Field of the invention The invention relates to the field of food products, more specifically to a process for the preparation of a food product wherein the preparation of said food product comprises a fermentation step. The invention further relates to a food product obtainable by such process.

Background of the invention

A process for food preparation relates to a set of methods and techniques used to transform raw ingredients into food or to transform food into other forms for consumption by humans or animals either in the home or by the food processing industry. Food processing typically takes clean, harvested crops or butchered animal products and uses these to produce attractive, marketable and often long shelf-life food products. Similar processes are used to produce animal feed

Different type of food processing exists such as fermenting, sun drying, preserving with salt, cooking.

The deliberate fermentation of foods by man predates written history and is possibly the oldest method of preserving perishable foods. Preserving by fermentation not only made foods available for future use, but more digestible and flavorful. The nutritional value produced by fermenting is another benefit of fermenting.

Fermentation is an important process in food preparation. It is the controlled decay of material using microorganisms which results in a more desirable product. Technically, fermentation is the biochemical conversion of sugars, starches, or carbohydrates, into alcohol, and organic acids, by microorganisms and (their) enzymes. The desirable microorganisms may prevent deterioration of the food by inhibiting the growth of the spoiling types of microorganisms. Most fermentation processes lower the pH of foods preventing harmful microorganisms to live with too acidic an environment. Controlled fermentation processes encourage the growth of desirable bacteria which starves, or fights off, the "bad" microbes. Depending on what is fermented, or the manner of fermentation, foods can remain consumable for prolonged periods, up to years. During the fermentation process of milk, such as in the production of most cheeses, yoghurt and buttermilk, the bacteria or yeast in the fermentation culture produces enzymes that break down the lactose. The organisms use lactose as a nutrient, usually producing acidic (by) products from the digestion of the lactose.

Moreover, fermentation enhances the flavors of some foods, such as in the extended fermentation of black teas, aged cheese, wine, and beer, which creates their distinctive flavors. Fermentation increases nutritional values with the biochemical exchange it produces, and may allow man to live healthier lives. For example fermented dairy products, like cheese, yogurt, and kefir, can be consumed by those not able to digest the raw milk, and aid the digestion and well-being for those with lactose intolerance and autism.

Cheese flavor is very important in dairy industry. Cheese manufacturers aim to improve the flavor of cheeses without substantially modifying the manufacturing technology and without extending the ripening time.

It is known that amino acid catabolism is a major process for flavor formation, especially in cheese prepared with only lactic acid bacteria (LAB). Lactic acid bacteria are the most commonly used microorganisms for preservation of foods. Their importance is associated mainly with their safe metabolic activity while growing in foods, utilizing available sugar for the production of organic acids and other metabolites. Their common occurrence in foods and feeds coupled with their long-lived use contributes to their GRAS status (Generally Recognized As Safe) for human consumption.

Lactic acid bacteria convert the carbohydrate energy source of food, such as lactose in milk, to lactic acid; examples are yogurt and cheeses from milk, and pickles from fruits and vegetables. Alternatively, yeasts, including but not limited to Saccharomyces species, may convert glucose to ethanol and carbon dioxide in leavened breads, or the sugars in grain or fruit beverages into alcohol and carbon dioxide in beers and wines. Molds or filamentous fungi also can be active in certain fermentations, such as Stilton cheese and soy sauce. It is estimated that about one-third of all food consumed has been fermented.

The conversion of amino acids to flavor compounds by lactic acid bacteria is essentially initiated by a transamination reaction. Amino acid transamination is catalyzed by aminotransferases and results in the formation of alpha-ketoacids, which are then degraded, via one or more additional steps, into the different aroma compounds, whereas the alpha- ketoacid acceptor is transformed into the corresponding amino acid. Alpha-Ketoglutarate (aKG) has been reported as the best alpha-ketoacid acceptor for amino acid transamination by Lactococcus lactis; other alpha-ketoacids, especially pyruvate, can also be used, but the aminotransferase activities have been reported to be up to 40 times lower with pyruvate than with aKG. However, for some lactic acid bacteria strains, it appeared that pyruvate may be as efficient an acceptor as aKG (Tanous C. et al. 2005). A few years ago, Yvon, Berthelot, and Gripon (1998) showed that the conversion of amino acids to aroma compounds by lactic acid bacteria in cheese was limited by the lack of an alpha-ketoacid acceptor for transamination reactions (Tanous C. et al. 2005).

Flavour compounds are formed from the central metabolite pyruvate in the cytoplasm. Citrate fermentation is a strain-specific trait amongst prokaryotic lactic acid bacteria that is associated with the production of carbon dioxide and C4 flavor compounds (diacetyl, acetoin and butanediol). During carbohydrate/citrate co-metabolism, additional pyruvate from citrate added to the central pyruvate pool in the glycolytic pathway is converted to acetoin. Citrate is transported into the cell by the secondary transporter CitP. Inside, citrate is converted to acetate and oxaloacetate catalyzed by citrate lyase (CitL). Acetate leaves the cell while oxaloacetate is decarboxylated to pyruvate by a soluble oxaloacetate decarboxylase termed CitM. alpha-acetolactate synthase converts two molecules of pyruvate to one molecule of alpha-acetolactate while releasing carbon dioxide. The majority of alpha-acetolactate is decarboxylated to acetoin by alpha-acetolactate decarboxylase. A small part of the chemically unstable alpha-acetolactate results in the formation of diacetyl in a nonenzymatic oxidative decarboxylation reaction (Pudlik, A.M. and Lokema, J.S., 2011).

