University of Puerto Rico

Mayagez Campus
Technology and Food Science Department

Food Chemistry Workshop

HIS Workshop 2012

TABLE OF CONTENTS

EXPERIMENT I: NUTRITIONAL LABELING USING A COMPUTER PROGRAM ....3

EXPERIMENT II: DETERMINATION OF WATER ACTIVITY (aw) USING THE DECAGON AQUALAB ......9 EXPERIMENT III: VITAMIN C DETERMINATION BY INDOPHENOL METHOD...12

EXPERIMENT IV: TITRATABLE ACIDITY DETERMINATION BY TITRATION METHOD.......17 EXPERIMENT V: ACIDS, PROTEINS, AND MAILLARD BROWNING..20 EXPERIMENT VI: PIGMENTS27

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EXPERIMENT I NUTRITIONAL LABELING USING A COMPUTER PROGRAM

INTRODUCTION The 1990 Nutrition labeling and Education Act mandated nutritional labeling of most foods. As a result, a large portion of food analysis is performed for nutritional labeling regulations and these are available on-line at http:// vm.cfsan.fda.gov/~dms/fgl-toc.html. However, interpretation of these regulations and the appropriate usage of rounding rules, available nutrient content claims, reference amounts, and serving size can be difficult. Additionally, during the product development process the effect of formulation changes on the nutritional label may be important. As an example, a small change in the amount of an ingredient may determine if a product can be labeled low fat. As a result, the ability to immediately approximate how a formulation changes will impact the nutritional label and can be valuable. In some cases, the opposite situation may occur and a concept called reverse engineering is used. In reverse engineering the information from the nutritional label is used to determine a formula for the product. Caution must be used during reverse engineering. In most cases, only an approximate formula can be obtained and additional information not provided by the nutritional label may be necessary. The use of nutrient databases and computer programs designed for preparing and analyzing nutritional labels can be valuable in all of the situations describe above. In this laboratory you will use a computer program to prepare a nutritional label from a product formula, determine how changes in the formula affect the nutritional label, and observe an example of reverse engineering.

OBJECTIVE Prepare a nutritional label for a yogurt formula, determine how formulation changes will affect the nutritional label

MATERIALS TechWizardTM Version 4: Formulation and Nutrition Labeling Software PC with Microsoft Excel 97 or Excel 2000.

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METHOD A: PREPARING NUTRITION LABELS FOR SAMPLE YOGURT FORMULAS PROCEDURE 1. Start TechWizardTM program and select Enter Program (click on Ok). Enter the Nutrition Labeling section of the program (Program will open on Formula Development, Look for labeling tab and select labeling section). 2. Enter the ingredients for formula #1 listed in Table 1-1. (Click on the add ingredient button, then select each ingredient from the ingredient list window and click on the Add button, click on close after all ingredients have been added.) Table 1-1 Ingredient Milk Skim milk Skim milk, Condense Sugar liquid Modified starch Stabilizer, emulsifier Formula #1 (%) Formula #2 (%) 38.201 48.201 35.706 25.706 12.888 12.888 11.905 11.905 0.800 0.800 0.500 0.500

3. Enter the percentage of each ingredient for formula #1 in the % (wt/wt) column-selecting the Sort button above the column will sort the ingredients by the % (wt/wt) in the formula. 4. Enter the serving size (common household unit and the equivalent metric quantity) and number of servings. (First click on the Serving Size button under Common Household Unit, enter 8 in the window, click on Ok, select oz from the units drop down list; next click on the Serving Size button under Equivalent Metric Quantity, enter 227 in the window, click on Ok, select g from the units drop down list; and finally click on the Number of Servings button, enter 1 in the window, click on Ok.) Note by clicking on the show ref. table button, a summary of the CFR 101.12 Table 2 Reference Amounts Customarily Consumed Per Eating Occasion will be displayed. 5. Enter a name and save formula #1. (Click on the Formula Name window, enter food analysis formula#1 and in the top of window click on the save icon. 6. View the nutritional label and select label options (click on the View Label button, select the label type you want to display-the standard, tubular, linear, or simplified format can be displayed). 7. Click label options and select the voluntaries nutrients you want to declare-you may want to select protein-show ADV since yogurt is high in protein; the daily value footnote and calories conversion chart will be displayed unless Hide Footnote and hide Calorie

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Conversion Chart are select; when you have finished selecting the label options select apply and then close to view the label). 8. Edit the ingredient declarations list (click on the View/Edit Declaration button, click Yes when asked-Do you wish to generate a formula declaration using individual ingredient declarations?-Each ingredient used in formula can be selected in the top window and the ingredient declaration can edited in the middle window). *Note the rules for ingredient declaration are found in the CFR 101.4 9. Print the nutrition label for formula #1 (click on the print label button, click print, to return to the label format menu, click on the close button). 10. Return to the Nutrition Info & Labeling section of the program (click on the return button). 11. Enter the percentage of each ingredient for formula #2 in the % (wt/wt) column. 12. Go to file, then save as, enter a name and save formula #2. 13. View and print the nutrition label for formula #2 (click on the View label button, click on the Print label button, then click on print;; to return to the label format menu, click on the close button).

