Research Question: To what extent does the application of different citrus juices affect the oxidation levels of Malus domestica ‘Gala’?Hypothesis:Citrus fruits containing more acidic juices will prevent, or slow down, oxidation in Gala apples the best. As for what will be best for baking, an acidic juice that is also not too tart to the taste would be suitable. Variables:Table 1.1: Independent and dependent variablesIndependent VariableType of citrus juice applied to applesFive trials of each of the following conditionsLemon juiceLime juiceOrange juice Grapefruit juiceNo juiceDependent VariableOxidation (browning) of apple slicesTable 1.2: Controlled variablesControlled VariablesWhy was it controlled?Method for ControlAmount of juice applied to apple slices. To ensure that no apple slices are exposed to more or less acidity. Place each apple slice into a cup containing 80 mL of the given juice. 2. Mass of each apple slice.So the amount of juice spreads throughout the apple slices evenly. Cut, peel, and measure (on a scale) each apple slice to be 21.5 g. 3. Time each apple slice spent submerged in juice.To ensure that the apple slices have enough time to process a significant level of enzymatic browning, while having enough time to absorb the acidic juice which will lower the pH. Each apple slice will spend 1 hour in the given juice. 4. Time each apple spent in transition (being cut and peeled before entering juice).So that no uneven browning occurs while exposed to oxygen before submergence in juice, that would in-turn throw off the end results. (chart continued on next page)I will take exactly 5 minutes and 30 seconds to peel, cut, and measure the mass of each apple slice. 5. Amount of distilled water added to blender with each apple slice to ensure smooth blending into a liquid substance.Same amount of distilled water blended with each apple to ensure that each samples’ level of browning remains consistent. Each individual apple slice will be blended into a liquid substance along with 14.8 mL (one tablespoon) of distilled water which will help bring it into a liquid state. 6. Time spent in blender. To ensure that the cells of each apple slice are broken down the same amount to prevent any uneven browning. Each individual apple slice (with its distilled water) will spend exactly 5 seconds in the blender. 7. Absorption measured at same wavelength.So that accurate and consistent results are recorded, that were all measured under the same conditions.The spectrophotometer will be used to measure the absorption of 610 nm ± 0.001 in each liquid sample. Background Information:A highly present enzyme in fruits such as apples, peaches, pears, and bananas, polyphenol oxidase. When this enzyme comes becomes exposed to oxygen, plant phenolic compounds turn into brown pigments called melanins. When the fruit in question is damaged in any way, whether it be by cutting or bruising, browning occurs. This is a common occurrence in cutting and eating fruit every day. This reaction is known as enzymatic browning and occurs when the pH of the fruit is somewhere between 5.0 and 7.0.Enzymatic browning can be slowed or prevented in several ways. First, by denaturing the enzyme with heat, for example by cooking. Next, by decreasing the pH of the fruit, for example by adding lemon juice or other acids. Enzymatic browning can also be prevented by reducing the enzymatic browning reaction rate. This can be accomplished by storing the fruit in the refrigerator where it is not subjected to heat, or reducing the amount of available oxygen by putting cut fruit under water or vacuum packing it, or by treating the fruit with certain preservative chemicals like sulfur dioxide.The method of slowing the process of enzymatic browning that I will be putting to the test in this IA is that of applying lemon juice and other acids to the apple slices to reduce their pH. Because I do not have the equipment, test materials, or access to the ability to test specifically for levels of enzymatic browning, or oxidation, I will be testing the next most reliable marker for oxidation in apples; physical browning. To measure the level of browning, I will use a spectrophotometer which detects how much of a certain color, or wavelength, your sample absorbs or reflects. In my case, I will be testing for absorbance of a dark orange color. This method of experimentation is unique and shows resourcefulness, specifically relating to gearing one’s experiment towards what materials are at their disposal. This type of experimentation is completely new to me, because of the use