Lab Exercises/Demonstrations ........

A Laboratory Experiment, Based on the Maillard Reaction, Conducted as a Project in Introductory Statistics
Olena Kravchuk, Antony Elliott, and Bhesh Bhandari

Introduction
ABSTRACT: A simple laboratory experiment, based on the Maillard reaction, served as a project in Introductory Statistics for undergraduates in Food Science and Technology. By using the principles of randomization and replication and reflecting on the sources of variation in the experimental data, students reinforced the statistical concepts and techniques introduced to them in lectures before the experiment. The experiment was run simultaneously by several student groups, using the same materials. Comparing the results of their analyses of variance, students became aware of the difference between P values and significance levels in making statistical decisions. In the experiment, the complete randomized design was applied; however, it is easy to adjust the experiment to teach students simple regression and randomized block designs. The benefits of incorporating laboratory-based experiments in the curriculum of an introductory statistics course for teaching non-statistics majors are well recognized in statistical education literature (Smith 1998; Martinez-Dawson 2003). Laboratory experiments in statistical courses improve student appreciation of the relevance and importance of statistics in their programs and enrich student research skills in their major areas. In Food Science and Technology programs, there is no shortage of simple and inexpensive experiments as students have extensive practical classes in chemistry, biochemistry, food processing, and other disciplines. However, as Martinez-Dawson (2003) noticed, statistical design and analysis of experiments are often left outside practical courses where students concentrate only on performing the experiment; the statistical analysis of the results is not emphasized. There is an immediate appeal to students to have a statistical component added to the practical aspects in their major disciplines. We would also argue that when a project in introductory statistics is related to the major disciplines, it helps students retain statistical knowledge in a long-term perspective as if the statistical concepts are not integrated with their discipline knowledge, it may be difficult for student to recall and apply these concepts later (Boyle 1999). The level of complexity of a laboratory experiment to be incorporated into introductory statistics depends on several factors: the place of the statistical course in the program, the time available, and the learning objectives associated with the experiment. In many introductory statistics courses, the material is taught almost exclusively in transmission mode in lectures with students having little more independence in tutorials/computer classes. A course project often provides an opportunity to balance such transmission learning by utilizing the learning-in-action approach; for the project to be a success, it is crucial to give student the advantage of gathering data themselves, not merely viewing a demonstration of research-grade instrumentation (Stanley and Baker 2002). In many applied science programs, an introductory statistics course is commonly the only service course that teaches how to design an experiment and manage experimental data. The number of methods covered in such a course may be large; moreover, students often experience difficulties with statistical thinking. According to Smith (1998), a way to help student develop their statistical reasoning is to incorporate active-learning strategies that allow student to supplement what they have heard and read about statistics by actually doing statisticsdesigning studies, collecting data, analyzing their results, preparing written reports, and giving oral presentations. We argue that all these goals may be fulfilled in a course project based on a laboratory experiment. This article demonstrates an example of the action-learning approach exercised in the School of Land and Food Sciences, Univ. of Queensland, Australia, in the introductory statistics course (STAT2701, Biometrics 1) by undergraduate student in Food Science and Food Technology programs. The article is structured as follows: in the Study Description section, we indicate the present state of the students knowledge
MS 20050334 Submitted 6/3/05, Revised 7/1/05, Accepted 8/25/05. The authors are with School of Land and Food Sciences, Univ. of Queensland, Brisbane, 4072, QLD, Australia. Direct inquiries to author Kravchuk (E-mail: o.kravchuk@uq.edu.au).

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An experiment in introductory biometrics . . .

Table 1Assessment scheme for the project
Assessment criteria Individual performance during experiment (total 30) Expectations A student is actively involved in every stage of experiment and demonstrates an understanding of the procedures (proposed scheme of randomization and experimental protocols). The student also takes responsibility for and fulfils a certain function in the group. The student is actively involved in peer discussion. Project report (total 50) A student reflects honestly and thoroughly on all the observed errors in the experiment, including materials, methods, and people involved. The project report is prepared in Minitab following the criteria for statistical report available on the Blackboard site of the course. In the report, the student demonstrates a good understanding of formal statistical techniques and the corresponding Minitab procedures. Group presentation (5-min oral presentation) (total 15) A team presents its results in an engaging way, explains the observed variability, interprets the statistical conclusions of the project, and proposes possible improvements to the experiment. Team-work (total 5) A team respects and supports its members and works effectively within the given time. The collected results are distributed to every member of the team.

