A method of producing organic acids or salts thereof.

Abstract

A method of producing organic acids or salts thereof from a dairy side steam is provided. The method comprises an upstream processing process to remove minerals from the dairy side stream and subsequent fermentation using a bacterial strain capable of converting lactose to propionic acid, to provide a fermentation broth comprising one or more organic acids or organic acid salts.

Description

[0001] Title of the invention

[0002] A method of producing organic acids or salts thereof.

[0003] Field of the Invention

[0004] The present invention relates to methods of producing organic acids or salts thereof. Also contemplated is organic acids and organic acid salts produced according to the method of the invention and their use.

[0005] Background of the Invention

[0006] The salts of organic acids (also referred to as “organic acid salts”) produced during bacterial fermentation are known to inhibit the growth of spoilage microorganisms, especially fungi, in food products. Therefore, the end-product of bacterial fermentation, containing a high level of organic salts, can be used as a food preservative to extend the shelf-life of food including baked goods. They are also used as animal feed preservatives.

[0007] Propionic acid is one such organic acid. It is a carboxylic acid with the chemical formula CH3CH2CO2H. Propionibacterium sp., are known to produce metabolites from sugar, including propionic acid, by fermentation. Propionibacteria are pleomorphic catalase- and grampositive, anaerobic, aerotolerant bacteria that produce propionic acid as the main product via fermentation together with other products such as acetic acid. Traditionally Propionibacteria have been used as one of the starter cultures of Swiss-type cheese to produce the characteristic flavour and eyes. More recently, Propionibacteria are used in the production of food products. Both lactose and lactate are suitable carbon sources for propionic acid fermentation. The fermentation metabolism of Propionibacteria differs depending on the carbon source used. When sugar is available, the Embden-Meyerhof-Parnas (EMP) pathway is used as described in Antone II, et al, (Antone II et al., Propionic Acid Fermentation — Study of Substrates, Strains, and Antimicrobial Properties. Fermentation. 2023; 9(1):26).

[0008] One of the major limitations to current methods of producing propionic acid is the low yield. Large scale production by Propionibacteria can have severe inhibition of end products during cell growth. The formation of by-products, such as acetic acid and succinic acid, can also be a challenge. Organic acids, such as propionic acid and their salts are generally produced via petrochemical routes, which are not sustainable and increase the environmental pressure associated with the production of the organic acids and salts. Organic acid synthesis via microbial pathways during the fermentation of sugars such as lactose from dairy side streams is a more sustainable production route. Propionic acid is a known by-product of biological fermentation for vitamin B12. EP3140416 discloses a method to produce a biotechnological product, such as vitamin B12, using cocultivation of Propionibacterium and a yeast cell, from sweet or sour whey.

[0009] Various preservatives are available on the market that increase the shelf life of bread and other bakery products. Examples of these preservatives include calcium propionate, sodium propionate, potassium propionate, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate and potassium benzoate. A disadvantage of some preservatives is that they change the organoleptic properties that could be unsatisfactory, such as taste, of the product.

[0010] EP3917334 discloses an anti-mould bakery additive, that comprises propionate and other natural organic acid salts, which is produced by microbial fermentation of carbohydrates, such as grain flours, using a consortium of three Propionibacterium strains, namely Propionibacterium theonii NCIM 2932, Propionibacterium freudenreichii NCIM 2111 and Propionibacterium shermanii NCIM 5137.

[0011] WO2018 / 029219 describes a process for producing pure lactic acid from a whey by-product rich in lactose and minerals. The process described includes processing the whey by-product including a neutralisation step to precipitate calcium and phosphate salts, prior to fermentation to produce lactic acid.

[0012] It is an aim of the current invention to solve one or more problems of the prior art and provide a method of producing an organic acid, such as propionic acid.

[0013] Summary of the Invention

[0014] The Applicant has developed a process, typically a three-stage process, for producing organic acids or salts thereof, from dairy side streams, such as dairy whey side streams. The method involves upstream processing of the dairy side stream to remove minerals (monovalent and / or multivalent ions) and provide a substrate, bacterial fermentation of the substrate to provide a fermentation broth comprising one or more organic acid salts and optional downstream processing of the fermentation broth to recover the organic acid salts.

[0015] Dairy side streams naturally contain a large amount of monovalent ions, such as sodium, potassium and chloride. Multivalent ions, such as calcium, phosphorus, sulphur, and magnesium are also present. The method of the invention comprises an upstream processing step. This upstream processing step removes ions and provides a suitable fermentation substrate by reducing the overall mineral content and thus facilitating microbial fermentation. This involves pH adjustment to a suitable pH, e.g. neutralisation,, separation, and optional membrane fractionation and ultra-heat treatment which may be used as alternative steps or used in combination.

[0016] The pH of the dairy side stream is adjusted to the required pH using a suitable base. Neutralisation of the dairy side stream is carried out with a suitable base, such as a metal hydroxide base. This step precipitates insoluble salts, typically calcium and phosphate salts, typically 50% or more, which can be removed by a suitable separation process, such as centrifugation. After removal the product is typically called a clarified product or substrate. This step may be used on its own or it may be followed by filtration.

[0017] A membrane fractionation method, such as nanofiltration / diafiltration, removes a large proportion of monovalent ions from the dairy side stream. This step may be used on its own, or it may follow a pH adjustment step in which the clarified substrate undergoes filtration. After filtration, the product is typically called a demineralised product. This may be fully or partially demineralised depending on whether or not clarification has taken place.

[0018] This upstream processing procedure generates a more suitable fermentation substrate by reducing the mineral content and increasing the lactose content. Additional minerals generates a higher osmotic pressure environment in the fermentation substrate that may inhibit bacterial cell growth, decrease organic acid productivity and lead to incomplete fermentations. Therefore, upstream pre-treatment can enhance the bioconversion of lactose to organic acids and their salts during fermentation improving the overall fermentation performance such as lactose to organic acid productivity, yield and conversion.

[0019] As aspect of the invention provides a method to produce organic acids, or salts thereof, from a dairy side stream, comprising the steps of: incubating the dairy side stream with a bacterial strain capable of converting lactose to propionic acid, to provide a fermentation broth comprising one or more organic acids selected from propionic acid, acetic acid and succinic acid, or salts thereof.

[0020] In an embodiment, the method comprising the steps of: adjusting the pH of the dairy side stream to a pH of from about 5 to about 10, to precipitate multivalent ions, separating the precipitate to provide a clarified product, optionally filtering the clarified product to provide a demineralised product and / or heat treating the product, incubating the product with a bacterium capable of converting lactose to propionic acid to provide a fermentation broth containing one or more organic acids or organic acid salts.

[0021] In an embodiment, the method does not include a pH adjustment step or separation of the precipitate step prior to fermentation. Instead, the upstream processing of the method includes taking the dairy side stream directly to the filtering step and / or the step heat treatment. In this method, the dairy side stream is (i) filtered to provide the demineralised product, optionally followed by heat treatment, or (ii) passed through a step of ultra-heat treatment.

[0022] In an embodiment, the incubation step further comprises adding one or more suitable bases to convert the organic acids to organic acid salts, to provide a fermentation broth containing one or more organic acid salts, i.e. one or more of propionates, acetates, succinates and lactates.

