum.

The alcohol may be ethanol.

Optionally, the process may be an integrated fermentation process.

According to a second aspect of the invention, there is provided a foodstuff obtainable, obtained or directly obtained by the process described in the first aspect. According to a third aspect of the invention, there is provided a process for increasing the essential amino acid content of a feedstuff, the process comprising:

(i) performing a first fermentation to produce an essential amino acid, the first fermentation comprising the steps of:

a. providing a first feedstock; b. introducing the first feedstock to a first vessel;

c. introducing an essential amino acid-producing microorganism to the first vessel; and

d. fermenting the first feedstock to produce a nutritionally

enhanced mixture comprising the essential amino acid and the essential amino acid-producing microorganism;

(ii) performing a second fermentation to produce ethanol, the second fermentation comprising the steps of:

a. providing a second feedstock;

comprising ethanol and a residue, the residue comprising a feedstuff; and

(iii) isolating the feedstuff from the ethanol and residue mixture;

wherein the nutritionally enhanced mixture is introduced into at least one of stage (ii) and stage (iii) of the process, and wherein the feedstuff is thereby nutritionally enhanced.

According to a fourth aspect of the invention, there is provided a feedstuff obtainable, obtained or directly obtained by the process described in the third aspect.

According to a fifth aspect of the invention, there is provided a process for increasing the lysine content of a feedstuff, the process comprising:

(i) performing a first fermentation to produce lysine, the first fermentation comprising the steps of:

a. providing a first feedstock;

b. introducing the first feedstock to a first vessel;

c. introducing a lysine-producing microorganism to the first vessel; and d. fermenting the first feedstock to produce a nutritionally enhanced mixture comprising lysine and the lysine-producing microorganism;

a. providing a second feedstock;

comprising ethanol and a residue, the residue comprising a feedstuff; and

(iii) isolating the feedstuff from the ethanol and residue mixture;

According to a sixth aspect of the invention, there is provided a feedstuff obtainable, obtained or directly obtained by the process described in the fifth aspect. The alternative features and different embodiments as described apply to each and every aspect and each and every embodiment thereof mutatis mutandis.

Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example, with reference to the drawings, in which:

Figure 1 is a flow diagram which illustrates a process in accordance with one embodiment of the invention; Figure 2 is a flow diagram which illustrates a process in accordance with a second embodiment of the invention; and

Figure 3 is a flow diagram which illustrates a process in accordance with a third embodiment of the invention.

Detailed Description

In a typical ethanol biorefinery, biomass feedstock, such as wheat or maize, undergoes anaerobic fermentation to yield ethanol as a first product, and a fermentation residue. The fermentation residue comprises unfermented grain residues and moisture, commonly referred to as wet distiller’s grains (WDG) and thin stillage, respectively. This fermentation residue is then processed to yield dried distiller’s grains with solubles (DDGS), which can be used as an animal feed ingredient.

The process for increasing the nutrient content of a foodstuff is broken down into three main stages, namely performing a first fermentation to produce a nutrient, performing a second fermentation to produce an alcohol and isolating the foodstuff from the alcohol and residue mixture. The product of the first fermentation is introduced into at least one of the second fermentation and the isolation of the foodstuff to yield a

nutritionally enhanced foodstuff. The process as further described below is summarised in the flow diagram of Figure 1.

Referring to Figure 1 , there is shown a process for producing nutritionally enhanced foodstuff from an ethanol biorefinery. A biomass feedstock that is rich in starch, e.g., wheat or maize, is milled or ground in a feedstock processing tank 10 to generate a flour. The flour is added to a mash tank 20 and mixed with water and enzymes, e.g., amylases, to generate a mash, which is then heated to hydrolyse the starch from the feedstock into fermentable sugars. The mash tank 20 is heated in two stages; the mash is heated to 85 °C for two hours, the temperature is then lowered to 60 °C and maintained at 60 °C for four hours. The resulting hydrolysed mash, which is rich in glucose, is then used as the feedstock for the aerobic fermentation reaction.