Only a few strains of lactic acid bacteria are able to ferment citrate. The ability to metabolize citrate by L. lactis is invariably linked to endogenous plasmids that contain the gene encoding the transporter that is responsible for uptake of citrate from the medium. Citrate transporters have been found in strains belonging to the genera Lactococcus and Leuconostoc. Both are gram-positive bacteria and used in fermentation process of food products. In the dairy industry, these Lactococcus and Leuconostoc strains are used in mixed cultures, and metabolism of citrate is important in many fermentation processes because of the formation of carbon dioxide, diacetyl, acetoin, and 2,3-butanediol, compounds that contribute to the organoleptic properties of the fermentation products (Bandell, M. et al, 1998). Species of Lactococcus lactis have been classified in three industrially significant phenotypes: L. lactis subsp. lactis (lactis phenotype), L. lactis subsp. cremoris (cremoris phenotype), and L. lactis subsp. lactis biovar diacetylactis (di acetyl actis phenotype). Within all Lactococcus species, citrate is fermented only by L. lactis subsp. lactis biovar diacetylactis. The genes encoding the citrate metabolic pathway are located on the chromosome, in contrast to the gene encoding the citrate uptake system that is encoded by plasmids (Kelly & Ward, 2002; Rademaker et al, 2007). Most of diacetylactis strains used in laboratory work are cured of own plasmids which results in lack of citrate uptake system.

Citrate fermentation in Leuconostoc mesenteroides results in the formation of an electrochemical proton gradient across the cell membrane (proton motive force) by a secondary mechanism in which the citP plays a crucial role. The transporter catalyzes exchange of divalent anionic citrate and monovalent lactate, which results in the generation of a membrane potential with physiological polarity (i.e., the inside is negative). Lactate is a product of citrate fermentation during cometabolism with a carbohydrate (citrolactic fermentation). Upon entering the cell, citrate is cleaved by citrate lyase, which yields acetate and oxaloacetate. Decarboxylation of the latter compound yields carbon dioxide and pyruvate. Pyruvate functions as a sink for the reducing equivalents produced in the heterofermentative carbohydrate degradation pathway and is converted to the end product lactate, which leaves the cell in exchange for citrate (precursor-product exchange). The decarboxylation of oxaloacetate plays an additional role in metabolic energy generation since it consumes a cytoplasmic proton, which results in the generation of a transmembrane pH gradient with physiological polarity (i.e., the inside is alkaline). Together, the charge translocation catalyzed by the transporter and the proton consumption in the decarboxylation step are equivalent to the pumping of a proton out of the cell. In citrate-fermenting Lactococcus species the initial steps of citrate breakdown proceed through the same intermediates as those observed in Leuconostoc species, and the homofermentative carbohydrate metabolism in lactococci is likely to produce enough lactate for the CitP to function as a citrate-lactate exchanger (Bandell, M. et al., 1998).

The genes coding for the citrate carriers of Lactococcus lactis (citPll) and Leuconostoc lactis (citPlcl) have been found to be almost identical, suggesting that the energetics of the citrate metabolic pathway described above for Leuconostoc mesenteroides is also valid for lactococci. Thus, the citrate metabolism in Lactococcus lactis results in the generation of a proton motive force (Bandell, M. et al., 1998).

The presence of a functional pathway for the formation of aKG by L. lactis appears to be essential for flavor formation from amino acids. There are three main natural pathways for the formation of aKG by bacteria from components present in cheese or milk (glutamate, citrate, pyruvate). Firstly, the glutamate dehydrogenase (GDH) pathway produces aKG directly by oxidative deamination of glutamate, utilizing NAD, NADP or both as a cofactor. GDH activities have recently been revealed in lactic acid bacteria. NADP dependent activity was detected in most Lactobacillus plantarum strains and in several strains of L. lactis from vegetal origin, Lb. paracasei and Streptococcus thermophilus strains, whereas NAD- dependent activity was observed in only a few L. lactis and S. thermophilus strains. Moreover, it has been demonstrated that the ability of lactic acid bacteria to produce aroma compounds from amino acids is closely related to their GDH activity (Tanous et al, 2005).

The second possible pathway (citrate-oxaloacetate pathway) leads to the production of aKG from citrate and glutamate by requiring the successive action of citrate permease (CitP), citrate lyase (CitL) and aspartate aminotransferase (Asp- AT). CitP allows citrate uptake inside the cells and CitL initiates citrate catabolism by transforming citrate to oxaloacetate (OAA). OAA can then be transformed into aspartate and aKG, in the presence of glutamate, by an Asp-AT. In L. lactis species, only the diacetylactis subspecies possesses CitP and CitL; but, in this subspecies, OAA is mainly decarboxylated to pyruvate, which is then transformed to lactate, acetate and diacetyl (Cocaign- Bousquet, Garrigues, Loubiere, & Lindley, 1996). However, the conversion of OAA to Asp has never been investigated. Finally, the third pathway for the formation of aKG (citrate-isocitrate pathway) utilises the oxidative branch of the tricarboxylic acid (TCA) cycle and leads to the production of aKG from either pyruvate or citrate. The production of aKG from pyruvate necessitates the action of pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), citrate synthase (CS), aconitase (ACN) and isocitrate dehydrogenase (IDH). PDH and PC are necessary to degrade pyruvate into AcetylCoA and OAA, respectively, both used by CS to synthesize citrate. Citrate is then transformed by ACN into isocitrate, which is finally oxidized to aKG by IDH. For the production of aKG from citrate, only CitP for citrate uptake and the action of ACN and IDH are necessary (Tanous et al., 2005)..

Previously it has been proposed to enhance the proteolysis in cheeses so as to increase the quantity of free amino acids. The proteolytic system of lactococci has been widely studied, several peptidases have been purified and characterized and their genes have been cloned and sequenced. However, although the over-expression of peptidases increases the accumulation of the free amino acids, it does not significantly affect the development of flavor. It therefore appears that the factors limiting the development of flavor do not exist at the level of the production of free amino acids, but are also involved in their degradation (US 2002/0127300). Previous attempts to improve flavor compound production by adding aKG to the fermentation medium were shown to be very ineffective. US 2002/0127300 relates to a process for the production of a cheese or of a cheese flavored food product, characterized in that a preparation additive comprising at least one keto acid chosen from the group consisting of aKG, and the keto acids which are direct precursors of flavor compounds, such as alpha- ketoisocaproate, ketoisovalerate, and phenylpyruvate, is used to enhance the flavor of the said product. The preparation of the said product comprises a maturation (pre-ripening) step in the presence of at least one lactic acid bacterium, and the said additive is added to the said product, prior to the said maturation (pre-ripening) step or during it.