[0023] In an embodiment, the method further comprises recovering the organic acid or organic acid salts from the fermentation broth. This can include removing the bacterial biomass from the fermentation broth and concentrating and / or drying the organic acid or organic acid salts from the fermentation broth. This recovered product is called an organic acid product or an organic acid salt product.

[0024] Typically, the one or more organic acid is one or more of propionic acid, acetic acid, succinic acid and lactic acid. Typically, the one or more organic acid salts is one or more of propionate, acetate, succinate and lactate. Typically, the organic acid salts can be calcium, sodium or potassium based propionate, acetate, succinate and lactate.

[0025] In an embodiment, the pH is adjusted with the addition of one or more suitable bases. The desired pH may be from 6 to 9, 6 to 8 or around 7.

[0026] In an embodiment, the dairy side stream is neutralised, i.e. adjusted to a neutral pH of 7, by addition of a suitable base. Preferably, a basic metal hydroxide.

[0027] Preferably, the basic metal hydroxide is calcium hydroxide. Other metal hydroxides may be employed to neutralise the substrate and precipitate the salts, for example sodium hydroxide, magnesium hydroxide, and potassium hydroxide.

[0028] The metal hydroxide may be a liquid or in dry form, such as a dry powder.

[0029] In an embodiment, the dairy side stream is a dairy whey side stream comprising at least 10 g / L lactose. In an embodiment, it may be 30 g / L lactose. In an embodiment, the dairy side stream is selected from a dairy whey side stream, milk protein concentrate, whey protein concentrate or milk permeate or a combination thereof.

[0030] Typically, the dairy whey side stream is selected from whey permeate (WP), whey permeate by-products such as delactosed permeate (DLP) and delactosed concentrate (DLC) and concentrated whey permeate (CWP).

[0031] The dairy side stream can be a powder or a liquid.

[0032] In an embodiment, the filtration step to remove monovalent ions may be by means of n an of i I trati o n / d i af i I trati o n .

[0033] In an embodiment, the product is diluted 1 :3 to 1 :5 prior to filtration.

[0034] In an embodiment, the nanofiltration / diafiltration employs a membrane having a molecular weight cut-off of 100-400 Daltons.

[0035] In one embodiment, the nanofiltration / diafiltration employs a membrane having a molecular weight cut-off of about 150-300 Daltons.

[0036] The step of heat treatment prior to fermentation is used to reduce the microbial load or sterilise the dairy side stream prior to fermentation. This may be by pasteurisation to remove or reduce microorganisms present in the product. In an embodiment, the heat treatment is an ultra-heat treatment step.

[0037] The ultra-heat treatment step is carried out at a temperature of from about 90°C to about 150°C, or from about 110°C to about 125°C, and typically for a period of from about 1 second to 180 seconds, e.g. 60 seconds. However, it will be appreciated that any time may be used suitable to provide the desired function. In an embodiment, the treatment step is carried out at a temperature of about 105°C for about 30 seconds.

[0038] In an embodiment, the dairy side stream is heated before, during, or after pH adjustment and prior to separation. Typically, it is before pH adjustment. For example, the product may be heated at 50°C to 80°C, e.g. 70°C, and incubated for about 10 to 60 minutes, e.g. 30 minutes before neutralisation. Typically, the product is mixed during heating to ensure homogenous heating.

[0039] Typically, the upstream processing steps, i.e. prior to fermentation, reduce the calcium levels in dairy side stream by at least 40%, 50%, 60% or 70%. In one embodiment, the upstream processing reduces the calcium levels by at least 40% to 80%. In one embodiment, when the stream is delactosed whey permeate, the upstream processing reduces the calcium levels in the whey by-product by 65 to 85%, ideally 70 to 75%. In one embodiment, when the stream is whey permeate, the upstream processing reduces the calcium levels by 50 to 70%, ideally 55 to 61 %. % values provided are % dry weight unless otherwise indicated.

[0040] In one embodiment, the upstream processing reduces the phosphate levels in the dairy side stream by at least 50, 60%, 70% or 80%. In one embodiment, the upstream processing reduces the phosphate levels by at least 50% to 95%. In one embodiment, when the stream is delactosed whey permeate, the upstream processing reduces the phosphate levels in by 75 to 95%, ideally 80 to 90%. In one embodiment, when the stream is whey permeate, the upstream processing reduces the calcium levels in the whey by-product by 60 to 80%, ideally 65 to 71%.

[0041] The % reduction values provided above for calcium and phosphate are based on a mg / kg parameter. Thus, if liquid DLP contains 1000 mg calcium per kg pre-treatment, and 300mg per kg post-treatment, this correlates with a 70% reduction in calcium.

[0042] In an embodiment, the bacterial strain used in fermentation is from the genus Propionibacterium. In an embodiment, the strain is from the species Propionibacterium freudenreichii. In an embodiment, the strain is from the species Propionibacterium acidipropionici. In an embodiment, the strain is Propionibacterium freudenreichii ssp. shermanii NCI MB 8099, Propionibacterium freudenreichii ssp. freudenreichii NCI MB 5959, Propionibacterium freudenreichii ssp. shermanii NCI MB 10585, Propionibacterium acidipropionici NCI MB 8070 or other Propionibacteria that can produce propionic acid from dairy streams containing lactose. Two or more strains may be used in combination.

[0043] In an embodiment, after the fermentation step, the method further comprises a step of removing bacterial cells from the fermentation broth. Typically, removal is by centrifugation but it will be appreciate that any suitable means may be used. This step provides a supernatant that comprises one or more organic acids or organic acid salts , depending on the incubation conditions used.

[0044] The supernatant comprising the organic acids or organic acid salts can be further treated or processed if desired.

[0045] In an embodiment, the supernatant is concentrated. Concentration may be by any suitable means such as evaporation or reverse osmosis.

[0046] In an embodiment, the supernatant is pasteurised and concentrated.

[0047] The end product is an organic acid (comprising propionic, acetic, succinic and lactic acid) or organic acid salt (comprising propionate, acetate, succinate and lactate) concentrate.

[0048] In an embodiment, the organic acid salt concentrate is mixed with a powder carrier. In an embodiment, the powder carrier is maltodextrin.

[0049] In an embodiment, the organic acid salt concentrate is dried. This may be by processing through spray drying technology. The product is an organic acid salt powder.

[0050] In an embodiment, the method has a lactose to organic salt yield of at least 50%, 60%, 70%, 75% or 80%.

[0051] In an embodiment, the method has a substrate conversion (lactose consumption) of at least 70%, 80%, 95% or 99%.

[0052] The bacterial cells removed from the fermentation broth may be further concentrated, e.g. via drying, to produce a vitamin B12 product.

[0053] In an embodiment where it is desired to produce a vitamin B12 product as a by-product of the method of the invention, the incubation step further comprises addition of one or more precursors of vitamin B12, such as cobalt chloride and 5,6 dimethylbenzimidazole (DMBI).

[0054] In such an embodiment, the conditions are switched from anaerobic or microaerophilic conditions to aerobic conditions after producing the organic salts.

[0055] An organic acid product produced by the current invention is provided.

[0056] An organic acid salt product produced by the current invention is provided.

