A Method of Enhancing Ethanol Fermentation
20230112015 · 2023-04-13
Assignee
Inventors
Cpc classification
C12N1/38
CHEMISTRY; METALLURGY
Y02E50/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
C12N1/38
CHEMISTRY; METALLURGY
Abstract
A method of forming an ethanol fermentation enhancement mixture is provided and involves: hydrating a dried yeast with at least 0.1% Baclyte or a banana extract by volume, and a yeast growth media to produce a pre-fermentation mixture; and maintaining the pre-fermentation mixture at a temperature between 20° C. and 40° C. for between 30 minutes and 8 hours. Alternatively, the method involves: providing a solution of hydrated activated yeast; supplementing the solution of hydrated activated yeast with 0.1% to 25% BacLyte or banana extract by volume; and maintaining the solution of hydrated activated yeast at a temperature between 20° C. and 40° C. for between 30 minutes and 8 hours. A fermentation method involves preparing an ethanol enhancement mixture; adding the mixture to a bulk fermentation mixture containing a sugar source; and maintaining the bulk fermentation mixture at temperature of between 2° C. and 40° C. to allow fermentation of the sugar source to ethanol.
Claims
1. A method of forming an ethanol fermentation enhancement mixture comprising the steps of: hydrating a dried yeast with at least 0.1% Baclyte or 0.1% of a banana extract by volume, and a yeast growth media to produce a pre-fermentation mixture; and maintaining the pre-fermentation mixture at a temperature between 20° C. and 40° C. for between 30 minutes and 8 hours to form the enhancement mixture.
2. The method according to claim 1, wherein the method comprises the step of: hydrating a dried yeast with at least 0.1% Baclyte.
3. The method according to claim 1, wherein the method comprises the step of hydrating a dried yeast with at least 0.1% of a banana extract.
4. The method according to claim 1, wherein the yeast growth media is a YPD media.
5. The method according to claim 4, wherein the YPD media comprises 10% yeast extract, 20% peptone, and 20% dextrose, with the remainder being water.
6. The method according to claim 1, wherein the yeast is Saccharomyces cerevisiae.
7. The method according to claim 1, wherein the mixture is maintained at a temperature between 32° C. and 38° C.
8. The method according to claim 1, wherein the amount of Baclyte or banana extract in the pre-fermentation mixture is between 0.1% and 25% by volume.
9. (canceled)
10. The method according to claim 9, wherein the amount of BacLyte or banana extract in the pre-fermentation mixture is between 2% and 10% by volume.
11. (canceled)
12. The method according to claim 1, wherein the pre-fermentation mixture is maintained at temperature for between 2 and 8 hours.
13. The method according to claim 12, wherein the pre-fermentation mixture is maintained at temperature for between 3.5 and 4.5 hours.
14. A method of forming an ethanol fermentation enhancement mixture comprising the steps of: providing a solution of hydrated activated yeast; supplementing the solution of hydrated activated yeast with 0.1% to 25% BacLyte by volume or 0.1% to 25% of a banana extract; and maintaining the solution of hydrated activated yeast at a temperature between 20° C. and 40° C. for between 30 minutes and 8 hours to form the enhancement mixture.
15. The method according to claim 14, wherein the yeast is Saccharomyces cerevisiae.
16. The method according to claim 14, wherein the solution of hydrated activated yeast is maintained at a temperature between 32° C. and 38° C.
17. The method according to claim 14, wherein the solution of hydrated activated yeast is supplemented by 0.5% to 1.0% by volume of BacLyte.
18. The method according to claim 14, wherein the solution of hydrated activated yeast is supplemented by 2% to 10% by volume of BacLyte.
19. (canceled)
20. The method according to claim 14, wherein the solution of hydrated activated yeast is maintained at temperature for between 2 and 8 hours.
21. (canceled)
22. A fermentation method comprising the steps of: iv) preparing an enhancement mixture by carrying out the method according to claim 1; v) adding the enhancement mixture to a bulk fermentation mixture containing a sugar source; and vi) maintaining the bulk fermentation mixture at temperature of between 2° C. and 40° to allow fermentation of the sugar source to ethanol.
