Use of by-products from the alcoholic beverage manufacturing industry

Abstract

Thraustochytrids can be cultured using by-products from the alcoholic beverage production process. By-products can include residues from the distillation step used in malt whiskey production, such as pot ale. The culture can be used to provide a product with enhanced amounts of lipids, oils, or fatty acids, including omega-3 fatty acids.

Description

[Technical Field]

[0001] The present invention relates to the cultivation of microorganisms using by-products of the alcoholic beverage manufacturing industry and the production of useful compounds, including, for example, oils and fatty acids, through such cultivation. [Background technology]

[0002] The production of alcoholic beverages generates by-products that are often simply treated as waste and need to be disposed of or have uses found in an environmentally friendly and cost-effective manner.

[0003] For example, the production of malt whisky produces several such by-products. One of the main whisky by-products is pot ale, which is the residue left after the first distillation, which takes place in a pot still.

[0004] The amount of pot ale produced in a distillery amounts to about two-thirds of the pot still's capacity - typically, 8 litres of pot ale are produced for every litre of whisky produced - thus creating a significant amount of this nutrient-rich by-product liquid that needs to be disposed of.

[0005] The production of whiskey involves two distillations, with the product of the first distillation proceeding to the second distillation. The residue left after the second distillation in the whiskey production process is called spent lees. The amount of spent lees is typically one-third of the initial charge volume of the second distillation. Although the volume of spent lees produced is significantly less than the volume of pot ale, it is still an important by-product of the whiskey production process that needs to be dealt with.

[0006] Another example of a nutrient-rich by-product is found in the beer production process. Brewing beer often produces a wet colloidal debris called trub. In the brewing process, trub is the material remaining after the wort is boiled and transported for brewing. Traub also refers to the sediment remaining at the bottom of the fermenter after fermentation. This, along with wash water and other industrial waters in the beer production process, produces a nutrient-rich wastewater that generally cannot be simply discharged into municipal sewer systems without incurring costs.

[0007] By-products of alcoholic beverage production often have little economic value themselves. They are currently disposed of or used in several ways. For example, pot ale can be used as a base for ruminant livestock feed. Pot ale can also be used as a field spread, similar to brewery wastewater. Each of these disposal methods comes at a cost. Livestock feed production requires large amounts of energy. For example, pot ale must be significantly reduced in water content, which requires energy. Typically, over 90% of the water content of pot ale is removed in livestock feed production. The unavoidable costs associated with transporting by-products to treatment or disposal sites can also be significant. Whisky distilleries are often located in remote locations, which contributes to transportation or shipping costs.

[0008] Due to the significant amount of by-products from the production of alcoholic beverages, and the significant amount of organic material present in these by-products, alternative disposal routes that reduce economic and environmental costs are sought.

[0009] One recent use of pot ale is as a nutrient source for fermentation processes to produce butanol and / or acetone (WO 2012 / 001416). Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention addresses the problem of finding alternative and useful disposal routes for by-products of the production of alcoholic beverages. [Means for solving the problem]

[0011] In a first aspect of the present invention, there is provided the use of a composition for culturing Thraustochytrids, the composition comprising a by-product from an alcoholic beverage manufacturing process.

[0012] The present inventors have found that compared to other microorganisms, Thraustochytrids are particularly effective and efficient in treating alcoholic beverage by-products.

[0013] Thraustochytrids are heterotrophic microorganisms, meaning that they require an external supply of energy contained in complex organic compounds to maintain their existence. Thraustochytrids can act as scavengers and decomposers of organic material, from which they obtain the complex organic compounds they need to survive. In their natural habitat, Thraustochytrids generally live in marine (saline) environments.

[0014] By growing on alcoholic beverage by-products, Thraustochytrids accumulate significant amounts of useful substances, including lipids, oils, fatty acids, such as omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and antioxidants, which can be, or be incorporated into, foods, supplements, or dietary supplements for humans, animals, or fish.

[0015] One area in which the present invention is particularly useful is in the field of aquaculture. Fish farming is a process that produces large amounts of omega-3 fatty acids. Contains fatty acids Food Use: The present invention provides a safe and sustainable food product, in contrast to some conventional methods that source fishmeal and fish oil from pelagic fish or other unsustainable sources.

[0016] The present invention provides a method for the preparation of a pharmaceutical composition for human consumption. Living There is also an increasing demand for foods and other beneficial nutrients.

[0017] The present invention not only converts distillery and brewery waste by-products into useful materials, but also purifies the waste by-products so that they can be safely released back into the environment.

[0018] The materials produced in the present invention may be extracted or purified from Thraustochytrid cultures, or alternatively, compositions containing the Thraustochytrids themselves and the materials produced may be used directly in some applications. The Thraustochytrids and spent medium, along with any by-product residues, may be centrifuged and dewatered (e.g., dried in an oven or spray-dried).

[0019] The composition used to cultivate Thraustochytrids may have a BOD5 value within the range of 1 to 50 g / L, optionally 1 to 40 g / L, optionally 5 to 30 g / L, and optionally 10 to 20 g / L, as measured according to standardized test method 5210 B of "Standard Methods for the Examination of Water and Wastewater" (Clesceri et al., 2005, Washington DC: American Public Health Association, American Water Works Association, and the Water Environment Association; www.standardmethods.org).