METHOD B: ADDING NEW INGREDIENT TO A FORMULA AND DETERMINING HOW THEY INFLUENCE THE NUTRITION LABEL Sometimes it may be necessary to add additional ingredient to a formula. As an example, let us say you decided to add an additional source of calcium to yogurt formula #1. After contacting several suppliers you decided to add Fieldgate Natural Dairy Calcium 1000, a calcium phosphate product produce by First District Association (Litchfield, MN), to the yogurt formula. This product is a natural dairy-based whey mineral concentrate and contains 25% calcium. You want to determine how much Dairy Calcium 1000 you need to add to have 50% and 100% of the Daily Value (DV) of calcium in one serving of your yogurt. The composition of the Dairy Calcium 1000 you will add is show in Table 1-2.

PROCEDURE 1. Add the new ingredient to the database. (From the Edit Ingredient tab, select edit current files). 2. Enter the name of the new ingredient and its composition. (From the edit ingredients menu select Add ingredient, enter the ingredient name-Dairy calcium 1000, click Ok, enter the amount of each nutrient in the appropriate column, click on the Finish Edit button).

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3. Edit the ingredient declaration (what will appear on the ingredient list) for the new ingredient. (From the declaration tab, declaration section, add, choose ingredient, ok. Then select edit declaration, enters the ingredient declaration Whey Mineral Concentrate in the bottom Ingredient Declaration window, click Ok, and click on the close button). Table 1-2 Component Ash Calcium Calories Lactose Phosphorus Protein Sugars Total Carbohydrate Total Solids Water Amount 75% 25,000mg/100g 40 cal/100g 10% 13,000mg/100g 4.0% 10g/100g 10g/100g 92% 8.0%

4. Save the change to the ingredient file. (From the file menu select save ingredient file, return to the Formula Development section by selecting Close ingredient file from the file menu). 5. In the Formula Development section of the program, from the File menu select Open Formula and select food analysis formula #1, click on the Open button, click on Yes for each question). 6. Add the new Dairy Calcium 1000 ingredient to food analysis formula #1. (Click on the add ingredient button, then select Dairy Calcium 1000from the ingredient list, click on the Add button, click on the X to close the window). 7. Calculate the amount of calcium (mg/100g) required to meet 50% and 100% of DV (see example below).

Calcium required= (DV for calcium/serving size) * 100g * %DV require Calcium required for 50% of the DV= (1000mg/227g) * 100g * 0.50 Calcium required for 50% of the DV = 220mg/100g

8. Go to Form Dev and Batching, Formula Dev, Add Properties (Find calcium in the Properties column and enter 220 in the minimum and maximum columns for calcium-this lets the program know that you want to have 220mg of calcium per 100g). Enter the amount of calcium required in the formula except skim milk and Dairy Calcium 1000. In both the HIS Workshop 2012 6

Min and Max columns of the formula ingredients enter 38.201 for milk, 12.888 for condensed skim milk, 11.905 for sugar liquid, 0.800 for modified starch and 0.500 for stabilizer-this lets the program adjust the amount of skim milk and Dairy Calcium 1000 (calcium phosphate) and keeps the amount of all the other ingredients constant. Click on the Formulate button, click Ok). 9. Enter a name and save the modified formula. (Click on the Formula name window, enter food analysis formula#1 added calciumin the top formula name window, click on the X to close the window, select Save Formula from the file menu). 10. Transfer the new formula (with added calcium phosphate) to the Nutrition label section of the program. (From the Formula Dev menu select crearte Nutr Label Section, click yes for each of the question). 11. Make sure your have the correct serving size information. (See Method A, step 4). 12. View and print the nutrition label for the new formula for 50% of the calcium DV. (Click on the View label button, click on the Print label button, and then click on print; click on the return button to the Formula development Section). 13. Produce a formula and label that has 100% of the calcium DV. (Repeat Steps 7-12 except using the calculate amount of calcium required to meet 100% of the calcium DV-you will have to perform this calculation yourself by following the example in Step 7).