[0057] The product is typically made up of sodium or calcium propionate (25-50% TS), acetate (15- 30% TS), succinate (5-10% TS) and lactate (0-10% TS). Other components such as minerals (5-15%), protein (2-8% TS) and residual sugars including glucose, galactose, and lactose (0- 5% TS) are also present. This product may be a fermentation broth.

[0058] An aspect of the invention provides an organic acid salt product comprising (or consisting of) sodium or calcium propionate, acetate, succinate and lactate. Typically the product comprises from about 15 to 50%, e.g, 25 to 50% TS sodium or calcium propionate, e.g. 30%, 35%, 40%, 45%, from about 10 to 35%, e.g. 15% to 30% TS acetate, e.g. 20% or 25%, from about 5% to 10% TS succinate, e.g. 6% or 8%, from about 0 to 10% TS lactate, e.g. 2%, 6% or 8%. The product may further comprise other components such as minerals (5-15% TS), protein (2-8% TS) and residual sugars including glucose, galactose, and lactose (0-5% TS). In an embodiment the product is a dry product such as a powder. In an embodiment the product is a liquid. The product may be one produced by the method of the current invention.

[0059] An aspect of the invention provides an organic acid product comprising (or consisting of) propionic acid, acetic acid, succinic acid and lactic acid. The product may be one produced by the method of the current invention. Typically the product comprises from about 15 to 50%, e.g. 25-45% TS propionic acid, e.g. 30% 35%, 40%, from about 10% to 40% 10% to 25% TS acetic acid, e.g. 15% or 20%, from about 1 to 15%, e.g. 5% to 10% TS succinic acid, e.g. 6% or 8%, from about 0 to 15%, e.g. 0 to 10% TS lactic acid, e.g. 2%, 6% or 8%. The product may further comprise other components such as minerals (5-15% TS), protein (2-8% TS) and residual sugars including glucose, galactose, and lactose (0-5% TS).

[0060] A preservative comprising the organic acid salt product produced by the method of the current invention is provided.

[0061] A vitamin B12 product produced by the method of the current invention is provided. A calcium phosphate product produced by the method of the current invention is provided.

[0062] A nutritional food supplement comprising (or consisting of) the calcium phosphate product is also provided.

[0063] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0064] Definitions

[0065] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0066] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0067] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0068] As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g., a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps. The term “dairy side stream” is a dairy-based product containing lactose that is produced from the production of primary dairy product such as milk, milk proteins, cheese, lactose and whey proteins. In an embodiment, it is one that contains at least 10g / L lactose. The term includes dairy whey side stream.

[0069] “Dairy whey side streams” or “whey by-product” means a liquid by-product of whey processing that contains at least 10 g / L lactose. It may also contain other organics and at least 3 g / L minerals. Generally, the whey by-product is produced when liquid whey is fractionated to remove protein leaving a permeate rich in lactose and minerals. The term includes whey permeate (WP), delactosed permeate (DLP), delactosed concentrate (DLC) and concentrated whey permeate (CWP). In the method of the current invention, these substrates are pretreated to reduce the mineral content. When not pre-treated to reduce the mineral content, the whey by-product typically has a high mineral content. In one embodiment, the dairy whey stream contains at least 3 g / L, 6 g / L, 10 g / L, 15 g / L, 30 g / L, 60 g / L, 100 g / L, 200 g / L, 300 g / L minerals. Mineral concentrations for calcium, magnesium, sodium, potassium, sulphur, and phosphorus after diluting and digesting incoming samples with 5% HNO3 and processing the test samples through an Agilent 720 inductively coupled plasma-optical emission spectroscopy (ICP-OES) instrument. The analytical wavelengths (nm) chosen for each element were as follows: Ca (318.127 and 422.673), Mg (280.270), Na (568.821 and 589.592), K (769.897), P (213.618) and S (181.972). Chloride concentrations were determined via titration using a Mk II 926 chloride analyser. In one embodiment, the dairy whey stream contains at least 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, lactose, and other organics. Lactose, glucose, galactose, and organic acid concentrations were determined on a high-performance liquid chromatography (HPLC, Agilent series 1200, Japan) system equipped with a refractive-index detector. The HPLC column used was Hi-Plex H 8 pm (300 mm x 7.7 mm) with 8.5 mM sulphuric acid as the mobile phase at a flow rate of 0.6mL / min whilst the column temperature was maintained at 50 °C.

[0070] The bacterium used in the method of the invention is one capable of converting lactose to propionic acid. Typically, it can utilise lactose to also produce other organic acids, such as acetic, succinic and lactic acid. In an embodiment, the bacterial strain used in fermentation is from the genus Propionibacterium. Examples of bacteria capable of bioconversion of lactose in substrates into organic acids, including propionic acid, are described in the literature, and that can be used in the method of the invention include but are not limited Propionibacterium acidipropionici (Jiang et al.; Enhanced propionic acid production from whey lactose with immobilized Propionibacterium acidipropionici and the role of trehalose synthesis in acid tolerance. Green Chem., 2015, 17, 250), Propionibacterium freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii (Colomban et al.; Sequential production of propionic acid from whey permeate by sequential fermentation, ultrafilration and cell recycling, Biotechnology and Bioengineering, vol.42, (1993)), Acidipropionibacterium acidipropionici, Acidipropionibacterium cyclohexanium, acidipropionibacterium jensenii and Acidipropionibacterium thoenii (Antone et al.; Propionic acid fermentation - study of substrates, strains, and antimicrobial properties. Fermentation 2023, 9, 26). The bacterium is one with a minimum bioconversion activity of lactose to propionic acid of 10%. Method to test this activity are known in the art, for example lactose and propionic acid can be quantified via HPLC analysis of fermentation test samples.

[0071] When used herein the term “whey permeate” is taken to mean the following: Whey is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacture of cheese and casein. It can exist as sweet whey or acid whey. The whey may be obtained from bovine milk or milk from other mammals such as goats or sheep. Preferably, the milk is bovine milk. Whey permeate is produced by removing protein and other solid components from whey. It is generally produced by treating liquid whey to ultrafiltration. Whey permeate typically contains at least 30 g / L lactose.

[0072] When used herein “concentrated whey permeate” (CWP) refers to a product derived from whey permeate evaporation. Typically, concentrated whey permeate contains 200-240 g lactose per litre.

[0073] When used herein “delactosed whey permeate” (DLP) is a by-product of processing of whey permeate to remove lactose. However, it has a higher amount of lactose post-filtration compared with whey permeate, and higher amounts of salts and minerals, especially chlorides and phosphates. Typically DLP contains at least 180 g lactose per litre.

[0074] When used herein the term “neutralising” means adding a base to the dairy side stream to bring the pH at or close to neutral, at which pH divalent calcium and phosphate ions in side stream and can be removed from the substrate. In a preferred embodiment, the base is a basic metal hydroxide which includes alkali metal hydroxides and alkaline earth metal hydroxides. Exemplary metal hydroxides include sodium hydroxide, potassium hydroxide, magnesium hydroxide and calcium hydroxide.