[0020] The BOD5 value obtained using the above test method is one way to quantify the biochemical oxygen demand of materials used in culturing Thraustochytrids. The BOD5 value provides an indication of the amount of biodegradable organic matter present. It is the amount of dissolved oxygen required by aerobic organisms to decompose the organic material present in a given aqueous sample over a specific period of time at a certain temperature. Unpolluted rivers typically have a BOD of less than 1 mg / L. Moderately polluted rivers vary between 2 and 8 mg / L. Raw sewage averages 200 to 600 mg / L, while efficiently treated municipal sewage has a BOD of less than 20 mg / L.

[0021] Thus, it can be seen that the present invention is surprisingly capable of dealing with waste by-products that have very high biochemical oxygen demands and would otherwise generally require some other treatment or disposal.

[0022] Another way to define a composition used for culturing Thraustochytrids is by its chemical oxygen demand (COD). COD is a measure of the amount of oxygen consumed when a strong oxidizing agent is used to oxidize the materials present. A given sample may have a higher COD value than its BOD value because the former depends on the total organic content, while the latter depends on the organic content that can be consumed under certain biological conditions. The standard test method for determining COD is ASTM D1252-06(2012)e1, Test Method B, Standard Test Methods for Chemical Oxygen Demand (Dichromate Oxygen Demand) of Water, ASTM International, West Conshohocken, PA, 2012, www.astm.org). Compositions used to cultivate Thraustochytrids in accordance with the present invention may have a COD according to this test method in the range of 1 to 100 g / L, optionally 1 to 50 g / L, optionally 1 to 40 g / L, optionally 5 to 30 g / L, optionally 10 to 20 g / L, optionally 2 to 100 g / L, optionally 2 to 80 g / L, optionally 10 to 60 g / L, optionally 20 to 40 g / L.

[0023] The present invention is surprisingly capable of dealing with waste by-products that have very high chemical oxygen demands and would otherwise generally require some other treatment or disposal.

[0024] The composition for culturing Thraustochytrids used in the present invention may be or may comprise a distillery by-product, which may be optionally selected from one or more of draft, pot ale, pot ale syrup, spent lees, barley dark grain, spent wash, spent wash syrup, wastewater, or wheat or maize dark grain.

[0025] The distillation by-products may be from a malt distillation process or a grain distillation process. The distillation by-product may be a whisky by-product, such as a malt whisky by-product, e.g., a Scotch whisky by-product. The whisky by-product may be pot ale.

[0026] The present invention may utilize pot ale produced during whiskey production as well as pot ale from other sources, i.e. alcohol production processes.

[0027] Pot ale may be provided by a process that includes distilling a wash, optionally in a copper still, to produce a low wine distillate (a material that is further processed by further distillation and other steps to produce an alcoholic beverage) and the pot ale. The wash, which includes alcohol and water and other materials, may be provided by a previous fermentation step that includes fermentation of wort using yeast. The wort includes one or more carbohydrates, such as glucose and / or other sugars. The wort may be obtained by known processes, such as malting and mashing, which are well known in the malting art.

[0028] The composition used in the present invention to cultivate Thraustochytrids can be or include a brewery by-product. The brewery by-product can be brewery waste liquor. The brewery by-product can be trav. Trav is a sediment formed from non-fermentable products in wort. The brewery by-product can be brewery wastewater.

[0029] During distillation, a liquid that has already undergone a fermentation process is heated to produce alcohol, thus separating the ethanol from diluent components such as water. Other volatile components, generally organic matter, are also extracted from the fermentation liquid during distillation. Any distillation that is part of the production of an alcoholic beverage leaves behind a residual liquid and / or solid by-product. This residual by-product contains organic matter, which may include yeast or yeast residues from fermentation, and can act as a nutrient source for Thraustochytrids.

[0030] The solids content of the composition used to cultivate Thraustochytrids can optionally be up to 30% w / w, optionally up to 20% w / w, optionally up to 15%, optionally up to 10% w / w, optionally up to 5% w / w, optionally up to 3% w / w, or optionally up to 1% w / w. The solids content of the composition used to cultivate Thraustochytrids can optionally be at least 0.1% w / w, optionally at least 0.5% w / w, optionally at least 1% w / w, or optionally at least 3% w / w. For example, the range can be 1-15%. One advantage of the present invention arises from the ability of Thraustochytrids to process pot ale or other alcohol industry by-products, which may have some solids content. Nevertheless, the present invention does not require the presence of solids, and waste by-products with no or minimal solids content, including those that have undergone process steps such as filtration or other processes to remove solids, can be effectively processed by Thraustochytrids.

[0031] The composition used to cultivate Thraustochytrids may include yeast or yeast residue. Optionally, the amount of yeast or yeast residue present is from 0.1% to 20% by weight, for example from 1% to 15% by weight.

[0032] The composition used for culturing Thraustochytrids may contain carbohydrates. Optionally, the carbohydrate content is 5 to 100 g / liter, for example, 10 to 70 g / liter, for example, 20 to 50 g / liter, for example, 5 to 40 g / liter, for example, 50 to 100 g / liter.

[0033] The composition used for culturing Thraustochytrids may contain nitrate. Optionally, the nitrate content is in the range of 500 to 1,500 mg / L, or 750 to 1,000 mg / L, or 500 to 750 mg / L, or 1,000 to 1,500 mg / L.