The basis of cheese making relies on the fermentation of lactose by lactic acid bacteria and the production of lactic acid which lowers the pH and in turn assists coagulation, promotes syneresis, helps prevent spoilage and pathogenic bacteria from growing, contributes to cheese texture, flavor and keeping quality. Lactic acid bacteria also produce growth factors which encourages the growth of non-starter organisms, and provides lipases and proteases necessary for flavor development during curing.

The production of aKG, which is required for amino acid transamination, by lactic acid bacteria is often a limiting factor in the conversion of amino acids to flavor compounds in cheeses. Since many attempts to improve flavor compound production by adding aKG to the medium were very ineffective, there is still a need to for the development of an improved process for the preparation of a food product, in particular for improving the flavor of a food product.

Description of the invention Surprisingly, it has been demonstrated that the low transamination activity using alpha-ketoglutarate (aKG) as precursor and ultimately resulting in the formation of flavor compounds during fermentation of food products is limited by the lack of transport of aKG into the cell and not due to low transaminase activity in the cytoplasm. The inventors have arrived at the surprising feature that the citrate transporter citP is capable of transporting both aKG into the cell and the produced keto acid flavor compound out of the cell. Introducing the citrate transporter citP in a strain of lactic acid bacteria (LAB) resulted in higher production of keto acid flavor compounds than observed in its absence. Therefore, lactic acid bacteria strains comprising an aKG transporter, preferably the citrate transporter citP, may conveniently be used in the preparation of food product using LAB.

Accordingly, the present invention provides a process for the preparation of a food product comprising a fermentation step in the presence of a lactic acid bacterium expressing an aKG transporter, preferably citP, wherein aKG is present in said fermentation step. Preferably, lactate is also present in the fermentation step since this enhances the citP mediated uptake of aKG.

Preferably, in the process according to the invention, at the start of the fermentation step at least 0.1%, 0.5%, 1%, 5% 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the bacteria present are lactic acid bacteria expressing an aKG transporter, preferably citP. Preferably, at most 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% of the bacteria present are lactic acid bacteria expressing an aKG transporter, preferably citP. Preferably, 0.1% - 99.9%, 1% - 99%, 5% - 95%, 10% - 95%, 20% - 95%, 30% - 95%, 40% - 95%, 50% - 95%, 60% - 95%, 70% - 95%, 80% - 95%, 90% - 95% of the bacteria present are lactic acid bacteria expressing an aKG transporter, preferably citP. Preferably, 0.1-90%, 1% - 90%, 5% - 90%, 10% - 90%, 20% - 90%, 30% - 90%, 40% - 90%, 50% - 90%, 60% - 90%, 70%) - 90%), 80%) - 90%), of the bacteria present are lactic acid bacteria expressing an aKG transporter, preferably citP. Preferably, 10% - 95%, 10% - 90%, 10% - 80%, 10% - 70%, 10% - 60%, 10% - 50%, 10% - 40%, 10% - 30%, 10% - 20%, 5% - 10%, 1% - 10%, 0.1% - 10%) of the bacteria present are lactic acid bacteria expressing an aKG transporter, preferably citP.

Preferably, in the process according to the invention, at the start of the fermentation step at least lE+04/ml, 5E+04/ml, lE+05/ml, 5E+05, lE+06/ml, 5E+06/ml, lE+07/ml, 5E+07/ml, lE+08/ml, 5E+08/ml, lE+09/ml, 5E+09/ml viable lactic acid bacteria expressing an aKG transporter, preferably citP are present. Preferably, in the process according to the invention, at the start of the fermentation step at least lE+04/ml - 5E+09/ml viable lactic acid bacteria expressing an aKG transporter, preferably citP are present.

The food product in the process according to the invention may be any known food product. Preferably, the food product is a product wherein the preparation comprises a fermentation step. The food product may comprise compounds of plant or animal origin, and may contain essential nutrients, such as carbohydrates, fats, proteins, vitamins, and/or minerals. The food product may be intended for ingestion by an organism and subsequently assimilation by the organism's cells to produce energy, maintain life, and/or stimulate growth. The food product in the process according to the invention includes but is not limited to a product selected from the group consisting of a dairy-, grain-, vegetable-, fruit-, fish-, or meat- based product. The term "based product" is herein defined that the food product is produced from a specific raw material such as dairy, grain, vegetable, fruit, fish, or meat. The food product may be based on a mixture of different raw materials e.g. a mixture of dairy and grain or a mixture of meat and fruit.

The fermentation step in the process according to the invention may be any type of fermentation known to the person skilled in the art as described earlier herein. Preferably, the fermentation step involves fermentation with a lactic acid bacterium expressing an aKG transporter, preferably citP. During the fermentation step also other microorganisms such as Propionibacterium, Brevibacterium, Pediococcus, Enterococcus and/or Oenococcus may be present.

In the process according to the invention, the fermentation process preferably affects the maturation of the food product by enhancing the flavor formation.

The process according to the invention may comprise a separate maturation step wherein the food product matures during a period which may be from hours to years wherein the texture and flavor of the food product develop to an extent desired. The maturation may also be comprised in or overlap another step in the process according to the invention; the maturation may e.g. already start during the fermentation and continue after the fermentation. The maturation may even continue until the food product is consumed or prepared further. Since the process of flavor formation is more efficient in the process according to the invention, said maturation step may preferably be shortened in time from about 10 to 90%, from about 20 to 90%, from about 30 to 90%, from about 40 to 90%, from about 50 to 90%, from about 60 to 90%, from about 70 to 90%, from about 80 to 90%, from about 85 to 90%.