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Figure 1. Examples of Nutritional Labeling (Nutritional Fact)

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EXPERIMENT II DETERMINATION OF WATER ACTIVITY (aw) USING THE DECAGON AQUALAB

INTRODUCTION Water activity plays an important role in the quality, stability and preservation of food. According to the current Good Manufacturing practices- a Safe-moisture level is a level of moisture low enough to prevent the growth of undesirable microorganisms in the finished product under the intended conditions of manufacturing, storage, and distribution. The maximum safe moisture level for a food is based on its water activity (aw). An aw will be considered safe for a food if adequate data are available that demonstrate that the food at or below the given aw will not support the growth of undesirable microorganisms. Water activity plays also an important role to control the rate of chemical reactions such as maillard, enzymatic reaction and lipid oxidation. The Decagon AquaLab models CX-2 use the chilled-mirror dewpoint technique to measure the water activity of a product.

OBJECTIVE Determine the water activity of different food to determine if they are potentially hazardous food.

MATERIALS AquaLab Models CX-2 Reference humidity standards Disposable sample cups from Decagon or equivalent AquaLab cleaning kit (optional) Food Samples: Soy sauce, marmalade, crackers

PROCEDURE 1. Calibrate the instrument before sample measurements. 2. For analysis of food, use 6M NaCl, which has aw = 0.760 or choose a humidity standard with a value higher than 6M NaCl as long as it is of a lower value than the anticipated a w of the sample being measured. The aw reading obtained by measuring the Decagon Devices humidity standards must be within 0.003 aw units of the stated value of the standard. 3. Measure in duplicate the aw of 6M NaCl or of another chosen humidity standard

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4. If readings of the humidity standard are within the stated ranges, take duplicate readings of distilled water. If the readings for distilled water are within 1.000 0.003, the calibration is complete. Hint : Always start calibration / verification with the lowest aw slush. Samples should be left intact if possible to ensure that the aw is not altered. If necessary, cut the sample material to fit the sampling cup. Fill the plastic sample cups half full in such manner that most of the bottom of the cup is cover. Take care not to use your hands to directly handle the sample material. If needed, clean the exterior surfaces (sides and rim) of the cup to avoid contamination of the reading chamber. 5. Turn the sample drawer knob to the "Open/Load" position and open the sample drawer. Place the prepared sample cup in the drawer and slide the drawer closed being careful not to spill or splash liquid samples. 6. Turn the knob to the "Read" position to seal the sample cup with the chamber and start the read cycle. When the instrument begins to beep and/ or the decimal points or the LCD flash, the reading is complete. The display will show a final aw and temperature measurement. 7. Measure the aw of each sample in duplicate by taking 2 reading of the same sample cup. 8. Record sample aw reading and the temperature. Hint: To minimize reading time, always start the readings with the dryer food and finish with the most humid. 9. Report sample aw values using all three decimal places. Determine the mean of multiple readings using three decimal places, and then report the final mean value rounded to two decimal places.

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DATA AND RESULTS Table 1. Water activity and temperature measurement Trial 1 2
Ave. =

Aw

Temperature (C)

Soy sauce

Marmalade

1 2
Ave. =

Crackers

1 2
Ave. =

Figure 1. Water activity meter

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EXPERIMENT III VITAMIN C DETERMINATION BY INDOPHENOL METHOD

INTRODUCTION Vitamin C is an essential nutrient in the diet, but is easily reduce or destroyed by exposure to heat and oxygen during processing, packaging, and storage of food. The U.S. Food and Drug Administration require the vitamin C content to be listed on the nutrition label of foods. The instability of Vitamin C makes it more difficult to ensure an accurate listing of vitamin C content on the nutrition label. The official method of analysis for Vitamin C determination of juices is the 2,6-dichloroindophenol titrimetric method (AOAC Method 967.21). The principle of the method involves the oxidation of ascorbic acid and the reduction of the indicator dye to a colorless solution. At the endpoint of the titration ascorbic acid containing sample will react with the dye and it will turn colorless, when

there is a lack of ascorbic acid the excess of dye will fall in the acid solution and turn rose pink. The titer of the dye can be determined using a standard ascorbic acid solution. Food sample in solution then can be titrated to calculate the ascorbic acid content. While this method is not official for other types of food products, it is sometimes used as a rapid, quality control test for a variety of food products, rather than the more time-consuming microfluorometric method (AOAC Method 984.26). The procedure outline below is from AOAC Method 967.21.

OBJECTIVE Determine the Vitamin C content of various orange juice products using the indicator dye 2,6dichloroindophenol in a titration method.