[0075] When used herein the term “metal hydroxide” refers to metal hydroxides that are basic such as sodium hydroxide, potassium hydroxide, magnesium hydroxide and calcium hydroxide. The term includes alkali metal hydroxides and Alkaline earth metal hydroxides. Specific hydroxides include sodium hydroxide, calcium hydroxide, barium hydroxide, magnesium hydroxide, and potassium hydroxide. Typically, the metal hydroxide is employed in a dry form such as a dry powder form or in solution. Typically, a sufficient amount of metal hydroxide is added to neutralise the product. When used herein the term “Nanofiltration / Diafiltration” refers to a process of nanofiltration in which the feed is diluted prior to nanofiltration. In one embodiment, the feed is diluted 1 :2, 1 :3, 1 :4, 1 :5 or 1 :6 times. In one embodiment, nanofiltration employs a membrane having a molecular weight cut-off of 100-400 Daltons, preferably 150-300 Daltons. In one embodiment, the nanofiltration / diafiltration step employs a GE Osmonic Membrane with a 150-300 Dalton cut-off.

[0076] Total solids (TS) is the sum of total dissolved solids (TDS) and total suspended solids (TSS) in a liquid or powder, i.e. , dry solids content.

[0077] Brief Description of the Figures

[0078] The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:

[0079] Figure 1 : Summary of method to produce organic acid salts from dairy side streams.

[0080] Detailed Description of the Invention

[0081] The current invention provides a method to produce organic acids from a dairy side stream using bacterial fermentation. If desired the fermentation conditions can be such that one or more organic acid salts are produced in the fermentation broth.

[0082] Dairy side streams, such as whey used as feedstocks, for microbial fermentation in the method of the current invention, are supersaturated in the disaccharide sugar, lactose, typically containing at least 10 g / L or 30g / L lactose. It may be from 10 g / L to 150 g / L, or 30 to 100 g / L. It will be appreciated that the dairy side stream that naturally has a larger amount of lactose may be diluted to provide a lower amount of lactose.

[0083] The method can be adapted for fed-batch fermentation.

[0084] The method can be adapted for continuous fermentation. This provides a substrate for organic acid production.

[0085] The organic acids or organic acid salts produced by the method of the invention can be used as a preservative in bakery products.

[0086] As shown in Figure 1 , the current method includes upstream processing step of the dairy side stream prior to fermentation. The removal of salts and ions in this step enhances bacterial fermentation. The upstream processing steps are what takes place in the method prior to fermentation, i.e. incubation with a suitable bacterium.

[0087] The upstream processing can include one step of pH adjustment, such as, neutralisation, or one step of filtration and / or ultrafiltration, or the steps can be carried out sequentially. For example, in an embodiment, the upstream processing is a pH adjustment step. In a further embodiment, the upstream processing is a filtration step. In a further embodiment, the upstream processing includes a step of pH adjustment and a step of filtration. In a still further embodiment, the upstream processing includes a filtration step and a pH adjustment step. In a still further embodiment, the upstream processing includes a pH adjustment step and a heat treatment step. In another embodiment, the upstream processing includes a step of pH adjustment, a filtration step and a heat treatment step. In an embodiment, the upstream processing is a heat treatment step.

[0088] The pH adjustment step is carried out to precipitate the salts, such as calcium and phosphate salts, from the dairy side stream substrate.

[0089] This can involve a dilution and heat-treatment step at around 60 °C and the addition of a suitable based, such as a basic metal hydroxide, such as Ca(OH)2, NaOH, Ba(OH)2, Mg(OH)2 or KOH, to increase the pH of the substrate ideally to neutralise the dairy side stream at or close to around pH 7 to promote the precipitation of the mineral salts e.g. calcium and phosphate salts. It will be appreciated that any suitable base capable of providing a pH suitable to neutralise the substrate can be used. Such a base is known in the art.

[0090] Elevated temperatures, such as between 50°C to 80°C lowers the solubility of calcium and phosphate salts. Typically, this step is carried out at around 60°C. A pH range of 6 to 10 can also be targeted by adding a sufficient amount of base depending on the desired final and phosphate removal requirement.

[0091] Typically, the base is employed in a dry form or in solution. Ca(OH)2 addition has a higher phosphate removal potential than NaOH addition as more calcium ions introduced into the dairy side stream are readily available for insoluble calcium phosphate formation. For NaOH addition, theoretically less calcium and phosphate salts are removed as the Na+ions introduced from NaOH have the potential to form e.g., sodium phosphate or sodium chloride which generally remain in the soluble phase of the dairy side stream. The metal base hydroxides listed herein provide the chemical precipitation of calcium and phosphate salts, such as calcium phosphate and magnesium phosphate. Any suitable incubation period is applied to the pH-adjusted dairy side stream to support the nucleation, crystallisation and precipitation of calcium and phosphate salts. Typically, this incubation period is about 20 minutes, or any suitable time to have the intended result.

[0092] The precipitated calcium and phosphate salts are removed by a separation process, such as centrifugation and membrane fractionation. The removal of insoluble calcium and phosphate salts by means of chemical precipitation followed by separation reduces the demineralisation that is required during the downstream process (post-fermentation). The solid by-product mainly consisting of calcium, phosphate and lactose is a suitable source of calcium and phosphate minerals for nutritional, functional, chemical, and biological applications, such as a nutritional food supplement. Once separated from the solid by product, the product is typically called a clarified product. Thus the combination of a neutralisation step and a separation step is generally referred to as a clarification process.

[0093] Following removal of the precipitated calcium and phosphate salts, the clarified product, or “supernatant” is subjected to nanofiltration / diafiltration to remove monovalent ions, if a second step is included in the upstream processing. The step facilitates the subsequent microbial fermentation and downstream processing of the fermentation broth. After ion removal, the product is typically called a demineralised product.

[0094] In an embodiment of filtration, the substrate is subjected to a volume concentration factor VCF of from 2-6 volumes, typically 3-5 volumes, or 4-5 volumes, of diafiltration water at from about 20 to 35 bar, e.g. 25 to 30 bar, transmembrane pressure. In an embodiment, the transmembrane pressure is 30 bar. In an embodiment, the temperature range for this step is from about 8°C to about 12°C, e.g. 10°C. In an embodiment, the temperature is from about 45°C to about 50°C, e.g. 48°C. A D-Series Dairy GE DK8038 C30 membrane with a molecular weight cut-off point of 150-300 Dalton for uncharged organic molecules is used for this demineralisation step.

[0095] Prior to fermentation, the substrate, which is a demineralised product, may undergo ultra-heat treatment. This is an optional step.

[0096] The ultra-heat treatment (UHT) may be by any known mean of UHT.

[0097] One example is “indirect” UHT which is a UHT process configuration where the liquid or product is not in direct contact with the heating medium. In this embodiment, a heat exchanger may be used. “Tubular” or a tubular heat exchanger (THE) is a type of heat exchanger that is part of a UHT processing unit used. Other examples of heat exchangers possible on a UHT unit include a plate and frame heat exchanger and a scraped surface heat exchanger.

[0098] Other examples of UHT include but are not limited to steam infusion and direct steam injection. In an embodiment, the pasteurisation step is at a temperature of from about 60°C to about 90°C for a suitable amount of time. This may be any time sufficient for purpose to inactive nonspore forming bacteria and a sufficient amount of vegetative microorganisms as well as inhibiting microbial and enzyme activity. These parameters are within the remit of a person skilled in the art.