The maturation may preferably be shortened in time at least 10%, at least 15%, at least

20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%), or most preferably 99%.

The aKG transporter may be any transporter that is capable of transporting aKG into the cell of a lactic acid bacterium. Preferably, the aKG transporter is citP.

CitP may be any citP. Preferably citP according to the invention is a citP able to transport aKG. Preferably citP is from a prokaryotic cell, wherein said prokaryotic cell is a bacterium; more preferably, said bacterium is a lactic acid bacterium; a preferred lactic acid bacterium is Leuconostoc or Lactococcus lactis, more preferably Lactococcus lactis ssp diacetyllactis.

In the process according to the invention, a preferred citP is a citP with the amino sequence of SEQ ID NO: 1 or an equivalent thereof. An equivalent citP may be any polypeptide with citP activity including transport of aKG into the cell with an amino acid sequence that has preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 1.

In the process according to the invention, a preferred citP encoding nucleic acid is a nucleic acid with the sequence of SEQ ID NO: 2 or an equivalent thereof. An equivalent citP may be any nucleic acid sequence with at least that has preferably at least 30%, 35%, 40%, 45%, 50%, 55%, 60% 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 2. The citP encoding nucleic acid may also be a homologous citP encoding nucleic acid.

Sequence identity is herein preferably defined as a percentage of identity and is determined by calculating the ratio of the number of identical nucleotides/amino acids in the sequence divided by the length of the total nucleotides/amino acids of said sequence, preferably minus the lengths of any gaps.

The terms "homologous" gene or polypeptide are herein defined as a gene or polypeptide that is obtainable from a strain that belongs to the same species, including variants thereof, as does the strain actually containing the gene or polypeptide. Preferably, the donor and acceptor strain are the same. Fragments and mutants of genes or polypeptides are also considered homologous when the gene or polypeptide from which the mutants or fragments are derived is a homologous gene or polypeptide. Also non-native combinations of regulatory sequences and coding sequences are considered homologous as long as the coding sequence is homologous. It follows that the term heterologous herein refers to genes or polypeptides for which donor and acceptor strains do not belong to the same species or variants thereof.

Expression is herein defined as any step involved in the production of the citP polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post- translational modification, and if appropriate, secretion. The aKG transporter, preferably citP, may already be present in the lactic acid bacterium. The encoding nucleic acid may e.g. be present in the genome or may be present extrachromosomally, e.g. on a separate vector such as a plasmid. The nucleic acid encoding the alpha keto glutarate transporter, preferably citP, may also be introduced into the lactic acid bacterium using classical genetic means (non-recombinant) and recombinant means known to the person skilled in the art. Such means include, but are not limited to: an expression construct comprising the encoding nucleic acid and if required control fragments such as a promoter and terminator fragment such as to induce expression of the encoding nucleic acid; a vector known to the person skilled in the art comprising the encoding nucleic acid or the expression construct, such as a plasmid or cosmid; a bacteriophage used as a vector. Expression may require amplifying, sequencing and/or cloning of the nucleic acid encoding the aKG transporter, preferably citP. The person skilled in the art knows how to perform these techniques since they are well known in the art.

The citrate transporter citP plays a pivotal role in the kinetics of the citrate metabolism pathway. CitP is a member of the 2-hydroxycarboxylate transporter (2-HCT) family (transporter classification [TC] 2. A.47), in which the malate/lactate exchanger mleP, which functions in the malolactic fermentation pathway in lactic acid bacteria, and the Na ⁺ -citrate symporter citS, which functions in citrate fermentation in Klebsiella pneumoniae, are also found. The name of the family refers to the 2-hydroxycarboxylate motif, i.e., HO-CR ₂ -COO ^" , shared by the substrates of the transporters in the family. Kinetic studies of citP revealed two modes of transport, symport of divalent citrate with one proton and exchange of divalent citrate with monovalent lactate. Since the former was much slower than the latter, it was concluded that citP is a symporter that was optimized to catalyze exchange, which would be the physiological function. Chase studies with membrane vesicles loaded with radiolabeled citrate showed a remarkable tolerance of citP (and mleP, but not citS) to the R substituents of 2-hydroxycarboxylate substrates. CitP accepted R groups differing in size and charge, ranging from H atoms in glycolate to acetyl groups in citrate and everything in between in physiological or nonphysiological substrates. The specificity of citP for substrates carrying different charges forms the mechanistic basis for membrane potential generation (Pudlik, A.M. and Lolkema, J. S., 201 1).

The activity of citP can be measured by the activity of the citrate fermenting pathway on plates and by resting cells and by direct transport assays using membrane vesicles and radiolabeled citrate. Using plates, cit+ and cit- are selected as blue and white colonies, respectively, on Kempler and McKay plates (Kempler and Mckay, 1980).

Citrate consumption by resting cells are assayed in a total volume of 1.5 ml. Resting cells at an OD ₆ 6o of 6 in 50 mM potassium phosphate pH 5.8 buffer are incubated at 30 °C without agitation for 10 min. At t=0, citrate iss added at a concentration of 2 mM together with glucose. Samples of 100 μΐ of the cell suspensions are taken every 5 min and immediately centrifuged for 0.5 min at maximum speed in a table top centrifuge. The supernatant is stored on ice until further analysis by the enzymatic assays using a citrate assay kit and/or by HPLC (see below). Measurements of the concentrations of citrate are usually in good agreement between the two methods.

