Food Chemistry Laboratory FDSC 402

Fall 2004

Professor in Charge John Coupland

103, Borland Lab 865-2636

Coupland@psu.edu

Office hours: Wednesday 3:30-5:00 PM

http://www.courses.psu.edu/fd_sc/fd_sc400_jnc3/FDSC402/fdsc402_web_page.htm

Lab Coordinator Emily Furumoto

203C Borland Lab. 863-3106

ejf4@psu.edu Office hours: 11-12 AM Thurs

Teaching Assistant Teaching Assistant

Annette Evans 8B Borland Lab

863 8670 axe145@psu.edu

Office hours: 5-6 PM Wed

Trupti Palav 102 Borland Lab

863 2954 tsp126@psu.edu

Office hours: 5-6 PM Thurs

(additional office hours are available by appointment)

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You have taken several lab courses already at Penn State and will take several more before you graduate. The laboratory is somewhere some of you will spend the bulk of your future careers and others will never return to one after graduation. Whatever your goals, this course will offer you opportunities to improve your applied science skills and will also be a useful reinforcement for other FDSC courses. More importantly, food chemistry experiments very often do not work out as we planned. In chemistry labs the chemicals are pure, the conditions are controlled and you can be expected to get a “right” answer. In food chemistry we often have poorly defined starting materials and many reactions occurring in parallel under non-ideal conditions. Unsurprisingly the data we get is often noisy and hard to interpret. Wherever you go in life you will be trying to make difficult decisions on poor data. The machine is broken – you don’t have a good schematic or time to pull it apart but its making a squeaking noise and drawing too much power. Can you infer the cause and propose a solution without a more thorough diagnosis? Developing this type of skill should be a benefit of this class. Come to the lab prepared – know what you will be asked to do and have an idea of the results you expect. Be observant – what do your samples look like? What do they smell like? Do you think your measurement technique is capable of giving meaningful results for your sample? Be thorough – record your observations. Make sure you get the data you were asked for but note your other observations. Be imaginative – once you have your observations can you construct a scientific explanation including not only what you expected (and why) but also what happened (and why). Have fun – remember there isn’t a final! Course Objectives: The goal of this class is for students to make scientific measurements of some of the important chemical reactions occurring in foods. Students successfully completing this class will:

1. Recognize the important reactions in food chemistry and their consequences. 2. Be familiar with methods to measure these reactions. 3. Be capable of reporting their results in an appropriate format. 4. Be capable of designing and conducting an experiment to understand a simple

food chemistry problem.

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Class Hours: Unless otherwise announced, the class will meet in Room 201 or 4 Borland Lab. between 10-1 each Friday of semester. Attendance is required. The class will start with a lecture/discussion before beginning the practical work. Academic Integrity: The measurements you make as a working scientist will be used by yourself and others to make decisions that will affect the profitability of companies and the lives of individuals. It is therefore essential that you truthfully and accurately report both what you did and what you saw and measured. The University policy on academic integrity applies in this course (http://www.psu.edu/ufs/policies/47-00.html#49-20). You are encouraged to work together within your research group to conduct laboratory exercises and discuss the interpretation of results but the actual presentation of the report is your individual responsibility and you are not permitted to copy each others work. Safety Considerations: You will be working with materials and techniques that could be harmful to you and others in the lab. Instructors will attempt to inform you of unusual risks but it is your responsibility to follow typical good laboratory safety practices (the Penn State manual is available online http://www.ehs.psu.edu/safety/ Safety_Manual.doc). If you are unsure about the safety of a procedure or your capacity to perform it safely, ask an instructor before proceeding.

• You must wear a lab coat, safety glasses, and close toed shoes at all times

• Dispose of waste carefully. Many reagents can be washed down the sink with plenty of water but organic solvents and some other materials must be put in designated storage bins for safe disposal. If in doubt, ask your instructor.

• Other classes use the lab. At the end of each exercise you must leave all your glassware clean and put away and the bench surfaces cleaned down.

• No mouth pipetting • Label all reagents

(name/date/contents) • No eating or drinking allowed in

the laboratory

If you do not behave safely and responsibly in the lab

you will not be allowed to continue the exercise.

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Grading Policy: Credit will be available as follows:

Lab Reports 160 Pre-lab exercises 20 Participation and performance in lab 20 Project work 50 Total 250

Letter grades will be assigned based on the percentage total score according to the typical PSU system, i.e.: 100.0-93.0=A, 92.9-90.0=A-, 89.9-87.0=B+, 86.9-83.0=B, 82.9-80.0=B-, 79.9-77.0=C+, 76.9-70.0=C, 69.9-60.0=D, 0-59.9=F. Lab Reports. Reports are due the Friday following completion of the exercise (as noted in the timetable). All reports are to be typed (Times New Roman font, 12 point, 1” margins, line spacing 1.5). Figures must be computer generated (or equivalent quality) and may either be imported into the text or attached on separate sheets. The report must contain (1) Title, name, date, (2) Introduction. In 2-3 paragraphs describe the basic science relevant to the work and your goals/hypotheses for the study. Make references to at least 2 pieces of literature to support your basic science (e.g., lab manual, textbooks, journals). List your references at the end of the text. (3) Methods and Materials. Describe what you did and how you did it. Very often the methods section can be brief: “The experiment was conducted as described in the laboratory manual (Coupland, 2002).” but be sure to list any deviations from the procedure set out in the manual. The methods section should be written in the past impersonal form (e.g., “A standard protein solution was prepared by weighing 20.0 g of soy powder…”). (4) Results. In this section you must introduce all of your results and describe them, calling particular attention to the relevant details. All your Figures should be numbered, and in the text work through the data systematically. First, state what the Figure is supposed to show (e.g., “The absorbance of the solutions was measured as a function on protein concentration and the results are reported in Figure 3”) then describe the data and any important observations (e.g., “Absorbance increased with protein concentration and a linear regression fit well with the data (r2=0.989)”). Comment on the quality of your data and also include other observations that may be relevant. Link your paragraphs together so the text flows from one idea to the next. (5) Discussion. Use this section to answer any questions provided with your exercise. (6) Conclusion. In 1-2 sentences state how the results fit with your expectations and hypotheses set out in the introduction. Each lab reports will be graded out of a possible 20 points. There are n lab reports and your grade will be calculated from your n-1 best results. An important goal of this class is for you to see the reactions first hand. Therefore I will not accept lab reports for students who do not attend the lab. Late work will be penalized 1 point plus an additional 1 point per additional working day late.

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Pre-Labs. To get the most of the lab you must come prepared. This will include carefully reading the lab manual and the relevant sections of a food chemistry textbook. You should arrive at each lab knowing what you plan to do, why, and what results you can expect. To help with your reading, you will be provided with a question sheet to complete and hand in at the start of each lab where we start a new exercise. Project Work. As your project, you will be given a problem to work on as a group (see below). The exercise is designed to be more open-ended than a typical lab and is intended to improve your capacity to plan and conduct your own experiments. The project will be graded as a group exercise. Only one report is necessary for the whole group. Meet with instructors to set out experimental design and equipment needs 5 Demonstrate competence with measurement method(s) 5 Data review with instructors 5 Oral presentation of project 15 Written presentation of project 20

TOTAL 50

Lab Groups: For much of this class you will be working in the following groups: 1 Baer, Bickerstaff, Carmichael, Enslin

2 Erdman, Fetherolf, Francl, Gevin

3 Grant, Grasso, Hileman, Kelley

4 Krall, Lamm, Lehr, Livingston

5 Mc Pherson, Miller, Saengruangkit, Soika

6 Stoltzfus, Sutton, Yeo

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Provisional Timetable

Date Exercise Work due Aug 31st

Orientation and basic lab protocols

Sept 3rd

Data reporting, statistics and computer skills (ASI building computer lab)

Sept 10th

Chemical kinetics (alternative exercise) Exercise 1: Basic

techniques (kinetics prelab)

Sept 17th

i. Candy manufacture ii. Writing workshop

Exercise 2: Kinetics (draft version) (candy prelab)

Sept 24th

i. Set up water activity experiment (moisture

sorption isotherms and texture analysis) ii. Set up protein functionality (edible films) iii. Candy evaluation

Exercise 2: Kinetics (final version) (moisture sorption prelab)

Oct 1st Protein functionality

Exercise 3: Candy (protein functionality prelab)

Oct 8th i. Moisture sorption ii. Starch functionality

Exercise 5: Protein functionality (starch prelab)

Oct 22nd Flavor chemistry (Exercise 9) Exercise 4: Moisture sorption (flavor prelab)

Oct 29th Nonenzymatic browning Exercise 6: Starch (in class test on browning)

Nov 5th Lipid oxidation

Exercise 7: Browning (lipid oxidation prelab)

Nov 12th

Project work (preliminary measurements) Exercise 8: Lipid

oxidation

Nov 19th Project work (preliminary measurements) Dec 3rd Project work (main data generation) Dec 10th Project presentations, clean-up Project report

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Exercise 1a: Basic Laboratory Procedures The goal of this exercise is to learn to use the basic equipment necessary in the rest of the class and to generate some data for use in the next exercise. Purdue University maintains a website giving more detailed guidance on many of these procedures: http://chemed.chem.purdue.edu/genchem/lab/equipment/index.html. In this class you will learn:

• Lab safety essentials • Accurate measurement of mass, volume, pH and absorbance.