[0034] The composition used for culturing Thraustochytrids may contain phosphate. Optionally, the phosphate content is in the range of 100-250 mg / L, or 150-200 mg / L, or 100-150 mg / L, or 200-250 mg / L.

[0035] The composition used for culturing Thraustochytrids may contain protein. Optionally, the protein content is from 1 to 100 g / l, or from 5 to 75 g / l, or from 8 to 50 g / l.

[0036] The composition may include a salt, for example sodium chloride, in the range of 0.1M to 0.8M. The composition may contain salt, for example, artificial sea salt, in the range of 0.13 M to 0.6 M. The artificial sea salt may contain sodium chloride as a major component, for example, in an amount of sodium chloride greater than 60 mol%, or 60 to 90 mol%, or 65 to 70 mol%, or about 66 mol%. The artificial sea salt has the following composition:

[0037] NaCl 66.1% MgSO4.7H2O 16.3% MgCl2 12.7% CaCl23.3% KCl 1.6% The composition may contain salt in the range of, for example, 5 to 50 g / l, or 10 to 40 g / l, or 15 to 30 g / l, or 5 to 20 g / l, or 25 to 50 g / l, or 8.5 to 43 g / l, or 8.75 to 42.5 g / l.

[0038] The composition used to cultivate Thraustochytrids may have a pH of 5 to 9, optionally 6 to 8, optionally 6.5 to 7.8, optionally 6.8 to 7.5.

[0039] The by-product (e.g., pot ale) can be diluted with, for example, water, such as tap water, to provide a composition for use in culturing Thraustochytrids. This dilution can result in a composition that achieves the properties outlined herein, such as BOD value, COD value, solids content, or other properties. Optionally, the pot ale can be diluted 2-5 times. Optionally, the pot ale can be diluted to comprise 15%-60%, optionally 20%-50%, optionally 25%-37.5%, or optionally 25%-35% of the volume of the composition for use in culturing Thraustochytrids.

[0040] The inventors anticipated that pot ale would need to be diluted to a significantly greater extent. The ability of the method of the present invention to process relatively concentrated compositions with significant COD and BOD values ​​is surprising.

[0041] The inventors also anticipated that pot ale would require some form of purification, such as centrifugation or filtration, to remove solids and potential microbial inhibitors. Surprisingly, the process of the present invention is robust enough to handle whole by-products. Thus, the present invention provides the use of a composition for culturing Thraustochytrids, including unpurified by-products from an alcoholic beverage production process.

[0042] Pot ale may also be suitably diluted using wash water from a distillery or other water. This provides the added benefit of affecting the pH of the composition, thereby reducing the need for other ingredients. Wash water from a distillery may be alkaline, and alkaline wash water may be useful for correcting the acidic nature of pot ale. The use of spent lees may also provide the necessary dilution for pot ale. Spent lees is the residue from the second distillation in whiskey production and is low in nutrients compared to pot ale. Water separated from the Thraustochytrids after growth according to the present invention may also be used to dilute pot ale.

[0043] The by-products used in the present invention, such as pot ale, may contain significant amounts of copper. This may be due to the presence of copper vessels, such as pot stills, or copper equipment. The copper content may be such that pot ale-based feeds cannot be used for sheep, which are sensitive to excessive dietary copper. In the context of the present invention, one might expect that the copper content in pot ale would inhibit the efficiency of microbial growth. However, the inventors have surprisingly found that Thraustochytrids are not affected by this at the concentration levels used in the present invention.

[0044] Other materials may be added to the by-product to provide a composition for use in culturing Thraustochytrids.

[0045] Although the by-product, e.g., pot ale, is nutrient rich, it may lack sufficient carbohydrate content for optimal growth of heterotrophic Thraustochytrids. Thus, the growth medium may contain additional amounts of carbohydrate.

[0046] Suitable carbohydrates for additional addition may include monosaccharides such as glucose, fructose, or galactose, or their oligosaccharides. Other suitable carbohydrates may include disaccharides such as lactose, maltose, or sucrose. Other complex carbon or carbohydrate sources, such as glycerol, may also be suitable for inclusion in the growth medium. Other possible components are compounds from biodiesel production (e.g., crude glycerol), sodium acetate, fructose, and potato starch. Other possible components are cellulosic biomass, carboxylic acids and their salts, fatty acids, amino acids, alcohols, esters, and other derivatives. These chemicals may provide the carbon source necessary for Thraustochytrid growth.

[0047] The process of culturing Thraustochytrids is: (i) a first step in which the Thraustochytrids are propagated from a small amount or inoculum of Thraustochytrids; and (ii) the stress response of the Thraustochytrid is one or more omega-3 fatty acidOr, to enhance the accumulation of other useful products, a second phase may be included in which the nutrient supply differs from that of the first phase.

[0048] The present invention provides omega-3 without the need for such a two-step process. fatty acid and other useful materials, but it has been found that the use of such a two-stage process can provide additional benefits, increasing the amount of useful product and the efficiency with which the product can be produced.

[0049] Optionally, the first stage itself may be considered to include two phases: a sporulation phase and a reproductive phase. During the sporulation phase, conditions, e.g., nutrient supply, may be selected to enhance, promote, or optimize spore production. During the reproductive phase, conditions, e.g., nutrient supply, may be selected to enhance, promote, or optimize Thraustochytrid reproduction.