Transport studies are performed in 50 mM potassium phosphate pH 6, at a membrane protein concentration of 0.25-0.3 mg/mL and usually at 30 °C. Membrane vesicles prepared from cells containing CitP were preincubated at 30 °C for 10 min in 50 mM potassium phosphate pH 7 containing ionophores and the radiolabeled substrates. An artificial pH gradient was generated by adding a small aliquot of H ₂ SO ₄ 0.2 N resulting in a pH drop of 1 unit. The generation of an inverted membrane potential (positive inside) was obtained by a thiocyanate diffusion potential. Membranes were concentrated in 50 mM potassium phosphate pH 6, containing 100 mM potassium thiocyanate. At the zero time point, concentrated hybrid membranes were diluted 100-fold into the same buffer without SCN- containing 4.5 μΜ [l,5-14C]citrate. Uptake assays were performed by the rapid filtration method. Samples (100 μΐ.) were taken at subsequent time points, transferred into 2 mL ice- cold 0.1 mM LiCl to stop the reaction and filtered through 0.45 μπι pore-size cellulose-nitrate filters (Schleider & Schull, GmbH). Filters were rinsed with 2 mL ice-cold 0.1 mM LiCl, transferred to scintillation vials and the internalized radioactivity was determined.

In the process according to the invention, a preferred host organism expressing an aKG transporter, preferably citP, is a lactic acid bacterium. Other organisms expressing an aKG transporter, preferably citP that are capable of converting aKG into flavor compounds may also be used. Such organism may be a prokaryotic or a eukaryotic cell. The host organism expressing an aKG transporter, preferably citP, used in the process according to the invention may have an impaired or partly impaired citrate metabolism in order to reduce C0 ₂ production during fermentation when C0 ₂ would be an undesired product.

When the host cell is a eukaryotic cell, the cell may be any eukaryotic cell and is preferably a fungus. Said fungus can be a yeast or a mold such as Penicillium. When said fungus is a yeast, a preferred yeast is a yeast selected from Saccharomyces and Kluyveromyces, preferably Kluyveromyces lactis. Another preferred yeast is selected from Geotricum candidum, Yarrowia lipolitica, Pichia fermentans and Debaryomyces hansenii.

When the host cell is a prokaryotic host cell, the host cell is preferably a lactic acid bacterium. Lactic acid bacteria are widely used in the preparation of food products using fermentation, including the production of cheese, cider, sausages, pickled vegetables, beer or wine, some breads, and other fermented foodstuffs, such as soymilk, kefir, buttermilk, and others. Accordingly, the process according to the invention can conveniently be used in the preparation of cheese, cider, sausages, pickled vegetables, beer or wine, some bread, and other fermented foodstuffs, such as soymilk, kefir, buttermilk, soured milk, yoghurt, acidophilus milk, quark, ymer, filmyolk, viili, kumis, sour cream, creme fraiche, pavlaka.

Formula 1

Molecular Weight: 146.09814 [g/mol]

Molecular Formula: C ₅ H ₆ 0 ₅ aKG (formula 1) is a keto acid produced by de-amination of glutamate, and is an intermediate in the Krebs cycle. aKG is used for enhancing the flavor of a food product whose preparation comprises a step of maturation (ripening) in the presence of lactic acid bacteria and in particular of lactococci and is an important biological compound. The enhancement of the flavor by aKG preferably results in an increase of catabolism of amino acids mediated by said citP expressing lactic acid bacteria of at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or 100 fold.

In the process according to the invention, the lactic acid bacterium expressing an aKG transporter, preferably citP may be any lactic acid bacterium. Preferably, the lactic acid bacterium is a Lactococcus species and/or a Leuconostoc species such as Leuconostoc mesenteriodes, Leuconostoc citreum, Leuconostoc mesenteriodes ssp. cremoris, Leuconostoc mesenteriodes ssp. dextranicum and Leuconostoc mesenteriodes ssp. lactis. More preferably, the lactic acid bacterium is a Lactococcus species. A preferred Lactococcus species is Lactococcus lactis.

Lactococcus lactis is a gram-positive bacterium used extensively in the production of food products, including but not limited to buttermilk and cheese. L. lactis cells are cocci that group in pairs and short chains, and, depending on growth conditions, appear ovoid with typically 0.5 - 1.5 μιη in length. L. lactis does not produce spores (non-sporulating) and are not motile (non-motile). The capability to produce lactic acid is one of the reasons why L. lactis is one of the most important microorganisms in the dairy industry. L. lactis is of crucial importance for manufacturing dairy products, such as buttermilk and cheeses.

Lactococcus lactis is a food-grade bacterium that is widely used in food production and more preferably in the dairy industry. During the last two decades, significant advances have been made in the field of lactococcal genetics and protein expression systems. Consequently, we have seen the emergence of new areas for the application of engineered L. lactis for protein expression, gene delivery, vaccine delivery, and therapeutic drug delivery. L. lactis is considered an advantageous host for protein expression and delivery. The bacterium has an established safety profile through its long use in fermented dairy products and is considered as a GRAS (generally recognized as .safe) microorganism.

Preferred subspecies of L. lactis are, but are not limited to a species selected from the group consisting of J. lactis ssp. cremoris, L. lactis ssp. hordniae, L. lactis ssp. lactis, andL. lactis ssp. diacetylactis.

More preferably, the L. lactis in the process according to the invention is L. lactis ssp. diacetylactis. Accordingly, preferably, in the process according to the invention the lactic acid bacterium expressing an aKG transporter, preferably citP is a Lactococcus lactis and/or a Leuconostoc. More preferably, in the process according to the invention the lactic acid bacterium expressing an aKG transporter, preferably citP is a L. lactis ssp. diacetylactis. In the process according to the invention, a mixture of two or more lactic acid bacteria expressing an aKG transporter, preferably citP, and as described herein may be used, such a Leuconostoc and a Lactococcus, preferably Lactococcus lactis. Preferred Leuconostoc species are selected from the group consisting of Leuconostoc mesenteriodes, Leuconostoc citreum, Leuconostoc mesenteriodes ssp. cremoris, Leuconostoc mesenteriodes ssp. dextranicum and Leuconostoc mesenteriodes ssp. lactis; preferred Lactococcus lactis species are but are not limited to a species selected from the group consisting of L. lactis ssp. cremoris, L. lactis ssp. hordniae, L. lactis ssp. lactis, andL. lactis ssp. diacetylactis.