MATERIALS Beaker, 150ml Beaker, 250ml 2 Bottles, glass, 200-250ml, one color amber and one clear, both with lids. Burette, 50 or 25ml 9 Erlenmeyer flasks, 50 or 125ml Fluted filter paper, 2 pieces Funnel, approximately. 6-9cm diameter 2 Glass stirring rods Graduated cylinder, 25ml 12

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Graduated cylinder, 100ml Pipette bulb Ring stand 3 Spatulas Volumetric flaks 50, 200 and 250ml 2 Volumetric pipettes, 2ml Volumetric pipette 5, 7 and 10ml Weighing boats or paper

PROCEDURE Standardization with dye 1. Pipette 5 ml metaphosphoric acid-acetic acid solution into each three 50ml Erlenmeyer flasks. 2. Add 2.0 ml ascorbic acid standard solution to each flask. 3. Using a funnel, fill the burette with the indophenol solution (dye) and record the initial burette reading. 4. Place the Erlenmeyer flask under the tip of the burette. Slowly add indophenol solution until a light but distinct rose-pink color persists for >5 s (takes about 15-17ml). Swirl the flask as you add the indophenol solution. 5. Note final burette reading and calculate the volume of dye used. 6. Repeat step 3-5 for the other two standard samples. Record the initial and final buret readings and calculate the volume of dye used for each sample. 7. Prepare blanks: Pipette 7.0ml metaphosphoric acid-acetic acid solution in each of three 50ml Erlenmeyer flasks. Add to each flask a volume of distillated water approximately equal to the volume of dye used above (i.e., average volume of dye used ti titrate three standard samples). 8. Titrate the blanks in the same way as step 3-5 above. Record the initial and final burette readings for each titration of the blank, and calculate the volume of dye used.

Analysis of juice sample 1. Pipette 5 ml metaphosphoric acid-acetic acid solution into each three 50ml Erlenmeyer flasks. 2. Place into each of three 50 ml Erlenmeyer flasks 5 ml metaphosphoric acid-acetic acid solution and 2 ml orange juice. HIS Workshop 2012 13

3. Titrate each sample with the indophenol dye solution (as you did in step 3-5 above) until a light but distinct rose-pink color persists for >5 s. 4. Record the initial and final burette readings and calculate the difference to determine the amount of dye used for each titration.

DATA, CALCULASTIONS AND RESULTS Table 1. 2,6-dicloroindophenol (dye) used during titration Trial Ascorbic acid 1 2 3 1 2 3 1 2 3 Burette start (ml) Burette end (ml) Vol. Titrant (ml)

Blank

Sample

Using the data obtained in standardization of the dye, calculate the titer using the following formula:

Titer = F = (mg ascorbic acid in volume of standard solution titrated)/(average ml dye used to titrated standards - average ml dye used to titrated blank)

**mg ascorbic acid in volume of standard solution titrated = (mg of ascorbic acid/50 ml) * 2 ml

Calculate the ascorbic acid content of juice sample in mg/ml, using the equation that follows and the volume of titrant for each of your replicates. Calculate the mean and standard deviation of the ascorbic acid content for your juice (mg/ml). Obtain from other lab members the mean ascorbic acid content (mg/ml) for other type of juice. Use these mean values of the juice sample to express the

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Vitamin C content of the juice sample as milligram ascorbic acid/100ml, and as milligram ascorbic acid/8fl. oz. (29.56 ml/fl. oz.).

mg ascorbic acid/ml = (X-B) * (F/E) * (V/Y) Where: X= average ml for sample titration B= average ml for sample blank titration F= titer of dye (= mg ascorbic acid equivalent to 1.0 ml indophenol standard solution) E= ml assayed (=2ml) V= volume of initial assay solution (= 7 ml) Y= volume of sample aliquot titrated (= 7 ml)

Table 2. Ascorbic acid (AA) content for replicates of orange juice sample Replicate 1 2 3 X= SD= mg AA/ml

Table 3. Summary of ascorbic acid content of orange juice sample Sample identity 1 2 3 4 mgAA/ml mgAA/100ml mgAA/8fl. oz

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Figure 1: Ascorbic acid Titration: 2,6-dicloroindophenol method