of new equipment (spectrophotometer), and because it is the first experiment I had to design myself, so the entire project is an uncharted frontier for me. This project is also great because it applies to the real everyday needs of people who frequently manage fresh produce. I have a strong love for baking and cooking as a hobby, but also for real purposes such as preparing dinner for my family or crafting a creative dish for a party. My admiration for baking and cooking derived from my late great grandmother who used to bake for my siblings and I every time we saw her. As a frequent baker and chef, I deal with oxidation of fresh produce in all kinds of recipes. More specifically, in recipes such as an apple pie or an apple crisp, the enzymatic browning of apples occurs fairly quickly and is regarded as unfavorable in these desserts. In order to keep the apples fresh and presentable for consumption, a common technique is to treat the apples with lemon juice, although I personally have some discrepancies with this method. The application of lemon juice to apples tends to turn the sweet taste to a more sour and tart flavor, which seems rather counterintuitive to the purpose of baking a sweet treat. This is a widely known dilemma in the cooking world, which I intend to further research in this IA. I would like to know if any other acidic citrus juices offer the same effects of slowing enzymatic browning as lemon juice, without disturbing the beloved sweet taste of apples. Materials:5 peeled and sliced Gala apples (Malus domestica ‘Gala’) KnifeApple peelerCutting surfacesOne small sterile cutting boardOne large sterile cutting boardCitrus juice400mL lemon juice400mL lime juice400mL orange juice400mL grapefruit juiceCitrus Juicer25 plastic cups500 mL measuring cup (± 12.5 mL)Magic Bullet blender370 mL distilled waterScale (± 0.05 g)Labquest tabletSpectrophotometer Spectrophotometer substance cuvettes Stopwatch (± 0.01 s)Risk Assessment:All materials are safe to use except for the knife and apple peeler. These objects are sharp and should be handled with caution. Protective clothing/gear is not necessary.Percent Uncertainties:Table 2: Percent uncertainty calculationsApparatusPercent UncertaintyMeasuring cup1500*100=0.2%Scale0.0521.5*100=0.233% (3 s.f)Stopwatch0.01330*100=3.0310-3(negligible) Total % uncertainty = 0.433%Procedure: Clean all materials well before beginning Cutting boardsKnife and apple peelerBlender cup and bladeCitrus juicerMeasuring cupSpectrophotometer substance cuvettes Juice citrus fruit to acquire 400 mL of each type of juice (lemon, lime, orange, and grapefruit).Remove all seeds from juice if any fall inClean the citrus juicer and measuring cup thoroughly after juicing each different type of citrus.(Limit the following cutting and peeling process for every apple to 5:30.)Peel 1 Gala apple.Cut the first apple evenly into fifthsUse the scale to measure the mass of each slice, and make sure each slice has the same mass (21.5g)Simultaneously place one slice in a cup with 100 mL of lemon juice, one slice in a cup with 100 mL of lime juice, one slice in a cup with 100 mL of orange juice, and one slice in a cup with 100 mL of grapefruit juice.Place the fifth apple slice in an empty cup to act as the control group. Set a timer for 1 hour. After 1 hour is over, remove the apple slices from the cups of citrus juice. Gently pat the surface of the apple slices dry to remove any excess citrus juice. Blend each individual apple slice separately for 5 seconds with 14.8 mL (one tablespoon) of distilled water added to the blender to to aid in liquefying the apple. Next, place a small liquid sample of each blended apple into individual spectrophotometer cuvettes. Measure each samples’ absorbance of 610 nm ± 0.001 separately in the spectrophotometer. “Connect the sensor directly to the USB port of the… LabQuest.” On labquest, “select the unit or data type you wish to measure.” (absorbance) “Calibrate the SpectroVis Plus””choose Calibrate ? Spectrophotometer from the Experiment menu. Note: For best results, allow the Spectrophotometer to warm up for a minimum of five minutes.” “Fill a cuvette about 3/4 full with distilled water (or the solvent being used in the experiment) to serve as the blank. After the Spectrophotometer has warmed up, place the blank cuvette in the Spectrophotometer. Align the cuvette so the clear side of the cuvette is facing the light source.””Follow the instructions in the dialog box to complete the calibration, and then click OK.” “Collect Data with LabQuest : Measurement vs. Wavelength (Generate a