In an embodiment of the process according to the invention, a lactic acid bacterium expressing an aKG transporter, preferably citP, does preferably not comprise or does preferably not consist exclusively of Lactococcus lactis strain C RZ157 and/or aKG transporter (preferably citP) expressing derivatives of Lactococcus lactis strain C RZ157.

Preferably, in the process according to the present invention, the food product is a dairy fermentation product. A preferred dairy fermentation product is cheese. A dairy product is herein defined as any food product, whether liquid or solid at room temperature, that has milk or a part thereof as an ingredient or is made with or from milk or a part thereof. A part of milk may be any part, such whey, casein, milkfat, milkprotein and includes any product that can be derived from milk by any process.

Cheese in the context of the present invention is a food product known to the person skilled in the art that is typically made from coagulated curds that are pressed together to form a more or less solid texture that is usually allowed to ripen. Cheese is a generic term for a diverse group of milk-based food products; as such the raw material used to prepare cheese is also referred to as cheese-base; a preferred cheese-base is milk. Cheese comprises proteins and fat from milk. Suitable milk used to prepare cheese is from cows, buffalo, goats, or sheep or mixes thereof, but is in the context of the invention not limited thereto. Milk may have higher or lower fat contents. Suitable vegetable or animal fat may be added to the cheese base, e.g. palm oil. The person skilled in the art knows what fat is suitable for making cheese.

A conventional method of making cheese from milk is given here as a non-limiting example and typically comprises at least several of the following basic steps:

a) a fermentation step;

b) coagulation by means of modification of micelles of milk casein in order to provide a three-dimensional protein network, the coagulum;

c) slicing the coagulum and eliminating lactoserum in order to provide the curd; d) grinding the curd, optionally pressing the curd in moulds;

e) acidification, optionally salting or brining the curd, and

f) refining the curd.

Cheese styles, textures and flavors may depend on the origin of the milk, temperatures, times, target pH for different steps, the sequence of processing steps, the use of salting or brining, block formation, and aging. All these parameters may vary considerably between cheese types. Typical steps that contribute to the development of flavor and texture are a fermentation step and a maturation (ripening) step.

In the process according to the invention the cheese may e.g. be but is not limited to Gouda, Cheddar, Edam, Brie, Camembert, Stilton, Gorgonzola, Blue cheese, Goat cheese, Swiss cheese, Emmental, Gruyere, Brick and Mozarella.

A preferred cheese in the process according to the invention is a cheese selected from the group consisting of Gouda, Cheddar, Edam, Brie, Camembert, Stilton, Gorgonzola, Blue cheese, Goat cheese, Swiss cheese, Emmental, Gruyere, Brick and Mozarella. A most preferred cheese is a Gouda type cheese.

The process according to the invention may comprise a step wherein the cheese base is subjected to a thermal and mechanical processing step of the initial cheese base (preferably milk), optionally after fractionation thereof, at a temperature T, in order to obtain a limited destructuring of the protein network of the initial cheese base, wherein T is maximum of 60° C, 70° C, 80° C, or 90° C. Preferably T is 80° C.

When the food product in the process according to the invention is cheese, fermentation is preferably a step wherein the dairy product, preferably milk is pre-treated, possibly pre-ripened comprising addition of a lactic acid bacteria culture expressing an aKG transporter, preferably citP, appropriate to the desired type of cheese and mixed with a coagulant, such as a rennet. Rennet is an enzyme derived from the lining of the stomachs of calves.. Calf rennet contains two active components: the enzymes chymosin and pepsin. A rennet may also be from a non-animal source, such a vegetable rennet from plants or a microbial rennet from bacteria or fungi. Said non-animal rennet may be a recombinant rennet; e.g. the gene encoding calf chymosine may be cloned into a yeast or a filamentous fungus.

In the process according to the invention, a lactic acid bacterium expressing an aKG transporter, preferably citP, may be added in any stage during the preparation of the food product, as long as fermentation occurs and thus flavor development is enhanced. When the food product is cheese, a lactic acid bacterium expressing an aKG transporter, preferably citP, may be added in any stage during the preparation of the cheese, e.g. be added to the coagulum, or to the curd after slicing the coagulum, or after grinding the curd, or while pre- ripening the cheese. A preferred stage in the process according to the invention wherein a lactic acid bacterium expressing an aKG transporter, preferably citP, is added to the coagulum. A lactic acid bacterium expressing an aKG transporter, preferably citP, may be added in any amount effective to initiate fermentation. Preferably, at least 1E+04 viable bacteria per ml are added. When the food product is cheese, preferably 1E+04 viable bacteria per ml are added.

In the process according to the invention a lactic acid bacterium expressing an aKG transporter, preferably citP may be added once of multiple times or even continuously during each of the steps of the process according to the invention.

The process according to the invention for the preparation of a food product comprising a fermentation step in the presence of a lactic acid bacterium expressing an aKG transporter, preferably citP, may further comprise a thermal and mechanical processing step in order to obtain a limited destruction of the protein network in the dairy product base. Such a thermal and mechanical processing step preferably has the following parameters. After inoculation with the starter culture, the food product is typically held for 45 to 60 min at 25 to 30° C to ensure the bacteria are active, growing and have developed acidity. This stage is called pre-ripening the food product and is preferably done prior to renneting if the food product is milk and the food product to be prepared is cheese. Accordingly, a process according to the invention for the preparation of a food product preferably comprises a pre- ripening step wherein the lactic acid bacterium expressing an aKG transporter, preferably citP, is actively growing. Preferably, when the food product to be prepared is cheese, the pre- ripening step is done prior to renneting.

