Intramuros, Manila College of Engineering and Technology Department of Chemical Engineering Introduction to Biotechnology: Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Prepared by: AMADO, Rosenn B. DANDAMUN, Benbellah Ali Y. EBREO, Mencyn Michelle Kellie GONZALES, Belinda Jean A IGAYA, Deanne May G. JUAGCO, Cris-Anne III LOPEZ, Charmaine A. TORRES, Angela Jane R. BSCHE-IV Submitted to: Engr. Ronald Allan Co Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 2 ACKNOWLEDGEMENT We acknowledge the presence of Almighty God on our side that guide and enlighten our minds in coping up with such great pressure that has been put on our shoulders. And also, the thorough help of Engr. Ronald Allan S. Co for the review of our study. We consider his advises as our prior objective to fulfill. Lastly, to our parents whom unconditionally understand what we were going through and for the financial support. Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 3 Chapter I: INTRODUCTION BACKGROUND OF THE STUDY Apple juice is a fruit juice made by the maceration and pressing of apples. The resulting expelled juice may be further treated by enzymatic and centrifugal clarification to remove the starch and pectin, which holds fine particulate in suspension, and then pasteurized for packaging in glass, metal or aseptic processing system containers, or further treated by dehydration processes to a concentrate. In the study, apple juice was used as our primary material for the fermentation. Fermentation typically refers to the conversion of sugar to alcohol using yeast. In its strictest sense, fermentation is the anaerobic metabolic breakdown of a nutrient molecule, such as glucose, without net oxidation. Fermentation is also used much more broadly to refer to the bulk growth of microorganisms on a growth medium. Alcohol is formed when yeast feeds on sugar. Alcohol is ethanol or ethyl alcohol. There are many informal names for alcohol (e.g. booze, bevvy, drink), and there are many different types of alcoholic drink (e.g. beer, lager, wine, spirits, cider). But they all contain ethanol. Ethanol is made from the sugar in grain or fruit and it is a colorless, clear liquid. Yeast is a one-cell fungus which reproduces by budding off new cells. This tiny organism grows and multiplies by feeding on sugar in foods such as grain and fruits. As the yeast feeds on the sugar, carbon dioxide and alcohol are produced. They are the waste products of the yeasts anaerobic respiration thats respiration without oxygen. Sugar energy + alcohol + carbon dioxide C6H12O6 energy + 2C2H5OH + 2CO2 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 4 When yeast respires anaerobically, only a small amount of energy is released from the sugar and so most of the sugar stays in the alcohol. When the alcohol concentration reaches about 15% of alcohol by volume, the yeast cells die. Ethanol concentration can be based by determining the density measurement of the ethanol. Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 5 OBJECTIVES The objective of this study is to determine the wine and ethanol concentrations using bio-chemical reaction and stabilization process. Specific Objectives: It aims to determine ethanol concentration based by the density measurement of ethanol Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 6 Chapter II: Methodology Materials Needed (28) 500 ml PET bottles (14) bending straws (2) 1L TIPCO Apple Juice 1 gallon distilled water 10 g yeast (Not all will be consumed) Funnel Analytical Balance Thermometer (2) beakers Vials or any glass bottles for weighing sample Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 7 Procedure Preparation: 1. Bottles obtained were of the same size and brand. Labels were removed. 