A portion (5-10% w/w) of the hydrolysed mash is removed from the mash tank 20 and provided to a first fermentation reaction vessel 130. The hydrolysed mash is optionally filter sterilised prior to being added to the first fermentation reaction vessel 130. The filter sterilisation process involves centrifugation of the hydrolysed mash, followed by filtration, optionally using 0.2 pm filters. The liquid phase is then added to the first fermentation reaction vessel 130.

The hydrolysed mash is then mixed with salts, at least one source of nitrogen, water and vitamins to generate a fermentation media. The glucose rich hydrolysed mash is utilised as the carbon source in the fermentation media. The salts added to the fermentation media are typically selected from calcium chloride dihydrate (CaCl2.2H20), ammonium sulfate ((NH₄)₂S0₄), magnesium sulfate heptahydrate

(MgS0_4.7H₂0), sodium chloride (NaCI), manganese (II) sulfate

monohydrate (MnS0_4.H₂0), iron sulfate heptahydrate (FeS0_4.7H₂0), potassium phosphate monobasic (KH2P0₄) and potassium phosphate dibasic (K₂HP0₄). Other components that are optionally added to the fermentation media include, but are not limited to, urea, yeast extract, peptone, L-serine, thiamine and D-biotin.

The fermentation media is cooled to 30 °C and inoculated with a nutrient-producing microorganism. The nutrient-producing microorganism used is dependent on the target nutrient required. Target nutrients are typically essential amino acids, which can enhance the nutritional value of the animal feed ingredient generated as a co-product in the process. The target nutrients can be essential amino acids selected from the group consisting of lysine, methionine, threonine, tryptophan, valine, histidine, isoleucine, leucine and phenylalanine, and are typically L-lysine, L- methionine, L-threonine or L-tryptophan. In one embodiment, the nutrient is an essential amino acid, typically L-lysine. Alternatively, the essential amino acid is methionine and/or leucine. Further amino acids can be the target nutrients, such as non-essential and/or conditionally non-essential amino acids. For example, the amino acid may be arginine.

It will be appreciated that the target nutrients could be more than one amino acid such that the nutritional value of the animal feed ingredient is enhanced by more than one amino acid. When the target nutrients are more than one amino acid, this may comprise a combination of one or more essential amino acids, non-essential amino acids and/or

conditionally non-essential amino acids. The nutrient-producing microorganisms can be selected from

Corynebacterium glutamicum, Bacillus megaterium, Bacillus brevis, Brevibacterium heali, Corynebacterium lilium, Candida biodini,

Kluyveromyces lactis, Methylomonas sp., Providencia rettgeri,

Pseudomonas sp., Saccharomyces cerevisiae, Brevibacterium flavum, Corynebacterium acetoacidophilum, Escherichia coli, Aerobacter aerogenes, Serratia marcescens, Aureobacterium flavescens,

Micrococcus glutamicus, Paracolobacterum coliforme, P. intermedium, Bacillus anthracis, and Bacillus cereus, and is typically Corynebacterium glutamicum. Aerobic conditions are maintained by aerating and agitating the media. Optimal conditions are dependent on the nutrient-producing

microorganism used. The product of the aerobic fermentation is a nutritionally enhanced fermented broth 140, which comprises target nutrient and the target nutrient-producing microorganism.

In one embodiment, as shown in Figure 1 , the nutritionally enhanced fermented broth 140 is introduced into the mash tank 20. This results in a nutritionally enhanced wheat mash, which is used as the carbon source for the anaerobic fermentation reaction in the ethanol biorefinery.

There is no requirement to process the fermented broth 140, e.g., to inactivate the nutrient-producing microorganism using a heat treatment step, prior to introducing the broth to the mash tank 20 of the ethanol biorefinery. This is because the mash tank is heated to a temperature that is sufficient to thermally inactivate the microorganism. Inactivation of the nutrient-producing microorganism at this stage is crucial to ensure that the microorganism does not interfere with the normal growth of yeast in the anaerobic fermentation.

The nutritionally enhanced mash is then provided to a second

fermentation reaction vessel 30. Anaerobic fermentation reaction conditions are introduced to the second vessel by lowering the

temperature to 30 °C and inoculating the nutritionally enhanced mash with an alcohol-producing microorganism, such as Saccharomyces cerevisiae.