of food chemistry and statistics, provide the study description, state the learning objectives, and explain the assessment criteria; in the Laboratory Experiment section, we describe the experiment and list the materials, methods, and time requirements; in the Student Learning section, we discuss the outcomes of the experiment in the context of student learning and suggest possible modifications to the experiment; in the Conclusions section, we argue that the experiment was a success in the outcomes achieved and suggest that a standard laboratory practicum may be a valuable source of projects for a corresponding introductory statistics course. In the Appendix, we present an example of a student report.

Study Description
In the School of Land and Food Sciences, Univ. of Queensland, introductory statistics (Biometrics I) is taught in the 1st semester of the 2nd y of the 3-year Bachelor of Applied Science (Food Science and Nutrition) and 4-year Bachelor of Food Technology programs. Food chemistry is taught in the 2nd semester of the 2nd y following the introductory statistics course. One of the goals of Biometrics I is to ensure that, upon completing the course, students will be able to communicate effectively with biometricians . . . about how to design the experiment; what variables to measure and how often; how to organize the data; what sort of data analysis is required; possible problems and limitations of the experiment; research questions that may be answered (Course outline, STAT2701, 2005). The concepts of statistical importance and significance, and their corresponding measures, R2 (coefficient of determination) and P value, are often difficult for students to understand; nevertheless, these concepts are essential to the data analysis for their final year projects. For the Food Technology students in the School, analysis of variance (ANOVA) is the most common type of statistical analysis in their final-year projects; hence, efforts are made in the introductory statistics course to enhance the students understanding of various experimental designs. At the time of this experiment (week 9 of a 13-wk course), the students had been taught the concepts of t tests and ANOVAs and the elementary principles of experimental design (randomization, replication, and control of experimental error). The timeframe of the course allowed us to allocate two 2-hour tutorials and a 1hour lecture for the course laboratory project: the 1st tutorial was supervised laboratory work; the 2nd was a computer class and the lecture time allowed students to present their results. The following learning objectives were associated with the experiment: 1. Enhance students understanding of the basic principles of
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statistical design: replication, randomization, and control of experimental error. 2. Improve students experience in handling the standard techniques of 1-way ANOVA: computations and discussion of the assumptions (normality, independence, and homoscedacity of the experimental error). 3. Accentuate that the P value and coefficient of determination (R2) of the experiment are statistics, that is, random values that depend on the data observed in an experiment. The students were provided with an elementary background of the Maillard reaction, the description of the experiment, a laboratory safety manual, and a simple statistical questionnaire before the experiment. They worked in groups (4 to 8 students) for 2 h in a food science laboratory under the supervision of a technician and biometrician. After the experiment, the students performed the data analysis in Minitab 14 and in the lecture, a fortnight after the experiment, delivered short oral group presentations. The students worked on the data analysis and presentations in the same group in which they conducted the laboratory experiment. However, the final reports (including Minitab project files) were submitted individually. Improvement in the students understanding of the statistical concepts was evaluated by short interview, the statistical report, and group presentation. To facilitate group work in the project, the assessment scheme included students individual participation, as well as cooperation within the groups. This scheme worked well, with students being actively engaging to participating in group discussions. The assessment scheme of the project is shown in Table 1. To emphasize the statistical component of the experiment, the students were asked to discuss in groups and complete simple questionnaires before and after the experiment. They were shown how to set up data files and perform the data analysis in Minitab. Instructions were also provided to the students on the expected format of the presentation of results in the final report. However, the discussion section of the report and group presentations were not guided, with the students only being asked to connect what they experienced in the experiment to what they had learned in STAT2701 and to reflect on the accuracy of their experimental techniques in connection to the R2 observed. The students were also asked to comment on the experiment. The general feedback was very positive, with students commenting that they liked the opportunity to apply statistical techniques in practice. Some minor suggestions were provided for the time management before and during the experiment. Additional comments about student feedback is further discussed in the Student Learning section.
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Laboratory Experiment
Materials