As scientists we are concerned with controlled measurement of the systems that interest us. There are an almost unlimited number of things we could measure about a food product; the difficult bit is to use our experience and basic chemical knowledge to determine which are likely to be the most important. For example in the yogurt product we know a sour flavor is due to acid, probably lactic acid made by the culture. We might therefore want to measure pH and look at the growth of the fermentation organisms. We can then use our quantitative measurements to improve the product either by changing the fermentation time or otherwise reducing the amount of acid produced. In this class we will look at our basic tools of measurement and learn to use them efficiently. We will measure:

• Mass: The four-figure balance is one of the most precise tools in the laboratory. Disadvantages are that it is fairly slow, very expensive and quite delicate. It can also be difficult to measure hot or otherwise volatile product. The mass limit on the balance is 200 g, which is quite easy to exceed with the combined weight of the sample and container. Other balances offer lower precision (e.g., 2-figure or even 1-figure balances) but can handle greater masses.

It is not always necessary to measure to the highest precision available. In general the maximum error should be small (~<1%) of the total measurement. If for example my balance weighed to 1-figure and the reading was 1.0 g the actual weight could be anywhere between 1.05 and 0.95 g. The maximum percentage error in my reading is then (0.05/1 x 100%=) 5%. On the other hand my reading were 100.0 g, my error would be (0.05/100 x 100% =) 0.05% - much better. Therefore I should weigh a smaller weight on a more accurate balance to get the same level of precision.

• Volume: It is far easier to transfer and measure liquids by volume. The available ways to do this are by pipette or burette. A pipette is a calibrated volume of glass or plastic that can be filled to contain a known volume of liquid. If used correctly they can be very precise, but it is easy to get wrong!

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A constant volume pipette (one of the older glass models) can deliver only the volume etched on the side. Carefully place a bulb over the blunt end and slowly suck up the sample to just beyond the mark. Remove the bulb and quickly cap the blunt end with a finger. Slowly release sample until the liquid meniscus lies on the marker line. Touch the tip to the wall of the beaker to release any hanging drops. Transfer the pipette to a new container and release the liquid. Let it flow out under gravity then touch the tip to the edge of the glass to let the small remaining volume flow out. Do not: • Mouth pipette • Allow liquid to be sucked up into the

bulb • Force the liquid to flow too fast • Contaminate sample or standard with a

dirty pipette • Try to blow out the last drop in the tip

after emptying

An adjustable pipette (e.g., Gilson, Autopipettor etc.) can deliver any volume of liquid within the range available. Place a clean plastic tip over the end of the pipette. Set the dial to the volume required. Push the plunger down to the first stop, place the tip in the sample and slowly release to allow the tip to fill. Transfer the pipette to the new container and slowly press the plunger down to the first then the final stop to squeeze out all the liquid. There may be a small droplet left in the tip – that’s OK. Do not: • Let the pipette sit horizontal on the

bench with a tip on – liquid can flow up into the barrel

• Change the dial setting during a series of repeated transfer operations.

• Release the plunger too fast – again liquid can flow into the barrel.

A burette is a good way of transferring variable volumes of liquid, its often less precise but offers great flexibility. A burette is a differential measurement, record the volume at the start of an experiment and the volume at the end and know how much you have added by difference.

• Release liquid slowly and be careful it is mixed well with the sample you are delivering into.

• To measure volume, line up the meniscus with the scale. Keep your eye level with the point of measurement to avoid parallax errors.

• Be careful there are no air bubbles in the burette • Try to use the smallest volume (therefore highest precision) burette that you can

complete a titration in one filling. Extra filling means more error.

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• pH: The pH scale is a logarithmic measure of the concentration of hydrogen ions:

][log10

+−= HpH

so a change in pH scale of 1 unit means a 10 fold change in the concentration of hydrogen ions. pH is measured with a glass electrode. The electrode measures the ionic concentration but it is hidden behind a semi-permeable membrane designed to allow only hydrogen ions to pass and be detected. A pH meter is only reliable when calibrated and should be recalibrated several times a day or whenever making a measurement at a different temperature.

• Absorbance. The absorbance of a solution is a measure of how much light it

absorbs. If you shine 100 units of light in and get 90 out you have a transmittance of 90/100, 90%. You can calculate absorbance as:

Absorbance=2 -log (%Transmittance)

The logarithmic scale is confusing because very small changes in the transmitted light can lead to large changes in absorbance. So small errors at low transmittance lead to large errors in absorbance (avoid absorbance >1) and small measurements are difficult for the reasons discussed under “Mass” (avoid absorbance <0.1).

Calibration is important for a spectrophotometer. It needs to be told the zero and 100% intensity reading. Zero can be measured simply by blocking the light beam. Full scale (the maximum light the detector can ever get) is done by putting everything into the light beam that will go there during an experiment except the sample. So make a measurement with cuvette+ solvent (usually water) and set the dial to 100% transmittance (0 absorbance). Absorbance depends on the wavelength of light used so set the wavelength dial, and don’t touch it again through calibration and measurement.

0

0.5

1

1.5

2

2.5

3

0% 20% 40% 60% 80% 100%

Transmitance

Abso

rban

ce

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Exercise 1a Procedure 1a.1 pH of Foods Goal: To determine the pH of a range of foods.

Materials Apparatus

5 sample foods pH 4 and 7 buffers

0.1 M HCl1

pH meter

Procedure

Calibrate the pH meter.

• Calibrate the pH meter as described in the handout

Note when moving the electrode between solutions rinse with distilled water and dab (not rub) dry with a paper towel. Be careful not to contaminate buffers or samples. Make sure you use the same stirring conditions to make your measurements as you did for your calibration. Never leave the electrode dry for any extended period of time.

i. Transfer about 20 ml of sample to a suitable container ii. Measure the pH; allow the probe several minutes to come to equilibrium with the

sample before taking a reading. iii. Repeat for all samples. Run the milk sample last as dairy proteins can

contaminate the electrode. After the experiment leave the electrode soaking in 0.1 M HCl to clean off the proteins. The TAs will dispose of HCl before the next class.

1a.2 Pipetting Goal: To calibrate the autopipetter

Materials Apparatus Gilson automatic pipette & tips

Weighing bottle

1 Hydrochloric acid is caustic. In case of spillage, flood with plenty of water and contact an instructor.

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Procedure i. Set the autopipettor to 1 mL. ii. Accurately weigh a measuring bottle (or any suitable container) using the 4-figure

balance. iii. Remove the bottle from the balance, add 1 mL of distilled water and re-weigh.

Knowing the density of water at 25°C is 0.997 g mL-1 calculate the volume of water delivered.

iv. Empty the vial and repeat for 5 measurements. 1a.3 Spectrophotometry Goal: To measure the optical properties of different beverages

Materials Apparatus Gatorade 10 test tubes

Gilson automatic pipette & tips

Procedure

Operating the Spectrophotometer.

• Plug in and turn on the Spec 20. It must warm up for 10 minutes before use.

• Set the instrument to the proper wavelength by turning the knob located on the right hand surface of the spectrophotometer. The wavelength setting can be seen through the window next to the knob.

• Obtain a properly cleaned cuvette and fill it about ¾ full of the reference solution (usually water). Tubes in the Spec 20 are easily scratched and imperfections in the glass can lead to differences in the reading. Wipe outer tube surface with a Kimwipe to remove fingerprints and insert tube into the spec. Attempt to line the cuvette up in the spec the same way for each reading. The vertical line on the tube should line up with the mark on the spec.

• With no cuvette in the sample holder, close the cover and rotate the zero light control knob (left front knob) to display a reading of 0.0% transmittance. Provided that the instrument is not turned off and this knob is not moved, no other adjustments to this control are needed.

• Place the reference solution cuvette in the sample holder, close the cover, and rotate the light control knob (front right knob) to display a reading of 100.0% transmittance. This procedure must be repeated every time measurements are taken at a new wavelength or if several measurements are made at the same wavelength. Fill a properly cleaned cuvette 3/4 full of you sample solution.

• Place your sample cuvette in the sample holder and close the cover and read either the absorbance or percent transmittance as needed.

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i. Mark 10 test tubes and add water/beverage as described in the table

Tube 1 2 3 4 5 6 7 8 9 10 Beverage /mL 0 1 2 3 4 5 6 7 8 9 Water /mL 9 8 7 6 5 4 3 2 1 0

ii. Measure the absorbance of the 10 beverage solutions [Yellow -- 400 nm, Red –

500 nm, Orange – 480 nm, Green – 380 nm] iii. Select the sample with an absorbance (bottom scale) closest to 1, and for this

sample only measure the absorbance at 20 nm intervals between 400 and 600 nm.

Exercise 1b: Data reporting, statistics and computer skills. In the previous exercise you learnt to make measurements. In this class you will learn the basics of manipulating that data for presentation. You will learn:

• Use of Microsoft Excel for data reporting and analysis • Basic statistics (mean, standard deviation, regression analysis) • Rules for drawing good graphs and tables

In all steps use the data from the corresponding 1a experiments. For this exercise it is only necessary to supply the Figures and Tables listed below and answers to Question

1b.2.vi. 1b.1 Tables Tables are the preferred way of reporting precise numerical data in an organized manner. The reader can readily see your numerical results but trends may not be obvious and the lack of graphical cues can make the results less compelling. i. In Microsoft Word, generate a formatted table listing the measured pH of your

food samples. ii. Format the table using Table>>Autoformat>>Simple, and uncheck “color”. Add

a title and make sure you are using appropriate units. 1b.2 Basic Statistics Excel is a powerful program for handling your calculations and doing basic statistics. In this exercise you will take the data from Exercise 1.1 and write an Excel spreadsheet to calculate the mean and standard deviation of the data. i. Enter your mass data in one column ii. Look up the density of water at lab temperature on the web and enter this data on

the same worksheet iii. Use an equation to calculate a column of volumes next to the column of masses iv. At the bottom of the column of masses use the MEAN and STDEV functions to

calculate the mean and standard deviation of the mass. v. Export the data to Word and format the table appropriately. Add a title. vi. In one or two sentences describe what you can conclude from this data.