The anaerobic fermentation reaction yields a nutrient rich fermented mash comprising ethanol and a residue, which is transferred to a distillation vessel 40. The distillation vessel 40 is operated at 63 °C under vacuum to separate the bioethanol 50 from the nutritionally enhanced fermentation residue 70. Carbon dioxide 60 is generated as a co-product of the distillation reaction.

The target nutrient remains in the post distillation fermentation residue 70, which comprises unfermented grain residues (wet distiller’s grain) and an aqueous solution comprising water and the target nutrient (thin stillage). The fermentation residue 70 is centrifuged to separate the nutritionally enhanced wet distiller’s grain (WDG) 100 and the nutritionally enhanced thin stillage 80. The thin stillage 80 is then concentrated via evaporation at 75 °C under vacuum using a rotary evaporator to remove at least part of the water and increase the concentration of target nutrient. The

concentrated thin stillage 90 is then mixed with the WDG to produce a nutritionally enhanced WDG slurry 110, which is dried at 85 °C to yield nutritionally enhanced dried distiller’s grains with solubles 120.

Referring to Figure 2, there is shown a second embodiment of the invention, wherein the nutritionally enhanced fermented broth 140 is introduced into the distillation vessel 40 in the ethanol biorefinery.

At this stage, inactivation of the microorganism is not critical because fermented mash in the distillation vessel 40 contains no glucose that can act as a carbon source for the microorganism. However, if inactivation of the microorganism was required, the distillation vessel 40 is heated to a temperature that is sufficient to inactivate the microorganism.

In the distillation vessel 40, the nutritionally enhanced fermented broth 140 is mixed with the fermented mash provided from the second fermentation reaction vessel 30. In this embodiment, the fermented mash would not be nutritionally enhanced prior to being mixed with the nutritionally enhanced fermented broth 140 in the distillation vessel 40.

As described above, the distillation vessel 40 is operated at 63 °C under vacuum to separate bioethanol 50 from the fermentation residue 70. Carbon dioxide 60 is also generated as a co-product of the distillation reaction.

The target nutrient remains in the post distillation fermentation residue 70 and is further processed as described above to yield nutritionally enhanced DDGS 120.

Referring to Figure 3, there is shown a third embodiment of the invention, wherein the nutritionally enhanced fermented broth 140 is introduced into the thin stillage component 80 of the fermentation residue 70.

In this particular embodiment, the WDG 100 component of the

fermentation residue 70 provided from the ethanol biorefinery will not be nutritionally enhanced. The nutrient is added to the DDGS via the nutritionally enhanced thin stillage component 80 of the fermentation residue 70.

The nutritionally enhanced fermented broth 140 is mixed with the thin stillage 80 in the rotary evaporator that is used to increase the

concentration of target nutrient in the thin stillage. The concentrated thin stillage 90 comprising target nutrient is then mixed with the WDG to produce a nutritionally enhanced WDG slurry 110, which is dried at 85 °C to yield nutritionally enhanced dried distiller’s grains with solubles 120. As described above, inactivation of the microorganism at this stage of introduction is not critical, however, the rotary evaporator is heated to a temperature that is sufficient to inactivate the microorganism. In addition, the drying of the nutritionally enhanced slurry 110, in any of the embodiments described above, is carried out at a temperature that is high enough to inactivate the microorganism. Therefore, the DDGS produced in the above described process will not contain any active microorganism and is deemed safe for use as a feed ingredient in all animal feeds.

Example 1 : L-lvsine as Target Nutrient

The process is exemplified using L-lysine as the target nutrient. Wheat is milled in a feedstock processing tank 10 to generate a flour, which is introduced into the mash tank 20 of the ethanol biorefinery process. In the mash tank 20, the flour is mixed with water and enzymes in a two stage process to generate wheat hydrolysate, which is a hydrolysed mash comprising fermentable sugars. In the first stage, the flour is mixed with water and an endoamylase, such as SPEZYME® CL WB. The mash tank 20 is then heated to 85 °C for two hours to hydrolyse the a-1 ,4-glucosidic bonds of the starch contained in the wheat feedstock into soluble dextrins and oligosaccharides. In the second stage, the hydrolysed mash is mixed with a saccharifying enzyme, such as OPTIDEX L 400. The temperature of the mash tank 20 is then lowered to 60 °C and maintained at 60 °C for four hours to convert the soluble dextrins and oligosaccharides into glucose.