The eggs (50 g each from Williams Eggs, Warwick, QLD Australia) used in this experiment were purchased from a local store; Dglucose monohydrate was purchased from Unilab (Seven Hills, NSW, Australia), and filter papers were from Filtech (Armidale, NSW, Australia). All aqueous solutions were prepared with highpurity water produced with a Millipore (Billerica, Ma., U.S.A.) Milli Q system. All reagents were of analytical reagent grade unless otherwise stated.
Equipment

Each student group of no more than 8 students needed the following: 2 eggs; 1 mL pipette; 10 mL graduated pipette; pipette dispensers; fifteen 20-mL screw-top test tubes with lids; fifteen 20-mL test tubes; fifteen 4.5-cm funnels; filter paper, Filtech 1893-090; container; 2 test tube racks; marker pen; 4 different-colored beads. The class needed the following: vortex mixer (Ratek Instruments, Boronia, Victoria, Australia); covered water bath (Ratek Instruments shaking water bath); UV/Vis spectrophotometer (Pharmacia LKB, Alemeda, Calif.); 1 cm3 plastic or quartz spectrophotometer cells and distilled water for the reference cell.
Preparation of glucose solutions

These solutions were prepared before and made available for the experiment: 1. 1% Glucose solution: 5 g of glucose dissolved in 500 mL of distilled water; 2. 2% Glucose solution: 10 g of glucose dissolved in 500 mL of distilled water; 3. 3% Glucose solution: 15 g of glucose dissolved in 500 mL of distilled water; 4. 6% Glucose solution: 30 g of glucose dissolved in 500 mL of distilled water.
Measurement of absorbance

cose solutions (discussed subsequently) for the groups of 3 tubes randomly assigned to the glucose concentrations. Mixture of egg white with glucose solutions. Students added 9 mL of the prepared 1%, 2%, 3%, or 6% glucose solution to 1 mL of egg white into a test tube assigned to the concentration. This process was repeated twice more for each of the concentrations. 3. Marking the solutions As students prepared each solution, they placed a cap on the test tube and marked the concentration on the lid by marker pen; then they placed the solutions into a test tube rack. 4. Mixing the solutions In a random order, students took each solution separately out of the test tube rack and thoroughly vortex mixed it. Students then replaced the solutions into the rack. 5. Heating the solutions The laboratory assistant placed the rack of 15 test tube solutions into a boiling water bath and the water bath was then covered. The egg white solutions were heated for 20 min. 6. Cooling the solutions After this heating time, the laboratory assistant removed the rack from the boiling water bath (CAUTION: HOT) and placed the test tubes into ice to cool the egg white solutions to about 5 C. 7. Mixing the solutions Once each test tube was cooled, the students thoroughly vortex-mixed each solution and then placed the tubes back into the test tube rack in a random order. 8. Filter the solutions Students filtered each solution through filter paper into another test tube to produce a transparent solution. (As an alternative to the filtration step of the experiment, the samples could be centrifuged instead with no adverse affect to the samples.) 9. Measure the absorbance Student measured the absorbance of each solution at 420 nm on a UV/vis spectrophotometer and recorded the results.
Time required

The absorbance and hence browning of the egg white/glucose samples was measured on a Pharmacia LKB Ultrospec III UV/vis spectrophotometer at 420 nm (any spectrophotometer capable of making measurements at 420 nm can be used for this experiment). Approximately 2.5 mL of each filtered egg white/glucose solution was placed into a 1 cm3 plastic or quartz spectrophotometer cell for the absorbance measurement; distilled water was used as the reference.
Experimental method

Students required 2 h to perform the experiment: 15 min of preparation and initial discussion and 1 h 45 min of the laboratory work with 20 min group discussion during the experiment (while the tubes were being heated). We also allocated 2 h for doing the Minitab analysis during computer/tutorial class, and enough time for group presentations (8 min per group). Students were expected to spend 6 to 8 h outside class working on their project reports.
Technical support and supervision

1. Preparation of egg whites Students separated egg whites from egg yokes and placed the egg whites into a container and mixed thoroughly. Students then pipetted egg whites into 15 screw-topped test tubes by placing 1 mL of egg white into each of the test tubes. 2. Preparation of egg white solutions Preparation of the control solution. Students prepared the control solution by adding 9 mL of distilled water to 1 mL of egg white into a screw-topped test tube randomly selected out of the 15 tubes. This process was repeated twice more to produce 3 separate solutions. Randomization of mixtures of egg white and glucose solutions. To randomly allocate the 12 tubes with egg white to glucose solutions, student used 4 colored beads, where each glucose concentration was assigned 1 color. Beads were selected without replacement, 1 at a time, at random. Once all 4 beads had been selected, the process was repeated to allocate the next 4 tubes to the glucose solutions and so on. Students then prepared the mixtures of egg white and glu72

Technical assistants and biometricians supervised student in the laboratory, with teaching load of approximately 5 student per supervisor.