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1b.3 Graphing A graph is an excellent way to display data. Trends become much more visually obvious than in a table but the precise numeric data is lost. i. Enter your data from Exercise 1.2 is a worksheet in three columns (volume of

beverage, volume of water and absorbance) ii. Calculate in a separate column the concentration of beverage and arrange a copy

of the absorbance data alongside it iii. Plot a graph of beverage concentration vs. absorbance. iv. Add linear regression line to the data and make a note of the equation of the line

and the correlation coefficient. v. Assume a 5% error in the absorbance data and add error bars to the graph. vi. Format the graph and add a title. vii. Enter the wavelength and absorbance data into a separate worksheet. viii. Plot and format a graph of absorbance vs. wavelength. Format appropriately.

Figures and Tables A key part of the scientific process is communicating your results with others. In oral communication you have some flexibility – you can see if your audience is bored or uncomprehending and they may ask you questions to clarify a difficult issue. However the standard mode of communication is often a report which will take on an existence independent of you and must be able to survive on its own. A key aspect of your report is the logical presentation of your data. Data is the selected measurements of reality you have made. It does not represent the whole picture but merely your selection as a scientist of what you think is important. In selecting which measurements to make you have made a value judgment that others may (or perhaps should) disagree with. Furthermore the data you report is convoluted by experimental and sampling inaccuracy. Whatever the weaknesses of your data set you now have a responsibility to communicate it to your audience as cleanly as possible. Another goal of data presentation is to help see and communicate patterns in data (e.g., linear regression) not immediately obvious. The basic methods of data presentation are Figures (including graphs, drawings, photographs) and Tables. You have a lot of flexibility to choose the format that best suits your purposes but your choice may put a “spin” on your data that you should be aware of.

• Drawings. Line drawings are used sparingly in most publications but can be an excellent way of illustrating a complex apparatus, a complex physical mechanism or to sketch a graph you have no hard data for but merely wish to illustrate a trend. Be very careful with drawings – if all the scales and distances do not correspond to the story you are trying to tell then change them.

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• Photographs. A photograph is one of the most compelling ways to present qualitative2 data. A well-used photograph can show the obvious differences between samples not adequately captured by raw numbers. However it is easy to use photographs dishonestly and let their persuasive power infer statistical significance that does not exist or cover up the poor quantitative measurements. Some photographs reproduce poorly in black and white. Always include a scale.

• Scatter Plots. The simple graph is the most commonly used method of data

presentation. (In Excel avoid “line plots” – they look like graphs but are not the same). Scatter plots can be used when both x and y are continuous variables (i.e., can take on any value within the range). Conventionally x is the controlled variable and y is measured. Error can be easily incorporated as error bars (typically one standard deviation). Scatter plots provide a good visual representation of data making it easy to pick out trends but it is hard to extract the raw numbers should they be needed at a later date.

• Bar Charts. Bar charts are the best ways of representing non-continuous data. X

is now a label not a value and y the measured value (sometimes with an error bar).

• Tables. Tables are the best way of listing hard numbers in a coherent way. It can

be difficult to pick out trends in data but the numerical information is preserved. Error can be acknowledged as x±y or sometimes using superscripts to indicate statistical significance. All columns and rows must have a heading (often with a unit).

Other general rules:

1. It is rarely permissible to present the same data twice (e.g., a Figure and a Table) in the same report.

2. All Figures and Tables must be numbered in the order they are referred to in the text.

3. All Figures and Tables need a legend (i.e., a title) that describes what is being shown. The legend should be long enough to stand alone (e.g., “Calibration curve” is insufficiently descriptive).

4. Acknowledge all units. 5. Describe numbers to the precision you know them. 6. Do not use color or shading. (Sometimes in a presentation).

2 It is possible to extract quantitative information from photographs through careful image analysis.

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Example of a Good Figure

Example of a Good Table Table 3: Effect of pH and temperature on the rate of pea chlorophyll degradation

First order rate constant /min-1

pH 80°C 90°C 100°C5.5 0.046 0.080 0.1606.2 0.022 0.046 0.0826.8 0.008 0.016 0.0347.9 0.004 0.008 0.017

Report data to the precision you are confident in, if possible use “±” to describe standard deviation

Use a unit for all numbers that have one

Give a title to all columns and to the table.

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Exercise 2: Kinetics of Color Extraction Kinetics. Change is inevitable but it is useful to know how fast it’s going to happen. We might be interested in how the texture of a potato changes as it is cooked, the vitamin content of baby food changes with storage or, in this case, the color of spinach during cooking. Chemists are rather strict about measuring the concentration of one chemical as it changes. Food scientists can be more relaxed and measure the rate of change of any measurable property (A). The rate of change of A is then given as:

nAkdtAd ][][

−=

where k is the rate constant and t is the time. n is the order of the reaction, chemically the number of molecules taking place in the rate limiting step of the reaction but in our case a parameter that can be known from published research or calculated by curve-fitting. This is a differential equation, it tells us the rate of change of A at any time but really we want the actual value of A. There are published integrals for various values of n (or you could just work it out), for a first order reaction (n=1):

ktAA −= 0lnln or ktAA

−=0

ln

or, for zeroth order kinetics

A = A0-kt

where A0 is the initial value of the measured parameter. The slope of an appropriate graph will be the rate constant of the reaction. Knowing rate, we can use the equations to calculate the value of the measured parameter at any time. Reaction rates depend on many of the other chemicals present (catalysts or inhibitors) and on the temperature. We can describe at the temperature dependency of reaction rate using the Arhennius equation:

)exp(0 RTE

kk a−= or ln k = ln k0 -Ea/R (1/T)

where k is the rate at absolute temperature T, Ea is the activation energy of the reaction, R is the gas constant, and k0 is a constant. This equation implies that ln (k) is proportional to 1/T. By measuring the rate at various temperatures and fitting a straight line to an Arhennius plot we can calculate the activation energy from the slope.

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A kinetic approach can be taken to the investigation of physical processes. One of the difficulties your instructor faces living in the US is an absence of well-made tea. The secret is to use really hot water and in this alternative experiment we will show the effects of water temperature on the extraction kinetics of tea. We will track the extraction through measurements of color released although bear in mind that other components (e.g., flavor, caffeine) may be extracted at different rates. We will describe the reaction as follows:

Color (bound) Color (free) or Cb Cf So the rate of the reaction is:

dtCd

dtCdRate fb ][][

=−=

We will assume the reaction is first order, so:

][][][][

fbfb CkCk

dtCd

dtCdRate =−==−=

where k is the rate constant of the reaction. Integrating we get:

ktCCtotal

b −=][

][ln

We are measuring absorbance (a) which is, according to the Beer-Lambert law, proportional to concentration. We also know the maximum extractable amount of color is [Ctotal] so [Cb]=[Ctotal]-[Cf] (this is a good step – we are not measuring Cb but only the released stuff – Cf). Putting these two ideas into the above equation:

kta

aa−=

−

max

maxln

So plotting the natural log of (amax-a)/amax against time should give a slope of -rate constant. The rate constant should increase with temperature according to the Arhennius equation so if we plot ln(rate constant) against reciprocal absolute temperature we should get a straight line with slope –Ea/R.

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Exercise 2: Procedure

Materials Apparatus Berry or black tea

Waterbaths (40, 60 & 80°C)

Conical flask (3) Spectrophotometer

Spectrophotometer tubes

Procedure i. Number the tubes. In this experiment you will capture a lot of data and you must

be organized to keep track of all the information. Take time to make a table to compile all of your data before starting work.

ii. Weigh 200.0 g of water into 3 Erlenmeyer flasks. Seal with foil and heat in the waterbaths (40, 60, 80°C).

iii. Pre-weigh 0.5 g of tea onto a piece of foil. When the water reaches temperature, quickly add the tea and swirl to mix. Reseal with foil and return to the waterbaths. Mix the tea again every few minutes throughout the experiment

iv. At time intervals shown below, transfer a few milliliters of tea to a cuvette using a 10 ml pipette. (Try not to transfer tea leaves – if necessary strain through filter cloth). Measure the absorbance of the solutions (415 nm). If it is more convenient, store the strained tea until you have time to measure. If you miss a time or decide to make a measurement at another time be sure to note the actual time the sample was taken. Note – it is not strictly necessary to make measurements at these exact times but note the actual times you use in your experiments.

Recommended times to take samples

40°C sample 60°C sample 80°C sample 0 min* 0 min* 0 min* 2 min 1 min ½ min 5 min 2 min 1 min

10 min 5 min 2 min 15 min 10 min 5 min 30 min 20 min 10 min 40 min 30 min 20 min

*For your time zero measurement, quickly add the tea to the hot water, stir, and separate. v. Data presentation

• Plot absorbance as a function of time. Plot all temperatures on the same axes and clearly differentiate between them in the legend.