23. The fermentation method according to claim 22, wherein the bulk fermentation mixture is not further supplemented with BacLyte beyond that present in the enhancement mixture.
24. The method according to claim 22, wherein the sugar source is a potato, molasses, grain, sugar cane or any other suitable fermentation feedstock.
Description
DRAWINGS
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FIRST INVESTIGATION
[0056] The results of a first investigation into the use of BacLyte to form an enhancement mixture are shown in
Preparation of Enhancement Mixture
[0057] A Saccharomyces cerevisiae distiller's yeast (Safsprit HG-1 produced by Fermetis) was used to prepare four samples of enhancement mixture in triplicate. Each sample was subject to different hydration regimes, along with a control sample in which the yeast was not hydrated. The hydration regime of each sample was as follows:
TABLE-US-00001 TABLE 1 Sample Components Control (C) Dry yeast only Water (W) Dry yeast & distilled water Water & BacLyte (WB) Dry yeast, distilled water, 5% BacLyte by volume Water & YPD (WY) Dry yeast, YPD Water, YPD, & BacLyte (WYB) Dry yeast, YPD, and 5% BacLyte by volume
[0058] Each sample was hydrated for 4 hours and maintained at a temperature of 35° C. Each sample contained 0.5 g of Safsprit HG-1 yeast. The resulting enhancement mixtures were then utilised in fermentation of a fermentation mixture containing sugar. In the samples containing YPD, the sample consisted of 10% yeast extract, 20% peptone, 20% dextrose (by volume), excluding any BacLyte component.
Preparation of Fermentation Mixture
[0059] Maris Piper potatoes were used as the base sugar source for the fermentation mixture. The potatoes were rinsed and scrubbed thoroughly to remove any dirt or debris from their surface. They were then finely chopped and then crushed using a blender. A potato and water mixture was produced with a ratio of 1 kg of potato per litre of water. The mixture was homogenised and decanted into 2 litre bottles.
[0060] In order to gelatinise the starch in each bottle, each bottle was heated at 115° C. for 14 minutes. Exogeneous enzymes were then added according to accepted dosage instructions. Specifically, distiller's high temperature α-amylase was added at 1 g per litter, stirred into each bottle at 85° C. and the bottle was then held at between 85° C. and 90° C. for 1 hour. After cooling to 60° C., distiller's glucoamylase was then added to each sample at 1.4 g per litre and the bottles were then held at this temperature at 1 hour. The bottles were then cooled to 30° C. to form the fermentation mixture.
Fermentation
[0061] The enhancement mixtures were added to the bottles at 30° C. and the bottles were maintained at this temperature. Fermentation was monitored by recording the weight changes of each bottle and by recording the specific gravity of each fermentation mixture during fermentation.
Subsequent Processing
[0062] Upon completion of the fermentation the solid matter was removed from each fermentation mixture utilising a laboratory sieve stack containing wire mesh sieve layers with holes of 1400 μm and 710 μm respectively. From each fermentation mixture, one litre of liquid was obtained. At this point, the liquid from the triplicates of each sample was combined to produce three litres of liquid, one for each different sample.
[0063] Each liquid was then distilled in a 5 litre copper still to produce low wines. The alcohol content of the low wines produced from the distillation of the liquids was monitored and distillation continued until the alcohol production of the low wines reached 1% ABV.
[0064] Subsequently the low wines were then distilled in a 2 litre copper still. The first 10 ml was collected as foreshots. The alcohol content of each foreshot was measured using an Anton-Paar DMA 100 handheld density meter. The following portion of the distillate was collected as hearts. The percentage of alcohol was monitored throughout the distillation and the hearts were collected until there was a 12.5% decrease in ABV from the ABV of the corresponding foreshot. The residual liquid was discarded.