a. Titration set up

b. Titration end point demonstration

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EXPERIMENT IV TITRATABLE ACIDITY DETERMINATION BY TITRATION METHOD

INTRODUCTION The titratable acidity or total acidity of a food is determinate by acid-base titration to measure the total concentration of acids. This acids are mostly organic acids (citric, malic, lactic, tartaric), but phosphoric acid is an inorganic acid sometimes added to food. The organic acids present in foods influence the flavor, color, microbial stability and quality. The titratable acidity of fruits is used, along with sugar content, as an indicator of maturity. While organic acids may be naturally present in the food, they also may be formed by fermentation or added during formulation and processing. Increasing the acidity of foods, either through fermentation or the addition of weak acids, has been used as a preservation method since ancient times. Organic acids are more effective as preservatives in the undissociated state. There are two fundamentally different conventions for expressing acidity: titratable acidity and hydrogen ion concentration, or pH. The former expresses total acidity but does not measure the strengths of the acid, while pH indicates acid strength. pH is defined as the logarithm of the reciprocal of the hydrogen concentration. It may be also defined as the negative logarithm of the molar concentration of hydrogen ions. Thus, a [H3O+] concentration of 1 x 10-6 is expressed simply as pH 6. The [OH-] concentration is expressed as pOH and would be pOH 8 in this case. It is important to understand that pH and tritatable acidity are not the same. To determine the titratable acidity, a known volume (or weight) of a food sample is titrated with a standard base, to either a pH or phenolphthalein endpoint. The volume of titrant used, along with the normality of the base and the volume (or weight) of sample, are used to calculate the titratable acidity, expressed in terms of the predominant organic acid.

MATERIALS Caprisun juice and natural juice (lemon, grapefruit) Pipettes 10 ml Burette 50 ml Burette clamp Clamp support Magnetic stirring plate Erlenmeyer flask 125 ml 0.1N NaOH 17

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Phenolphtalein 1%

PROCEDURE 1. Pipette 10 ml juice sample into each three 125 ml Erlenmeyer flasks. 2. Measure the pH of the samples with a pH meter and record the value. 3. Add three drop of phenolphthalein and a magnetic stir to each flask. 4. Assemble the apparatus for carrying out the titration as shown in Figure 1. 5. Titrate each sample with the 0.1N NaOH solution until phenolphthalein endpoint. 6. Record the initial and final burette readings and calculate the difference to determine the amount of NaOH used for each titration.

DATA, CALCULATIONS AND RESULTS Table 1. NaOH volume used during titration of the blank Blank 1 2 3 4 Table 2. NaOH volume used during titration of the sample Sample identity 1 2 3 4 Formula % tritable acidity = (Vsample Vblank) L x NNaOH (eq.g/l) x Acid Factor (88.06g/eq.g) x 100 Sample weight (g) Initial lecture NaOH (ml) Final lectura NaOH (ml) Total Vol. used in simple titration Initial lecture NaOH (ml) Final lectura NaOH (ml) Total vol. used in blank titration

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Figure 1: Titratable acidity

a. Titration setup

b. Titration end point

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EXPERIMENT V ACIDS, PROTEINS, AND MAILLARD BROWNING

INTRODUCTION

Proteins are the most abundant macrocomponent in cells making more than 50% of the dry weight. They are very important in foods due to the nutritional properties and the functional properties. The challenge for food scientists is to develop new processes and products that maintain both properties: their nutritional value and functionality. Functional properties of proteins determine their behavior in foods and strongly affect their texture and quality. The amino acid composition of proteins influences the functional qualities of individual proteins. Some proteins may act as surfactants, have foam and emulsion-stabilizing ability; others have coagulating, foaming and leaving capacity, and others have good water binding or holding properties that allow them to coagulate and form gels under certain conditions; and some are important for their enzymatic activity. Many foodstuffs possess distinctive color and odor characteristic as a result of reactions between amino groups and reducing compounds (Maillard reaction or Strecker degradation). Depending on the extent of formation, these pigments and odors may be desirable or undesirable. In addition, free amino acids influence taste sensations. Denaturation of proteins causes changes that might affect the quality and stability of food.

EXPERIMENT A: MAILLARD REACTION INTRODUCTION AND OBJECTIVE Under certain conditions, reducing sugars may react with compounds bearing a free amino group and undergo a sequence of reactions known collectively as the Maillard reaction. As a part of this, alpha-dicarbonyl compounds produced in the Maillard reaction can react with amino acids and produce aromatic pyrazines. While a certain amount of browning and flavor generation is desirable in many foods, excess browning and aroma are undesirable. The objective of this exercise is to evaluate the aroma and color of heated amino acid-glucose solutions.

MATERIALS D-Glucose- 50 mg L-Aspartic acid- 50 mg L-Lysine- 50 mg 20

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L-Phenylalanine L-Valine- 50 mg L-Methionine- 50 mg L-Leucine- 50 mg L-Proline- 50 mg L-Arginine- 50mg

PROCEDURE 1. To 50 mg of D-glucose in a test tube add 50 mg of an amino acid; add 0.5 ml of distilled water. Mix thoroughly. 2. Smell each mixture and record any sensations. Place a piece of heavy aluminum foil over each test tube top and heat the solutions in a water bath at 100 C for 45 minutes. Cool the contents to about 25 C in a water bath. Record the odor sensations for each solution (e.g.,chocolate-like, popcorn-like). Record the color as 0 = none, 1 = light yellow, 2 = deep yellow, 3 = brown. (Note: Color formation can be measured quantitatively if the solutions are diluted to 5 ml, except for arginine and lysine, which need to be diluted to 500 and 1000 ml, respectively). The transfer the samples to colorimeter tuber and determine their absorbance at 400 nm. At 400 nm, the pigmentation or degree of browning is measured. What factors influence the degree of Maillard browning?