• Plot afinal vs. temperature. In your discussion, comment on what you think the figure tells you about the system.

• Plotting the natural log of (amax-a)/amax against time (all temperatures on the same set of axes). Fit a straight line to the data and calculate the rate constants. In practice, the first data point is often unreliable as the

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absorbance is changing too fast at that time so it is better to not use this point in your line fitting. Also the final point cannot be calculated [(amax-amax)/amax=?]

• Show rate as a function of temperature in an Arhennius plot. (Plot -ln(k) as a function of 1/(absolute temperature). Calculate the rate of extraction at 30°C and the activation energy of the reaction.

• In summary you should have 4 Figures, absorbance vs. time for all temperatures, final absorbance vs. temperature, first order plot for all temperatures and an Arhennius plot.

Grading

• Introduction Set up background (1), Describe goals (1), Cite literature (1)

• Methods and Materials (1)

• Data Presentation (9). Points will be deducted for errors including use of an inappropriate title, use of the correct data, spacing of the axes, units, axis labels, clear symbols and lines, overall legibility

• Results and Discussion Introduce all Figures and Tables (1), describe the data (1), be critical of data (1), Why is it a 1st order reaction? (1), Compare to zeroth order (1), Calculate an activation energy (units!) (1), Critical assessment of analysis (1)

• Overall format (1)

19

Exercise 2: Pre-Lab Questions a. Why is it necessary to cap the Erlenmeyer flasks? (2 points)

b. On the following graph sketch how you expect the absorbance to change as a function of time at (i) a high temperature, and (ii) a low temperature (4 points)

Change in absorbance

e

c. What are the axes of an Arhennius plot (2 points)

i. x-axis?

ii. y-axis? d. Why was it necessary to make sure all the tea leaves w

spectrophotometer reading? (2 points)

20

tim

ere extracted before making a

Exercise 3: Physical Chemistry of Candies The goal of this exercise is to understand some of the ways that the physical chemistry of solutions is relevant in food science and to critically evaluate the relationships between microstructure and texture for a simple food product. Much of the chemistry of candies can be understood in terms of the state diagram of sucrose (the major ingredient). A state diagram illustrates the relationship between composition (water content, x-axis), temperature (y-axis) and physical state. At the start of the experiment the sugar is dissolved in water and heated to the boiling point.

x

Figure: State Diagram of Sucrose. In the region marked “sugar crystal” the solution will only crystallize if given sufficient time and in this case will remain a supersaturated solution. X=typical start position in candy cooking. Figure reproduced from Hartel (2002). Boiling sugar. As the mixture is heated the temperature rises and the solution begins to boil. The boiling point of a sugar solution is higher than pure water. As boiling progresses, pure water leaves as steam so the sugar is concentrated and the boiling point further increases. Elevation of boiling point of a solution is a colligative property, i.e., ideally depends on the number of molecules in solution rather than the type of molecules. The elevation of boiling point can be calculated from the Clausius-Clapeyron relationship:

mKT bb ⋅=∆ where ∆Tb is the elevation of boiling point, m is the molarity of the solvent and Kb is the ebullioscopic constant (a function of the boiling point of the solvent, the enthalpy of vaporization of the solvent and the molar mass of the solute). Therefore a solution boiling at a higher temperature is more concentrated than one boiling at a lower temperature. However, we cannot expect a quantitative Clausius-Clapeyron relationship because we do not know the molar mass of the corn syrup and because of two chemical reactions that may change the number of molecules present.

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(i) Inversion. Sucrose can break down to glucose and fructose particularly in the

presence of acid and catalytic enzymes. There is some sucrose inversion during boiling of a sugar syrup.

C12H22O11(sucrose) + H2O + H+ => C6H12O6(fructose) + C6H12O6(glucose) + H+

(ii) Maillard browning. Reducing sugars (i.e., those with a free or accessible

aldehyde group) can react with amines at high temperatures to form brown colors and bitter and meaty flavors. Reactive proteins are available in the dairy ingredients. Note that sucrose can also caramelize at very high temperatures (>200°C) in the absence of amines to also produce bitter and buttery flavors and brown colors. Both Maillard browning and caramelization are separate reactions but they are both non-enzymatic browning.

As the solution is boiled, it will become supersaturated, i.e., the concentration of dissolved sugar is greater than the solubility limit. Given sufficient time the sugar would crystallize out to give a mixture of crystals in a saturated sugar solution. The sample is not allowed time to crystallize and also the larger carbohydrates present in the corn syrup serve to inhibit crystallization. Boiling is continued to produce the desired final moisture content. The sample is then poured into a mold to cool. As the solution is cooled, it becomes much more viscous, and the reduced molecular mobility prevents the molecules arranging themselves in a crystal lattice in the time available. The viscous, cooled sugar solution solidifies without crystallizing. The amorphous solid formed is structurally similar to a liquid (i.e., no long range structure) but is so viscous that it has mechanically solid like properties. The viscosity of the solution (or the inverse of the molecular mobility) increases with (i) cooling – less thermal energy means less molecular motion, and (ii) decreasing moisture content – the small water molecules act as a molecular lubricant (or plasticizer) to ease the diffusion of the sugar molecules. (Molecular mobility is also a function of the molecular weight of the solute; see Fennema text for details). The limit of high viscosity occurs at the glass transition line (see state diagram). To the right and below the glass transition line the amorphous sugar solution changes from a rubbery to a glassy state and there is a dramatic drop in molecular mobility. A rubbery solid tends to be chewy with slow chemical reactivity. A glassy solid tends to be brittle and crunchy with almost no chemical reactivity. By boiling the candy to different temperatures samples are generated with different moisture contents. The supersaturated solutions are cooled to the same temperature and when they are cooled may either be glassy or rubbery depending on their moisture content.

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Analytical Methods Thermal analysis (not used in the current version of this exercise). Thermal analysis is a very powerful tool to identify the physical properties of the system by measuring the temperatures when structures melt and reform and the heat absorbed and released when they do. There are many methods of thermal analysis but we will use differential scanning calorimetry (DSC). In this method the sample is sealed into a small aluminum pan. The sample is typically very small (~10 mg) to allow very rapid and uniform heat transfer. The sealed pan is then loaded into a tiny furnace and an empty pan is loaded into an adjacent similar furnace. The same heating is then applied to both furnaces and the samples begin to warm up (or alternatively cool) and the temperature of each is measured. The heaters are computer controlled and can be programmed to heat at a given rate. The furnace containing the sample will typically require more heat than the adjacent reference furnace because of the extra energy to warm the sample. When the sample melts, extra energy is needed by the sample furnace to overcome the latent heat. The instrument records the differential energy required as a measure of the thermal properties of the sample. The major physical transitions detectable in a DSC are illustrated in the figure (i) Melting. Heat is absorbed by the sample (i.e., endothermic process) as the sample melts leading to a peak. (ii) Crystallization. Heat is released by the sample as it crystallizes (i.e., exothermic process) as the sample crystallizes leading to a peak. (iii) Glass transition. The glassy and rubbery states have different specific heats (i.e., require different amounts of energy to heat the same mass of material) and so tinstrument baseline will be higher for one than the other. There is a second-order transition between a high and low value on heating as the sample goes through a glass transition (i.e., a sigmoidal function). All of the meatransitions occur over a finite time and hence, because we are changing temperature constantly, a finite temperature range. We often describe peaks by an onset or maximum temperatures and glass transitions as the temperature when the heat flux is midpoint between the high and low temperature plateaus.

he

surable

as

Texture analysis is a set of methods used to characterize the rheological properties of (typically) solids. The TA-XT2 Texture Analyzer (http://www.stablemicrosystems.com) has a robot arm that can be programmed to move up or down at a fixed rate and a force transducer that measures how hard it hto push to move. The output is a force vs. distance or time plot. Different attachments can be fitted to the armso it can push or pull at food in different ways and createa characteristic force/deformation curve. In most cases

23

the results are very empirical (i.e., depending on the instrumental configuration) and arein some way designed to simulate actual use of the food. It is possible to make a fundamental measurement of food texture but to do this it is necessary to know not only the force acting but also the dimensions of the food the force acts upon so it is important to strictly control the shape of the sample. Whatever the shape of the force/deformation curve it is often useful to decide on some characteristic and feature from the curve (e.g., force to fracture, initial slope, change in shape after compression).

Color. To understand color measurement it is useful to first consider human perception of color. Light entering our eyes usually comes from a reflection of sun or artificial light from the surface of whatever we look at. The color of the light entering our eyes will then depend on the color of the ambient light, and the efficiency that the surface reflects each of these colors. That light is focused onto three types of light-sensitive nerve cells, cones, in our eyes each sensitive to a different set of wavelengths. A colored light contains different intensities of different frequencies and these will stimulate all of the optically sensitive nerves. If the nerve cell is particularly sensitive to the wavelengths in the light it will be more stimulated. For example, the nervous signal gained by the brain might therefore be something like: Type 1 cones = strong response, Type 2 cones = less strong response, Type 3 cones = very strong response. For each color the eye will receive three independent pieces of information and transmit them to the brain. The brain interprets the precise balance of these three responses as a color perception. Because our sensory response to color is based on three pieces of information about the light we can say that color is a three dimensional variable. (One consequence of our relatively simple color vision is that it is possible to generate a huge variety of colors on a TV screen using just three types of colored pixel. The three primary colors are mixed to different degrees to stimulate our three types of cone cells in different ways).