Results
[0065] The mean cumulative weight loss of each pre-fermentation sample is shown in
TABLE-US-00002 TABLE 2 Time (hrs) C W WB WY WYB 0-2 0.198 ± 0.121 0.121 ± 0.078 0.309 ± 0.128 0.503 ± 0.084 0.764 ± 0.135 2-3 0.114 ± 0.045 0.073 ± 0.035 0.138 ± 0.035 0.753 ± 0.101 0.778 ± 0.017 3-19 2.662 ± 0.005 2.906 ± 0.236 2.595 ± 0.236 3.548 ± 1.073 5.115 ± 1.585 19-21 0.010 ± 0.005 0.014 ± 0.005 0.016 ± 0.003 0.033 ± 0.001 0.017 ± 0.004 21-23 0.016 ± 0.007 0.017 ± 0.004 0.013 ± 0.004 0.015 ± 0.006 0.019 ± 0.008 24-44 0.030 ± 0.003 0.044 ± 0.006 0.040 ± 0.006 0.032 ± 0.010 0.023 ± 0.004
[0066] The difference in the rate of weight loss in the WY and WYB samples in the first two hours as compared to the C, and W samples was found to be statistically significant (p<0.05). Furthermore the weight loss of the WYB samples was significantly higher than the rates of the weight loss of WB and WY samples in the first two hours. The weight loss in the third hour of fermentation decreased in the C, W, and WB samples but increased in the WY and WYB samples. The weight loss in the third hour was significantly higher in the WY and WB samples than those of the C, W, and WB samples. Weight loss increased significantly in all samples in the 3-19 hour time period. Again, the highest weight loss occurred in the WYB sample and this weight loss was significantly higher than any other sample. After 19 hours the hourly rate of weight loss decreased markedly. This reduced hourly weight loss continued in the subsequent time periods.
[0067] From the measured weight loss it is possible to calculate a theoretical yield value of ethanol for each sample from the cumulative weight loss. In particular, a theoretical equation for the partial oxidation of glucose through the fermentation pathway, taking into consideration the molecular weight of the respective components, states that per kg of glucose, there is a theoretical yield of 0.489 kg of CO.sub.2 and 0.511 kg of glucose (Daoud & Searle, 1990). Using these yield values, ethanol and CO.sub.2 production can be calculated from the cumulative weight loss. The results of this calculation are shown in
TABLE-US-00003 TABLE 3 Sample Mean Ethanol Yield C 37.8 g W 40.5 g WB 40.2 g WY 38.1 g WYB 51.6 g
[0068] The WB and WY samples showed no more ethanol production than the W sample and hardly any more ethanol production than the C sample. Surprisingly, the WYB sample produced significantly more ethanol than any other sample, showing the advantageous use of the combination of YPD media and BacLyte in pre-fermentation over the use of either separately.
[0069] The volume of low wines produced during the subsequent processing and the alcohol by volume of the low wines is set out in table 4:
TABLE-US-00004 TABLE 4 Sample Volume (ml) Alcohol by Volume (%) C 950 13.9% W 920 13.0% WB 995 13.4% WY 905 13.9% WYB 910 14.9%
[0070] The WYB sample produced a low wine of significantly higher alcohol by volume than any other sample. It is noted that the WB and WY samples produced low wines with an alcohol by volume no higher than that of the control sample.
[0071] The volume of the hearts produced during the subsequent processing and the alcohol by volume of the hearts is set out in table 5:
TABLE-US-00005 TABLE 5 Sample Volume (ml) Alcohol by Volume (%) Alcohol volume (ml) C 81 58.3% 47.2 W 102 54.9% 56.0 WB 77 59.9% 46.1 WY 79 59.9% 47.3 WYB 95 59.8% 56.8
[0072] It is believed that the result for the W sample in table 5 is anomalous. This is because the volume of liquid produced and the alcohol by volume of this sample is not in keeping with any of the WB, WY, or C samples. Further, in contrast to all of the other samples, the alcohol by volume of the W low wine sample is significantly below 60% and the volume of low wine is similar to that of the W, WY and WYB samples and significantly lower than either the C or WB samples. As such one would not reasonably expect such a high volume for W in Table 5. In addition, in contrast to all of the other samples, the amount of alcohol in the W sample is not in keeping with the theoretical alcohol yield set out above in table 3. This indicates that there could have been methodological issues in producing the hearts from the W sample and the result of the W sample shown in table 5 is not correct.
