USE OF BY-PRODUCTS FROM THE ALCOHOLIC BEVERAGE MANUFACTURING INDUSTRY

Abstract

A by-product from an alcoholic beverage manufacturing process may be used to cultivate a thraustochytrid. The by-product may comprise a residue from a distillation step used in malt whisky manufacture, such as pot ale. The cultivation may be used to provide a product with enhanced amounts of lipids, oils, or fatty acids, including omega-3 fatty acids.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. A method for cultivating a thraustochytrid in a vessel containing a composition, the method comprising: combining the composition and the thraustochytrid; and applying a process to the combined composition and thraustochytrid for cultivation of the thraustochytrid, wherein the composition comprises a by-product from an alcoholic beverage manufacturing process.

10. The method as claimed in claim 9, wherein the process comprises a step of sterilizing said vessel prior to charging it with said thraustochytrid and said composition.

11. The method as claimed in claim 10 wherein said sterilizing comprises cleaning with an alkali followed by rinsing with water followed by applying an antimicrobial agent to coat the interior surfaces of said vessel.

12. The method as claimed in claim 9, wherein the process comprises a step of pasteurizing said composition or said by-product prior to charging said vessel with the same.

13. The method as claimed in claim 9, wherein said cultivation results in a product comprising one or more of lipids, oils, or fatty acids.

14. The method as claimed in claim 13, wherein said product comprises omega-3 fatty acids.

15. The method as claimed in claim 9, wherein said cultivation comprises: a first stage of proliferating thraustochytrid from an inoculum of thraustochytrid; and a second stage of causing a stress response of the thraustochytrid due to a differing nutrient supply from that of the first stage, which enhances the accumulation of one or more omega-3 oil or other useful product.

16. The method as claimed in claim 9, wherein said cultivation comprises: a first stage of proliferating thraustochytrid from an inoculum of thraustochytrid in media that promotes sporulation; a second stage of utilizing alcoholic by-products as a carbon source for cell growth and wherein a nutrient supply differs from that of the first stage; and a third stage, wherein fermentation parameters in the third stage differ from the first stage and second stage causing a stress response of the thraustochytrid to enhance the accumulation of one or more omega-3 oil or other useful product.

17. A product comprising one or more lipids, oils, fatty acids, omega-3 fatty acids, eicosapentaenoic acid and/or docosahexaenoic acid, obtained by or obtainable by the method of claim 9.

18. A method of processing a by-product from an alcoholic beverage manufacturing process, the method comprising: providing a by-product from an alcoholic beverage manufacturing process; applying a thraustochytrid to said by-product; and allowing said by-product to be used as a growth medium for said thraustochytrid.

19. The method of claim 18, wherein the by-product comprises pot ale.

20. The method of claim 18, wherein the by-product comprises a residue from a distillation step used in malt whisky manufacture.

21. The method of claim 18, wherein the product comprises one or more of lipids, oils, or fatty acids.

22. The method of claim 9, wherein the composition comprises pot ale.

23. The method of claim 9, wherein the composition comprises a residue from a distillation step used in malt whisky manufacture.

Description

[0086] FIG. 1 shows the growth of thraustochytrid strains using various carbon sources;

[0087] FIGS. 2 to 4 show the growth of non-thraustochytrid strains;

[0088] FIGS. 5 to 8 show the change in dissolved oxygen content and pH over time, in growth media comprising, respectively, 12.5%, 25%, 37.5% and 50% (by volume) of pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp.;

[0089] FIGS. 9 and 10 show the change in dissolved oxygen content and pH over time, in growth media comprising 25% (by volume) of pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp., where additional glucose is added; and

[0090] FIG. 11 shows the change in cell count and dry cell weight during cultivation of the thraustochytrid Aurantiochytrium in media comprising pot ale and glucose, with further glucose added post-inoculation.