The term "food product being prepared" is defined as the total amount of food product present at the moment. During fermentation, this will be the total amount of non-matured food product present during fermentation; i.e. the entire fermentation substance present.

During the preparation of a food product using a method according to the invention, a food preparation additive may used. Food preparation additives include substances added to food to preserve flavor or enhance its taste and appearance. Some additives have been used for centuries; for example, preserving food by pickling (with vinegar), salting, as with bacon, preserving sweets or using sulfur dioxide as in some wines. A preferred food preparation additive in a process according to the invention is aKG. In most cases aKG will already present in sufficient amounts at the beginning of the fermentation since aKG is already present in many of the raw materials described previously herein. Furthermore, during the fermentation, aKG is actively produced by the microorganisms used in the fermentation. According to an embodiment no additional aKG is added to the fermentation. It may however be desired to add additional aKG to the fermentation. Accordingly, in the process according to the invention, aKG may be added as a preparation additive. aKG may be added in any convenient amount and from any source, such as pure synthetic aKG, or viable or non-viable microorganisms comprising aKG. A preferred amount is from O.OOlmM to lOOmM aKG added relative to the food product being prepared. More preferably, from 0.0 lmM to lOOmM aKG, from 0. lmM to 20mM aKG, from 0.5mM to 20 mM aKG, or most preferably, from 2 to 10 mM aKG is added.

The aKG as a preparation additive can be added once of multiple times or even continuously during each of the steps of the process according to the invention. Preferably, aKG is added at the start of the fermentation step in the process for the preparation of a food product. Another preferred preparation additive is lactate since lactate enhances the citP mediated uptake of aKG. In most cases lactate will already present in sufficient amounts at the beginning of the fermentation since lactate is already present in many of the raw materials described previously herein. Furthermore, during the fermentation, lactate is actively produced by the microorganisms used in the fermentation. According to an embodiment no additional lactate is added to the fermentation. It may however be desired to add additional lactate to the fermentation.

Since flavor compounds production from aKG in the medium of L. lactis is limited by the uptake of the substrate into the cell, the inventors showed that introducing the citrate transporter citP that transports aKG in a strain of L. lactis resulted in a higher production of the keto acid flavor compounds in the cell. According to the process of the invention, introducing the citrate transporter citP in a strain of L. lactis preferably results in at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold higher production of keto acids than observed in its absence. Preferably introducing the citrate transporter citP in a strain of L. lactis resulted in at least 10 fold higher production of keto acid flavor compounds than observed in its absence. The amount of aKG and keto acid flavor compounds in a food product or any matrix is preferably determined using HPLC/RP-HPLC analysis, preferably as described in the examples herein. The process according to the first aspect of the invention can conveniently be used for the preparation of a food product.

Accordingly, in a second aspect, the present invention provides a food product obtainable by a process according to the first aspect of the invention. Preferably, said food product is obtained by a process according to the first aspect of the invention. Preferably the food product according to the invention comprises at least a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold higher flavor compounds than a food product prepared by process without a citP expressing lactic acid bacterium.

The food product according to this aspect may be any food product described in the first aspect of the invention. Preferably, the food product is selected from the group consisting of cheese, cider, sausages, pickled vegetables, beer or wine, some bread, and other fermented foodstuffs, such as soymilk, kefir, buttermilk, soured milk, yoghurt, acidophilus milk, quark, ymer, filmyolk, viili, kumis, sour cream, creme fraiche and pavlaka. Preferably, the food product is a dairy fermentation product. A preferred dairy fermentation product is cheese, such as soft cheese, semi soft cheese, semi-hard cheese, hard cheese, freash cheese, wey cheese, stretched curd cheese, soft ripened cheese, washed rind cheese, blue cheese, and processed cheese. A preferred cheese is a cheese selected from the group consisting of Gouda, Cheddar, Edam, Brie, Camembert, Stilton, Gorgonzola, Blue cheese, Goat cheese, Swiss cheese, Emmental, Gruyere, Brick and Mozarella. A most preferred cheese is a Gouda type cheese.

In this document and it its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the element. The indefinite article "a" or "an" thus usually means "at least one". All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures

Figure 1 : Production of alpha-keto-isocaproic acid (black) and alpha-keto-isovaleric acid (grey) from 2 mM L-leucine (black) or 2 mM L- valine (grey) in the presence of 2 mM alpha- ketoglutarate by resting cells of L. lactis strain IL1403 (A), IL1403 treated 0.15 % Triton X- 100 (B), and IL1403(pFL3) (C).

Figure 2: FIPLC detection of alpha-keto-isocaproic acid (A) and alpha-keto-isovaleric acid (B). Elution of standard solutions of 0.2 mM alpha-keto-isocaproic acid (A, track 1) and 0.2 mM alpha-keto-isovaleric acid (B, track 1), supernatant of L. Lactis IL1403(pFL3) (track 2) and L. Lactis IL1403 (track 3). Other tracks show incubation in the presence of only alpha- ketoglutarate or L-leucine or L-valine.

Figure 3 : Concentration of alpha-ketoglutarate consumed and alpha-keto-isocaproic acid (A,C) or alpha-keto-isovaleric acid (B,D) produced from L-leucine (A, C) or L-valine (B, D), respectively, by resting cells of L. Lactis IL1403(pFL3) in the presence (A, B) and absence (C, D) of 0.15 % Triton X-100.

Examples HPLC RP-HPLC analysis.