[0073] If the W sample is accepted to be anomalous, the hearts of the WYB sample have an alcohol volume more than 20% greater than the other samples. Most surprisingly, the hearts of the WYB sample have an alcohol volume 20% greater than either the WY or the WB sample, which is something that would not be expected from the disclosure of the prior art.
[0074] Subsequently the contents of each of the hearts was analysed for higher alcohol content using gas chromatography with flame ionisation detector (GC-FID). The results of this analysis is set out in table 6. This table shows the contents of each heart in ng/μl:
TABLE-US-00006 TABLE 6 Component C W WB WY WYB Acetaldehyde 235.89 290.13 440.21 232.50 167.81 Ethyl acetate 1176.68 827.29 1814.29 1940.09 4235.89 Isoamyl acetate 4305.99 3983.07 5883.56 6019.06 5915.97 n-Butanol 196.97 172.68 185.78 193.34 159.31 Pentanol 5361.71 5715.15 6248.46 6127.21 5926.81 Furfural 665.16 930.98 441.22 535.93 538.65 n-Propanol N/D N/D N/D 3099.78 385.327
[0075] Most significantly, the amount of ethyl acetate is more than double in the WYB sample than any other sample, and four times that of the W sample.
[0076] In
[0081] One of the reasons for the limitation on ethanol yields from bulk fermentations is the toxicity of alcohol on yeast. In conventional fermentations when the alcohol content of the bulk fermentation mixture reaches 8.5% the bulk fermentation mixture becomes toxic to the yeast and fermentation ceases. Therefore, as shown in
[0082] BacLyte appears to have mode of action that acts upon the stress response of yeast. In particular, BacLyte appears to allow yeast to tolerate higher alcohol concentrations of up to 10%. This is shown in the maximum ethanol concentrations of the bulk fermentation mixtures of
[0083] The effect of the use of the pre-incubation is also shown by comparing mixture i) with mixtures ii), iii), and iv). In particular, in mixture i) there is an initial time-lag of around 6 hours whilst the yeast propagates within the bulk fermentation mixture. If an enhancement mixture is used in accordance with the present invention then this time-lag is eliminated as the yeast has already propagated.
[0084] In addition it is expected that pre-incubation of yeast with BacLyte results in increased fermentation rates of bulk fermentation mixtures as compared to fermentation of bulk fermentation mixtures without BacLyte. This is because of the increased metabolic activity of yeast in the presence of BacLyte. This can be seen by comparing the fermentation rates of mixtures iii) and iv) with the fermentation rates of mixtures i) and ii).
[0085] In summary, fermentation according to the present invention utilising an enhancement mixture prepared according to the present invention is expected to provide higher ethanol yields at a quicker rate, as compared to either traditional fermentation methods or prior art fermentation methods utilising enhancement mixtures.
[0086] In particular, as shown in the data above at table 3, the theoretical ethanol yield of a bulk fermentation mixture utilising an enhancement mixture according to the present invention has been found to be 25% higher than the theoretical ethanol yield than bulk fermentation mixtures utilising hydrated yeast according to the prior art. Hydrated yeast according to the prior art includes yeast hydrated in water alone, yeast hydrated with only BacLyte and water, and yeast hydrated with only YPD media.
SECOND INVESTIGATION
[0087] The results of a second investigation into the use of BacLyte and a banana extract to form an enhancement mixture with alternative sugar sources are shown in
[0088] Yeast was hydrated in accordance with the hydration protocol set out in Hornig, J (2019) “A study into the efficacy of hydration regimes & novel nutrient-rich media on yeast propagation, fermentative activity and distillate composition in potato-based Spirit” MSc dissertation [2018-2019] The School of Engineering and Physical Sciences (“Hornig et al.). The second investigation was performed with a different sugar source to the potato feedstock utilised in Hornig et al. with these tests looking at the effects of “Propagreater” and a banana extract in grain and sugar cane fermentations
Materials:
[0089] Yeast Pinnacle MG+ (fresh crumbles); [0090] “Propagreater”: a supplement consisting of a 5× concentrate of Yeast Peptone Dextrose (YPD) media and 25% Baclyte; [0091] Banana extract; [0092] Yeast Peptone Dextrose (YPD) media solution; [0093] Feedstock: [0094] Grains (rye, wheat and barley) or, [0095] Sugar cane.