Spectrum)””Fill a cuvette about 3/4 full of the solution to be tested and place it in the spectrophotometer.””Start data collection by tapping on the Start button in the lower left corner of the screen. Tap the Stop button to end data collection.””Select wavelength. Note: The wavelength of maximum absorbance (? max) is automatically selected. This ? max will be used for any subsequent data collection, such as a Beer’s law experiment (abs vs. conc.) or a kinetics experiment (abs vs. time). If you wish to choose another wavelength, you can tap on the graph to select a new wavelength. Another way to change the wavelength is to navigate to the Meter screen, tap on the meter, and select Change Wavelength. Enter the wavelength of your choice and select OK. If the wavelength you type in is not measured by the unit, LabQuest will automatically choose the wavelength closest to your choice.” 14. Record results calculated by spectrophotometer. 15. Repeat this process 4 more times for a total of 5 sets of data. Figure 1: Lab SetupData Collection:Figure 2: Example Wavelength SpectrumAll the samples resulted to have some level of a dark orangey-brown color, although the simultaneous treatment of apples with different citrus juices side-by-side revealed which juices caused the treated apples to become the least dark, and the most dark. Table 3: Qualitative ObservationsCitrus Juice Observed darkness of apple slice on a scale of 1-5 (1= lightest, 5= darkest) LEMON1LIME3ORANGE4GRAPEFRUIT2NO JUICE 5Qualitative Analysis:Because all the samples resulted to be a dark orange-to-brown color, I chose a wavelength of 610 nm at which to measure the absorption of all my samples (610 nm is located on the darker side of the orange section on the wavelength spectrum). Theoretically, a more truly orange-toned sample that measures close to 610 nm would have an absorption of close to 0.00 or 0% at 610 nm, because the color that orange absorbs is blue. Therefore, to get the same results, I could measure each sample at about 450 nm (located in the blue section of the wavelength spectrum) and hope to see an absorption closer to 1.00, or 100%. Because I am searching for the type of juice that causes the least oxidation, I am looking for the type of juice that causes the apple slices to be the lightest, and therefore least orangey color. So, the closer to 0.00 the following values are, the more oxidation is present, and the values farther away from 0.00 are the ones which provide the least oxidation, which are the ones I will value. Table 4: Raw DataCitrus JuiceSet 1Set 2Absorption at 610nm ± 0.001 Set 3Set 4Set 5Lemon0.3560.3490.3170.3120.336Lime0.2080.2120.2070.2030.201Orange0.0120.0230.0080.0160.019Grapefruit0.3140.3270.3540.3060.321No Juice0.0050.0130.0170.0050.006 Graph 1: Line chart depicting effect of citrus juice on oxidation: citrus juice (treatment group)This graph shows the significant difference in effect of the different treatment groups; lemon juice, lime juice, orange juice, grapefruit juice, and no juice. The colored lines represent the different data sets. I felt that the use of this line chart would make the difference between the treatment groups more evident. Graph 2: Column chart depicting effect of citrus juice on oxidation: trialsThis graph shows the variation of data between all five sets. The colors represent each type of citrus juice; lemon=yellow, lime=green, orange=orange, grapefruit=purple, no juice=blue. I felt that the use of this column chart would highlight the consistency between all five data sets, as well as provide an overall visual representation of the data. Data Processing:The data collected above had to be processed in order to draw meaningful conclusions from it, so I determined that the best test to conduct would be an ANOVA test, which performs an analysis of variance among the data. Because the ANOVA test is meant to detect variation, calculating standard deviation was not necessary. The only other calculation performed was an average of each treatment group to obtain one solid benchmark which could be used to form a conclusion about the data. Table 5: Means of each treatment group Citrus JuiceMean Absorption at 610nm ± 0.001Lemon0.334Lime0.206Orange0.016Grapefruit0.324No Juice0.009To further prove the significance of my data, as previously stated, I performed an ANOVA test: Analysis of Variance. For the sake of completely understanding the test, and the significance of the test, I performed it by hand instead of in an automatic program. My steps were as follows: ANOVA Test Step 1: State HypothesesH0= there is no