A portion (5-10% w/w) of the hydrolysed mash comprising approximately 250 g/L glucose is removed from the mash tank 20, filter sterilised and provided to a first fermentation reaction vessel 130. The filter sterilisation process consists of centrifugation, followed by filtration using 0.2 pm filters. The resulting filtrate is then added the first fermentation reaction vessel 130.

The filtered mash is then mixed with salts, at least one source of nitrogen, water and vitamins to generate a fermentation media. In particular, the fermentation media in the first reaction vessel 130 comprises: filtered mash, calcium chloride dihydrate (CaCl2.2H20), ammonium sulfate ((NH₄)₂S0₄), magnesium sulfate heptahydrate (MgS0_4.7H₂0), sodium chloride (NaCI), manganese (II) sulfate monohydrate (MnS0_4.H₂0), iron sulfate heptahydrate (FeS0_4.7H₂0), potassium phosphate monobasic (KH₂P0₄), potassium phosphate dibasic (K₂HP0₄), urea, yeast extract, peptone, L-serine, thiamine and D-biotin.

The fermentation media is cooled to 30°C and inoculated with

Corynebacterium glutamicum (ATCC 21543), which is an

L-lysine-producing microorganism. Aerobic fermentation conditions are maintained by aerating and agitating the media to maintain a dissolved oxygen concentration value of 30%.

The product of the aerobic fermentation is a nutritionally enhanced fermented broth 140, which comprises L-lysine and Corynebacterium glutamicum (ATCC 21543). The fermented broth 140 is introduced into the mash tank 20. This results in a nutritionally enhanced wheat mash comprising approximately 185 g/L glucose and approximately 13.5 g/L L- lysine.

The whole fermented broth comprising Corynebacterium glutamicum (ATCC 21543) cells can be added directly to the mash tank because the mash tank is heated to a temperature that is sufficient to thermally inactivate the microorganism. This removes the requirement for an additional heat treatment processing step, which is typically required for current lysine feed supplements.

The nutritionally enhanced mash comprising approximately 13.5 g/L L-lysine is then provided to a second fermentation reaction vessel 30. Anaerobic fermentation reaction conditions are introduced to the second vessel 30 by lowering the temperature to 30 °C and inoculating the nutritionally enhanced mash with Saccharomyces cerevisiae grown overnight in yeast peptone dextrose.

The anaerobic fermentation reaction yields an L-lysine rich fermented mash, which is transferred to a distillation vessel 40. The distillation vessel 40 is operated at 63 °C under vacuum to separate bioethanol 50 from the L-lysine rich fermentation residue 70. Carbon dioxide 60 is generated as a co-product of the distillation reaction.

The L-lysine is retained in the post distillation fermentation residue 70, which consists of unfermented grain residues (wet distiller’s grain) and an aqueous solution comprising water and L-lysine (thin stillage). This fermentation residue is then processed as described below to obtain L- lysine enriched DDGS 120. The L-lysine enhanced fermentation residue 70 is centrifuged to separate L-lysine enhanced wet distiller’s grain (WDG) 100 from L-lysine enhanced thin stillage 80. The thin stillage 80 is then concentrated via evaporation at 75 °C under vacuum using a rotary evaporator to remove at least part of the water and increase the concentration of L-lysine. The concentrated thin stillage 90 is then mixed with the WDG to produce an L-lysine rich WDG slurry 110, which is dried at 85 °C to yield dried distiller’s grains with solubles 120 comprising approximately 11.3 g/L L-lysine.