A colorimeter exposes the sample to a pulse of light designed to simulate normal daylight (or artificial light). The spectrum of the reflected light is measured (just as you measured the spectrum of light transmitted through Gatorade samples in the last experiment). The spectrum measured is characteristic of the way the sample reflects different wavelengths (colors) of light and therefore contains similar information to the human perception of color. However a spectrum is hard to interpret in terms of a human perception of color, we need to simplify the spectrum and interpret it as a three-dimensional variable just as our brains and eyes work to perceive color.

24

Converting a complex spectrum to three parameters is a data reduction operation. There are various methods to make this transformation but we will use the Lab conventions. The complex spectrum is reduced to three numbers (L, a, and b). These three numbers represent the position of our color in three-dimensional space: L= light vs dark, a= red vs green, b= yellow vs. blue.

Bibliography Fennema OR. Water and ice. In: Fennema OR, ed. Food chemistry. New York: Marcel Dekker, Inc., 1996, 17-94. Hartel R. Crystallization in foods. Gaithersburg: Aspen, 2001. Any Physical Chemistry text on colligative properties

25

Exercise 3: Procedure

(i) The TAs will weigh the following ingredients into a stirred candy cooker and cook at heat setting 5.

Ingredient Per 1000

g batch /g Water 82 Sucrose 40.6 Corn syrup 15.9 Sweetened, condensed milk 23.5 Unsalted butter 11.5 Salt 0.3

(ii) Once the mixture is homogeneous, record temperature as a function of time.

Make notes describing the changing appearance and behavior of the sample as it cooks.

(iii) Remove five samples at different temperatures between 105-125°C at the

instructor’s recommendation (collect two samples at each temperature). At each temperature carefully remove a ladle of sample (~20 g) and pour slowly into a numbered aluminum weighing boat. Note the time of sampling and photograph each sample being poured into the mold. Collect a smaller sample (a few grams residual on the ladle for thermal analysis). Chill the samples until required for analysis (next week). All samples will be identified by the temperature at which they were taken.

Following week

(iv) Measure the color of each candy sample using the Lab colorimeter. Record

the L, a, and b values in a table along with a brief (2-5 word) description of the appearance of the sample. Photograph the samples.

(v) Measure the texture of the samples using a TA-XT2 Texture analyzer. Note

what is happening to the sample (e.g., bending, cracking) as it is being compressed. (TA-XT2 protocol to be provided). Also measure the texture profile of a soft caramel at low temperatures. Example TA-XT2 settings: Mode: Force in Compression, Option: Return to Start, Pre-test Speed: 1mm/sec, Test speed: 0.5 mm/sec, Post-test speed: 10 mm/sec, Distance: 2mm, Trigger Type: Auto, 20 g. Data Acquisition: 50 pps

(vi) Data presentation.

• Prepare the following Figures and Tables for your lab report.

• Table 1 will describe mix composition (use the data provided for ingredients above). Add an extra column labeled “Ingredient

26

functionality” and use it to briefly (1 sentence) describe the role that ingredient plays in facilitating manufacture or in the finished product.

• Table 2 will list the temperatures samples were taken, a description of the appearance of the hot (liquid) and cold (solid) sample and the L, a, and b values.

• Figure 1 is a time-temperature plot with the data taken during cooking. • Figure 2 (optional) is a compilation of the photographs of hot and cold

samples • Figure 3 is a compilation of the force/distance texture analysis data for all

of the samples. Plot all data on the same set of axes and clearly identify the temperature each sample was taken. In the text describe how the different shapes of the curves

• Figure 4 is a scatter plot with the x-axis the temperature the sample was taken and the apparent elasticity3 of the sample on the y-axis.

(vii) In your methods describe the precise experimental conditions used in the

texture analysis measurements. (viii) In your results and discussion describe your data and comment on the physical

and chemical reactions leading to the results you see. In particular include in your text: Why does temperature change with cooking time? Why does the sample go brown? What do the shapes of the texture analysis curves reveal about the sample properties? How did the texture of the soft caramel change on cooling and why? How can you explain your results in terms of glass transition theory?

(ix) Explain your results in reference to an annotated state diagram (include as

Figure 5). Grading

• Introduction Set up background (1), Describe goals (1), Cite literature (1) • Methods and Materials (1) Must include conditions of TA-XT2 operation. • Data Presentation (8). Deduct 0.2 points for each error in: title, use of the

correct data, spacing of the axes, units, axis labels, clear symbols and lines, overall legibility. Deduct 0.2 points for incorrect ingredient functionality in the Table or for missing the main purpose of the ingredient.

• Results and Discussion Introduce all Figures and Tables (1), describe the data (1), be critical of data (1), Why does temp increase with time? (0.5), Why does it go brown? (0.5), Critical assessment of shape of texture curves (0.5), Effect of cooling on a soft candy (0.5), Explanation of process using a state diagram (2.0)

• Overall format (1)

3 The slope of the initial rise in force with distance.

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Exercise 3: Pre-Lab Questions

1. Sucrose is not a reducing sugar. So how does it (indirectly) take part in the Maillard reaction? (2 points)

2. What is the approximate moisture content of a hard candy? (2 points)

3. Do you think the glass transition in the low-boiling candy will be higher or lower than the glass transition in the high boiling candy? (2 points)

4. On the following graph sketch the force-deformation curves expected on forcing a sharp cone probe into a hard and soft sample. (4 points)

force

deformation

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Exercise 4: Water Activity and Food Texture In this lab we will explore the relationships between the moisture content of the environment and the moisture content and texture of the food. A piece of food will gain or lose moisture to the environment but not necessarily in a linear manner. At a give level of atmospheric moisture one food may bind more or less water than another. The relationship between food water content and atmospheric moisture content is given by a moisture sorption isotherm. We can measure moisture sorption by the mass gained by a piece of dry food on coming to equilibrium with a known humidity. The easiest way to make a known humidity is to use a saturated salt solution. Any solution has some tendency to bind water and thus there is an equilibrium moisture content above it (the water activity aw). Typically the more concentrated a solution, the lower the equilibrium moisture above it. If the moisture is constantly removed, for example leaving a pan of salt water out on a sunny windy day, the solution will dry out. If the solution is left in a moist environment it will gain water until it has dried out the environment and come to equilibrium again. We use a supersaturated solution (i.e., with crystals left at the bottom) so that when it absorbs moisture from the environment, more crystals dissolve but the solution concentration remains the same. Similarly if the solution is dried by the environment more crystals form but the solution concentration remains the same. Some examples of the water activities provided by different saturated solutions at 25°C are given below: Salt: Drierite LiCl K-acetate MgCl2 KCO3 NaBr NaCl KCl aw: <0.01 0.112 0.227 0.328 0.432 0.576 0.753 0.843 So if we put a piece of food in a container with (not in) a ssolution, the food will gain or lose moisture until it is in equilibrium wthe solution. If we start with a dry piece of food, the mass gain is the moisture gain and we can calculate thequilibrium moisture content of thefood. Different solutions have different moisture binding capacitiso by repeating the experiment with different solutions we can generate a moisture sorption isotherm. A typisotherm for a polymeric food is sigmoidal and can be divided into three zones. Zone 1: Often >95% of the water, very loosely bound. Zone 2: More tightly bound but gradually removed as the food is dried. Zone 3: Very tightly bound water.

0

10

20

30

40

50

0.0 0.2 0.4 0.6 0.8 1.0

Aw

Moi

stur

e

Zone 1 Zone 2 Zone 3

upersaturated

ith

e

es

ical

We will mainly be dealing with dry and semi-dry foods where the bulk of the water is already missing (e.g., dry fruits, most powders, pasta, hard candies, cookies) so typically only Zone 1 and Zone 2 water will be present. (What would happen if you left any of

29

these foods in a very moist environment?) For these foods very often, big changes in behavior occur at the changeover between Zone 1/Zone 2. If the water content less than that, the food is stably and crunchy. If the water content is greater than that the food becomes progressively soft and unstable. We can calculate the monolayer value (the junction point) by fitting the BET model to a measured moisture sorption isotherm.

Mk k a

a a k aw

w w

=− − +

0 1

11 1( )( w )

where M is the water content of the food and k0 and k1 are constants. The monolayer value is given by k0. Analytical Methods Water Activity Measurement. Water activity could be measured by placing samples in equilibrium with a number of salt solutions and calculating the one where there is no mass change. This is too laborious and slow to be useful in practice, so instead various methods exist to measure the moisture content of the air in equilibrium with the sample. The Decagon water activity meter used in this lab brings the sample into a small chamber where the headspace is in contact with a mirror. The mirror is chilled and eventually reaches the dew point of the air (i.e., the temperature when water starts to condense on surfaces). The mirror “fogs up” and the temperature at which this occurs is measured and the relative humidity of the gas (and hence the food) calculated automatically. References:

• Ted Labuza’s website: http://fscn.che.umn.edu/Ted_Labuza/Pages_Folder/aw.html

• Decagon website: http://www.wateractivity.com/ • Fennema OR. Water and ice. In: Fennema OR, ed. Food chemistry. New York:

Marcel Dekker, Inc., 1996, 17-94. • Hartel R. Crystallization in foods. Gaithersburg: Aspen, 2001.