[0050] In the first stage, a composition may be used that includes an alcoholic beverage process by-product (e.g., a composition that includes pot ale) and, optionally, other ingredients, such as additional carbohydrates (e.g., glucose) and dissolved oxygen and salts.

[0051] The sporulation stage within the first stage may use a composition comprising a defined medium that includes a carbohydrate source (eg, glucose), salts, and inorganic nitrate sources to promote sporulation.

[0052] The propagation phase within the first stage may use a composition comprising an alcoholic beverage process by-product (e.g., a composition comprising pot ale) and, optionally, other ingredients, such as additional carbohydrates (e.g., glucose) and dissolved oxygen and salts that favor cell growth.

[0053] The second stage can be distinguished from the first stage in that at least a portion of the nutrients from the alcoholic beverage process by-products (e.g., pot ale-containing compositions) are depleted, and optionally, additional amounts of carbohydrates (e.g., glucose) are added at the start of the second stage. The additional amount of carbohydrates can be utilized by the Thraustochytrids under depleted nutrient conditions to produce fatty acids, e.g., omega-3 fatty acids. fatty acid can be generated.

[0054] The amount of carbohydrate (e.g., glucose) added in the first stage can optionally be 5-60 g / L, optionally 5-50 g / L, optionally 10-40 g / L, optionally 10-30 g / L, optionally 15-25 g / L. The amount of carbohydrate (e.g., glucose) added in the second stage can optionally be the same. These amounts are applicable when a range of alcoholic by-products is used, such as pot ale (e.g., pot ale diluted to 20-55% by volume, e.g., 30-45% by volume, e.g., 35-40% by volume).

[0055] According to the present invention, the Thraustochytrid microorganisms may first be allowed to grow at a temperature selected to optimize growth (e.g., room or ambient temperature or slightly higher, e.g., 20-40°C, e.g., 25-35°C, e.g., about 30°C), typically for several days (e.g., 1-5 days, e.g., 2-4 days, e.g., 3-4 days, e.g., 78-90 hours). The amount of inorganic nitrate added may optionally be 1-10 g / L. This may maximize spore production and allow for higher cell densities during fermentation.

[0056] After this initial growth period, the cells can be transferred to a medium consisting of an alcoholic beverage process by-product (e.g., a composition containing pot ale) for growth. After several days (optionally 3-7 days), a further amount of additional carbohydrate can be added. This can help the microorganisms maximize their consumption of nutrients provided by the pot ale or other by-product. The amount of carbon source added at this stage depends on the amount or density of Thraustochytrids formed. At this point after the initial growth period, the operating temperature can be lowered, for example, to about 5-20°C, e.g., 10-15°C.

[0057] The change in growth conditions introduced several days after the start of the culture (optionally 3-4 days) is intended to induce a stress response in the microorganisms that leads to increased fatty acid production by Thraustochytrids. Nutrient depletion can help generate this stress response. Lowering the temperature can also help promote the stress response.

[0058] Growth conditions may include temperature, salinity of the growth medium, pH of the growth medium, and the amount of additional carbohydrate present in the growth medium. One or more of the following adjustments may be made to the growth conditions while the Thraustochytrids are being cultured:

[0059] The temperature can be increased or decreased. · The salinity of the growth medium may be adjusted (e.g., increased).

[0060] The pH of the growth medium can be adjusted. Additional carbohydrates may be added to the growth medium.

[0061] Typically, sterile air may be supplied to the culture vessel during growth. The air may be supplied through a sparger. The air may be filtered.

[0062] Optionally, by-products from the alcoholic beverage production process can be subjected to an enzymatic pretreatment step. Without wishing to be bound by theory, a portion of the organic matter in distillation by-products is less bioavailable and cannot be fully utilized during high-density (i.e., added carbon source) culture. This portion may contain intact yeast cells and grain proteins, as well as carbohydrates that could not be hydrolyzed to a yeast-fermentable form. Enzymatic pretreatment of the by-products can convert such compounds into simpler, more easily assimilated materials. Enzymes that can be used include the following commercially available enzymes: amyloglucosidase, prolyl endopeptidase, cellulase, papain, subtilisin, and lyticase.

[0063] The methods of growing Thraustochytrids disclosed herein have been found to result in about a 50-95% reduction, such as about a 75-95% reduction, e.g., about a 90% reduction, in some cases, in the initial BOD or COD values ​​of pot ale or other by-products.

[0064] After cultivation is complete, the cultivated Thraustochytrid microorganisms can be separated from the growth medium solution. This separation can be achieved, for example, by centrifugation, sedimentation, chemical flocculation, biological flocculation, flotation, or membrane filtration. Dehydration of the product of the growth process reduces its volume by 5% to 20%, for example, 8% to 10%, and reduces the omega-3 fatty acid An enriched algal paste may be produced. The final product may contain Thraustochytrids and separated pot ale solids. The product may be in the form of an algal paste.

[0065] The aqueous solution resulting from this separation step can be reused to dilute a fresh amount of pot ale or other by-products to produce fresh growth medium. Use of such separated water in subsequent Thraustochytrid cultures can help enhance growth rates.

[0066] Further processing of the algae paste can produce an oil or powder. The oil or powder contains omega-3 fatty acid It is also rich in vitamins and minerals and can be used as feed for aquaculture.

[0067] In a further aspect of the invention, there is provided a method for accumulating lipids produced by Thraustochytrids cultured as described herein.