As described above, an initial concentration of 13.5 g/L L-lysine in the mash tank 20 yields a final concentration of 11.3 g/L L-lysine in the DDGS 120. Therefore, throughout the ethanol biorefinery process an 84% recovery of L-lysine is achieved.

HPLC Analysis

The L-lysine concentration of the samples was obtained using HPLC. HPLC was also used to confirm that the structural integrity of the L-lysine was maintained throughout the process. Sample Preparation for HPLC Analysis

DDGS samples were crushed to a fine power using a motor and pestle. The resulting powder (0.6 g) was mixed with approximately 6 mL of HCI (6 M) and incubated at 80 °C for approximately 24 hours. After incubation, the samples were filtered using Whatman grade 1 filter paper to separate the suspended solids from the mixture. The resulting filtrate was then completely evaporated on a hot plate set at 50 °C and the resulting pellet was re-suspended in MilliQ water. This process was repeated three times to remove any trace amount of HCI present. The final pellet was then dissolved in 6 mL of borate buffer (0.4 M) and centrifuged for 15 mins at 9000 rpm. The supernatant was filter sterilised using 0.2 pm filters and made up to a final volume of 10 mL. The samples were stored at -20 °C until they were required for HPLC analysis. HPLC Parameters The HPLC samples were analysed on an Agilent 1290 UPLC using the conditions detailed in the below table.

Table 1 : HPLC Parameters

HPLC Results A range of samples from various stages throughout the process were analysed to determine the structural integrity of L-lysine throughout the process.

The following samples were analysed by HPLC:

(i) fermented broth comprising L-lysine and Corynebacterium glutamicum (ATCC 21543);

(ii) wheat mash comprising L-lysine; and (iii) DDGS comprising L-lysine.

The results for all three samples showed a consistent peak at 14.4 minutes. This peak was shown to represent L-lysine using an L-lysine standard that was analysed using the same experimental conditions. The consistent peak observed at 14.4 minutes from samples obtained at various stages throughout the process indicated that the structural integrity of the L-lysine was maintained during the ethanol biorefinery process. Ethanol Yield from Biorefinerv Process using L-Lysine Enriched Wheat

Mash

A comparison study was performed to determine if the use of L-lysine enriched wheat mash would have any detrimental effect on the ethanol yield from the biorefinery process.

The anaerobic fermentation reaction of the ethanol biorefinery process was carried out using standard wheat mash, i.e. , wheat mash that had not been enriched with the L-lysine fermented broth, and wheat mash comprising L-lysine enriched fermented broth.

The ethanol yield was determined by calculating the concentration of ethanol (g/L) produced relative to the concentration of glucose (g/L) in the hydrolysed mash used in the ethanol biorefinery process. The

concentration of glucose in the feedstock and the concentration of ethanol produced were measured using HPLC.

The HPLC samples were analysed on an Agilent 1200 with Refractive Index Detector (RID) using the conditions detailed in the below table.

Table 2: HPLC Parameters for Glucose and Ethanol Analysis

The net loss of CO2 during the ethanol biorefinery process was also calculated by measuring difference in the weight of the hydrolysed mash at the beginning and end of the fermentation reaction.

The results of the comparison study are shown in Table 3 below.

Table 3: Ethanol Yield from Biorefinerv Process using Standard Wheat

Mash and Wheat Mash comprising L-lvsine fermented broth The differences in the ethanol yield using the L-lysine mash compared to the standard mash were found to be minimal and were deemed

insignificant. Therefore, the use of L-lysine enriched mash comprising L-lysine and Corynebacterium glutamicum (ATCC 21543) was found to have no detrimental effect on the anaerobic fermentation reaction in the ethanol biorefinery process. The applicability of the process described herein to other amino acids was exemplified by the addition of pure amino acid at the start of the process. This obviated the need to source and culture different specific

microorganisms that are high producers of different amino acids. These further examples are described in below and are designated as Examples 2 (L-arginine), 3 (L-leucine) and 4 (L-methionine). HPLC analysis and parameters were identical to Example 1.