[0099] Bacteria (propionibacteria or acidipropionibacteria) provide a suitable inoculum for the production fermentation containing dairy side streams. In the production fermentation, the bacteria allows the bioconversion of lactose contained in the dairy side streams to organic acids, such as propionic acid.

[0100] Means of fermentation are known in the art and the methods described herein are exemplary only. It will be appreciated that the specific parameters and conditions may be optimised or changed from the exact conditions discussed in the Examples. Generally, the bacterial fermentation in the current invention is performed by a batch mode operation but can also be fed batch or a continuous fermentation operation. Methods of fermentation are known in the art. The fermentation conditions are anaerobic or microaerophilic fermentation conditions to produce the organic acids and their salts.

[0101] During bacterial fermentation organic acids are produced. The organic acids produced during bacterial fermentation can be converted into their salt form by reacting the organic acids with appropriate bases during bacterial fermentation. The appropriate bases include but are not limited to NaOH, Ca(OH)2, Mg(OH)2 or KOH. This is illustrated in the steps of Figure 1. The preferred organic salts are calcium and sodium organic salts produced by reacting the organic acids during bacterial fermentation with NaOH or Ca(OH)2. Sodium and calcium propionate are the main organic salts produced during fermentation after the addition of NaOH or Ca(OH)2 to the fermentation broth to generate these organic salts from propionic acid. Other organic salts are also produced such as calcium or sodium acetate, succinate and lactate salts after the addition of NaOH or Ca(OH)2 to the fermentation broth to generate these organic salts from acetic, succinic and lactic acid. A combination of calcium, sodium, potassium and / or magnesium organic salts can also be produced from organic acids using more than one of the metal hydroxide bases.

[0102] After completion of the fermentation step, the fermentation broth contains mostly organic acids or organic acid salts, and bacterial cells. To remove the bacterial cells, the fermentation broth passes through a separation step, such as a clarification step, to yield a supernatant that is rich in organic acids or salts thereof. This can be called a “clarified broth”.

[0103] Propionibacterium sp. are known producers of vitamin B12, and therefore, the removed bacterial cells can be further processed, e.g. via spray drying technology, to concentrate the vitamin B12 produced into a finished powder containing the vitamin B12. It will be appreciated that methods of concentrating bacterial biomass are known in the art. Thus, the invention also provides a method to prepare vitamin B12. In an embodiment of the method of the invention where it is desired to produce a vitamin B12 product as a by-product of the method of the invention, the incubation step may further comprise addition of one or more precursors of vitamin B12, such as cobalt chloride and 5,6 dimethylbenzimidazole (DMBI). Cobalt chloride and DMBI function as precursors for microbial vitamin B12 biosynthesis in the fermentation broth. An aerobic fermentation step is also introduced after the anaerobic or microaerophilic fermentation step. Means to introduce such a step during fermentation are known in the art. The aerobic fermentation step allows for DMBI synthesis and the formation of vitamin B12 due to the aerobic biosynthetic pathway of the microorganism. The cobalt chloride is generally added during the anaerobic fermentation step during the production of organic acids such as propionic acid as described in the method of the invention. This increases the cobalt ion concentration for cobalt chelation during vitamin B12 biosynthesis. Cobalt is required as it forms the central ion of the corrinic ring in all cobalamins (vitamin B12 family of compounds). The DMBI is generally added during the aerobic fermentation step to increase the rate of vitamin B12 biosynthesis. DMBI forms the lower ligand of cobalamin isoforms. The bacterial cells containing vitamin B12 can then be removed by centrifugation and further purified and concentrated to produce a vitamin B12 product.

[0104] The supernatant or “clarified broth” may be further processed to provide produce a concentrated liquid comprising of organic acid salts or organic acids. The clarified broth can vary in dry matter content depending on the fermentation process to produce the fermentation broth. Typically, the clarified broth is generally a diluted liquid, generally 8% to 12% TS, containing the organic salts. The clarified broth dry matter content may also be at least 1% TS, at least 2% TS, at least 4% TS, at least 6% TS, at least 10% TS or at least 12% TS. A concentration step is needed in the form of evaporation to provide a concentrated broth as a feed at the appropriate dry matter content for the drying process. This may be by falling film evaporation. As an alternative to evaporation, the supernatant can be passed through reverse osmosis. It will be appreciated that methods of concentrating supernatants are known in the art.

[0105] In an embodiment, the method of the invention provides the following:

[0106] Organic acids or their salts titre: 5 to 100 g / L.

[0107] Organic acids or their salts productivity: 0.1 to 2 g / L / h.

[0108] Substrate conversion: 80 - 100%. Lactose to organic acids or their salts yield: 40 to 95%.

[0109] The retentate stream from reverse osmosis contains the concentrated organic salt solution.

[0110] After evaporation, the concentrated organic salt liquid can be mixed with a powder carrier, such as maltodextrin and spray dried, e.g., with an inlet temperature set at 170 to 190°C, e.g., 180 °C, and an outlet temperature set at 80 to 90°C, e.g., 85 °C. A powder containing the organic salts with a moisture content of around 3% mass is achieved after spray drying. The ideal moisture content range is <4.0% mass.

[0111] One example of the powder carrier is maltodextrin with a dextrose equivalent (DE) value of from 4 to 20, typically from 8 to 15, or a DE of 12. A further example of the powder carrier is whey protein isolate or concentrate. Another example of the powder carrier is calcium phosphate or a starch-based polymer. These powder carrier examples can be added separately or can be added as a blend of any combination.

[0112] As described herein the product of the method of the invention is an organic acid salt product or an organic acid product. This product comprises a number of organic acid salts or organic acids. In an embodiment, the method of the invention comprises a step of recovering one organic acid from this product, such that the recovered product contains from about 75% to 100% recovered organic acid. In an embodiment, it contains about 85% or more recovered organic acid. It may have a purity of 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or 100%. The organic acid may be any organic acid in the product of the method of the invention. For example, if a pure propionic acid product is desired that has a purity of from 95% to 100%, the method of the invention further comprises a step of recovering propionic acid from the organic acid salt or organic acid product. Methods of recovery are well known in the art. For example, if a pure acetic acid product is desired that has a purity of from 95% to 100%, the method of the invention further comprises a step of recovering acetic acid from the organic acid salt or organic acid product. Methods of recovery are well known in the art. If a pure succinic acid product is desired that has a purity of from 95% to 100%, the method of the invention further comprises a step of recovering succinic acid from the organic acid salt or organic acid product. Methods of recovery are well known in the art.

[0113] The invention further provides an organic acid salt product or concentrate produced by the method of the invention.

[0114] The invention also provides a method of making a bakery product, comprising the method of the invention. The invention provide use of the organic acid salt product disclosed herein or produced by the method of the invention as a preservative in food products, such as bakery goods Therefore, the invention also provides a preservative (herein “preservative” of the invention”).

[0115] The invention also provides a method of making a food product, such as a bakery product, the method comprising a step of adding the preservative of the invention to the bakery product. This step of adding the preservative may take place at any step during the preparation of the bakery product.