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Exercise 4 Procedure

Materials Apparatus • Various dry baked goods (e.g., breakfast

cereal, crackers, rice cakes, cookies). • Similar samples previously freeze-dried

to zero moisture and are sealed into plastic bags prior to use.

• Set of 6 dessicators containing saturated salt solutions for maintaining known water activity.

• Texture analyzer with a range of sample holders

• Water activity meter and cups.

i. With the assistance of an instructor, measure the water activity of each sample using the Decagon aw meter and record the results in a table along with a description of the texture of the product.

ii. Prepare 6 dessicators containing a saturated solution of the water-activity modifying salt4 and a support for the samples.

iii. Select one product per group and weigh pieces of product (4 figure precision) onto pieces of labeled aluminum foil and carefully place into the desiccators. Three samples per dessicator.

iv. Store for approximately 2 weeks to allow the samples to come to moisture equilibrium. (Be careful to keep the jars somewhere safe where they won’t be knocked over.

2 weeks later

v. Reweigh each sample. Because the samples will gain or lose moisture rapidly in

contact with the lab atmosphere, you will need to move quickly. When you are finished, return the samples to the dessicator as quickly as possible. Calculate moisture content on a dry weight basis (i.e., change in mass over initial mass). Note that you will need the mass of the foil from two weeks ago to make your calculation.

vi. Use the TA-XT2 to measure the texture of the products. (Work with an instructor to use the texture analyzer). Rather than use a standard protocol you are required to use the fittings available for the instrument to design a test to measure crispness of the product. Practice with commercial samples before using your relatively few experimental samples. When you are ready to start your real measurements you will again need to work quickly when the samples are removed from the dessicator. With the help of an instructor identify a characteristic feature of the curve (e.g., force to fracture, deformation to fracture etc.) that you will report as your measure of crispness.

vii. Data Presentation. • Table 1 is a description of each product and a measurement of its water

activity.

4 Caution: Some of these salts may be toxic. Your instructors will inform you of any unusual risks.

31

• Figure 1 is a sketch of the texture analysis method you developed. Show the size of the sample and the way the probe was designed to interact with it. List in the Figure legend all relevant test conditions (speeds, times, distances etc.).

• Figure 2 is force-deformation graphs for each sample. It is only necessary to plot a single texture curve from each water activity; pick representative data. If possible plot all samples on the same axis, if not separate them as Figure 2a, Figure 2b etc. Identify on your Figures the characteristic crispness feature you have selected.

• Figure 3 is a moisture sorption isotherm for your sample. Include error bars equal to one standard deviation from your triplicate measurements at each water activity.

• Plot the characteristic texture feature for crispness for each sample on a secondary y-axis alongside the sorption data in Figure 3. Include error bars equal to one standard deviation from your triplicate measurements at each water activity.

viii. Describe your method for texture analysis in the methods and materials. Also

include a rationale for selecting your measure of crispness from the texture profile.

ix. Relate the moisture sorption isotherm to the food texture data.

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Exercise 4 Pre-Lab Questions

1. What is the typical aw for a cracker? (2 points)

2. What other methods are used to measure the moisture content of air in commercial water activity meters? (2 points)

3. Draw a labeled diagram for a test-configuration using the TA-XT2 to measure the crispness of a cracker. (2 points)

4. Sketch the expected force-deformation curves using the test you suggest in Figure 4 for a crispy product stored at very low and a similar product stored at very high water activities. (4 points)

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Exercise 5: Protein Functionality The goal of this experiment is to demonstrate some of the functional roles of proteins in foods and how these can be modified through ingredient interactions. Proteins are important functional ingredients in foods. We depend on them to form gels and to stabilize emulsions, foams and films. In the present work we will investigate some of the structures that can be made from whey protein isolate (WPI). Whey protein is a mixture of globular proteins found in milk. They are a by-product of cheese manufacture and widely sold and used as a food ingredient. Whey protein is a mixture of globular proteins each with established primary, secondary, tertiary and quaternary structures maintained by non-covalent interactions. We are concerned with the inter-protein interactions that will hold the molecule in a given conformation and intra-protein interactions that may lead to aggregation. The most important of these here are:

• Hydrophobic interactions. Hydrophobic amino acids will try to avoid water by either coiling into the core of the polymer or adsorbing into a non-polar solvent.

• Electrostatic interactions. Proteins have ionizable amino acids that may carry a positive or negative charge depending on the pH. Like charges repel.

We can adjust the magnitude of the electrostatic interactions by titrating the protein through its isoelectric point and the hydrophobic interactions by denaturing the protein. Globular proteins can be thermally denatured by heating above a characteristic temperature. The globule unfolds and exposed some of the hydrophobic core amino acids to the aqueous solvent. If the structure does not regenerate, there will be a pressure to aggregate to reduce the hydrophobic interaction. Based on these simple rules of protein behavior we can try to understand the functional properties of whey proteins in film. Firstly to form a solid gel, a protein must aggregate and form a continuous (i.e., percolating) structure throughout the container. This can be seen as a partial precipitation of the protein because there are strong protein-protein interactions yet not so strong that they exclude interaction with solvent.

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Figure: (a) Soluble protein molecules (~ 2 nm) can (b) weakly associate and trap water in a gel, or (c) strongly aggregate and exclude water.

Figure: Electron micrograph of a WPI gel. Note the similarity to Figure (c) left.

Many proteins can stabilize emulsions because their hydrophobic amino acids partition into the surface of the freshly formed oil droplet (diameter typically~1 µm) and cause the protein to stick at the oil-water interface (i.e., adsorb). The adsorbed layer gives some stability by shielding the oil from the aqueous phase but the proteins now to a great extent control the functional properties of the system. If the protein tends to aggregate (e.g., following thermal denaturation or in the absence of strong electrostatic repulsion), the droplets they are attached to will aggregate also. Aggregation of the droplets in an emulsion can lead to gelation, but can also lead to creaming because the flocculated droplets are much larger. Proteins can also be dried to form flexible films in the presence of small hydrophilic molecules known as plasticizers. Film functionality is believed to be related to the ability of the plasticizer to reduce the glass transition temperature of the film and allow it to be flexible at room temperature. Edible films can be used to protect a food item from moisture or oxygen by restricting the diffusion of gas to the surface. One example might be coating nuts to reduce the amount of oxygen with access to the readily oxidizable lipids and thereby delaying rancidity. Another example would be coating a frozen pizza crust to prevent moisture diffusing from the tomato sauce and causing it to go soggy. Exercise 5 Procedure and Prelab: To be provided in a later handout.

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Exercise 6: Starch

In this lab we will investigate the effects of temperature and shear on the structure and functional properties of starch slurries. Starch is largely a mixture of amylose and amylopectin molecules. Both of these are glucose polymers with α1-4 linkages but amylopectin also has α1-6 branch points and is a much larger molecule. Plants use starch as a long-term energy store by locking the amylose and amylopectin into semicrystalline granules. A cross section of a starch granule and the arrangement of amylopectin within it are shown in the Figure. The location of the amylose is unknown but presumed to be in the amorphous region.

A starch granule will gelatinize if heated in the presence of water. The heat and water increases the molecular mobility of the amorphous regions (i.e., cause a glass to rubbery transition) and the added freedom allows the crystalline region to melt. The granule looses crystal structure, gains water and expands to many times its original size. The swollen granules can often overlap one another leading to a great increase in solution viscosity. Excess heating or shear can rupture the weak swollen granules leading to a decrease in paste viscosity. On cooling there is a progressive realignment and recrystallization first of amylose and then the amylopectin molecules as double helices. Recrystallization acts as linking points in the polymer matrix and can lead to the formation of a solid gel.

36

Exercise 6: Procedure

Materials Apparatus • Potato starch

• DSC & stainless steel pans • Viscoamylograph • Optical microscope with

polarizing filters and digital camera

Calorimetry (demonstration) i. Prepare a 10% suspension of both starch samples in water. ii. Heat from 20°C to 100°C at 10°C min-1 in the DSC. Cool and repeat the heating

cycle. Viscoamylograph and Microscopy iii. Weigh 2.5 g of starch and add cold water for a total weight of 28 g. iv. Process using a standard heating/shearing protocol in Dr. Seetharaman’s

laboratory and measure the changing temperature and viscosity over time. v. Remove samples (~0.5 ml) at intervals during processing for microscopic

investigation. vi. Visualize your samples using an optical microscope both under normal

illumination and polarized light and save representative microscopy images. Water holding capacity vii. Weigh 5 g of starch into a graduated cylinder and make up to 100 ml. Mix by

gently inverting 6 times viii. Allow the starch to settle for 1 hr and measure the height of the starch layer. ix. Repeat the experiment but first bring the 5 g of starch to a boil in 75 ml of water

in a beaker. Measure the temperature as the sample heats up. Estimate and report the temperature of starch gelatinization. How did you identify the onset of gelatinization? Cool the starch sample and transfer to a graduated cylinder for water holding capacity measurement. Use additional cold water to wash the residual starch from the boiling beaker into the cylinder and make up the volume to 100 ml.

Gelation properties x. Weigh 0.1, 0.5, 1, 2, 3, and 10 g of starch into labeled test tubes and make up to

20 g with water. (It may be necessary to adjust the absolute amounts depending on your test tubes but maintain the starch/water ratio). Cap the tubes with aluminum foil and shake well to mix.

xi. Place into a boiling waterbath for 10 min then cool in ice water. Describe the appearance and texture of the samples.