Student Learning
Assessment and discussion

There were 6 student groups participating in the experiment. The group size varied from 4 to 8 students. The students learning outcome was assessed individually and as a group based on their oral and written reflections and formal statistical analysis and discussions. Students were asked to report a standard statistical analysis: scatter plot of the experiment, the summary table (observed sample means and standard deviations), the ANOVA of the experiment and the corresponding interval plots, and the results of Tukeys multiple comparisons. All calculations were performed in Minitab 14 with the final report being set up in the Report Pad of Minitab. An example of the statistical report is given in the Appendix.
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An experiment in introductory biometrics . . .

Table 2Group results for the experiment
Nr of students Group Group Group Group Group Group 1 2 3 4 5 6 8 4 4 5 5 7 Observed R2 0.68 0.87 0.89 0.98 0.98 0.78 Observed statistic, F(4,10) 5.4 16.8 21.4 138.6 115.0 9.0 Reported P value 0.014 0.000 0.000 0.000 0.000 0.000 Estimated pooled variance, MSE 0.049 0.031 0.023 0.007 0.007 0.040

Before the experiment, students were asked to discuss in groups and to write individual answers to the following questions: 1. What is the research problem of the experiment? 2. What sources of variation do you expect to observe in the experiment? 3. What is a purpose of randomization in your experiment? Directly after the experiment, while still in the laboratory, the students were asked to discuss in groups and to write individual answers to the following questions: 1. Are you ready to answer the research question of your experiment? What do you expect for your answer? 2. What sources of variation have you observed in the experiment? 3. How many randomization steps have you performed in the experiment? What was the meaning of these steps? In the before-experiment questionnaire, the students easily answered the 1st question saying that they wanted to explore the relationship between glucose concentrations and browning (some of the groups even formulated the hypotheses set: to test the null hypothesis that there is no browning increase with increased glucose concentration versus the alternative hypothesis that there is a significant difference in browning with increased glucose concentration). The students also argued what type of relationship could be expected, and several groups pointed out that at the highest concentration a plateau effect may happen. It is worthwhile to notice that, at the time of the experiment, the students did not have a deep understanding of the Maillard reaction and relied on their general background in Chemistry and the elementary treatise of the Maillard reaction provided to them 2 d before the experiment. The 2nd question of the questionnaire caused general confusion and many students asked the instructors to interpret or restate the question. After they were asked to think about what may constitute experimental error, they indicated several obvious causes of variation in the data. The answers to the 3rd question demonstrated a lack of general understanding as almost all groups simply recalled a definition from the lectures and answered that randomization should eliminate personal bias in treatment allocations. In the after-experiment questionnaire, the student referred to the expected type of relationship between glucose concentration and browning, many groups sketched the experimental data and tried to fit a response curve. The 2nd question did not cause any difficulties. The students explained many sources of experimental error and even suggested some possible improvements to the experiment (they obviously used their previous experience in presenting data in laboratory reports). Interestingly, many students related their answers back to the 2nd question of the before-experiment questionnaire: The variation was initially thought to be only in the glucose concentration, but was observed to be present in some mechanical components of the practical including pipetting, human measuring and apparatus which can be concluded to attribute to some unknown error in the results. The answers to the 3rd question showed an improvement in the understanding of the principle of randomization. The students argued that by randomizing glucose concentrations, they prevented the possibility of making something very wrong just for 1 of the concentrations
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(that is, confounding people laboratory experience and treatments). Some groups noticed that the randomization prevented a possible spatial effect of the water bath. One group also concluded that randomization was performed to minimize undesired possible correlation between observations in the experiment. While preparing the group presentation, students used their discussions, results of the analysis, and personal reflections. Interestingly, the conclusions of the group discussion varied from research-related (the students noticed that the expected saturation curve did not eventuate and suggested another set of concentrations: 0%, 2%, 4%, 6%, and 10%) to business-related (the students argued that it was necessary to better control the experimental error as a food company would not be happy to have the results of the analysis from a laboratory experiment that shows a small R2) and even management-related (the students proposed that they could design and analyze an experiment but to minimize human error, a skilful technician should be the one who performs the experiment). In individual written reports, the students mostly related R2 to the experimental error: with a coefficient of determination at 89.53% our test was fairly accurate. The rest of 10% cannot be answered by the ANOVA test as errors like human and technical errors have occurred, although many noticed the influence of the data variation: this is a good example of how a single error can compound itself into a larger one affecting nearly all areas of the analysis. However, none of the students referred to the pooled variation as being a measure of experimental error. This flaw in their understanding was corrected in the lecture following the presentations when students discussed the comparative results of the experiment, as presented in Table 2. The experiment was designed to enhance students understanding of statistical principles by being encouraged to think independently and articulate their thoughts to a competent and appreciative judge. The following are some cautions we have formulated based on the observations during this exercise: 1. Do not use the experiment to lecture students, direct explanations have to be prohibited; instead, lead students to answers by asking them questions. 2. Make it clear to students that they will not be penalized for any flaw in their experimental technique; however, you expect that all flaws will be discussed in their reports. 3. If using the before- and after-experiment questionnaire, do not assess its context formally but allocate some marks for completion. Be sure that students are made aware of such an assessment scheme (some of the students commented that this scheme allowed them to answer the questions honestly and openly without hesitating about any lack in their knowledge). 4. Advise students before the experiment that a grade will be given for individual student participation in group discussion. Facilitate the discussion by asking groups provocative questions. For example, we asked them whether it was possible to extend the conclusion of their analysis further than the 2 eggs they used, the carton of eggs that was purchased, the eggs from the supplier, and so forth. Not having a deep understanding of the Maillard reacVol. 4, 2005JOURNAL OF FOOD SCIENCE EDUCATION 73