[0116] Typically, the product of the invention or preservative of the invention is added to the bakery product in an amount sufficient to inhibit mould growth for at least 5 days. It may be added in an amount sufficient to inhibit mould growth for at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 20 days, at least 25 days or at least 30 days. Mould growth can be determined by visual inspection during storage of the bakery product, e.g. storage in a sealed bag and ambient temperature and humidity. Typically, sufficient preservation results can be attained by applying the organic acid salt product (powder) or concentrate (i.e. the liquid) in a quantity based on the weight of the flour of the bakery product. For instance, said dosage of the preservative can be mixed in with flour during formulation or added at the dough stage of the bakery product and this dosage based on flour weight may be at least 0.1%, at least 0.2%, at least 0.3%, at least 0.5%, at least 1% or at least 2%.

[0117] The amount of preservative or organic acid salt product or concentrate added will vary depending on the bakery product type, the shelf-life requirements and what is acceptable. A larger addition rate to the bakery product will achieve a longer shelf-life. A lower addition rate will achieve a shorter preservation duration. This would be understood by a person skilled in the art.

[0118] Bakery products are prepared from flour or meal derived from grain. Examples of bakery goods include but are not limited to, breads, biscuits, leavened breads, pizza bases, bagels, flatbreads, tortillas, buns, cakes, muffins, crackers, pastries, doughnuts and pies.

[0119] A method to prepare a bakery good preservative is provided comprising the method of the invention is provided.

[0120] A bakery good comprising the organic acid salt product, or the organic acid product described herein or produced by the method of the invention is provided.

[0121] The invention provides a method of preserving a bakery product, the method comprising adding the organic acid product of the invention to the bakery product. This step of adding the preservative may take place at any step during the preparation of the bakery product. A method to prepare vitamin B12 comprising the method of the invention is provided.

[0122] In an alternative embodiment of the method of the invention, the upstream processing steps are omitted. In other words, there is no neutralising or clarification step and no filtering step. This method comprises incubating a dairy side stream with a bacterium capable of converting lactose to propionic acid to provide a fermentation broth containing one or more organic acids or organic acid salts. All embodiments and preferred features described herein regarding fermentation and subsequent steps apply equally to this method.

[0123] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0124] EXAMPLES

[0125] Upstream processing steps

[0126] The dairy side stream, such as a dairy whey side stream, substrate is diluted to 12% total solids (TS) (w / w). If the dairy whey side stream is <12% TS, no dilution is required. The substrate is then subjected to heat-treatment at 70 °C via a heat-exchanger. A basic metal hydroxide in the form of calcium or sodium hydroxide is added to the whey permeate to adjust to pH 7. After pH adjustment, the whey permeate is incubated for 30 minutes to assist with the precipitation of calcium and phosphate salts.

[0127] After incubation, the substrate is processed through clarification for the removal of the insoluble calcium and phosphate salts. Following clarification, the supernatant is retained for analysis and further processing.

[0128] Table 1 : Average composition of whey permeate before and after sodium and calcium hydroxide pre-treatment.

[0129] To remove monovalent ions, the clarified substrate was processed via nanofiltration / diafiltration. Feed streams are subjected to a VCF of 4 at a temperature of 10 °C or 50 °C and 30 bar transmembrane pressure. Chloride, sodium, and potassium monovalent ions have the lowest % rejection during nanofiltration.

[0130] Table 2: Average rejection of minerals and lactose after nanofiltration of clarified whey permeate.

[0131] The demineralised substrate was passed through an indirect tubular ultra-heat treatment step at 105 °C for 30 seconds to reduce the microbial load of the dairy whey side stream prior to fermentation.

[0132] Fermentation

[0133] Bacteria (propionibacteria or acidipropionibacteria) were grown on suitable culture media to allow for cell growth and multiplication to provide a suitable inoculum for the production fermentation of dairy side streams. Dairy side streams are supersaturated in the disaccharide sugar, lactose. In the production fermentation, the metabolites produced by the bacteria allows for the bioconversion of lactose contained in the dairy side streams to organic acids. Generally, the bacterial fermentation is performed by a batch mode operation but can also be fed batch or a continuous fermentation operation. The lactose concentration of the dairy whey side streams are variable due to the nature of its origin and type of dairy whey side stream used. To confirm the lactose content of the solution, each dairy whey side stream is assayed each time to determine the volumes required at each fermentation stage.

[0134] Bacterial fermentation allows for the bioconversion of lactose to organic acids. The organic acids produced during bacterial fermentation can be converted into their salt form by reacting the organic acids with appropriate bases during bacterial fermentation such as NaOH, Ca(OH)2, Mg(OH)2 or KOH. The preferred organic salts are calcium and sodium organic salts produced by reacting the organic acids during bacterial fermentation with NaOH or Ca(OH)2. A combination of calcium, sodium, potassium and / or magnesium organic salts can also be produced from organic acids using more than one of the metal hydroxide bases. After completion of the fermentation, a fermentation broth is produced that contains the bio-based organic salts for downstream processing.

[0135] A biotechnological process of the invention to grow the culture and to produce organic salts from dairy whey side streams may include the following steps:

[0136] 1) Primary inoculum stage to prepare culture medium.

[0137] 2) Secondary inoculum stage for seed fermentation inoculum.

[0138] 3) Seed fermentation stage for production fermentation inoculum.

[0139] 4) Production fermentation to produce a fermentation broth containing organic salts.

[0140] 5) Optional continuation of subsequent production fermentations

[0141] 1) Primary inoculum stage

[0142] The primary inoculum stage consists of a 500 mL shake-flask pre-culture grown on liquid media, inoculated from a thawed glycerol stock containing a propionibacterium (Propionibacterium sp.) or acidipropionibacterium (Acidipropionibacterium sp.) bacterial culture.

[0143] Table 3: Recipe for primary inoculum medium. Add 300mL water in a beaker. Add all media components and continuously mix until all media components are solubilized. Adjust media to pH 6.5 using 1 M NaOH or 1 M HCI. Sterilise the media using a 0.2pm filter and transfer to a sterile Erlenmeyer flask.

[0144] Place the Erlenmeyer flask in a shake-incubator and cultivate under the operating conditions as shown in Table 4.

[0145] When the transfer criterion is reached (duration), remove the flask from the incubator and immediately transfer the broth aseptically into the fermentation vessels that contains the secondary inoculum substrate at the suitable operating conditions.

[0146] Table 4: Operating conditions in primary inoculum stage in orbital shaker.

[0147] 2) Secondary inoculum stage

[0148] The secondary inoculum stage consists of a 3L non-aerated fermentation in a bioreactor. The medium composition for this stage is described in Table 5.

[0149] Filter sterilise the media components in water using a 0.2pm filter. The dairy whey side stream can also be aseptically transferred directly to the bioreactor if the solution has passed through an ultra-heat treatment step, thus, avoiding the need to filter sterilise dairy whey side stream.

[0150] Add all media aseptically to the pre-sterilised bioreactor and start agitation. Bring the medium to operating temperature. Initiate pH control using 15% (w / v) calcium hydroxide or 20% (w / w) sodium hydroxide at the desired pH set point. Aseptically inoculate the bioreactor using the primary inoculum broth (inoculum ratio » 10%).