DATA AND RESULTS Sample Odor sensation Absorbance @ 400 nm

Treatment L-Aspartic acid L-Lysine L-Phenylalanine L-Valine L-Methionine L-Leucine L-Proline L-Arginine

Color

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EXPERIMENT B: EFFECT OF HEAT ON PROTEINS

INTRODUCTION AND OBJECTIVE Heat is one common denaturing agent of proteins in foods. Most proteins denature (uncoil) at a specific temperature. Once denatured, they are susceptible to coagulation/gelation, and this can be detected as turbidity in the dispersion. The objective of this exercise is to determine the effect of heat on the denaturation of egg albumin in aqueous solution.

MATERIALS Egg 1 Water baths (set at 55, 60, 63, 65 and 68 C) Spectrophotometer

PROCEDURE 1. Prepare 100 ml of 10% dispersion (v/v) of egg white and distilled water. Filter to remove opaque membranes. 2. Place 5 ml of the albumin solution in each of five test tubes. Place tubes in a water bath. Save the remainder for unheated control. 3. Place one test tube at each of following temperatures: 55, 60, 63, 65, and 68 C. Heat samples for 30 minutes. Cool the samples in a tap water. 4. Determine the absorbance of the samples with the spectrophotometer at 450 nm. Use the unheated sample to zero the spectrophotometer. Be sure to mix samples prior to reading. DATA AND RESULTS Table 1. Absorbance of Albumin solution Tube 1 2 3 4 5 6 HIS Workshop 2012 Temperature (C) 0.00 55 60 63 65 68 22 Optical Density 450 Set at 0

EXPERIMENT C: FOAMING INTRODUCTION AND OBJECTIVE Denaturation of the protein occurs when it unfolds, changing its nature. These changes might be mild or extensive and may be reversible or irreversible. Raw eggs are translucent because light is refracted and passed between individual proteins. When eggs denatures such as when it is beaten or whipped or cooked, fried, boiled it changes it appearance from translucent to opaque or white. When eggs are beating vigorously denaturation occurs and its volume increases from the original volume. Factors that affect the extent of denaturation, the foaming capacity (FC) of the proteins and its stability (FS) are type and time of beating, pH, temperature, concentration and the presence of other ingredients such as salt, sugars, and fat. In this experiment your will see how several factors affect FC and FS of eggs: temperature, dilution of water, pH and how certain ingredients such as fat, sugar, acid affect the foam formation, volume, surface area, and appearance.

Materials: 1 dozen of eggs 8 Funnels, 8 glass measuring cup, 8 100 ml graduate cylinder, 8 plastic spatulas, 8 Glass 8 measuring spoons 8 Mixers 1/8 tsp Cream of tartar 1tbs Distilled Water 2 tbs Sugar 1/8 Salt

PROCEDURE 1. Each group of two students should take one egg, one funnel, one measuring cup, and a graduate cylinder. A plastic spatula, a glass and a spoon. 2. Carefully break an egg and separate the egg white and yolk without breaking the yolk. 3. Beat the egg white with a mixer for 1 minute. This will be your control. 4. Measure the foam capacity by reading how many cups the volume has increased. Pour the recently formed foam to the funnel that will be on the top of the 100ml graduated cylinder.

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5. Each group will add one of the following: cream of tartar, or sugar, or water, or egg yolk to the egg whites, cold eggs and do the same procedure. Then compare it to the control using the same method.

DATA Measurement Control Sugar Water Egg Yolk Cream of Tartar Overbeating Salt Temperature

Volume (Cups) Foam Capacity Appearance Bubbles size (Coarse or Finer) Color (Opaque or Shinny) Foam Stability: Coalescence time (sec) Volume Drip (ml)

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EXPERIMENT D: COAGULATION OF PROTEINS INTRODUCTION AND OBJECTIVE While heat is a common coagulating agent in food, other food components may also affect the extent to which proteins denature. Among these components are salts, sugars, and acids. The objective of this exercise is to investigate some of the factor that affect the coagulation of proteins. MATERIALS Egg white 1 Distilled water 0.1M sodium chloride solution 5 ml 0.1M calcium chloride solution 5 ml 0.1M ferric chloride solution 5 ml 0.1M sucrose solution 5 ml 1.0M sucrose solution 5 ml 0.001M hydrochloride acid solution 5 ml 0.1M hydrochloride acid solution 5 ml

PROCEDURE 1. Dilute one egg (slightly beaten) with three volumes distilled water; stir slowly but thoroughly and filter. 2. To each of a series of test tubes, add 10 ml of the albumin dispersion and 5 ml of each of the solutions listed above, including distilled water. 3. Record the pH of the solutions containing distilled water and 0.01 M and 0.1 M HCl. 4. Place all of the tubes in a beaker of water, heat slowly, and note the temperature at which opalescence (cloudiness) develops. Generally, samples will need to be heated for at least 20 minutes.