[0068] In a further aspect of the invention, fatty acids, such as omega-3 fatty acids, can be obtained from Thraustochytrids cultured as described herein. fatty acid A method for accumulating is provided.

[0069] In a further aspect of the present invention, there is provided a method for producing aquaculture feed from Thraustochytrids cultured as described herein.

[0070] In a further aspect of the present invention, there is provided a method for culturing Thraustochytrids in a vessel containing a composition comprising a by-product from an alcoholic beverage production process, as defined above in relation to the first aspect.

[0071] An advantage of the present invention is that, even though the method can be used to produce foodstuffs suitable for consumption by fish, animals, and fish, the vessel does not need to be pressure-rated or pharmaceutical-grade. Standard brewing vessels (e.g., stainless steel brewing vessels) can be used. This results in significant cost savings. It is also convenient. The process can optionally be carried out at or near a distillery or brewery where alcoholic process by-products are produced and where access and supply of brewing vessels is already easy. The vessel can be a modified brewing fermenter. The vessel can be vented during use.

[0072] The method may include a preceding step of sterilizing the container. Additionally, ports and lines connected to the container, particularly those used to introduce or expel components from the container, may be sterilized. Sterilization may include one or more steps of rinsing with clean water. Sterilization may include one or more steps of washing with an alkali (e.g., sodium hydroxide solution), optionally at elevated temperatures (e.g., above 40°C, e.g., above 50°C, e.g., about 50-70°C, e.g., about 60°C). Sterilization may include one or more steps of applying an antimicrobial agent, e.g., a peroxide, e.g., peracetic acid, and allowing the antimicrobial agent to dry and coat the interior surface. Sterilization may include two or more or all of these steps. For example, sterilization may include washing with alkali, rinsing with water, and applying the antimicrobial agent. Optionally, there may be a rinsing or cleaning step (e.g., rinsing with clean water) before the alkali cleaning step. The container may be maintained under positive pressure during sterilization, e.g., by injecting an inert gas, e.g., sterile air, into the container, e.g., through a sparger at the bottom of the container.

[0073] Such sterilization procedures can be performed with the culture device assembled. This clean-in-place (CIP) method ensures high standards of hygiene, which the inventors have found to be advantageous when culturing Thraustochytrids. Sterilization can be performed using alcoholic beverage process by-products, such as pot ale, to ensure an environment in which Thraustochytrids can be cultured.

[0074] An additional advantage of the sterilization procedure of the present invention is that it eliminates the need for steam sterilization, which may require more expensive pressure-related vessels.

[0075] The seed organism may be harvested from a monoculture of Thraustochytrids. Subculturing from plates and liquid culture growth at this stage may each be possible. Each has its advantages. Subculturing from plates minimizes the risk of contamination, and liquid culture growth is generally faster. In either event, the purity of the inoculum may be tested before use.

[0076] According to the present invention, a container is charged with Thraustochytrids and a composition used to cultivate the Thraustochytrids, the composition comprising a by-product from an alcoholic beverage production process. The composition may be pasteurized before being placed in the container. Surprisingly, the inventors have found that pasteurization provides additional benefits, increasing effectiveness and reliability, despite the fact that the by-product may have already been subjected to significant heat treatment (e.g., pot ale may have been boiled for approximately three hours as part of the whiskey preparation process). Without wishing to be bound by theory, this may be because, for example, if spore-forming organisms are present in the port or line, contaminants (e.g., from such spore-forming organisms) may be introduced when the pot ale or other by-product is removed from the distillation or brewing equipment.

[0077] Pasteurization may involve heating the composition at a temperature and for a period of time sufficient to eliminate pathogens and other organisms other than yeast.

[0078] Pasteurization may involve heating the composition to a temperature of, for example, 70-125°C or higher, for example, 70-110°C, optionally 80-100°C, optionally 80-90°C, optionally 90-100°C, optionally 85-95°C. The composition may be heated to that temperature over a period which may optionally be from 10 seconds to 120 seconds or more, optionally from 10 seconds to 90 seconds, optionally from 20 seconds to 60 seconds, optionally from 20 seconds to 40 seconds, optionally from 25 seconds to 35 seconds. The heat treatment may optionally be a stepwise heat treatment, where the composition is heated to lower temperatures in preceding and / or subsequent steps.

[0079] The pasteurization can be in-line pasteurization. The pasteurization can be carried out by a heat exchange method, in which heat is transferred to the composition by a hot water or hot oil circuit via a hot plate or a heat exchanger.

[0080] The following examples summarize some of the experiments performed and features of some embodiments of the invention, it being understood that the scope of the invention is not limited to these details. [Brief explanation of the drawings]