[0151] Carry out the fermentation at a temperature of 30°C (+ / - 1 °C) and pH 6.5 (+ / - 0.1). Secondary inoculum completion occurs once there is exponential base demand / cell growth or there is >50% lactose is consumed. Table 5: Recipe for secondary inoculum substrate.

[0152] 3) Seed fermentation

[0153] The seed fermentation stage consists of a 20 L non-aerated fermentation in a bioreactor. The medium composition for seed fermentation is described in Table 6.

[0154] Filter sterilise the media components in water using a 0.2pm filter. The dairy whey side stream can also be aseptically transferred directly to the bioreactor if the solution has passed through an ultra-heat treatment step, thus, avoiding the need to filter sterilise dairy whey side stream.

[0155] Add all media aseptically to the pre-sterilised bioreactor and start agitation. Bring the medium to operating temperature. Initiate pH control using 15% (w / v) calcium hydroxide or 20% (w / w) sodium hydroxide at the desired pH set point. Aseptically inoculate the bioreactor using the bioreactor seed fermentation broth (inoculum ratio » 10%).

[0156] Carry out the production fermentation at a temperature of 30°C (+ / - 1 °C) and pH 6.5 (+ / - 0.1).. The seed fermentation can be used as an inoculum (inoculum ratio » 10%) for the production fermentation in a 200 L bioreactor using the same methodology once there is exponential base demand / cell growth or there is >50% lactose is consumed. Table 6: Recipe for seed and production fermentation substrate.

[0157] 4) Production fermentation

[0158] The seed fermentation stage consists of a 20 L non-aerated fermentation in a bioreactor.

[0159] Heat sterilise the media and sterilise the bioreactor in place at 121 °C for 15 mins. The dairy whey side stream .which has undergone upstream processing steps including the ultra-heat treatment step as per the above methodology can also be aseptically transferred directly to the bioreactor, thus, avoiding the need to filter sterilise the dairy whey side stream. The target starting lactose concentration for the production fermentation substrate is 60 g / L.

[0160] Add all media aseptically to the pre-sterilised bioreactor and start agitation. Bring the medium to operating temperature. Initiate pH control using 15% (w / v) calcium hydroxide or 20% (w / w) sodium hydroxide at the desired pH set point. Aseptically inoculate the bioreactor using the bioreactor seed fermentation broth (inoculum ratio » 10%).

[0161] Carry out the production fermentation at a temperature of 30°C (+ / - 1 °C) and pH 6.5 (+ / - 0.5).

[0162] Upon fermentation completion, base addition can be halted once approx. 90% lactose is consumed.

[0163] After completion of the 200 L production fermentation, the fermentation is harvested when the lactose is consumed, and base demand is no longer required. Otherwise, repeated batch fermentations can continue and scale-up can progress as per seed fermentation transfer criteria.

[0164] RESULTS

[0165] Fermentation performance

[0166] Typical fermentation performance is shown in Table 7. Table 7: Minimum, average, and maximum values for organic salts titre (g / L), organic salts productivity (g / L / h), substrate conversion (%), lactose to organic salts yield (%) and propionate, acetate, succinate, and lactate organic salt content of fermentation broth (% TS).

[0167] * Calcium or sodium organic acid salts.

[0168] Downstream processing description

[0169] Downstream processing of the fermentation removes the bacterial cells from the fermentation broth and concentrates the supernatant to further concentrate the clarified broth. After downstream processing, a final organic salt concentrate or powder is produced that can be used as a food preservative or anti-fungal agent to extend the shelf-life of foods such as baked goods.

[0170] Downstream processing steps

[0171] Following the completion of the production fermentation to effectively produce the organic salts at a concentration as shown in Table 7, the fermentation broth is sent for downstream processing. The fermentation broth contains mostly organic salts and bacterial cells. To remove the bacterial cells, the fermentation broth passes through a clarification step to yield a supernatant that is rich in organic salts. Propionibacterium sp. are known producers of vitamin B12, therefore, the bacterial cells can be further processed to dry the vitamin B12 produced and produce a finished powder containing vitamin B12.

[0172] To concentrate the clarified fermentation broth, the solution is processed through a falling film evaporator. The clarified fermentation broth is pasteurised at 72 °C for 15 seconds and cooled to 65 °C before evaporation. An anti-foam agent is added at 0.05% (v / v) before the feed solution is concentrated from 6-8% TS to 25% TS. Anti-foam is added to prevent foam formation during evaporation that can disrupt the operation of the unit operation and lead to unnecessary product losses. As an alternative, the clarified fermentation broth can be passed through reverse osmosis at a temperature of 20 °C, transmembrane pressure of 80 bar a volume concentration factor of 3-5 to concentrate the solution from 5-8% TS to 25% TS. The mentioned conditions for reverse osmosis is an example for optimal organic salt rejection and membrane flux. The retentate stream contains the concentrated organic salt solution. The concentrated fermentation broth can be further concentrated from 25% TS to 60% TS by evaporation.

[0173] After evaporation, the concentrated organic salt liquid can be mixed with a powder carrier such as maltodextrin and sent for spray drying with an inlet temperature set at 180 °C and an outlet temperature set at 90 °C. Generally, a multi-stage dryer with a belt drying, fluid-bed drying and sieving step is preferred. A powder containing the organic salts with a moisture content of <5% is achieved after the drying process.

[0174] An exemplary product used for the efficacy testing described is below. This is a powder product with 20 to 30% TS calcium propionate, 20 to 25% TS maltodextrin, 10 to 20% TS calcium acetate, 5 to 10% TS calcium succinate, 1 to 10% TS calcium lactate, 5 to 10% TS sugars, 3 to 5% moisture and around 0 to 1% TS lactose. The product has a pH of from 5.5 to 6.5. This is a calcium based organic acid salt product.

[0175] Efficacy testing for the product of the invention

[0176] The powder containing organic acid salts as described in this invention was assessed in white bread to determine the efficacy of the ingredient to function as a mould inhibitor to extend the shelf-life of bread and bakery products. The product used is a powder. A standard bread recipe containing white flour, salt, yeast and water was used for this assessment. This recipe consisted of 1000 g white flour, 557 g water, 28.8 g yeast and 15.3 g salt.

[0177] Test formulations were prepared with varying levels of organic acid salts product (0% (control), 0.5% and 1 % based on flour weight) added to the bread recipe. The organic acid salts product was mixed in with the dry ingredients during the bread dough formulation. The bread dough ingredients were mixed, separated into loaf tins at a scaling weight of 227 g for each white bread loaf and allowed to proof for 1 hour, followed by baking at 220 °C for 25 minutes. After baking, the bread loaves were cooled for 1 hour and stored in a bag that was heat sealed and added into another storage bag that was subsequently heat sealed to reduce the influence of potential contamination from the outside environment as much as possible. The bread loaves were stored at 22 °C for 30 days and a visual inspection for mould growth was carried out from Day 1 to Day 30. The rate of mould growth and mould inhibition performance of the organic acid salts product in the white bread loaves was based on a scoring system. The scoring ranged from 5 points (A): no visible mould on the white bread loaf, 4 points (B): 1 to 9 visible mould colonies or 1-9% mould surface area coverage observed on the white bread loaf, 3 points (C): 10-24% mould surface area coverage observed on the white bread loaf, 2 points (D): 25-49% mould surface area coverage observed on the white bread loaf and 1 point (E): 50-100% mould surface area coverage observed on the white bread loaf.