2. Bottles and covers were cleaned with soap and water. 3. After cleaning, it was sterilized with hot water. 4. Bottles and covers dried by wiping the excess water and turned the bottle down on a clean cloth for the condensate will run down and be absorbed by the cloth. 5. Bottles were paired, making up 14 pairs. 7 for the first batch. Another 7 for the second batch. 6. While it is still drying, covers were being bore with the use of cutter. 7. 2 straws were connected together and locked by scotch tape. And to be inserted on the bottle caps. 8. One end of the straw must be cut to make it shorter (for the Wine Mixture bottle). While, the other end of the straw must be long to have contact with the water. 9. Bottles were labeled from 1 to 7, on both batches. Preparation for Actual Set-up: 1. For basis, beaker was put on the analytical balance and set into zero. 2. 300 ml water were poured, and weighed. Record the data (mass, volume, density, average mass, average volume, average density) 3. For the next basis: beaker was put on the analytical balance and set into zero. Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 8 4. 150 ml apple juice were poured, plus one pinch of yeast and weighed. Record the data (mass, volume, density, average mass, average volume, average density) 5. After the preparation, bottles has to be placed in a dark room and shake free place. Actual Set-up: 1. Everyday, take a 3-trial of obtaining twenty (20 ml) for each bottle. (Everyday there should be four bottles to take samples from. 2 from the first batch (Wine mixture and Ethanol), and another 2 from the second batch (Wine mixture and Ethanol) 2. For every trial, It should be put on a dry vial, or any glass bottle to weigh the sample. 3. Record the data. (mass, volume, density, average mass, average volume, average density). Solving for Concentration: x = p winc solution (o:c. ) p winc (initiol) p ctonol p winc (initiol) Where: X= fractional concentration CEtOH = xEtOH Cwine = (1-x) wine(initial) %Et0E ConccntrotcJ (octuol) = xpEt0E Cwinc % EtOH Concentrated ( theo) = 100% - % wine mixture % winc mixturc = C winc p winc (initiol) (1uu%) % crror (Et0E Con:crsion) = %Et0E (tco) %Et0E (octuol) %Et0E (tco) (1uu%) Where: CEtOH = Concentration of Ethanol (g/ml) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 9 Cwine = Concentration of wine (g/ml) EtOH= density of pure ethanol (0.8059 g/ml)wine = density of wine @t=0, initial value Chapter III. Data and Results Table of Data DATA AND RESULTS: Apple Wine Basis: Volume (ml) Mass (g) Density (g/ml) Water 300 300 1 Wine Mixture 150 180 1.2 1 st batch (Ethanol) Day 1 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.2 1.06 Trial 2 20 20.8 1.04 Trial 3 20 21.2 1.06 Average 20 21.0667 1.0533 Day 2 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21 1.05 Trial 2 20 20.6 1.03 Trial 3 20 20.6 1.03 Average 20 20.7333 1.0367 Day 3 Volume (ml) Mass (g) Density (g/ml) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 10 Trial 1 20 20.9 1.045 Trial 2 20 20.9 1.045 Trial 3 20 20.6 1.03 Average 20 20.8 1.04 Day 4 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.7 1.035 Trial 2 20 20.7 1.035 Trial 3 20 20.5 1.025 Average 20 20.6333 1.0317 Day 5 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20. 6 1.03 Trial 2 20 20.6 1.03 Trial 3 20 20.8 1.04 Average 20 20.6667 1.0333 Day 6 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20. 