[0081] [Figure 1] FIG. 1 shows the growth of Thraustochytrid strains using various carbon sources. [Figure 2] Growth of non-Thraustochytrid strains is shown. [Figure 3] Growth of non-Thraustochytrid strains is shown. [Figure 4] Growth of non-Thraustochytrid strains is shown. [Figure 5] FIG. 5 shows the changes in dissolved oxygen content and pH over time in a growth medium containing 12.5% ​​(volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. [Figure 6]FIG. 6 shows the changes in dissolved oxygen content and pH over time in a growth medium containing 25% (by volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. [Figure 7] FIG. 7 shows the changes in dissolved oxygen content and pH over time in a growth medium containing 37.5% (by volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. [Figure 8] FIG. 8 shows the changes in dissolved oxygen content and pH over time in a growth medium containing 50% (by volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. [Figure 9] FIG. 9 shows the change in dissolved oxygen content and pH over time in a growth medium containing 25% (by volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. to which additional glucose was added. [Figure 10] FIG. 10 shows the change in dissolved oxygen content and pH over time in a growth medium containing 25% (by volume) pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. to which additional glucose was added. [Figure 11] Further glucose was added after inoculation. Figure 11 shows the changes in cell number and dry cell weight during cultivation of the Thraustochytrid Aurantiochytrium in a medium containing pot ale and glucose. DETAILED DESCRIPTION OF THE INVENTION

[0082] Example 1 - Experiments conducted to investigate the growth of various microorganisms utilizing feedstocks, demonstrating the superior performance of Thraustochytrids Various microorganisms were cultured in a basal medium (also called basal freshwater or freshwater basal medium) containing salts, nitrates, yeast extract, and a carbon source (glucose, glycerol, peptone, or acetate). Growth data from high-throughput experiments are summarized in the graphs in Figures 1-4. Percentage values ​​related to glucose, glycerol, peptone, or acetate refer to concentrations relative to a 40 g / L standard solution. Thus, for example, 200% glucose represents 80 g glucose / liter. The vertical axes in Figures 1-4 show growth rates assessed by optical measurements and estimated by NADH production. Figure 1 shows results using a Thraustochytrid strain (Aurantiochytrium). The strain was obtained from the Japan Biological Resource Centre (NBRC) (www.nite.go.jp / en / nbrc / index.html). The strains included Aurantiochytrium (NBRC No. 102614) and Schizochytrium (NBRC No. 102617). Comparative Figures 2-4 show results using the following non-thraustochytrid algal strains: Chlamydomonas sp. N. coccoides and C. protethecoides, respectively.

[0083] The Thraustochytrid strain was distinguished by reaching a maximum growth rate within approximately 24 hours from the time of inoculation (Figure 1), compared to the 60-80 hours required for other strains (Figures 2-4). In a large-scale fed-batch fermentation process, this could effectively reduce the overall run time of the batch by two days. Thraustochytrids were also effective at growing on a range of waste organic materials, as well as other materials, including fructose and potato starch. The Thraustochytrid strain had superior growth rates and was able to utilize a wide range of carbon sources and waste streams when compared to other strains.

[0084] Further experiments focused on culturing the microorganisms using by-products from the food and beverage industry, and it was found that Thraustochytrids performed particularly well using products from fermentation and related processes, including pot ale.

[0085] Example 2 - Growth of the Thraustochytrid Aurantiochytrium sp. using pot ale Microbial growth was monitored by observing changes in the dissolved oxygen (DO) content within the vessel. Figure 5 shows the progression of dissolved oxygen content (100% indicates saturation) in a growth medium containing 12.5% ​​(by volume) pot ale inoculated with a sample of the Thraustochytrid Aurantiochytrium sp. The Thraustochytrids grow and consume oxygen as they consume nutrients provided by the pot ale. This is reflected in a decline in dissolved oxygen. Once all nutrients are consumed, the Thraustochytrids can no longer consume oxygen. As the oxygen dissolved in the medium equilibrates with atmospheric oxygen, the DO value rises.

[0086] The changes in pH are thought to be due to different metabolic reactions occurring during fermentation. The initial drop in pH is thought to be due to the production of organic acids. As fermentation progresses, the availability of complex nitrogen sources within the pot ale can increase the concentration of ammonia, which can then stabilize or increase the pH.

[0087] Figures 6-8 differ from Figure 5 in that they relate to the use of initial concentrations of pot ale of 25%, 37.5% and 50% by volume.

[0088] In all cases, pot ale was found to act as an effective organic source for Thraustochytrids, although the 20-55% by volume range was particularly effective as it provided sustained growth.

[0089] Addition of additional nutrients, such as glucose, results in further enhancement. Figure 9 shows the growth of Aurantiochytrium sp. using 25% pot ale growth medium initially containing 10 g / L glucose. The Thraustochytrids are seen to utilize the carbon source within the first 24 hours of inoculation, as indicated by the drop in dissolved oxygen readings. In comparison, Figure 10 shows the growth of Aurantiochytrium sp. using 25% pot ale growth medium initially containing 20 g / L glucose, with an additional 20 g / L glucose added after 3 days. Due to the larger amount of carbon source, growth can be seen to be sustained at maximum levels for up to 144 hours.

[0090] Further experiments investigated the effect of adding additional glucose after inoculation. Two experiments were conducted using Aurantiochytrium sp. in defined medium and 25% pot ale medium. In the first experiment, the glucose concentration was set at 20 g / L. In the second experiment, the strain was first inoculated into medium containing 20 g / L glucose, and then an additional 20 g / L of glucose was injected three days after inoculation. The mass change per 100 ml in the former case was 2.52 g, and in the latter case was 4.48 g. Therefore, when an additional 20 g / L of glucose was added after inoculation, the productivity was 44.8 g / L. This is further illustrated in the following table and Figure 11.

[0091] [Table 1]

[0092] Cell counts were performed using a hemocytometer according to standard protocols: 1. A 10 μL sample of appropriately diluted fermentation medium was added between the coverslip and the counting chamber.