Products of a-ketoglutarate transamination (a-keto acids and its further metabolites) were determined by loading an aliquot of 10 μΐ of the supernatant on an Aminex FIPX-87H anion- exchange column with dimensions 300 x 7.8 mm (Bio-Rad Laboratories, Inc., Richmond, CA) operated at 30 oC in isocratic mode using 0.005 M H2S04 as the mobile phase and a flow rate of 0.8 ml/min. Amino acids were analyzed by RP-HPLC after DEEMM derivatization. Detection of aminoenone derivatives was performed in an Alltech Platinum EPS C18 column with dimensions 250 x 4.6 mm operated at 25 oC through the binary gradient shown in Table 1 with a flow rate 0.8 ml/min. 25 mM acetate pH 5.8 buffer with 0.02 % sodium azide was used as the eluent A, 80:20 mixture of acetonitrile and methanol was used as the eluent B. The target compounds were identified according to the retention times and were quantified using the internal standard method. Standard deviations were obtained from at least 3 different experiments.

Example 1 : Uptake of alpha-ketoglutarate limits transamination by L. lactis

L. lactis strain IL1403 was grown until the mid-exponential growth phase (OD ₆ 6o=0.6) in M17 broth medium at pH 7.0 supplemented with 0.5 % (w/v) glucose. The cells were harvested, washed twice, and resuspended at an OD ₆ 6o of 1.5 in 50 mM Kpi pH 5.8 buffer. The cells were incubated in the same buffer at 30 °C in the presence of 2 mM alpha- ketoglutarate, 0.05 mM PLP, and 2 mM of either L-valine or L-leucine.

Incubation of the cells in the same buffer at 30 °C in the presence of 2 mM alpha- ketoglutarate, 0.05 mM PLP, and 2 mM of either L-valine or L-leucine resulted in only minor amounts of alpha-keto-isocaproic acid, the keto acid formed from L-leucine, while no alpha- keto-isovaleric acid, the keto acid formed from L-valine could be detected (Figure 1A). In marked contrast, when the integrity of the cytoplasmic membrane was destroyed by treating the cells with the detergent Triton X-100 at a concentration of 0.15 % significant amounts of both keto acids were produced (Figure IB). In 3 hr of time, the cells produced 0.35 mM alpha-keto-isocaproic acid and 0.42 mM alpha-keto-isovaleric acid. It follows that the capacity of transamination in the cytoplasm is much higher than suggested by the whole cell experiments and that a transport step determines the rate of the overall process.

Example 2: Citrate transporter CitP transports alpha-ketoglutarate

L. lactis strain IL1403(pFL3) produces the citrate transporter CitP in trans from a non- native promoter which decouples citP expression from native control. The strain was grown as described above and resuspended in the same buffer. Under the same conditions as described above, the cells produced 62 μΜ of alpha-keto-isocaproic acid and 48 μΜ of alpha-keto- isovaleric acid (Figure 1C). The presence of CitP in the membrane boosted the production of alpha-keto-isocaproic acid by a factor of 10 and even much higher in case of alpha-keto- isovaleric acid. Example 3 : Production of keto acids follows from transamination activity

Incubation of resting cells of J. lactis strains IL1403 and IL1403(pFL3), i.e. the strain without and with CitP, respectively, with 2 mM of alpha-ketoglutarate resulted only in the production of alpha-keto-isocaproic acid when also L-leucine was present and only with the CitP expressing strain (Figure 2 A). No product formation was observed when only alpha- ketoglutarate or only L-leucine was present. The same results were obtained with L-valine as the amino donor (Figure 2B).

Example 4: alpha-Ketoglutarate is stoichiometrically converted to the keto acids

The yield of alpha-keto-isocaproic acid and alpha-keto-isovaleric acid produced from L- leucine and L-valine, respectively, when L. lactis IL1403(pFL3) incubated in the presence of 2 mM of alpha-ketoglutarate was determined. The yield was determined both for intact and permeabilized cells. In both cases and for both amino acids the yield was approximately 100 % (Figure 3). This is important as it shows that the transamination pathway is the only degradation pathway for alpha-ketoglutarate and all alpha-ketoglutarate is available for flavor compound formation.

Table 1 shows the absolute amounts of alpha-keto-isocaproic acid and alpha-keto-isovaleric acid produced by L. lactis IL1403(pFL3) and compares it to data from the literature. The low amounts produced over extended periods of time in previous experiments are most likely due to lysis of cells which releases the transaminases into the surroundings.

Table 1. Flavor compounds produced from L-leucine or L-valine with alpha-ketoglutarate as an amino acceptor.

Amino acid Concentration of flavor compounds

Reference Strain Conditions ¹

donor (μΜ) calculated per 3 h OD= 1.5 t= 3 Leu 62

Process according L. lactis subsp. lactis

OD

invention IL1403(pFL3)

pH Val 48 t= 40 h

L. lactis subsp. Cremoris

Kieronczyk et al. (2003) OD= 20 Leu

NCD0763

pH= 5.5

L. lactis subsp. Cremoris TIL46 t= 40 h Leu 16

Rijnen et al. (1999) (NCD0763 cured of its 2-kb OD= 10

plasmid) pH=8 Val 9

L. lactis subsp. Cremoris TIL46 t= 40 h Leu 19

Chambellon and Yvon

(NCD0763 cured of its 2-kb OD= 10

(2003) Val 11

plasmid) pH= 8

List of references

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Sun, J., Aluvila, S., Kotaria, R., Mayor, J. A., Walters, D. E., Kaplan, R. S., 2010. Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis. Mol Cell Pharmacol, 2(3): 101-110.

Tanous, C, Gori, A., Rijnen, L., Chambellon, E., Yvon, M., 2005. Pathways for a- ketoglutarate formation by Lactococcus lactis andtheir role in amino acid catabolism. International Dairy Journal, 15: 759-770.

Yvon, M., Berthelot, S., Gripon, J. C, 1998. Adding aketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. International Dairy Journal, 8: 889-898. Pudlik, A. M., and Lolkema, J. S., 2011. Citrate Uptake in Exchange with Intermediates in the Citrate Metabolic Pathway in Lactococcus lactis IL1403. Journal of Bacteriology, 193(3): 706-714.

US 2002/0127300. Use of keto acids to enhance the flavour of cheese products.

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