6 1.03 Trial 2 20 20.5 1.025 Trial 3 20 20.7 1.035 Average 20 20.6 1.03 Day 7 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.7 1.035 Trial 2 20 21 1.05 Trial 3 20 21 1.05 Average 20 20.9 1.045 1 st batch (Wine Mixture) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 11 Day 1 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.4 1.07 Trial 2 20 21.9 1.095 Trial 3 20 21.9 1.095 Average 20 21.7333 1.0867 Day 2 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.7 1.035 Trial 2 20 20.6 1.03 Trial 3 20 20.9 1.045 Average 20 20.7333 1.0367 Day 3 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.8 1.04 Trial 2 20 20.9 1.045 Trial 3 20 20.5 1.025 Average 20 20.7333 1.0367 Day 4 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21 1.05 Trial 2 20 21.6 1.08 Trial 3 20 21.8 1.09 Average 20 21.4667 1.0733 Day 5 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.5 1.075 Trial 2 20 21.1 1.055 Trial 3 20 21.6 1.08 Average 20 21.4 1.07 Day 6 Volume (ml) Mass (g) Density (g/ml) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 12 Trial 1 20 21.2 1.06 Trial 2 20 20.9 1.045 Trial 3 20 21 1.04 Average 20 21.0333 1.0517 Day 7 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.9 1.095 Trial 2 20 20.8 1.04 Trial 3 20 20.8 1.04 Average 20 21.1667 1.0583 2 nd Batch (Ethanol) Day 1 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.4 1.07 Trial 2 20 21.6 1.08 Trial 3 20 20.5 1.025 Average 20 21.1667 1.0583 Day 2 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21 1.05 Trial 2 20 20.8 1.04 Trial 3 20 21 1.05 Average 20 20.9333 1.0467 Day 3 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.9 1.045 Trial 2 20 20.5 1.025 Trial 3 20 20.3 1.015 Average 20 20.5667 1.0283 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 13 Day 4 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.6 1.03 Trial 2 20 20.6 1.03 Trial 3 20 20.4 1.02 Average 20 20.5333 1.0267 Day 5 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21 1.05 Trial 2 20 20.9 1.045 Trial 3 20 21 1.05 Average 20 20.9667 1.0483 Day 6 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.1 1.055 Trial 2 20 21.2 1.06 Trial 3 20 20.8 1.04 Average 20 21.0333 1.0517 Day 7 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.3 1.065 Trial 2 20 21.2 1.06 Trial 3 20 21.1 1.055 Average 20 21.2 1.06 2 nd Batch (Wine Mixture) Day 1 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.7 1.085 Trial 2 20 21.8 1.09 Trial 3 20 21.7 1.085 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 14 Average 20 21.7333 1.0867 Day 2 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 20.2 1.01 Trial 2 20 20.6 1.03 Trial 3 20 20.9 1.045 Average 20 20.5667 1.0283 Day 3 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.1 1.055 Trial 2 20 20.7 1.035 Trial 3 20 20.9 1.045 Average 20 20.9 1.045 Day 4 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.5 1.075 Trial 2 20 21.2 1.06 Trial 3 20 21.1 1.055 Average 20 21.2667 1.0633 Day 5 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.9 1.095 Trial 2 20 21.2 1.06 Trial 3 20 21.2 1.06 Average 20 21.4333 1.0717 Day 6 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21 1.05 Trial 2 20 21 1.05 Trial 3 20 21.5 1.075 Average 20 21.1667 1.0583 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 15 Day 7 Volume (ml) Mass (g) Density (g/ml) Trial 1 20 21.6 1.08 Trial 2 20 21.3 1.065 Trial 3 20 21.4 1.07 Average 20 21.4333 1.0717 Concentration Data Batch Time (EtOH) (Wine) x C EtOH C Wine 1 0 1 1.2 0 0 1.2 1 Day 1 1.0533 1.0867 0.28749 0.23169 0.855011 1 Day 2 1.0367 1.0367 0.41436 0.33393 0.702766 1 Day 3 1.04 1.0733 0.32149 0.25909 0.81421 1 Day 4 1.0317 1.0367 0.41436 0.33393 0.702766 1 Day 5 1.0333 1.07 0.32987 0.26584 0.804161 1 Day 6 1.03 1.0517 0.3763 0.30326 0.748439 1 Day 7 1.045 1.0583 0.35955 0.28976 0.768536 % EtOH (actual) %EtOH (theo) % Wine (mixture) % Error 0 0 100 --- 27.09771783 28.74904846 71.25095154 5.74395 47.51713966 41.43618371 58.56381629 14.6755 31.82109511 32.14920071 67.85079929 1.02057 47.51713966 41.43618371 58.56381629 14.6755 33.05786949 32.98655164 67.01344836 0.2162 40.51904326 37.63004314 