[0091] Example 1—Experiments carried out to investigate the growth of various microorganisms utilising feedstocks, indicating the superior performance of thraustochytrids

[0092] Various microorganisms were cultured in basal media (also referred to as basal freshwater or freshwater basal media) which contained salts, nitrates, yeast extract and a carbon source (glucose, glycerol, peptone or acetate). Growth data obtained from high-throughput experiments are summarized in the graphs of FIGS. 1 to 4. The percentage values associated with glucose, glycerol, peptone or acetate refer to concentration with respect to a standard solution of 40 g/l; therefore, for example, 200% glucose denotes 80 g glucose per litre. The vertical axes in FIGS. 1 to 4 indicate growth rates, assessed by optical measurement and approximated through NADH production. FIG. 1 shows results using Thraustochytrid strains (Aurantiochytrium). The strains were obtained from the Japanese Biological Resource Centre NBRC (www.nite.go.ip/en/nbrc/index.html). They include Aurantiochytrium (NBRC no. 102614) and Schizochytrium (NBRC no. 102617). Comparative FIGS. 2 to 4 show results with the following non-thraustochytrid algal strains: Chlamydomonas sp., N. coccoides and C. protethecoides, respectively

[0093] The thraustochytrid strains distinguished themselves by reaching the maximum growth rate within approximately 24 hours from the time of inoculation (FIG. 1) compared to 60-80 hours required for the other strains (FIGS. 2 to 4). In a large-scale fed-batch fermentation process this could effectively reduce the overall runtime for a batch by two days. The thraustochytrids were also effective at growing on a range of waste organic matter materials and other materials including fructose and potato starch. The Thraustochytrid strains had a superior growth rate and were capable of utilising a wider range of carbon sources and waste streams when compared to other strains.

[0094] Further experiments focused on the culturing of microorganisms using by-products of food and drink industries and it was found that Thraustochytrids performed particularly well using products from fermentation and related processes including pot ale.

[0095] Example 2—Growth of the thraustochytrid Aurantiochytrium sp. using pot ale

[0096] Microorganism growth was monitored by observing the change in dissolved oxygen (DO) content in a vessel. FIG. 5 shows the progression of dissolved oxygen content (100% indicates saturation) in a growth medium comprising 12.5% (by volume) of pot ale inoculated with a sample of the thraustochytrid Aurantiochytrium sp. As the thraustochytrid feeds on the nutrients supplied by the pot ale it grows, consuming oxygen. This is reflected in the drop in dissolved oxygen. Once all the nutrients have been consumed, the thraustochytrid is no longer able to consume oxygen. The DO value rises as the oxygen dissolved in the medium equilibrates with the atmospheric oxygen.

[0097] The changes in pH are believed to be due to the different metabolic reactions that occur during the fermentation. The initial decrease in pH is believed to be due to production of organic acids. As the fermentation progresses. utilisation of complex nitrogen sources within the pot ale may increase the concentration of ammonia which in turn may stabilise or increase the pH.

[0098] FIGS. 6 to 8 differ from FIG. 5 in that they relate to the use of initial concentrations of pot ale of 25%, 37.5% and 50% by volume respectively.

[0099] In all cases, it can be seen that the pot ale acts as an effective organic matter feedstock for the thraustochytrid. Nevertheless, the range of 20 to 55% by volume is particularly effective as this leads to sustained growth.

[0100] Addition of further nutrient, e.g. glucose, brings further enhancements. FIG. 9 shows the growth of Aurantiochytrium sp. using 25% pot ale growth media with 10 g/L glucose present initially. It can be seen that the thraustochytrid utilises the carbon source within the first 24 hours of inoculation, as indicated by the drop in the dissolved oxygen reading. In comparison, FIG. 10 shows the growth of Aurantiochytrium sp. using 25% pot ale growth media with 20 g/L glucose present initially, and with a further 20 g/L glucose added after 3 days: it can be seen that the growth is sustained at maximum levels for up to 144 hours due to the greater quantity of carbon source.

[0101] Further experiments investigated the effect of adding further glucose after inoculation. Two experiments were carried out with defined media and pot ale media at 25% concentration using Aurantiochytrium sp. For the first experiment the glucose concentration was set at 20 g/L and for the second experiment the strains were initially inoculated into media containing 20 g/L glucose and a further 20 g/L glucose was injected 3 days post inoculation. The mass change per 100 ml in the case of the former was 2.52 g and in the case of the latter was 4.48 g. Accordingly, in the case where a further 20 g/l of glucose was added post-inoculation the productivity was 44.8 g per litre. This is illustrated further in the table below and in FIG. 11.