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DATA AND RESULTS Table 1. Temperature at which cloudiness develop

Albumin solution mixed with Tube 1 2 3 4 5 6 7 8 Distilled Water 0.1M sodium chloride solution 0.1M calcium chloride solution 0.1M ferric chloride solution 0.1M sucrose solution 1.0M sucrose solution 0.001 HCl acid solution 0.1M HCl acid solution

pH

Temperature

---------------------

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EXPERIMENT VI PIGMENTS

INTRODUCCION Pigments contribute greatly to the aesthetic appeal of food. The chemical forms of some pigments are easily altered under conditions that may also affect the structural integrity of the tissue. Heating, pH changes, and oxidation reactions can affect pigment quality. The predominant meat pigment is myoglobin. Reactions of myoglobin determine the color of fresh and cured meats. Plant pigments may be categorized as carotenoids, chlorophylls, and flavonoids. Included in the flavonoid group are the phenolic compounds, which are the substrates in the enzymatic browning of fruits and vegetables. Preservation of desirable color, flavor, and textural qualities present at harvest of ripe fruits and vegetable depends greatly on control of the deteriorative changes caused by endogenous enzymes. Sometimes colorants are added to foods to enhance their marketability.

EXPERIMENT A: THE EFFECTS OF HEAT AND pH ON PLANT PIGMENTS

INTRODUCTION AND OBJECTIVE Many plant pigments, especially chlorophyll and the anthocyanins, are sensitive to heat and changes in pH. Under favorable acid conditions, these pigments will exhibit their correct colors, but when the pH is increased or decreased, the pigment may change to an undesirable color. This presents a sensory defect in the food. The objective of this exercise is to determine the effect of heat and pH on plant pigments.

MATERIALS Frozen peas - 25g Canned peas 25g Vinegar 10ml Grape juice- 10ml Cramberry juice 50ml 1N NaOH 100ml

PROCEDURE: CHLOROPHYLL 1. Heat 150 ml deionized water to boiling. 2. Add approximately 25 g frozen peas. HIS Workshop 2012 27

3. When the water returns to a boil, time for 7 min. 4. Remove the sample from the water and place in a beaker. 5. Add 10 ml vinegar to about 150ml deionized water and determine the pH of the solution. Boil the solution and repeat Steps 2 to 4. 6. Add 10 ml 1N NaOH to 150 ml deionized water and determine the pH of the solution. Boil the solution and repeat Steps 2 to 4. 7. Expose a fourth 25 g sample of peas to a cold mixture of 10 ml vinegar and 150 ml deionized water for 7 min without cooking. 8. Expose a fifth 25 g sample of peas to a cold mixture of 2 g NaHCO3 and 150 ml deionized water without cooking. 9. Set up a sixth beaker with canned peas. 10. Compare all the samples for color and texture.

RESULTS Table 1. pH, Color and texture of sweet peas pH solution ----------Color Texture

Beaker 1 2 3 4

Treatment frozen Boiled Boiled + Vinegar Boiled + NaOH

5 6 7

Cold H2O + vinegar Cold H2O + NaHCO3 canned ------

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Figure 1. Sweet peas color

a. Fresh

b. canned

PROCEDURE: ANTHOCYANINS 1. Mix 10 ml of grape juice and 90 ml of distilled water. 2. Determine the pH of the solution. 3. Remove 5 to 10 ml of this solution to a test tube. 4. Adjust the remaining solution to pH 5.0 with 1N NaOH. 5. Remove 5 to 10 ml of this solution to a test tube. 6. Adjust the remaining solution to pH 7.0 7. Remove 5 to 10 ml of the solution to a test tube. 8. Adjust the remaining solution to pH 10.0 9. Remove 5 to 10ml to a test tube. 10. Compare all the samples, especially noting the color. 11. Mix 50 ml cranberry juice and 50 ml of distilled water. 12. Repeat steps 2 to 10 with cranberry juice

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DATA AND RESULTS Table 2. pH and color of cranberries and grape juices