Example 2: L-arqinine as Target Nutrient Cereal is milled to generate a flour, which is introduced into the mash tank 20 for processing. In the mash tank 20, the flour is mixed with water, amino acid (L-arginine (at a concentration of 15 g/L)) and enzymes in a two stage process to generate a mash comprising fermentable sugars and the amino acid. In the first stage, the flour is mixed with water and an endoamylase, such as SPEZYME® CL WB. The mash tank 20 is then heated to 85 °C for two hours to hydrolyse the a-1 ,4-glucosidic bonds of the starch contained in the cereal feedstock into soluble dextrins and oligosaccharides. In the second stage, the hydrolysed mash is mixed with a saccharifying enzyme, such as OPTIDEX L 400. The temperature of the mash tank 20 is then lowered to 60 °C and maintained at 60 °C for four hours to convert the soluble dextrins and oligosaccharides into glucose.

This process results in a nutritionally enhanced cereal mash comprising approximately 200 g/L glucose and 15 g/L of amino acid (L-arginine).

The nutritionally enhanced mash generated is then diluted to provide a glucose concentration of 150 g/L and an amino acid (L-arginine) concentration of 11.25 g/L. This nutritionally enhanced mash comprising approximately 11.25 g/L of amino acid (L-arginine) is then provided to a fermentation reaction vessel 30. Anaerobic fermentation reaction conditions are introduced to the second vessel 30 by lowering the temperature to 30 °C and inoculating the nutritionally enhanced mash with Saccharomyces cerevisiae grown overnight in yeast peptone dextrose.

The anaerobic fermentation reaction yields ethanol and an amino acid (L- arginine) rich fermented mash, which is transferred to a distillation vessel 40. The distillation vessel 40 is operated at 63 °C under vacuum to separate bioethanol 50 from the amino acid rich fermentation residue 70.

The amino acid (L-arginine) is retained in the post distillation fermentation residue 70, which consists of unfermented grain residues (wet distiller’s grain) and an aqueous solution comprising water and amino acid (thin stillage). This fermentation residue is then processed as described below to obtain amino acid (L-arginine) enriched DDGS 120.

The amino acid (L-arginine) enhanced fermentation residue 70 is centrifuged to separate amino acid (L-arginine) enhanced WDG 100 from amino acid (L-arginine) enhanced thin stillage 80. This thin stillage 80 was then concentrated via evaporation at 75 °C under vacuum using a rotary evaporator to remove at least part of the water and increase the concentration of amino acid (L-arginine). The concentrated thin stillage 90 is then mixed with the WDG to produce an amino acid (L-arginine) rich WDG slurry 110, which is dried at 85 °C to yield dried distiller’s grains with solubles 120 comprising 8.5 g/L L-arginine.

As described above, an initial concentration of 11.25 g/L L-arginine in the mash tank 20 yields a final concentration of 8.5 g/L L-arginine in the DDGS 120. Therefore, throughout the ethanol biorefinery process a 76% recovery of L-arginine is achieved.

Example 3: L-leucine as Target Nutrient

Example 3 was carried out using identical parameters to Example 2. This resulted in dried distiller’s grains with solubles 120 comprising 6.6 g/L L- leucine.

As described above, an initial concentration of 11.25 g/L L-leucine in the mash tank 20 yields a final concentration of 6.6 g/L L-leucine in the DDGS 120. Therefore, throughout the ethanol biorefinery process a 59% recovery of L-leucine is achieved.

Example 4: L-methionine as Target Nutrient Example 4 was carried out using identical parameters to Example 2. This resulted in dried distiller’s grains with solubles 120 comprising 5.1 g/L L- methionine.

As described above, an initial concentration of 11.25 g/L L-methionine in the mash tank 20 yields a final concentration of 5.1 g/L L-methionine in the DDGS 120. Therefore, throughout the ethanol biorefinery process a 46% recovery of L-methionine is achieved.

All of the above-mentioned amino acids have been documented to be produced using Corynebacterium glutamicum and, as illustrated above, a microorganism that produces an amino acid can be introduced to the process, the microorganism being inactivated of as part of the process, and the amino acid passing through the process resulting in a nutritionally enhanced DDGS. Examples 1-4 demonstrate that the process of the present invention can apply to different amino acids, and it is clear that more than one amino acid could be used in the process concurrently.