significant variance between the groups: H0= 1=2=3=4=5H1= there is significant variance between the groups: H1= there is at least one difference among the groups: alpha level (significance level) = 0.05Step 2: Calculate FCRITICAL K = number of groups in studyK = 5N = total number of data values in studyN = 25df = Degrees of Freedom”Between” = between the groups”Within” = within each group”Total” = among all datadfBETWEEN= K – 1, 5 – 1dfBETWEEN= 4dfWITHIN= N – K, 25 – 5dfWITHIN= 20dfTOTAL= dfBETWEEN + dfWITHIN , 20 + 4dfTOTAL= 24Find FCRITICAL use F-Distribution table (see Graphic #3)Horizontal value = dfBETWEEN= 4Vertical value = dfWITHIN= 20Follow the chart to find the FCRITICAL value. FCRITICAL= 2.87Figure 3: F-Distribution Table Step 3: Analysis of the sum of the squares (sum of squared deviations from the mean): Find Variability for: Between groups, Within each group, Total data.Calculate the Sum of Squares for TOTAL data Sum of Squares TOTAL = SSTOTALCalculate Grand Mean = GMG = sum of all data values in study = 4.447N = number of data values in study = 25GM = GN= 4.44725= 0.17788 0.178SSTOTAL= (x-GM)2(0.356-0.178)2+(0.349-0.178)2+(0.317-0.178)2+(0.312-0.178)2+(0.336-0.178)2+(0.314-0.178)2+(0.327-0.178)2+(0.354-0.178)2+(0.306-0.178)2+(0.321-0.178)2+(0.012-0.178)2+(0.023-0.178)2+(0.008-0.178)2+(0.016-0.178)2+(0.019-0.178)2+(0.208-0.178)2+(0.212-0.178)2+(0.207-0.178)2+(0.203-0.178)2+(0.201-0.178)2+(0.005-0.178)2+(0.013-0.178)2+(0.017-0.178)2+(0.005-0.178)2+(0.006-0.178)2=SSTOTAL= 0.460389Calculate the Sum of Squares for WITHIN each group = SSWITHINSSWITHIN=(x1-x1)2+(x2-x2)2+(x3-x3)2+(x4-x4)2+(x5-x5)2(0.356-0.334)2+(0.349-0.334)2+(0.317-0.334)2+(0.312-0.334)2+(0.336-0.334)2+(0.314-0.324)2+(0.327-0.324)2+(0.354-0.324)2+(0.306-0.324)2+(0.321-0.324)2+(0.012-0.016)2+(0.023-0.016)2+(0.008-0.016)2+(0.016-0.016)2+(0.019-0.016)2+(0.208-0.206)2+(0.212-0.206)2+(0.207-0.206)2+(0.203-0.206)2+(0.201-0.206)2+(0.005-0.009)2+(0.013-0.009)2+(0.017-0.009)2+(0.005-0.009)2+(0.006-0.009)2=SSWITHIN= 0.003162Calculate the Sum of Squares for BETWEEN the groups = SSBETWEENSSBETWEEN= SSTOTAL – SSWITHIN SSBETWEEN= 0.460389 – 0.003162SSBETWEEN= 0.457227Step 4: Calculate variance between and within groupsMS = mean squaredCalculate variance BETWEEN groups = MSBETWEEN= SSBETWEENdfBETWEENMSBETWEEN = 0.4572274MSBETWEEN = 0.11430675Calculate variance WITHIN each group = MSWITHIN = SSWITHINdfWITHINMSWITHIN = 0.00316220MSWITHIN = 0.0001581Step 5: Calculate F-valueF=MSBETWEENMSWITHIN F=0.114306750.0001581F=723.0028463FCRITICAL= 2.87F>FCRITICAL, 723.0028463> 2.87Therefore, I will REJECT the null hypothesis, H0, that states: 1=2=3=4=5I will ACCEPT the alternative hypothesis, H1, that states: there IS a significant difference between the groups.Conclusion: Because orange juice-treated apples resulted in the lowest absorption of wavelength 610 nm, it is therefore the weakest choice for preventing oxidation. This makes sense, because out of all the citrus fruits, orange is definitely the sweetest and least acidic to the taste, with a pH around 2.8. Because lemon juice absorbed wavelength 610 nm to the most, it remains the best choice for preventing oxidation, with a pH around 2.0. The data collected significantly supports the hypothesis that more acidic citrus juices would best prevent oxidation in Gala apples, so lemon and grapefruit juice would be the best options for slowing down oxidation, which lime juice not too far behind. Unfortunately, none of the citrus juices applied to the Gala apples produce a desired taste that isn’t tart or sour. At this point, what citrus juice is applied to the apples is purely up to the opinion of the chef. Evaluation:The design of this experiment was very well suited to the type of information that needed to be acquired. Given the research question and lack of availability to equipment that directly tests for oxidation levels, all aspects of the experiment were well thought out and included in the experiment because they contributed to the accuracy and development of the data. The procedure was fairly straightforward, and not difficult to single-handedly carry out after studying and planning the desired course of action. All the equipment was easy to use, except for the spectrophotometer connected to the labquest tablet, which needed to be copiously studied before use in the actual experiment. I had to establish if I wanted to measure absorption or reflection of a wavelength, and which wavelength I wanted to measure from. After these determinations, I had to then read the spectrophotometer manual and learn how to conduct