[0096] The yeast was hydrated for 6 hours at 35° C. with a ratio of 1:50 (v/v) of yeast:water. The yeast used was fresh Pinnacle MG+ acclimatised to fermentation temperature, with 2 g/L pitching rate.
[0097] Six trials were carried out as follows: [0098] Trial 1 Direct yeast pitch [0099] Trial 2 6 hours incubation in water [0100] Trial 3 6 hours incubation in 1×YPD media [0101] Trial 4 6 hours incubation in 1× concentrate of YPD and banana supernatant 5% [0102] Trial 5 6 hours incubation in “Propagreater” supplement (a 5× concentrate of YPD and 25% Baclyte) diluted with 4 parts water to make it 1×YPD plus 5% Baclyte [0103] Trial 6 6 hours incubation in water and banana supernatant 5%
Grain Fermentation
[0104] Set 1 of fermentations was performed with a base of finely milled 70% rye, 20% wheat and 10% malted barley, saccharification at 62° C. for 1 hour with exogenous amylase, glucoamylase and cellulase, cooled down to 25° C. before pitching the yeast into the fermentation. pH 5.2 The results of this set is shown in
[0105] Comparing each trial, we can see trial 5 (hydrated with “Propagreater”) having a much shorter lag phase and overall fermentation profile as compare to pitching with either the dry yeast or water hydrated yeast controls. Similarly trials 3 and 4 show a noticeable, yet much less rapid, gravity decrease which suggests a positive impact of pre-treatment with YPD with or without the banana extract in hydration phase. Pre-treatment of yeast with banana extract (trial 6) also showed a positive effect but significantly less than either YPD alone or when it is used in combination with YPD.
[0106] Overall, this data demonstrates that “Propagreater” as having a significant impact in reducing the lag phase of the fermentation and the significantly accelerated drop in specific gravity is indicative of accelerated alcohol production. The data also suggests similar utility for the combination of YPD and Banana extract—albeit with significantly less dramatic improvements in the yeast's performance or rate of alcohol synthesis (as shown by decreasing specific gravity). In this grain fermentation the presence of banana in the pre-treatment effected a further drop in specific gravity which is indicative of a higher level of alcohol in the final fermentate.
[0107] Using the known calculation for conversion of specific gravity to percentage alcohol by volume:
[0108] Subtract the Original Gravity from the Final Gravity.
[0109] Multiply this number by 131.25.
[0110] The resulting number is your alcohol percent, or ABV %
[0111] We are able to calculate the theoretical values of final % ABV for “Propagreater” as being 6.43%, for YPD plus 5% Banana Extract as being 6.56% over the dry yeast and water hydrated control values of 6.04%. This gives a theoretical improvement of alcohol yield from these fermentations as being 6.4% with pre-treatment with “Propagreater” and 8.6% with pre-treatment with YPD plus Banana Extract.
Sugar Cane Fermentation:
[0112] These fermentations were performed with a base of sugar cane boiled yeast nutrient and citric acid for ph correction (pH 5.2), cooled down to 25° C. before pitching the yeast. Six trials were carried out, in accordance with the method for the grain fermentation set out above. The only difference being the sugar source for the fermentations being a sugar cane source, rather than a grain source. The results of the trials are shown in
[0113] Comparing each trial, it can be seen that trial 4 (hydrated with YPD and banana extract) and trial 5 (hydrated with “Propagreater”) demonstrate specific gravity reducing much more rapidly than the dry yeast (Trial 1) and water hydrated (Trial 2) controls. Trials 1, 2 and 3 show a longer lag phase than trails 4, 5 and 6—all of which see the yeast pre-treated with either Baclyte (in the “Propagreater” formulation) or the banana extract. This clearly demonstrates the effect that the presence of Baclyte or banana extract in the pre-treatment results in increased metabolic activity of the yeast.