The improved process as described herein provides an efficient, cost effective process for producing nutritionally enhanced animal feed ingredient, such as dried distiller’s grains with solubles, from an ethanol biorefinery. To date, DDGS has been nutritionally enhanced using nutrient supplements, which are added to the DDGS as an additional step before the feed ingredient is provided to the animals. This results in increased storage, handling and transportation costs which considerably adds to the animal feed ingredient cost and reduces the overall profit obtained from the DDGS co-product revenue stream in the ethanol biorefinery industry.

In addition, previous methods to enhance the nutrient content of the DDGS include modifying the anaerobic fermentation reaction in the ethanol biorefinery. This process requires the use of genetically modified microorganisms and the introduction of additional chemicals, which increases the cost and complexity of the process.

Therefore, the present invention provides an efficient, cost effective process for obtaining nutritionally enhanced DDGS from an ethanol biorefinery. The process can be incorporated into existing ethanol biorefineries and no additional chemicals or modifications to the existing process are required. Thus, the nutritionally enhanced DDGS produced can be provided directly for use as an animal feed ingredient, without the need for additional nutrient supplements. This results in a more efficient, cost effective process to increase the revenue obtained from the DDGS co-product.

While this invention has been described with reference to the sample embodiments thereof, it will be appreciated by those of ordinary skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.

Claims

1. A process for increasing the nutrient content of a foodstuff, the process comprising:

(i) performing a first fermentation to produce a nutrient, the first fermentation comprising the steps of:

a. providing a first feedstock;

b. introducing the first feedstock to a first vessel; c. introducing a nutrient-producing microorganism to the first vessel; and

d. fermenting the first feedstock to produce a nutritionally enhanced mixture comprising the nutrient and the nutrient-producing microorganism;

(ii) performing a second fermentation to produce an alcohol, the second fermentation comprising the steps of:

a. providing a second feedstock;

b. introducing the second feedstock to a second vessel; and c. fermenting the second feedstock to produce a mixture comprising an alcohol and a residue, the residue comprising a foodstuff; and

(iii) isolating the foodstuff from the alcohol and residue mixture; wherein the nutritionally enhanced mixture is introduced into at least one of stage (ii) and stage (iii) of the process, and wherein the foodstuff is thereby nutritionally enhanced.

2. A process as claimed in claim 1 , wherein the nutrient-producing microorganism is inactivated after it is introduced into at least one of stage (ii) and stage (iii) of the process, optionally wherein the nutrient-producing microorganism is thermally inactivated.

3. A process as claimed in claim 2, wherein the thermal inactivation occurs during the process without an additional heat treatment step.

4. A process as claimed in claim 2 or claim 3, wherein the nutrient- producing microorganism is thermally inactivated by at least one of stage (ii) and stage (iii) of the process, wherein at least one of stage (ii) and stage (iii) of the process comprises heating to a temperature that thermally inactivates the microorganism.

5. A process as claimed in any preceding claim, wherein the first

feedstock comprises a carbohydrate suitable for producing a nutrient and/or wherein the second feedstock comprises a carbohydrate suitable for producing an alcohol, optionally wherein the carbohydrate is a sugar, optionally wherein the carbohydrate is glucose or a source thereof.

6. A process as claimed in claim 5, wherein at least one of the first feedstock and the second feedstock is a biomass feedstock, optionally wherein at least one of the first feedstock and the second feedstock comprises a grain, optionally at least one of wheat, maize, buckwheat, rye, barley, millet and rice.

7. A process as claimed in any preceding claim, wherein at least one of the first fermentation and the second fermentation comprise the additional step of hydrolysing the first feedstock and/or the second feedstock, optionally before introducing the first feedstock into the first vessel and/or the second feedstock into the second vessel.

8. A process as claimed in claim 7, wherein the nutritionally enhanced mixture comprising the nutrient and the nutrient-producing microorganism is introduced into the second feedstock during the step of hydrolysing the second feedstock.