TABLE-US-00001 Final weight Preweight crucible crucible Productivity/ Productivity/ (g) (g) 100 m 1 L Strain 102614 Aurantiochytrium sp. grown on 25% pot ale and 40 g/L glucose: Productivity - 44.759 g/L 55.15 59.6259 4.4759 44.759 Strain 102614 Aurantiochytrium sp. grown on 25% pot ale and 20 g/L glucose: Productivity - 25.244 g/L 54.22 56.744 2.5244 25.244

[0102] The cell count measurements were carried out using a haemocytometer following a standard protocol: [0103] 1. A 10 μL sample of the fermentation media that had been appropriately diluted was added between the cover slip and the counting chamber. [0104] 2. Following a standard counting pattern, the cells were counted. [0105] 3. Depending on the type of haemocytometer used, a standard formula was used to calculate the cell density (this takes into consideration the average cells, dilution factors and the volume of the chamber, which is dependent on the type of haemocytometer).

Cell density=average cells*dilution factor/square volume

[0106] The dry cell weights gave an indication of the productivity of the fermentation. It was measured over the course of the fermentation and was an additional method used for the quantification of the cells. The below method was used to measure dry weight: [0107] 1. A receptacle was weighed and its mass recorded. [0108] 2. A defined amount of sample was placed in the receptacle and weighed and its mass recorded. [0109] 3. The sample was dried until there was no change in its mass after repeated drying/weighing. [0110] 4. The receptacle was weighed with the dried sample and its mass recorded.

[0111] Calculations were carried out as follows: [0112] 1. Subtract the weight of the receptacle from the 10 ml of sample with receptacle=Wet mass [0113] 2. Subtract the weight of the receptacle from the dried sample with receptacle=Dry mass [0114] 3. Dry mass/Wet mass*100=Dry weight (%)

[0115] All the dry weight calculations were carried out either in duplicate or triplicate and the value averaged to allow for error.

[0116] The cell count and dry weight are not linearly related. The dry weight also includes the mass of the pot ale solids whereas the cell count represents only the cells present in the sample. As the fermentation progresses the pot ale solids are depleted whilst simultaneously the cell numbers increase but still contributing to the overall dry weight differently compared to the pot ale solids. Therefore one value lags behind the other.

[0117] Having identified thraustochytrids as exhibiting surprisingly large growth rates compared to the other stains tested, the thraustochytrids were tested further to optimise growth conditions. In particular, the salinity requirements of the thraustochytrids were examined. Thraustochytrids are marine strains and live naturally in a saline environment. High salt concentrations may be disadvantageous however, as this encourages corrosion of metallic parts, for example vessels. In a series of experiments it was determined that thraustochytrids are capable of growing at 25% to 50% of normal sea water concentration without any significant reduction in growth rates. Nevertheless higher salt concentrations may be used if desired. The thraustochytrid growth was monitored by using a dissolved oxygen sensor.

[0118] Example 3—Detailed description of an example embodiment

[0119] A stainless-steel brewing vessel from a distillery was used as the vessel in which to cultivate thraustochytrid. The brewing vessel was first rinsed with clean water through a spray ball, or similar inlet device, located at the top of the vessel. The vessel was then cleaned with 2% sodium hydroxide for 10 minutes at a temperature of 60°, before rinsing again with clean water. The inside 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 a positive pressure. The cleaning and sterilization treatment was performed on every port and line attached to the vessel. This included in-line pasteurisation equipment which was to come into contact with the growth medium.

[0120] To prepare a growth medium for the thraustochytrid, pot ale from a Scotch whisky distillery was diluted to contain between 25% and 37.5% pot ale. The chemical oxygen demand was between 13.75 g/L and 20.63 g/L. The pH was adjusted to between 6.8 and 7.5 by adding sodium hydroxide. The following were then added to the pH adjusted, diluted pot ale solution:

[0121] 20 g/L of glucose;

[0122] 10.4 g/L of artificial sea salts (for example comprising 66% sodium chloride, 16% magnesium sulphate, 13% magnesium chloride, and smaller quantities of calcium chloride and potassium chloride.

[0123] An additional 20 g/L to 40 g/L of glucose was added at day 3 or day 4 depending on the density.

[0124] The growth medium prepared in the above manner was fed to the vessel through a flash pasteurizer in the form of a plate heat exchanger. The plate heat exchanger was used to heat the growth medium up to a temperature of between 70° C. and 125° C. The growth medium was maintained at this elevated temperature for between 10 s and 120 s. The medium was then cooled down 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 incoming medium entering the pasteurizer, preheating this incoming medium. A further heat exchanger then reduced the temperature of the pasteurized medium further down to the initial growth temperature of 30° C. The pasteurized growth medium was then passed into the cultivation vessel.