[0114] These tests prove the utility of “Propagreater” and banana extract plus YPD to improve fermentation efficiency and yield across multiple feedstocks. The improved effect occurring with both grain and sugar cane sources, in addition to the potato fermentation illustrated in
[0115] Pre-treatment with “Propagreater” brings about a beneficial effect in shortening lag phase and accelerating the fermentation. “Propagreater” demonstrated the highest performance in improving fermentation speed and the presence of the cruder banana extract in hydration phase also brought about greater decreases in specific gravity. Calculations of alcohol yield from final specific gravities demonstrates the effect of YPD plus banana extract or “Propagreater” (YPD plus Baclyte) as having a positive effect in terms of overall yield.
THIRD INVESTIGATION
[0116] A third investigation into the use of BacLyte and a banana extract to form an enhancement mixture was carried out.
[0117] Yeast was hydrated in accordance with the hydration protocol set out in Hornig, J (2019) “A study into the efficacy of hydration regimes & novel nutrient-rich media on yeast propagation, fermentative activity and distillate composition in potato-based Spirit” MSc dissertation [2018-2019] The School of Engineering and Physical Sciences (“Hornig et al.).
Materials:
[0118] HG-1 Yeast
[0119] “Propagreater”: a supplement consisting of a 5× concentrate of Yeast Peptone Dextrose (YPD) media and 25% Baclyte;
[0120] Banana extract;
[0121] Yeast Peptone Dextrose (YPD) media solution;
[0122] Feedstock: potato mash
[0123] Six trials were carried out as follows:
TABLE-US-00007 TABLE 7 Control Yeast HG-1 0.5 g Incubated for 6 Water (30 degrees) 5 ml hours at 30 degrees Enzyme Treated Mash 750 ml AntiFoam 0.2 ml Trial 1 Yeast HG-1 0.5 g Incubated for 6 YPD 1 ml hours at 30 degrees Water (30 degrees) 4 ml Enzyme Treated Mash 750 ml AntiFoam 0.2 ml Trial 2 Yeast HG-1 0.5 g Incubated for 6 Propagreater 1 ml hours at 30 degrees Water (30 degrees) 4 ml Enzyme Treated Mash 750 ml AntiFoam 0.2 ml Trial 3 Yeast HG-1 0.5 g Incubated for 6 20% Banana Extract 1 ml hours at 30 degrees YPD 1 ml Water (30 degrees) 3 ml Enzyme Treated Mash 750 ml AntiFoam 0.2 ml Trial 4 Yeast HG-1 0.5 g Incubated for 6 5% Banana Extract 250 microliters hours at 30 degrees YPD 1 ml Water (30 degrees) 3.8 ml Enzyme Treated Mash 750 ml AntiFoam 0.2 ml Trial 5 Yeast HG-1 0.5 g Incubated for 6 5% BacLyte 250 microliters hours at 30 degrees YPD 1 ml Water (30 degrees) 3.8 ml Enzyme Treated Mash 750 ml AntiFoam 0.2 ml
[0124] Each trial was incubated for 6 hours at 30 degrees in a water bath, in a 50 ml conical incubation vessel. After this incubation period the additions were pitched into a 1000 ml conical fermenter along with 750 ml of enzyme treated and crash cooled potato mash. These were then tested for density, pH, alcohol and sealed with one-way breathers that were sealed with antibacterial solution. From pitching the trials were then tested every 6 hours to determine the rate and growth of the ferments until completion. Upon completion, each ferment was stored at 2 degrees to stop any more malolactic fermentation or esterification before being vacuum distilled at 97 mbar to distil all the ethanol content to compare LPA yield from trial to trial.