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tion, the students were not able to point out the possible difference in the pH levels of egg whites. However, thinking about this question led to the general conclusion that it is important to know the property of experimental material, that is, the population of interest in statistical terminology, to be able to expand the results beyond the scope of a particular experiment. 5. Encourage students to send you a draft of their presentation, but comment on their drafts in a very limited way so as not to change the focus of their discussion.
Possible modifications of the experiment

ments and observations during and after the experiment. The 1st author also expresses her gratitude to Dr Gloria DallAlba and participants of EDUC6000 (Graduate Certificate in Higher Education for Experienced Teachers, Graduate School, UQ, Australia) for inspirational discussions. The authors are grateful to the Scientific and Associate Editors and 2 anonymous referees for their valuable suggestions that helped us improve the clarity of our discussions.

References
Boyle C. 1999. A problem-based approach to teaching biostatistics. J Stat Educ. Vol. 7, Issue 1. Available from: http://www.amstat.org/publications/ jse/secure/v7n1/boyle.cfm. Accessed Sep 23, 2005 Martinez-Dawson R. 2003. Incorporating laboratory experiments in an introductory statistics course. J Stat Ed. Vol. 11, Issue 1. Available from: http://www.amstat.org/publications/jse/v11n1/martinez-dawson.html. Accessed Sep 23, 2005 Smith G. 1998. Learning statistics by doing statistics. J Stat Educ. Vol. 6, Issue 3. Available from: http://www.amstat.org/publications/jse/v6n3/ smith.html. Accessed Sep 23, 2005 Stanley DW, Baker KW. 2002. A simple laboratory exercise in food structure/texture relationships using a flatbed scanner. J Food Sci Educ. Accessed Sep 23, 2005 Univ. of Queensland. 2005. Course outline of STAT2701. Available from: http://www.uq.edu.au/lafspa/. Accessed Sep 23, 2005