37

xii. Data Presentation a. Figure 1 is a viscosity vs temp/time plot from the viscoamylograph. Mark

points on the graph where samples were taken for microscopy (a-e). b. Figure 2a-e is a summary of the photomicrographs for potato starch. c. Table 1 is a description of the water holding capacity of the two starch

samples. d. Table 2 is a description of the gels formed by different concentrations of

potato starch.

xiii. The goal of your discussion is to explain functional properties of starch (Table 1 and 2, Figure 1) in terms of the changing structure on heating (Figures 1 and 3). In this case, I would suggest keeping the results and discussion sections separate. Start your discussion using references to text books and other sources to explain what Figures 1 and 2 in turn tell us about the changing properties of the starch granule (e.g., Figure 1 will show the granule melts at a given temperature, and Figure 3 the microstructures that cause the physical properties). How do these observations together help explain what is seen in Figure 2? Compare the gelatinization temperatures identified by the various techniques – are they the same? Do you expect them to be? Why? How does our improved understanding of starch behavior allow us to explain the differences seen in Tables 1 and 2? In this exercise I am particularly looking to reward your capacity to integrate information from a number of observations to tell one overall story.

38

Exercise 6 Pre-Lab Questions 1) What is the typical size of a native potato starch granule?

(2 points)

2) Identify a major US supplier of starch. (2 points)

3) Sketch the expected viscosity-temperature graph from the viscoamylograph. (4 points)

4) What is stabilized starch? (2 points)

39

Exercise 7: Nonenzymatic Browning

There are various reactions by which foods can go brown: • Enzymatic browning. The action of the enzyme polyphenoloxidase on phenols

and oxygen in plant foods to form brown colors. It only occurs in fresh cut plants where the enzyme is still active and has access to its substrates. Polyphenoloxidase requires copper as a co-factor, so copper chelators can limit enzymatic browning.

• Non enzymatic browning. There are three types:

o Maillard browning occurs between reducing sugars (i.e., those with a free aldehyde group) and amines at high temperatures. Leads to the formation of brown colors, meaty or caramel-like flavors as well as the loss of amino acids and the formation of potentially carcinogenic compounds.

o Caramelization. The reactions of sugars at very high temperatures (typically higher than Maillard). Formation of brown colors and buttery to bitter flavors.

o Lipid Browning. The polymerization of lipids after extensive heating (e.g., in old frying oils).

In this experiment we will be concerned with the Maillard reaction in foods and we will examine the ways different ingredients and processing conditions can favor and inhibit the reaction and the sensory consequences for the product. The first step of the Maillard reaction is the nucleophilic addition of an amine to an aldehyde on a reducing sugar. Any amine can participate but the side chain amine of lysine is particularly aggressive. The amine is eliminated and the products rearrange to form a 3-deoxyhexosulose (DH). DH can in turn breaks down to form hydroxymethylfurfural (HMF, sweet/buttery flavor note). See Figures 22 and 23 on p 172 of the Fennema text. Several dicarbonyl intermediates of this reaction (in particular DH) are highly reactive towards other amines (again especially lysine). The protein breaks down via the Strecker degradation (see below) to form (amongst other things) ammonia and an aldehyde based on the side chain of the reactive amino acid. Aldehydes are often powerful flavor volatiles that are sometimes responsible for nutty and meaty flavor notes.

NH2H

RCOOH

H O

NH R

COOH

H O

O

H O

OHR

O

H

CO2NH3

-H2O -H2O

-H2O

DH amineStecker aldehyde

The HMF and other products of the Maillard reaction can polymerize to form very large and ill-defined complexes. As the complexes grow they become darker brown and increasingly insoluble.

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The Maillard reaction can be inhibited by keeping the temperature low, reducing the accessibility of reagents or by adding sulfite ions (also known, inaccurately as sulfur dioxide). Sulfite ions are strong nucleophiles that can add to aldehyde groups to form an adduct that does not take part in browning. The mechanism of action of a sulfite is believed to be as follows, the browning intermediate DH reversibly binds a sulfite ion to form a sulfonated adduct. The product (DSH) is not capable of browning.

H O

O

HO

O

SO3-

Browning color and flavor

DH DSH

Because sulfite ion is readily volatile as sulfur dioxide gas and sulfur dioxide gas can cause attacks in a proportion of asthmatics, the additive has been prohibited by the FDA in foods that are meant to be eaten raw. Sulfites are allowed to a defined limit in some cooked foods because the additive escapes as gas during the cooking process. There is no widely accepted replacement for sulfites in foods although deprotonated thiols (R-S-) are capable of acting as nucleophiles in a similar manner to sulfite and, if they react suitably with DH, may form the basis for an anti-Maillard browning additive if flavor problems can be overcome.

Bibliography

“Food Chemistry” ed. O.R. Fennema (1996): Sections 4.1.4.6, 6.7.1.6, and 11.7.1.

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Exercise 7: Procedure

Materials Apparatus Glucose, glycine and lysine solutions (all 0.25 M prepared in 50 mM phosphate buffer, pH 8) Whey protein isolate (2 wt% in water raised to

pH 8 with a small volume of NaOH) Hydrolyzed WPI (2 wt% in water, pH dropped to 3 with a small volume of HCl, heated to 90°C for

30 min, cooled, pH raised to 8 with a small volume of NaOH)

Sugar cookie dough (4 tubes)

Spectrophotometer and cuvettes Hot plate & boiling water bath

Oven set at 200°C Baking trays Filter paper

Foil Frying pan and oil

Small paint brushes

Liquid Model System Browning i. Add 2 ml of glucose solution to a spectrophotometer tube and mix with 2 ml of

the different amine sources. Cap the tubes. ii. Prepare a boiling water bath by bringing a beaker of water to a boil on a hotplate

(add a few boiling chips to prevent knocking). iii. Heat the tubes, then at intervals (0, 10, 20, 40, 80, 100 min) remove from the bath,

dry, and quickly measure the absorbance (at 450 nm). iv. At the end of the experiment remove the caps and describe the aroma. Compare

the smell of the product with the smell of the starting compounds. Solid Model System Browning v. Mark two points on a piece of filter paper about 2 cm apart. vi. Apply one drop of glucose to one point and one drop of

each of the amino acid or protein solutions to the other. The liquid circles should expand and partly overlap.

vii. Fry the paper for a few seconds viii. Photocopy or photograph your papers onto a single

sheet and include it in your results. Browning of Baked Goods ix. Shape the dough into 25 g cookies as described on the packaging and arrange on

parchment paper on a cookie sheet. x. Use a glass rod to press two small lines

into the cookie surface to divide it into quarters (see figure)

xi. Paint half the cookie with amine solution and half with glucose as shown. Leave 5 min between paintings to let the surface dry. Try to distribute the solutions as evenly as possible. The idea is one quarter of the cookie gets glucose and amine solution, one just glucose, one just amine solution and one nothing. Try to

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distribute 3 drops of each solution on each quarter of the cookie. Mark the parchment paper under the cookie so you can identify which part has received which treatment. Prepare two cookies for each protein preparation.

xii. Bake each set of cookies together for 10 min and describe the color and aroma of the samples.

xiii. Data Reporting

a. Figure 1 is a plot of absorbance vs. time for the model systems. b. Table 1 is a summary of the aromas of the model systems and the

unheated reagents. c. Figure 2 is a copy of the browned filter papers. Use labels to identify the

reagents used in each case and describe your observations in the text d. Table 2 is a description of the cookies.

xiv. Discussion

• In the model systems (solutions and paper), which samples browned the fastest and why? Which samples did not go brown and why? Why did the paper brown so rapidly compared to the solutions? Did protein hydrolysis make any difference? Why?

• In this write up I am particularly looking for an integration of ideas across experimental models. What caused any significant differences between the model and real systems? In particular are there other ingredients in the cookie that could take part in the reactions or is the heating different in some way?

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Exercise 7 Pre-Lab Questions

1) Why are we conducting the model system experiment in pH 8 buffer rather than a more typical food pH (3-7)?

(2 points)

2) Which amino acid do you expect to react fastest? (2 points)

3) Draw the structure of the aldehyde formed from lysine via the Strecker degradation

(4 points)

4) Do you think the hydrolyzed protein will brown faster or slower than an intact

protein? Why? (2 points)

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Exercise 8: Lipid Oxidation

To be provided in a later handout.