[0125] The results of the third investigation are as follows:
TABLE-US-00008 TABLE 8 Final Gravity: Final ABV: mLPA: Final pH: Control 1.0026 7.14% 53.55 4.09 Trial 1 1.0027 7.13% 53.48 4.22 Trial 2 1.0017 7.52% 56.40 4.24 Trial 3 1.0014 7.56% 56.70 4.20 Trial 4 1.0011 7.60% 57.00 4.14 Trial 5 1.0020 7.48% 56.10 4.12
[0126] The specific gravity of the trials over time was as follows:
TABLE-US-00009 TABLE 9 Time 0 hrs 6 hrs 12 hrs 18 hrs 24 hrs 30 hrs 36 hrs 38.1 hrs Control 1.053 1.0498 1.0428 1.0326 1.0281 1.0167 1.0056 1.0026 Trial 1 1.053 1.0422 1.0356 1.0281 1.0267 1.0155 1.0082 1.0027 Trial 2 1.053 1.0392 1.0349 1.0273 1.0229 1.0122 1.0069 1.0017 Trial 3 1.053 1.0382 1.0331 1.0277 1.0237 1.0127 1.0049 1.0014 Trial 4 1.053 1.0378 1.0328 1.0269 1.0225 1.0094 1.0032 1.0011 Trial 5 1.053 1.0399 1.0347 1.0279 1.0233 1.0157 1.0057 1.0020
[0127] The fermentation ABV of the trials over time was as follows:
TABLE-US-00010 TABLE 10 Time 0 hrs 6 hrs 12 hrs 18 hrs 24 hrs 30 hrs 36 hrs 38.1 hrs Control 0.02% 1.21% 2.13% 3.46% 4.13% 5.55% 6.49% 7.14% Trial 1 0.04% 2.20% 3.07% 4.06% 4.24% 5.71% 6.67% 7.13% Trial 2 0.04% 2.60% 3.16% 4.16% 4.74% 6.14% 6.84% 7.52% Trial 3 0.02% 2.73% 3.40% 4.11% 4.63% 6.08% 7.10% 7.56% Trial 4 0.05% 2.78% 3.44% 4.21% 4.79% 6.51% 7.32% 7.60% Trial 5 0.02% 2.51% 3.19% 4.08% 4.69% 5.68% 7.00% 7.48%
[0128] As can be seen, the use of a fermentation enhancement mixture in the present invention is significantly advantageous: it produces a higher ABV more quickly than ethanol enhancement mixtures using YPD as an enhancer only (trial 1).
FOURTH INVESTIGATION
[0129] The results of a fourth investigation into the use of BacLyte and a banana extract to form an enhancement mixture with a molasses sugar source are shown in
[0130] Four trials were carried out with the following parameters
TABLE-US-00011 TABLE 11 Control Yeast C-70 0.5 g Incubated for 6 Water (30 degrees) 5 ml hours at 30 degrees Molasses 131.3 ml Citric Acid 1.5 g Sugar 42 g Water (80 degrees) 591 ml AntiFoam 1 ml Trial 1 Yeast C-70 0.5 g Incubated for 6 20% Banana Extract 1 ml hours at 30 degrees YPD 1 ml Water (30 degrees) 4 ml Molasses 131.3 ml Citric Acid 1.5 g Sugar 42 g Water (80 degrees) 591 ml AntiFoam 1 ml Trial 2 Yeast C-70 0.5 g Incubated for 6 “Propagreater” 1 ml hours at 30 degrees Water (30 degrees) 4 ml Molasses 131.3 ml Citric Acid 1.5 g Sugar 42 g Water (80 degrees) 591 ml AntiFoam 1 ml Trial 3 Yeast C-70 0.5 g Incubated for 6 5% Banana Extract 250 microliters hours at 30 degrees YPD 1 ml Water (30 degrees) 3.8 ml Molasses 131.3 ml Citric Acid 1.5 g Sugar 42 g Water (80 degrees) 591 ml AntiFoam 1 ml
[0131] In particular, the samples set out above were each incubated in 100 ml conical flasks for 6 hours at 30 degrees before being pitched into a specific molasses blend of feed grade molasses, citric acid, water, sugar and anti-foam set out above in Table 11 at 30 degrees before being sealed for fermentation with sterile seals. These were subsequently checked every 6 hours for SG, Brix, pH, ABV and internal temperature. Once fermentation was complete, these were distilled using a Buchi R300 Rotovapor at 97 mbar and 50 degrees to extract ethanol to calculate LPA yield.