9. A process as claimed in claim 7 or claim 8, wherein at least one of the first feedstock and the second feedstock is hydrolysed, optionally wherein the hydrolysed feedstock is wheat hydrolysate.

10. A process as claimed in any one of claims 7 to 9, wherein the

hydrolysed first feedstock comprises a solid phase and a liquid phase, and wherein the solid phase and liquid phase are separated, and wherein substantially only the liquid phase of the hydrolysed first feedstock is introduced to the first vessel.

11. A process as claimed in any preceding claim, wherein the second fermentation comprises the steps of:

(i) introducing alcohol-producing fermentation conditions to the second vessel; and

(ii) fermenting the second feedstock to produce a mixture

comprising an alcohol and a residue, wherein the residue comprises the foodstuff.

12. A process as claimed in any preceding claim, wherein isolating the foodstuff from the alcohol and residue mixture comprises the steps of:

(i) introducing the alcohol and residue mixture to a distillation vessel; and

(ii) distilling the alcohol, wherein the distillation process is

configured to substantially separate the alcohol from the residue.

13. A process as claimed in claim 12, wherein the nutritionally enhanced mixture is introduced into the distillation vessel before the step of distilling the alcohol.

14. A process as claimed in claim 12 or claim 13, wherein the residue comprising the foodstuff comprises a solid phase and a liquid phase, the liquid phase comprising a nutrient and a solvent, optionally an aqueous solvent.

15. A process as claimed in claim 14, wherein isolating the foodstuff from the residue further comprises the steps of:

(i) separating the solid phase and the liquid phase;

(iii) combining the concentrated liquid phase with the solid phase to provide a residue comprising the solid phase and the concentrated liquid phase; and

(iv) drying the residue comprising the solid phase and the

concentrated liquid phase to provide a foodstuff.

16. A process as claimed in claim 15, wherein the nutritionally

enhanced mixture is introduced into the liquid phase before the step of evaporating at least part of the solvent from the liquid phase.

17. A process as claimed in any preceding claim, wherein the foodstuff is a feedstuff.

18. A process as claimed in claim 17, wherein the feedstuff is selected from one or more of the group consisting of: distiller’s grains, wet distiller’s grains, wet distiller’s grains with solubles and dried distiller’s grains with solubles, optionally wherein the feedstuff is dried distiller’s grains with solubles.

19. A process as claimed in any preceding claim, wherein the nutrient is an amino acid, optionally wherein the nutrient is an essential amino acid.

20. A process as claimed in claim 19, wherein the essential amino acid is selected from one or more of the group consisting of: lysine, methionine, threonine, tryptophan, valine, histidine, isoleucine, leucine and phenylalanine; optionally wherein the essential amino acid is selected from one or more of the group consisting of: lysine, methionine and leucine; optionally wherein the essential amino acid is lysine.

21. A process as claimed in any preceding claim, wherein the first

fermentation is an aerobic fermentation and/or wherein the second fermentation is an anaerobic fermentation.

22. A process as claimed in any preceding claim, wherein the nutrient- producing microorganism is an amino acid-producing

microorganism, optionally wherein the nutrient-producing

microorganism is an essential amino acid-producing

microorganism.

23. A process as claimed in claim 22, wherein the nutrient-producing microorganism is selected from one or more of the group consisting of: Corynebacterium glutamicum, Bacillus megaterium, Bacillus brevis, Brevibacterium heali, Corynebacterium lilium, Candida biodini, Kluyveromyces lactis, Methylomonas sp., Providencia rettgeri, Pseudomonas sp., Saccharomyces cerevisiae,

Brevibacterium flavum, Corynebacterium acetoacidophilum, Escherichia coli, Aerobacter aerogenes, Serratia marcescens, Aureobacterium flavescens, Micrococcus glutamicus,

Paracolobacterum coliforme, P. intermedium, Bacillus anthracis, and Bacillus cereus, optionally wherein the nutrient-producing microorganism is Corynebacterium glutamicum.

24. A process as claimed in any preceding claim, wherein the alcohol is ethanol.

25. A foodstuff obtainable, obtained or directly obtained by the process of any one of claims 1 to 24.