Similar layouts of the experiment may be used to enhance students understanding of regression or blocking. In the following, we suggest regression and block designs. Regression experiment Concentrations of glucose (%): 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5. Instructions: Every concentration is to be prepared and measured once. Learning objectives: hypothesis testing, estimation of the regression coefficients, prediction and confidence intervals of regression. Statistical analysis: fitted-line plot, ANOVA of regression (test and diagnostics), confidence interval of the rate of reaction; confidence interval and prediction interval of absorbance. Report and disc ussion: the observed coefficient of determination, R2, P value, and the mean square error (in the context of experimental error). Randomized complete block design ANOVA (students as blocks) Concentrations of glucose (%): 0, 1.0, 2.0, 3.0, 4.0, 6.0. Instructions: Each student in a group should complete the experiment (1 replication per student). The results are to be combined within the groups for data analysis with students being analyzed as blocks. Learning objectives: experimental design, blocking effect, replication, and randomization. Analysis: scatter-plot, basic summary, ANOVA of Randomized Complete Block Design (tests and diagnostics), multiple comparisons of treatments. Report and discussion: the observed coefficient of determination, R2, P value, and mean square error (in the context of experimental error).

Appendix: Student statistical report of the experiment Minitab project reporta

In this project a CRD experiment was preformed and an ANOVA analysis under taken to examine the effect of glucose concentrations on the degree of the Maillard reaction in egg whites. The full statistical report on the recorded data is presented below.

Conclusions
It is possible to improve students understanding of statistical principles and techniques by introducing a laboratory experiment from the laboratory practicum of a major discipline into an introductory statistics course. In particular, in a Food Science and Technology program, the experiment based on the Maillard reaction may be a suitable choice. It is related to their major area of study, does not require expensive equipment, is doable in 2 h of supervised work, and is easy to analyze and comprehend. When an experiment is supervised by both a technician and biometrician, it provides a good opportunity to test and enhance students understanding of randomization, replication, and hypothesis testing and error estimation.

NB - Some unusual data present in concentrations 2 and 6.

Acknowledgments
The authors gratefully acknowledge the financial support for this research provided by the Faculty of Natural Resources and Veterinary Science, Univ. of Queensland from the Facultys fund for enhancement of student learning. The authors also thank the students enrolled in STAT2701, 1st semester of 2005, for their enthusiasm in the project and willingness to share their reflections and experiences. The authors are grateful to Del Greenway for her valuable com74

Descriptive Statistics: Absorbance (OD)

Variable Concentration (%) N 0 1 2 3 6 3 3 3 3 3 Mean StDev 0.01464 0.0231 0.0836 0.0410 0.0497

Absorbance (OD)

0.07067 0.1463 0.2170 0.1883 0.2337

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An experiment in introductory biometrics . . .

One-way ANOVA: Absorbance (OD) versus Concentration (%)
Source DF SS MS F P 0.014 Concentration (%) 4 0.05106 0.01276 5.37 (P value very small therefore significant) Error 10 0.02377 0.00238 Total 14 0.07483 S = 0.04876 R-Sq = 68.23% R-Sq(adj) = 55.53%

NB - R-Sq is very low and would normally be unacceptable in a food practical, technical errors have eventuated.

Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Concentration (%)
Individual confidence level = 99.18 Concentration (%)
NB - From graphs data appears to be both normal and random.

Lower

Center 0.07567 0.14633 0.11767 0.16300 0.07067 0.04200 0.08733

Upper 0.20656 0.27723 0.24856 0.29389 0.20156 0.17289 0.21823 0.10223 0.14756

Interval Plot of Absorbance (OD) vs Concentration (%)

Concentration (%) = 0 subtracted from: 1 0.05523 2 0.01544 3 0.01323 6 0.03211 Concentration (%) = 1 subtracted from: 2 0.06023 3 0.08889 6 0.04356

Concentration (%) = 2 subtracted from: 3 0.15956 0.02867 6 0.11423 0.01667

Concentration (%) = 3 subtracted from: 6 0.08556 0.04533 0.17623 Summary of Pairwise Comparisons Concentration 0:a Concentration 1:ab Concentration 2:bc (It would have been expected that if 0% is similar to 1 & 3 that 2 & 0 would be similar as well.) Concentration 3:abcd Concentration 6:bcde
NB - Concentration 2 displays unusual data, concluded to be attributed to large human error.
a This report is an original student work: the grammar, discussions and formatting are as presented by the student.

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