[0132] The results of the trials are set out below in Tables 12 and 13 and shown in
Specific Gravity:
[0133]
TABLE-US-00012 TABLE 12 Hours Passed 0 Hours 6 Hours 12 Hours 18 Hours 24 Hours 30 Hours 36 Hours 42 Hours 48 Hours Check No. Check 1 Check 2 Check 3 Check 4 Check 5 Check 6 Check 7 Check 8 Check 9 Control 1.095 1.092 1.0902 1.089 1.0870 1.0855 1.0837 1.0823 1.0794 Trial 1 1.095 1.0901 1.088 1.087 1.0835 1.0792 1.0755 1.0726 1.0717 Trial 2 1.095 1.089 1.0862 1.0833 1.0806 1.0773 1.0712 1.0669 1.0646 Trial 3 1.095 1.0881 1.0871 1.0859 1.0835 1.0792 1.0734 1.0712 1.0699
Fermentation ABV:
[0134]
TABLE-US-00013 TABLE 13 Hours Passed 0 Hours 6 Hours 12 Hours 18 Hours 24 Hours 30 Hours 36 Hours 42 Hours 48 Hours Check No. Check 1 Check 2 Check 3 Check 4 Check 5 Check 6 Check 7 Check 8 Check 9 Control 0.00% 0.40% 0.80% 1.23% 2.37% 3.98% 4.33% 4.85% 5.28% Trial 1 0.00% 1.79% 2.08% 2.20% 3.25% 4.85% 5.62% 6.13% 6.38% Trial 2 0.00% 1.97% 2.31% 2.66% 3.85% 5.28% 6.46% 7.30% 7.71% Trial 3 0.00% 2.08% 2.20% 2.31% 3.35% 4.85% 6.04% 6.46% 6.72%
[0135] As can be seen, the use of a fermentation enhancement mixture in the present invention is significantly advantageous: it produces a higher ABV than the control with molasses as the sugar source. This also validates the effect of the enhancement mixture of the present invention with molasses as a sugar source. The “Propagreater” mixture (trial 2) produced the highest alcohol ABV and, counterintuitively, the 5% of banana extract of trial 3 was found to be more effective than the 20% of banana extract of trial 1. Therefore, in accordance with the present invention it may be preferable to include an amount of banana extract that is 10% or less.
Preparation of Banana Extract
[0136] The banana extract discussed above and in the claims of the present invention may be generally prepared as follows: [0137] i) Peeled bananas stored at −84° C., are removed from the freezer. Keep the bananas in the frozen bag and gently break the bananas buy dropping them approximately a foot from a hard tabletop. The bananas will break easily due to their temperature. [0138] ii) Remove 250 grams of banana pieces for every 100 ml of water used and place on the lab bench onto absorbent paper toweling. [0139] iii) Bananas may remain at room temperature for 25-40 minutes, until softened. [0140] iv) Into a blender that has been cleaned and thoroughly rinsed with distilled water, add the weighed banana pulp and water so that one-half to three-quarters of the blender is filled. [0141] v) Blend the banana pulp/water mixture at the top high speed for 90 seconds. [0142] vi) Following blending, the blended banana puree is poured into centrifuge containers and spun at a speed of at least 3900 rpm, or a higher speed if allowed by the centrifuge, at a temperature of 20° C. for 30 minutes and then decelerated at the lowest rate to ensure gentle braking. [0143] viii) Collecting the supernatant into a large Corning autoclavable 1 L glass bottle with no more than 600 ml in the bottle. [0144] ix). Autoclaving at standard autoclave temperature (121° C., 25 minutes, 20 lbs. pressure) to achieve sterility. [0145] x) Allowing the supernatant to cool for 30-45 minutes and then pouring the supernatant into new, sterile 50 ml polypropylene tubes and subject to a round of centrifugation according to the same parameters for the initial processing of the blended fruit. [0146] xi) Collecting 40 ml of the supernatant into sterile, 50 ml polypropylene tubes and discarding any pelleted solid debris.
[0147] This method produces a banana extract in accordance with the present invention and in accordance with EP1945763B1, as discussed above. Any other suitable method for preparing the banana extract of EP1945763B1 disclosed in the patent elsewhere or in EP1945763B1 may be used as an alternative for the methods of the present invention.