[0125] An inoculum of thraustochytrid was prepared by growing a microscope confirmed monoculture of the microorganism on a plate culture for 4 or 5 days. The precise timing is dependent on growth conditions and the person skilled in the art will have no difficulty in judging this appropriately. This initial inoculum was next transferred to a suitable sterile borosilicate glass container with 3-way hosing cap. The glass container comprised a section of peristaltic hosing section attached for sterile transfer of the inoculum to the cultivation vessel. A glass syringe was attached to a check valve which was attached to the glass container, allowing for sterile sampling of the inoculum prior to it entering the cultivation vessel.

[0126] A mixture of thraustochytrid inoculum and pasteurized growth medium (a highly nutritious medium consisting of pot ale at 37.5%, glucose at 20 g/L and 10.4 g/L of artificial sea salts and high levels of dissolved oxygen) present in the cultivation vessel was maintained at a growth temperature of around 30° C. for a period of 3 to 4 days, during which the thraustochytrid proliferated rapidly. This resulted in a maximum cell number being achieved and the nutrients being depleted. In the next phase the cells entered the accumulation phase of growth: at this stage a further 20 g/L of glucose was fed to the vessel and the vessel temperature was reduced to 10-15° C. This change in the conditions is thought to induce a stress reaction in the thraustochytrid and thereby encourage the production of Omega-3 oils. The thraustochytrid was then cultured for a further 2 days.

[0127] Quality controls were carried out at appropriate stages to determine the purity of the starter culture, the efficacy of the in-line pasteurisation, the functioning of the air filter and the quality of the vessel sterilization.

[0128] At the end of the growth process, the resultant thraustochytrid was dewatered using conventional dewatering techniques. Dewatering was carried out to approximately 10% of the original volume. This produced an Omega-3 enriched algal paste. This paste can be further processed to produce an oil or a powder.

[0129] Example 4—Enzymatic pre-treatment of distillery by-product

[0130] It was found that enzymatic pre-treatment of distillery by-product could increase the glucose concentration by 480% in some experiments. On average, during the course of several fermentations carried out with enzymatic pre-treatment, it was found to increase the glucose utilisation of the thraustochytrid by up to 20% and the productivity by at least 25%.

[0131] To enable maximum conversion efficiency of the enzymes, prior to enzyme addition the pH of the pot ale media was adjusted to between pH 6-7 and enzyme addition was at a concentration range of between 0.01-0.05% v/v.

[0132] Algae (thraustochytrids) were aseptically inoculated from existing stock liquid cultures at 2% v/v within a laminar flow cabinet. Algal flask cultures were incubated at 30° C. with shaking (95 rpm) for a minimum of 2 days prior to enzymatic treatment experiments.

[0133] Enzymatic pre-treatment was performed in 50 mL conical flaks containing 25 mL fresh pot ale pH adjusted to pH 6-7 depending on the enzyme being used. All experiments were carried out in triplicate flasks along with triplicate control flasks. Preceding enzyme addition, pot ale flasks were autoclave sterilised using standard sterilisation temperature and duration.

[0134] Enzyme stock solutions were aseptically added at 0.02% v/v to sterilised pot ale flasks and filter sterilised water added to control flasks. Following enzyme addition, flasks were re-sealed and incubated at 30° C. with shaking (95 rpm) for 20 hours.

TABLE-US-00002 TABLE Adjusted pH used in each flask, chosen based on best fit reported for each enzyme optimum pH and working range. Enzyme Pot ale adjusted pH used Amyloglucosidase 6 Prolyl endopeptidase 6 Cellulase 6 Papain 7 Subtilisin 7 Lyticase 7 Control (pH 6) 6 Control (pH 7) 7

[0135] Enzymatic pre-treatment validation on soluble glucose levels were measured prior to and following enzyme treatment step, Preceding the incubation step, triplicate samples were taken aseptically from each sterilised flask and final post-treatment triplicate samples at the end of the incubation period. To carry out the glucose measurements the samples were centrifuged at 13,000 rpm for 10 minutes and the glucose was measured using SD Code free glucose monitor and corresponding glucose strips.

[0136] For the algal growth quantification, following a 5-day incubation period algal culture flasks were triplicate sampled at the start and end of the fermentation for glucose measurements. For algal productivity calculations, a 1 mL sample was aseptically taken from each flask and use for cell count measurements detailed earlier.