these actions and collect these measurements with the equipment. The experiment required undivided attention to the procedure, making sure to stay within time constraints, and to not contaminate substances or equipment. Despite the attention and efficiency required, the experimental process was manageable.Scientific Context:The findings of my experiment are widely supported by different varieties of sources that range from scientific studies, to classroom science lessons. It is a widely known fact in the world of food, and science, that lemon juice along with other acids have the ability to lower the pH of the food item they are applied to. Acidic compounds remove the necessary enzymes for browning to occur. It is also widely known phenolic acid is an extremely prevalent component in all different types of plants. More specifically, apples are one of the fruits with the highest phenolic acid content, and coincidentally, apples are an extremely popular food all around the world. Because of the prevalence of phenols in apples, correlations have been found between high phenolic content and enzymatic activity of apples, which theoretically means that apples with higher concentrations of phenols will oxidize faster and more intensely. The discovery of this correlation would justify the genetic modification of foods for the purpose of decreasing phenolic activity and thus decreasing browning. An example of these accomplishments in food engineering is in the production of Arctic Apples. Engineered by Okanagan Specialty Fruits Inc, these apples are a result of gene splicing, a technique that has allowed a reduction in polyphenol oxidase.Strengths:Consistency of controlled variables This allowed for more accurate resultsNo fruit (apple or citrus) was wasted in the processApple cores, stems, seeds, and citrus peels and seeds were composted afterwards.Uncertainty on measuring equipmentThe total uncertainty involved in this experiment was so small that the potential effects are negligible, which is ideal.Weakness/ LimitationSignificanceSuggestions for improvementLength of experimentBecause each set of apple slices needed to be submerged in the given citrus juice for one hour, put together with all the time required for preparation and processing, the time it took for one trial was well over an hour, which is far too long to conduct in one 50-minute biology class period. The experimental process had to be conducted outside of school. If I were to conduct this experiment again, I could perhaps cut down the time the apples are spent submerged in the citrus juice from one hour to 30 minutes. With that time cut down, I may have been able to fit the testing for each trial into a class period at school.Location of experimentationStemming from the fact that the experimental process took well over the length of a class period, the project then had to be conducted outside of school. The environment the experiment was conducted in, although clean and organized, was obviously not as sterile as a biology lab. Outside of a biology lab, other factors such as temperature control could not be as completely consistent.If the issue of experiment length were solved, then the project would be conducted in a biology lab, and therefore this limitation would also be solved. Lack of availability to equipment that tests specifically for oxidationThis is not so much a limitation than it is an area for expansion in this project. It would be interesting to see if the results of this experiment would be different if I was able to use equipment that tested specifically for oxidation, rather than testing for the next best marker. Rather than improving, this part of the experiment could purely be expanded upon. If more advanced equipment was available, I could attempt to answer questions such as, “to what extent do phenolic compounds affect oxidation,” or “to what extent is light a factor in oxidation.”Bibliography: Holderbaum,, Daniel (2010). “Enzymatic Browning, Polyphenol Oxidase Activity, and Polyphenols in Four Apple Cultivars: Dynamics during Fruit Development”(PDF). HortScience. http://www.raysahelian.com/phenolic.html http://www3.lib.cycu.edu.tw/exams_new/transfer_2nd/tps/92/tps9201/03.jpg https://www.wou.edu/las/physci/ch462/tmcolors.htm https://www.vernier.com/files/manuals/svis-pl/svis-pl.pdf http://www.seplessons.org/node/1690 el-Shimi, N. M. (1993-01-01). “Control of enzymatic browning in apple slices by using ascorbic acid under different conditions”. Plant Foods for Human Nutrition (Dordrecht, Netherlands). 43 (1): 71–76. ISSN 0921-9668. PMID 8464847.