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Exercise 9: Flavor Chemistry Lab5

The sensory response of flavor is clearly crucial to food product success but the molecular basis of flavors is extremely complex. Food flavor is a combination of a taste response (sweet, sour, bitter, salty) in the mouth along with a more complex aroma response in the nose. Aroma compounds are volatile. They have low vapor pressure, so when they are in the heated environment in your mouth, they volatize and travel up the retronasal passages to reach the olfactory receptors in the nose. Those compounds that stimulate a response from these receptors are odor active compounds. A given molecule may have a characteristic smell in solution and a given mixture of molecules may together provide a good analog for a real flavor. (Although there is huge variation between individuals both within a culture and between cultures.) However, once the relative concentration of the molecules changes, the strength and character of the flavor may also change. Only a proportion of flavors in a food will partition into the headspace gasses. This is given by the partition coefficient, Kp =[vapor phase]/[food]. A flavor with more affinity for the food will be less released into the headspace and therefore not perceived. On the other hand flavor released into the headspace prior to consumption will likely be lost and wasted. Changes in the food matrix can affect the partition coefficient (e.g., a lipophilic flavor will be better bound by a fatty food) as can changes in a flavor molecule (e.g., if a change in pH causes a flavor molecule to become charged – it may become more soluble in the aqueous phase of a food and so less released into the headspace. Consequently when we formulate a food flavor we must be careful that it actually works in our intended system. As one food scientist notes…

Traditionally, in the food industry, we develop products pretty much in this protocol - in some ivory tower someplace, the marketing people say, "This is the product we want; this is our product concept," and almost simultaneously they choose a flavor or flavors that they would like to sell this in. When the product is developed … we move to a prototype sensory loop in which when we develop a prototype, we take a sensory test, take it back to prototype because they never go through the first time, and this can loop thousands of times before you get the final product that you want. The drawback to this system is that you are trying to put a square peg into a round hole. You have decided the flavor early on, and it may not be the ideal flavor to use for this kind of product.6

The goal of this lab is to introduce you to some of the problems involved in flavor application. There are many reasons why flavors can change or not turn out the way you might want them. In this lab we will show examples of chemical changes in flavor compounds, flavor-base interactions and changes in flavor due to processing. The structure of the lab will differ from the previous labs in that most of the data will come from class observations made as you taste and smell the experiments. There is no write up required but there are some pre-lab questions. 5 based on a lab developed by Melis Cakirer, 2002 6 Robert Nelson. Senior Food Technologist, Flavors of North America. http://www.talksoy.com/health/t98Symposium4.htm

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Divide your group so 1 or 2 people are involved in the sample preparation for parts a, b, and c.

Exercise 9a: Effect of pH on Aroma Compounds The chemical structure of aroma compounds canchange depending on the pH of their environment; gaining or loosing protons according to their pK. The following demonstration shows how the aroma is either lost or changed depending on the pH of the environment and presents a practical application of this phenomenon.

Exercise 9a: Procedure

Materials Apparatus One fresh onion Onion extract Vanilla extract

Ground clove spice 0.1 M NaOH solution 0.1 M HCl solution

dH2O

Spectrophotometer tubes cloth

Sample preparation i. Add 1 ml of vanilla extract to a spectrophotometer tubes and mix with 1 ml of

NaOH solution, HCl solution or dH2O. Cap the tubes and mix well. ii. Make one set for each person in your group (3 tubes per person). iii. Add approximately 1g of clove spice to a spectrophotometer tube and mix with 1

ml of NaOH solution, HCl solution or dH2O. Cap the tubes and mix well. iv. Make one set for each person in your group (3 tubes for each person). v. Cut one fresh onion in half and rub the cut surface on a piece of cloth until a good

amount of the aroma has been transferred to the cloth evenly.

vi. Cut the cloth into 3 equal amounts and place each one in the bottom of a 250 ml beaker. Add enough of NaOH solution, HCl solution or dH2O to just cover the cloth.

vii. Cover the beakers with parafilm and allow it to sit for 10 minutes, gently agitating the solution to let it come in contact with all surfaces of the cloth.

viii. Bring all 3 demonstrations to room 202 and use them to set up tasting/smelling stations for all the members of your group.

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Exercise 9b: Flavor interactions with a Lipid Base Aroma compounds can be are either hydrophobic or hydrophilic. The extent to which they dissolve in fat vs. aqueous phases is reflected by the partition coefficient. This partition coefficient is important in product development because solubility characteristics of aroma compounds have important implications for flavor release and perception. Water soluble compounds volatize quickly in the mouth leading to high impact flavors. Fat soluble compounds are released more slowly from the matrix.

Exercise 9b: Procedure

Materials Apparatus Vanilla extract

Skim milk Whole milk Half & Half

Sugar

Sample cups

i. Delegate one member of your group to make milk samples in room 202. ii. In room 202, prepare 3 vanilla flavored milk samples, each containing 3c milk, 3 t

sugar, 3t vanilla extract. iii. Decide on a 3 number code to identify each of the different milk batches. Write

down your code system. iv. Distribute samples into sample cups for the entire class and label with the code

identifying the sample. Cap the sample cups and place at tasting stations. v. Make and record observations on these samples along with 2 commercial samples

as a class.

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Exercise 9c: Effect of Heating Method on Flavored Cake

Adding flavors to baked products is often a challenge since during the baking process, aroma compounds volatize and are lost to the environment. These losses may be compensated for by adding more flavor to the mix, realizing that some of the flavor will be lost during baking. Another way is to use a flavor which has been prepared in a non-volatile solvent. The idea is that lipid-based flavors should interact with the oil/fat phase of the food and be more reluctant to volatize. This laboratory demonstrates the effect of flavor solvent and type of baking on the perception of artificial lemon flavor in white cake mix. In lab, you will observe the flavor retention in 4 different formulations:

a) Conventionally baked cake with water-soluble flavor at 0.3% (by weight) in batter b) Microwave baked cake with water-soluble flavor at 0.3% in batter c) Conventionally baked cake with oil-soluble flavor at 0.3% in batter d) Microwave baked cake with oil-soluble flavor at 0.3% in batter

Exercise 9c: Procedure

Materials Apparatus

Water soluble lemon flavor Oil soluble lemon flavor

Jiffy white cake mix Water

Egg white Vegetable oil

Glass cake pans Aluminum cake pans Paper serving plates

Mixer Mixing Bowls

i. Prepare 4 treatments of flavoring + cake mix, incorporating flavoring into the white cake mix according to the following formulation

Jiffy white cake mix 255 g Water 100 g Egg white 30 g vegetable oil 10 g Oil or water soluble flavor 1.20 g

ii. For the water soluble flavor, mix the flavor first with water. Combine with cake mix, oil, and egg whites. Mix on low speed until moistened. Mix on high speed for 2 minutes.

iii. For the oil-soluble flavor, mix the flavor first with vegetable oil. Combine with cake mix, water, and egg whites. Mix on low speed until moistened. Mix on high speed for 2 minutes.

iv. Bake in a greased 8" x 8" glass cake pan for 20 minutes at 350 F, or cook 6 minutes in microwave (1/4 turn after 3 minutes). Cool 15 minutes and remove from pan.

v. After cakes are done cooking, take pans into room 202. Cut cakes into individual samples for the class, place on labeled paper plates.

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Exercise 9: Sensory Analysis

After all the samples have been prepared and set up in Room 202, your instructor will lead the class in making collective observations. Please don’t sample foods until asked to do so. a. Effect of pH Sniff (don’t taste!) the samples when directed to and record your description of the aroma. Note which is the “best” Acid Base Neutral Vanillin

Eugenol

Onion extract

b. Effect of fat content (vanilla milk) When directed to, rate the milk samples in terms of the intensity and duration of the vanilla flavor

Sample number Intensity Duration Identity of sample

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c. Effect of processing (cake)

Smell then taste the four cake samples. Record your observations on lemon flavor intensity and the presence of other flavors.

Microwave baked Oven baked

Oil soluble flavor

Water soluble flavor

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Exercise 9 Pre-Lab Questions

1) Capsaicin (the hot flavor in hot peppers) is a fat-soluble compound. Your tongue is burning up from eating too much chili -- which do you reach for, water or milk? Why?

(4 points)

2) Madeitupalymate provides a characteristic aroma to a lemon soda. It’s known that the molecule gains a proton and becomes uncharged at round about pH 5. Do you think it will be more or less volatile at higher pH? Will the partition coefficient go up or down with pH?

(6 pounts)

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Project Work The aim of the project work is to provide some guided experience in the design and conduct of a food chemistry experiment. The project is designed to take up only a relatively small part of the semester but give enough space for you to demonstrate your creativity. Remember planning and organization will be crucial. The capacity to think creatively as a scientist during the design, conduct and analysis of your experiments will be rewarded as much as the quality of your data. Some suggested projects are as follows. You are encouraged to propose another study subject to approval by the instructors.

Gelation. Show the effects of calcium and alginate concentration on the strength of an alginate gel Lipid Oxidation. Survey fresh fish from local markets. Does the concentration of a selected chemical marker of lipid oxidation correlate with your group’s sensory ranking of fishy aroma? Baking. Blend a hard and a soft fat to make fats with different SFC. Show how using these different fats changes the measured functional properties of cookies. Protein Functionality. Show how a mineral acid marinade can change the mass, area and thickness of a steak. Consider as a variable the molarity of the selected acid. Chlorophyll Chemistry. Show how metal ions can change the color of green vegetables and the absorbance spectra of the chlorophyll after extraction. Carbonation. Measure the release kinetics of gas from a can of soda after it is opened.

(note – each group is expected to do a different project. If you want one of these then claim it on a first come, first served basis).

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Project Timeline • Before November 5th: Meet with instructors as a group to discuss the basic ideas

of your project.

• On November 19th: Demonstrate to the instructors your competence in preparing samples and making measurements. Use this opportunity to make sure your methodology works. Identify 3-5 pieces of literature that you will use in your project write up.

• On December 3rd: Make the measurements needed for your report.

• Before December 7th: Review your data (in table or graphical format with an

instructor.

• On December 10th: Make a brief (15 min) presentation to the class to include (i) The basic science behind your project, (ii) Your goals, (iii) Methods and Materials, (iv) Results, (iv) Discussion in terms of the basic science involved. Explain why you saw what you saw.

On December 10th: Hand in a group report on your work. Your report should be an extended lab report (~twice the length of a normal lab report) including more background information and a detailed Materials and Methods Section.

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