Sample 1 2 3 4 5 6 7 8

Treatment Grape juice + H2O Grape juice, pH 5.0 Grape juice, pH 7.0 Grape juice, pH 10.0 Cranberry juice + H2O Cranberry juice, pH 5.0 Cranberry juice, pH 7.0 Cranberry juice, pH 10.0

pH solution

Color

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EXPERIMENT B: ENZIMATIC BROWINING INTRODUCTION AND OBJECTIVE

Some plant tissues contain phenolic compounds associated with their cells walls. Some of these also contain polyphenoloxidase (PPO), an enzyme that will convert the phenolic to a quinone, which will eventually be transformed into a brown melanoidin pigment. This reaction is generally undesirable when it occurs in tissues of fruit such as apple, banana, or pear. It is thus important to know how to control this browning reaction. The objective of this exercise is to assess the effect of various treatments on enzymatic discoloration of Red Delicious apples.

MATERIALS

1 Red Delicious apple 1% thiourea 60 ml Ascorbic acid 10 mg Sodium sulfite 10 mg Dipotassium phosphate 120 mg Spectophotometer Buchner funnel and filter flask Whatman No. 1 filter paper Blender

PROCEDURE

Note: It is important to work quickly once the apple tissue is cut the slices are placed into solution. Have the beakers labeled and solutions prepared in advance. 1. Peel and pare apples, cut into uniform thin slices, and divide into four lots of 30.0g each. Place one lot into a beaker containing 60 ml 1% thiourea, which will stop the browning reaction and serve as the control. Place the second lot into a beaker containing 60ml deionized water. Place the third lot into a beaker containing 60 ml deionized water with 0.01 g ascorbic acid. Place the fourth lot into a beaker containing 0.01 g sodium sulfite in 60 ml water, and after 45 seconds decant the solutions and replace with a solution containing 0.12g dipotassium phosphate in 60 ml water. HIS Workshop 2012 31

2. After the apple tissue has been in solution for 30 min, homogenize the contents of each beaker in a blender, and filter through a Buchner funnel into a filtration flask under aspirator vacuum using Whatman No. 1 filter paper. Attach the funnel to the filtration flask, turn on the aspirator, wet a piece of filter paper using a squeeze bottle of deionized water, and carefully place the filter paper in the Buchner funnel. Pour apple tissue from the blender into the Buchner funnel and continue filtration until several milliliters of filtrate have been collected. 3. Place 1 ml of each filtrate in four different test tubes each containing 5 ml water and mix. 4. Transfer contents of each test tube to a cuvette and read optical density at 475 nm using a spectrophotometer. Use deionized water to set the instrument to 0% T.

DATA AND RESULTS Table 1. Absorbance of filtrate

Sample 1 2 3 4

Apple Treatment 1% thiourea (control) Deionized water 0.01 g Ascorbic acid 0.01 g sodium sulfite + 0.12 g dipotassium phosphate

Optical Density (475 nm)

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EXPERIMENT C: PEROXIDASE ASSAY TO DETERMINE ADEQUACY OF BLANCHIG INTRODUCTION AND OBJECTIVE

Blanching is a heating process performed prior to freezing that inactivates enzymes that would otherwise cause deterioration in palatability, color and ascorbic acid content during storage. Polyphenol oxidase (PPO), which catalyzes the enzymatic browning reaction studied in experiment 3, is one target enzyme to inactive during blanching. Catalase and peroxidase are two endogenous enzymes that are frequently used as indices of the adequacy of blanching treatment because they are more heat resistant than PPO. It is assumed that if these enzymes are inactivated, enzymes that catalyze undesirable color, flavor, and texture changes will also be inactivated. The objective of this exercise is to test for the presence of peroxidase in raw and blanched samples of a vegetable.

MATERIALS

Broccoli-1 head Guaicol-1 ml 0.08% H2O2 (2.7 ml of 3% H2O2 diluted to 100 ml with deionized water)-10ml Watch glasses-2 Funnel Glass wool

PROCEDURE

PREPARATION OF SAMPLES

1. Divide broccoli into two lots. Remove large leaves and lignified parts of stalks. Cut broccoli lengthwise into uniform pieces. Blanch one lot by immersing in boiling water for 3 min. Remove from boiling water and plunge into cold water until cool. Let drain. 2. For each lot, blend one part vegetable with three parts distilled water (by weight) in a blender for 3 min. Filter through a funnel with a glass wool plug.

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ASSAY FOR PEROXIDASE

1. Put several milliliters of filtrate from the raw broccoli on a watch glass. 2. Add several drops of guiacol and several drops of 0.08% H2O2. A brick-red color indicates peroxidase activity. 3. Repeat Steps 2a and b for the blanched broccoli.

DATA AND RESULTS Table 1. Observation and Conclusion of enzyme presence

Brocoli Treatment Uncooked Blanched

Color

Presence or Absent of Enzyme

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