[0093] 2. Cells were counted according to a standard counting pattern. 3. Depending on the type of hemocytometer used, cell density was calculated using a standard formula (this takes into account the average cell, dilution factor and chamber volume which is dependent on the type of hemocytometer).

[0094] Cell density = average cells × dilution factor / volume squared Dry cell weight provided an indication of the productivity of the fermentation. It was measured over the course of the fermentation and was an additional method used to quantitate the cells. Dry weight was measured using the following method: 1. The container was weighed and its mass was recorded.

[0095] 2. A specified amount of sample was placed in a container, weighed, and the mass was recorded. 3. After repeated drying / weighing, the sample was dried until its mass remained unchanged.

[0096] 4. The container along with the dried sample was weighed and the mass recorded. The calculation was performed as follows: 1. Subtract the weight of the container from the weight of the 10 ml sample and container = wet mass 2. Subtract the weight of the container from the weight of the dried sample and container = dry mass 3. Dry mass / wet mass x 100 = dry weight (%) All dry weight calculations were performed in either duplicate or triplicate and values ​​were averaged to account for error.

[0097] Cell number and dry weight are not linearly related. Dry weight also includes the mass of pot ale solids, while cell number represents only the cells present in the sample. As fermentation progresses, the pot ale solids are depleted and simultaneously cell number increases, but they still contribute differently to the overall dry weight compared to the pot ale solids. Therefore, one value lags behind the other.

[0098] Compared to other dyes tested, Thraustochytrids demonstrated a surprisingly high growth rate, leading to further testing to optimize growth conditions. In particular, the salinity requirements of Thraustochytrids were investigated. Thraustochytrids are marine species that naturally inhabit saltwater environments. However, high salt concentrations can be disadvantageous because they promote corrosion of metal components, such as containers. In a series of experiments, it was determined that Thraustochytrids can grow at 25% to 50% of the normal salt concentration of seawater without significantly reducing growth rates. Nevertheless, higher salt concentrations can be used if desired. Thraustochytrid growth was monitored using a dissolved oxygen sensor.

[0099] Example 3 - Detailed Description of an Exemplary Embodiment A stainless steel brewing vessel from a distillery was used as a vessel for culturing Thraustochytrids. The brewing vessel was first rinsed with clean water through a spray ball or similar inlet device located on the top of the vessel. The vessel was then washed with 2% sodium hydroxide at 60°C for 10 minutes, and then rinsed again with clean water. The interior of the vessel was then coated with 0.1% peracetic acid, which was allowed to dry on the surface. Throughout this cleaning and sterilization process, sterile air was injected into the vessel through a sparger at the bottom of the vessel, maintaining the vessel under positive pressure. The cleaning and sterilization process was performed on all ports and lines attached to the vessel, including the in-line pasteurizer that came into contact with the growth medium.

[0100] To prepare growth media for Thraustochytrids, pot ale from a Scotch whisky distillery was diluted to contain 25% to 37.5% pot ale. The chemical oxygen demand was 13.75 g / L to 20.63 g / L. The pH was adjusted to 6.8 to 7.5 by adding sodium hydroxide. The following was then added to the pH-adjusted diluted pot ale solution: glucose 20 g / L; Artificial sea salt 10.4 g / L (e.g., containing 66% sodium chloride, 16% magnesium sulfate, 13% magnesium chloride, and smaller amounts of calcium chloride and potassium chloride)

[0101] Further, glucose was added at 20 g / L to 40 g / L depending on the density on the third or fourth day.

[0102] The growth medium prepared as described above was fed into a vessel through a flash pasteurizer in the form of a plate heat exchanger. The plate heat exchanger was used to heat the growth medium to a temperature of 70°C to 125°C. The growth medium was maintained at this elevated temperature for 10 to 120 seconds. The medium was then cooled to 30°C. This was done in two stages. In the first stage, heat from the medium was recovered in a further heat exchanger and transferred to the influent medium entering the pasteurizer, preheating this influent medium. A further heat exchanger then further reduced the temperature of the pasteurized medium to the initial growth temperature of 30°C. The pasteurized growth medium was then passed into the culture vessel.

[0103] Thraustochytrid inoculum was prepared by growing a microscopically confirmed monoculture of the microorganism on a plate for 4 or 5 days. The exact timing depended on the growth conditions, and one skilled in the art would have no difficulty in determining this appropriately. This initial inoculum was then transferred to a suitable sterile borosilicate glass vessel equipped with a three-way hose cap. The glass vessel contained a section of peristaltic hose attached for sterile transfer of the inoculum to the culture vessel. A glass syringe was attached to a check valve attached to the glass vessel, allowing for sterile sampling of the inoculum before it entered the culture vessel.

[0104] The mixture of Thraustochytrid inoculum and pasteurized growth medium (a nutrient-rich medium consisting of 37.5% pot ale, artificial sea salt, 20 g / L and 10.4 g / L glucose, and high levels of dissolved oxygen) present in the culture vessel was maintained at a growth temperature of approximately 30°C for 3-4 days, during which the Thraustochytrids rapidly multiplied. This resulted in maximum cell number and nutrient depletion. In the next phase, the cells entered an accumulation phase of growth; at this stage, an additional 20 g / L of glucose was supplied to the vessel, and the vessel temperature was reduced to 10-15°C. This change in conditions induced a stress response in the Thraustochytrids, thereby increasing their omega-3 fatty acid The Thraustochytrids were then cultured for an additional 2 days.