62.36995686 7.67738 37.70339078 35.95534128 64.04465872 4.86172 Batch Time (EtOH) (Wine) x C EtOH C Wine Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 16 2 Day 0 1 1.2 0 0 1.2 2 Day 1 1.0583 1.0867 0.28749 0.23169 0.8550114 2 Day 2 1.0467 1.0283 0.43568 0.35111 0.6771885 2 Day 3 1.0283 1.045 0.3933 0.31696 0.7280386 2 Day 4 1.0267 1.0633 0.34687 0.27954 0.7837605 2 Day 5 1.0483 1.0717 0.32555 0.26236 0.8093377 2 Day 6 1.0517 1.0583 0.35955 0.28976 0.7685359 2 Day 7 1.06 1.0717 0.32555 0.26236 0.8093377 % EtOH (actual) %EtOH (theo) % Wine (mixture) % Error 0 0 100 --- 27.09771783 28.74904846 71.25095154 5.74395 51.84840752 43.56762243 56.43237757 -19.007 43.5363516 39.33011926 60.66988074 -10.695 35.66644975 34.68662776 65.31337224 -2.8248 32.41690808 32.55518904 67.44481096 0.42476 37.70339078 35.95534128 64.04465872 -4.8617 32.41690808 32.55518904 67.44481096 0.42476 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 17 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 C EtOH C Wine Figure 1.1Line graph obtained from data on Batch 1 (Ethanol and Wine Concentration) Figure 1.2Line graph obtained from data on Batch 2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 C EtOH C Wine Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 18 y =0.0333x +0.1629 R =0.3713 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 2 4 6 8 10 Series1 Linear (Series1) (Ethanol and Wine Concentration) Zero Order Reaction (1) Time Xa Day 0 0 Day 1 0.28749 Day 2 0.414362 Day 3 0.321492 Day 4 0.414362 Day 5 0.329866 Day 6 0.3763 Day 7 0.359553 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 19 y =0.018x - 1.1081 R =0.0817 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0 2 4 6 8 Series1 Linear (Series1) First Order Reaction (1) Second Order Reaction (1) Time Xa 1/Xa Day 0 0 0 Day 1 0.28749 3.478376 Day 2 0.414362 2.41335 Day 3 0.321492 3.110497 Day 4 0.414362 2.41335 Day 5 0.329866 3.031538 Day 6 0.3763 2.657451 Day 7 0.359553 2.781228 Time Xa 1/Xa2 Day 0 0 0 Day 1 0.28749 12.0991 Day 2 0.414362 5.824257 Day 3 0.321492 9.675193 Day 4 0.414362 5.824257 Day 5 0.329866 9.190225 Day 6 0.3763 7.062046 Day7 0.359553 7.735229 y =-0.0601x +3.0811 R =0.1101 0 0.5 1 1.5 2 2.5 3 3.5 4 0 2 4 6 8 10 Series1 Linear (Series1) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 20 Third Order Reaction (1) Time Xa 1/Xa^2 Day 0 0 0 Day 1 0.28749 -1.24657 Day 2 0.414362 -0.88102 Day 3 0.321492 -1.13478 Day 4 0.414362 -0.88102 Day 5 0.329866 -1.10907 Day 6 0.3763 -0.97737 Day 7 0.359553 -1.02289 y =-0.3965x +9.7873 R =0.1412 0 2 4 6 8 10 12 14 0 2 4 6 8 10 Series1 Linear (Series1) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 21 Zero Order Reaction (2) First Order Reaction (2) Time Xa Day 0 0 Day 1 0.28749 Day 2 0.435676 Day 3 0.393301 Day 4 0.346866 Day 5 0.325552 Day 6 0.359553 Day 7 0.325552 Time Xa LN Xa Day 0 0 0 Day 1 0.28749 -1.24657 Day 2 0.435676 -0.83086 Day 3 0.393301 -0.93318 Day 4 0.346866 -0.10582 Day 5 0.325552 -1.12223 Day 6 0.359553 -1.10289 Day 7 0.325552 -1.12223 y =0.0269x +0.1881 R =0.2464 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 2 4 6 8 10 Series1 Linear (Series1) y =-0.0071x - 1.0195 R =0.0128 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0 2 4 6 8 10 Series1 Linear (Series1) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 22 Second Order Reaction (2) Third Order Reaction (2) Time Xa 1/Xa Day 0 0 0 Day 1 0.28749 3.478376 Day 2 0.435676 2.295282 Day 3 0.393301 2.542581 Day 4 0.346866 2.882955 Day 5 0.325552 3.071707 Day 6 0.359553 2.781228 Day 7 0.325552 3.071707 Time Xa 1/Xa^2 Day 0 0 0 Day 1 0.28749 12.0991 Day 2 0.435676 5.268332 Day 3 0.393301 6.464716 Day 4 0.346866 8.311432 Day 5 0.325552 9.435384 Day 6 0.359553 7.735229 Day 7 0.325552 9.435384 y =0.01x +2.8347 R =0.0031 0 0.5 1 1.5 2 2.5 3 3.5 4 0 2 4 6 8 10 Series1 Linear (Series1) y =-0.0031x +8.4052 R =9E-06 0 2 4 6 8 10 12 14 0 2 4 6 8 10 Series1 Linear (Series1) Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 23 DOCUMENTATION Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 24 Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 25 INTERPRETATION OF RESULTS The results of the experiment depicts how an ideal fermentation process would occur wherein the growing cells are consuming the substrate, and producing more cells as time passes. In this case, the concentrations of ethanol and wine increases and decreases respectively with time. The stages of yeast growth are noticeable in the graphs. From Day 0 to Day 2, the yeast undergoes an exponential growth. From Figure 1.1, the ethanol concentration increases from 0 to 0.333934 and the wine concentration decreases from 1.2 to 0.702766. From Figure 1.2, the concentration of ethanol increases from 0 to 0.351111 and the concentration of the wine decreases from 1.2 to 0.677189. Then from Day 2 to Day 5, the deceleration phase is seen. From Figure 1.1, the concentration of ethanol begins to decrease from Day 2, 0.333934, to Day 3, 0.25909 while the concentration of wine increases from 0.702766 to 0.81421. From Figure 1.2, the concentration of ethanol begins to decrease from Day 2, 0.351111, to Day 5, .262362 while the concentration of the wine increases from .728039 to 0.809338. The concentrations of wine and ethanol begin to become constant as the yeast approaches the stationary phase. As for the order of reaction, Zero Order Reaction yields an R 2 closer to 1 (In the first batch R 2 is equal to .3713 and in the second batch its value is .2464). For batch 1, the R 2 that is farther to 1 is from the First Order Reaction which yields an R 2 of .0817 while for batch 2; the R 2 from the Second Order Reaction is farther to 1 which is equal to .0031. Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 26 Chapter IV: CONCLUSION The fermentation of apple juice is relatively fast because it only takes 1-2 weeks for the juice to ferment. The lag phase (wherein the yeast matures and acclimates to the environment and no growth occurs) was not visibly seen in the graph. The cause of which may have been the variation of time (Day to Day). The rate of reaction in the exponential growth of the yeast follows first order kinetics. Parameters like the substrate and waste concentrations have an effect on the kinetics of yeast growth as seen in the deceleration phase of the yeast. The stationary phase (wherein no growth occurs) was only partially seen in the graph. Other factors that affected the fermentation process may have been the stabilization of the set-up, the thermal conditions, and the lighting in the area. The application of other kinds of yeast would also vary the fermentation process. The order that is best fit for the fermentation of apple juice is the Zero Order Reaction. The zero order reaction yielded a value of R 2 that is closer to 1 than the first, second, and third order reactions. Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 27 Chapter V: Recommendation The researchers recommend to have a better proportion of amount of yeast to the concentration of apple Juice. Enormous amount of yeast may lead the apple juice to ferment easily and flow out of the bottle. On the other hand, there are many fruits in the market that can be fermented, too. During obtaining of the sample, if using syringe, avoid making too much holes from the bottle so that air wont pass through. Make sure that bottles were sealed enough by packaging tapes. Use analytical balance to have an accurate data.
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