[0178] Mould growth was detected after 9 days of storage for the bread loaf containing no organic acid salts product (control), after 14 days of storage for the bread loaf containing 0.5% organic acid salt product and no mould was observed after 30 days in the bread loaf containing 1 .0% organic acid salt product. Bread loaves that contained the organic acid salts product exhibited anti-mould properties by delaying mould growth longer than the control.

[0179] The mould inhibition performance of the organic acid salts product in bread slices was also assessed by comparing with a control (no preservation ingredient added), calcium propionate and a wheat flour fermentate. White bread loaves were baked as described for the bread loaves and the bread loaves were sliced to obtain 5 slices for each batch and stored at three storage conditions: room temperature storage at 17-22 °C with an air hole and fully sealed, room temperature storage at 17-22 °C without any air holes and fully sealed and at controlled temperature storage in an incubator without any air holes at 22 °C fully sealed in bags. Each bread slice from all batches was stored in a separate heat sealed bag. Therefore, there was 4 batches containing 5 slices per batch where each slice per batch was assessed as per the scoring system used for the bread loaves. All 4 batches were assessed at 3 storage conditions: 1) control, 2) 0.5% organic acid salts product addition based on flour weight, 3) 0.5% wheat flour fermentate addition based on flour weight and 0.2% calcium propionate based on flour weight. Using the scoring system described for the bread loaves above, each individual slice was assessed each day for 30 days for each batch and storage condition. All storage conditions for each batch were averaged to determine an average score and to subsequently determine the performance (%) for mould inhibition performance with the 0.5% organic acid salts product batch. The control batches exhibited a -51 % mould inhibition performance to the 0.5% organic acid salts product. The 0.5% wheat flour fermentate batches exhibited a -35% mould inhibition performance to the 0.5% organic acid salts product. The 0.2% calcium propionate batches exhibited a -19% mould inhibition performance to the 0.5% organic acid salts product.

[0180] Equivalents

[0181] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

Claims1. A method to produce organic acids, or salts thereof, from a dairy side stream, comprising the steps of: incubating the dairy side stream with a bacterial strain capable of converting lactose to propionic acid, to provide a fermentation broth comprising one or more organic acids selected from propionic acid, acetic acid and succinic acid, or salts thereof.

2. The method of Claim 1 , wherein the dairy side stream is processed prior to incubating, the processing steps comprising:(a) adjusting the pH of the dairy side stream to a pH of from 5 to 10, to precipitate multivalent ions comprising calcium and phosphate, separating the precipitate to provide a clarified product, and optionally filtering the clarified product to remove monovalent ions and provide a demineralised product,(b) filtering the dairy side stream to remove monovalent ions to provide a demineralised product, or(c) heat treating the dairy side stream by ultra-heat treatment or pasteurisation.

3. The method of Claim 2, wherein step (a) comprises a further step of ultra-heat treating the clarified or demineralised product.

4. The method of Claim 2, wherein step (b) comprises a further step of ultra-heat treating the demineralised product.

5. The method of any one of Claims 2 to 4, wherein prior to, during, or after, pH adjustment in step (a) the dairy side stream is heat treated at a temperature of from 50°C to 80°C.

6. The method of any one of the preceding claims, wherein the incubation step further comprises adding one or more bases to convert the organic acids to organic acid salts to provide a fermentation broth containing one or more organic acid salts.

7. The method of any one of the preceding claims, further comprising recovering the organic acids or salts thereof from the fermentation broth.

8. The method of Claim 6, wherein the step of recovery comprises removal of the bacterial strains and concentrating the fermentation broth to produce a concentrated organic acid salt.

9. The method of any one of Claims 5 to 7, wherein the concentrated organic acid salt is dried to form a powder.

10. The method of Claim 9, wherein the concentrated organic acid salt is mixed with a powder carrier.11 . The method of Claim 9, wherein the powder carrier is maltodextrin.

12. The method of any one of Claims 2 to 11 wherein pH adjustment comprises addition of a basic metal hydroxide.

13. The method of Claim 12, wherein the hydroxide is selected from calcium hydroxide, sodium hydroxide, magnesium hydroxide, and potassium hydroxide.

14. The method of any one of the preceding claims, wherein the dairy side stream comprises at least 10 g / L lactose.

15. The method of any one of the preceding claims, wherein the dairy side steam is a dairy whey side stream selected from whey permeate (WP), whey permeate by-products such as delactosed permeate (DLP) and delactosed concentrate (DLC) and concentrated whey permeate (CWP).

16. The method of any one of the preceding claims, wherein the bacterial strain is from the genus Propionibacterium.

17. The method of any one of the preceding claims, wherein the bacterial strain is from the genus Acidipropionibacterium.

18. The method of Claim 16, wherein the strain is Propionibacterium freudenreichii or Propionibacterium acidipropionici.

19. The method of Claim 16, wherein the strain is Propionibacterium freudenreichii ssp. shermanii NCI MB 8099, Propionibacterium freudenreichii ssp. freudenreichii NCI MB 5959, Propionibacterium freudenreichii ssp. shermanii NCI MB 10585, Propionibacterium acidipropionici NCIMB 8070 or other Propionibacteria that canproduce propionic acid from dairy side streams containing lactose, or a combination thereof.

20. The method of any one of the preceding claims wherein the ultra-heat treatment is carried out at a temperature of from 90°C to 150°C.

21. The method of any one of the preceding claims, wherein filtration is nanofiltration / diafiltration.

22. The method of any one of the preceding claims, wherein the product is diluted 1 :3 to 1 :5 prior to filtration.

23. The method of any one of the preceding claims, wherein the product is heated after pH adjustment and prior to separation at a temperature of from 50°C to 80°C.

24. The method of any one of the preceding claims, wherein the separation step after pH adjustment is by centrifugation / clarification or ultrafiltration.

25. The method of any one the preceding claims for producing an organic acid, further comprising a step of recovery of propionic acid, acetic acid, or succinic acid, to provide a propionic acid, acetic acid, or succinic acid product with a purity of from 95% to 100%.

26. A preservative comprising an organic acid product, or organic acid salt product, produced by the method of any one of Claims 1 to 25.

27. A method of preserving a bakery product comprising the method of any one of Claims 1 to 25.

28. A fermentation broth comprising from about 15 to 50% TS propionic acid, from about 10% to 40% TS acetic acid, from about 1% to 15% TS succinic acid and from about 0 to 15% TS lactic acid.

29. A preservative product comprising from about 25 to 50% TS propionate, from about 10% to 35% TS acetate, from about 5 to 15% TS succinate and from about 0 to 10% TS lactate salts wherein the organic acid salts can be calcium, sodium or potassium based propionate, acetate, succinate and lactate.

30. The preservative of Claim 29, which is a powder.

31. The preservative of Claim 29, which is a concentrate (liquid).

32. A method of making a bakery product comprising the addition of the preservative of Claim 26, or 29 to 31 , or the broth of Claim 28 to the product.

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