[0105] Quality controls were performed at appropriate stages to determine the purity of the starter culture, the effectiveness of the in-line pasteurization, the function of the air filter and the quality of the container sterilization.

[0106] At the end of the growth process, the resulting Thraustochytrids were dehydrated using conventional dehydration techniques to approximately 10% of their original volume. fatty acid An enriched algae paste was produced, which can be further processed to produce an oil or powder.

[0107] Example 4 - Enzymatic pretreatment of distillation by-products Enzymatic pretreatment of distillation by-products has been shown to be capable of increasing glucose concentrations by up to 480% in some experiments. On average, over the course of several fermentations using enzymatic pretreatment, it was found to increase Thraustochytrid glucose utilization by up to 20% and productivity by at least 25%.

[0108] To allow for maximum conversion efficiency of the enzyme, the pH of the pot ale medium was adjusted to pH 6–7 before enzyme addition, and the enzyme addition was in the concentration range of 0.01–0.05% v / v.

[0109] Algae (Thraustochytrids) were inoculated aseptically from a 2% v / v existing stock culture in a laminar flow cabinet. Algal flask cultures were incubated at 30°C with shaking (95 rpm) for a minimum of 2 days prior to enzyme treatment experiments.

[0110] Enzyme pretreatment was performed in a 50 mL conical flask containing 25 mL of fresh pot ale, pH adjusted to pH 6-7 depending on the enzyme used. All experiments were performed in triplicate flasks, along with triplicate control flasks. Pot ale flasks were autoclaved using standard sterilization temperatures and times before enzyme addition.

[0111] The enzyme stock solution was aseptically added to sterile pot ale flasks at 0.02% v / v, and sterile water was filtered and added to the control flasks. After enzyme addition, the flasks were resealed and incubated at 30°C with shaking (95 rpm) for 20 hours.

[0112] [Table 2]

[0113] Table: Adjusted pH used in each flask, selected based on the best fit reported for each enzyme's pH optimum and working range.

[0114] The adequacy of the enzyme pretreatment on soluble glucose levels was measured before and after the enzyme treatment step. Triplicate samples were aseptically taken from each sterile flask prior to the incubation step, and final triplicate posttreatment samples were aseptically taken at the end of the incubation period. To perform glucose measurements, samples were centrifuged at 13,000 rpm for 10 minutes, and glucose was measured using an SD Code-free glucose monitor and corresponding glucose strips.

[0115] For quantification of algal growth, after a 5-day incubation period, algal culture flasks were sampled in triplicate at the beginning and end of fermentation for glucose measurements. To calculate algal productivity, 1 mL samples were aseptically taken from each flask and used for cell count measurements as detailed above.

Claims

1. 1. Use of a composition for culturing Thraustochytrids, said composition comprising residue from a distillation step used in the production of malt whisky.

2. The use according to claim 1 , wherein the composition comprises pot ale.

3. 3. The use according to claim 2, wherein the composition contains pot ale diluted to 20% to 50% v / v.

4. 4. The use according to any one of claims 1 to 3, wherein the composition has a chemical oxygen demand of 5 to 30 g / L, measured according to standardized test method ASTM D1252-06(2012) e1 (Test Method B).

5. 5. The use according to any one of claims 1 to 4, wherein the composition has a yeast content of 1 to 5 g / l, a carbon source in the range of 20 to 50 g / l, and salt in the range of 8.75 to 42.5 g / l.

6. 6. A method for culturing Thraustochytrids in a vessel containing a composition comprising residue from a distillation step used in the production of malt whisky, wherein the composition is as defined in any one of claims 1 to 5.

7. 7. The method of claim 6, comprising sterilizing the container before placing the Thraustochytrids and the composition into the container.

8. 8. The method of claim 7, wherein the sterilization comprises washing with alkali, followed by rinsing with water, followed by applying an antimicrobial agent to coat the interior surface of the container.

9. The method of any one of claims 6 to 8, comprising pasteurizing the composition before filling the container with the composition.

10. 10. The method of any one of claims 6 to 9, wherein the culturing produces a product comprising one or more of a lipid, oil, or fatty acid.

11. 11. The method of claim 10, wherein the product comprises omega-3 fatty acids.

12. 12. The method of claim 6, wherein the culturing comprises a first stage in which the Thraustochytrids are grown from an inoculum of the Thraustochytrids; and a second stage in which the nutrient supply is different from that of the first stage so that the stress response of the Thraustochytrids enhances the accumulation of one or more omega-3 fatty acids or other useful products.

13. 12. The method of claim 6, wherein the culturing comprises a first stage in which the Thraustochytrids are grown from an inoculum of the Thraustochytrids in a medium that promotes sporulation; a second stage in which nutrient supply differs from that of the first stage such that alcoholic beverage by-products are utilized as a carbon source for cell growth; and a third stage in which fermentation parameters differ from those of the first two stages to induce a stress response in the Thraustochytrids to enhance accumulation of one or more omega-3 fatty acids or other useful products.

14. 14. A method for producing a product comprising one or more lipids, oils, fatty acids, omega-3 fatty acids, eicosapentaenoic acid and / or docosahexaenoic acid, by the method of any one of claims 6 to 13.

Images