The World of Science in Food Workshop 2002

EXPECTED OUTCOMES

This experiment will illustrate that there are several factors which affect the growth of yeast. A beneficial microorganism, yeast has been used in the fermentation of foods for thousands of years. Bread, wine, beer, and fruit are processed and/or preserved using yeast in fermentation. The by-products of the fermentation process are carbon dioxide and ethyl alcohol (ethanol).

ACTIVITY OBJECTIVE

A yeast population is affected by a number of factors, the control of which is essential for optimal activity. These factors include pH, temperature, nutrient availability, and the concentration of available nutrients. By determining which factors affect the yeast activity, these variables can be controlled in the fermentation process.

This experiment will illustrate to the student that the growth of yeast is affected by pH, temperature, and nutrient level and that one natural by‑product of this fermentation process is carbon dioxide.

ACTIVITY LENGTH

It is suggested that students be put into collaborative groups for this activity for several reasons. First, this limits the amount of materials needed. Second, more activities can be conducted in a shorter amount of time. And third, students will experience working as a team. Depending on the amount of teacher preparation done prior to the activity, all of the lab experiments can be done in a typical classroom period of 30-45 minutes.

This activity can be conducted in several ways‑with all students in groups repeating all tests; dividing the class into four groups, with each group conducting one section of the tests; or dividing the class into groups of four students, with some groups repeating tests.

Solutions may be prepared by the students, or ahead of time by the teacher.

SCIENTIFIC PRINCIPLES

All living organisms, large and small, have one thing in common: the cell. This is a tiny living factory capable of converting simple food substances into energy and new cell material and of reproducing itself

Microorganisms are made up of a very few cells, or even a single cell, capable of carrying on all of life's processes. A basic understanding of cell structure and function is essential to understanding the actions of bacteria, yeast, and molds. Since the cell is the basic unit of all living things, one might think it is a simple structure. Nothing could be farther from the truth. The cell is complex in its makeup and its function. Many scientists have spent their lives studying it. The main parts of the cell are the nucleus, cytoplasm, and cell wall. The nucleus is the control center‑it directs cell division, the formation of new cells. The cytoplasm contains the parts which convert food material into energy and new cell materials. The cell wall or membrane holds everything together and controls the passage of material into and out of the cell.

Yeasts are small, single-celled plants. They are members of the fungus family (plural = fungi), which also includes mushrooms. Fungi differ from other plants in that they have no chlorophyll.

While bacteria thrive on many different types of food, yeasts require carbohydrates, such as sugar and starch. (However, the yeast used in this experiment, Saccharomyces cerevisiae, cannot utilize starch.) From these, they produce carbon dioxide (CO₂) gas and alcohol. Thus, they have been useful to man for centuries in the production of certain foods and beverages. They are responsible for the rising of bread dough and the fermentation of wines, whiskey, brandy, and beer. They also play the initial role in the production of vinegar.

Some yeasts are psychrotrophs, meaning that they can grow at relatively low temperatures. In fact, the fermentation of wines and beer is often carried out at temperatures near 7^oC. This also means that they can create a spoilage problem in meat coolers and other refrigerated storage areas.

Unlike bacteria, which multiply by binary fission (cell division), yeasts reproduce by a method called budding. A small knob or bud forms on the parent cell, grows, and finally separates to become a new yeast cell. Although this is the most common method of reproduction, yeasts also multiply by forming spores.

Because they can grow under conditions of high salt or sugar content, yeasts can cause the spoilage of certain foods in which bacteria would not grow. Examples are honey, jellies, maple syrup, and sweetened condensed milk.

Foods produced by the bacterial fermentation process, such as pickles and sauerkraut, can also be spoiled by yeasts which interfere with the normal fermentative process.

VOCABULARY

Aerobic-requiring oxygen or air for growth

Anaerobic-growth in the absence of oxygen

Budding-reproduction in which a small part of the cell wall swells out and a wall of cellulose soon shuts off this new growth from the parent plant. It becomes an independent cell, soon growing other buds.

By-product-one result of a chemical process.

Facultative anaerobes-microorganisms which can grow with or without oxygen

Fermentation-the process by which compound sugars and some starches are converted into simple sugars, which are further hydrolyzed into alcohol and C0₂.

Generation time-the time it takes for a microbial cell to double

Psychrotrophs-microbes which grow at refrigeration temperature

Yeasts-tiny, one-celled, oval-shaped, microscopic plants which can be found naturally in the nectar of flowers, on the surface of fruits, and in the soil.

MATERIALS REQUIRED

Safety goggles

125-mL Erlenmeyer flasks or small (8-oz) glass soft‑drink bottles

Balloons, 7.8-cm (7-inch) size

Table sugar (sucrose)

Fructose, lactose, and glucose from science supply catalog or health food store

pH paper

Wax pencil or marker

Masking tape

Large jar or 16 packages of rapid‑rise yeast

Vinegar

Ammonia

Clock or stopwatch

Warm water bath (40^oC and 80^oC)

Triple-beam balances or scales

100-mL graduated cylinders

Eyedropper

Thermometers

STUDENT

EXPERIMENTAL PROCEDURE

Group I-Temperature Experiment

Label flasks A through D. Add 80 mL of tap water (neutral pH only) to each flask and place the flasks in the following conditions:

Flask A-in ice bath.

Flask B-at room temperature.

Flask C-in 40'C water bath.

Flask D-in 80'C water bath.

Dissolve 5g of sucrose in each flask. Add 4 g of rapid-rise yeast to each flask and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly

Group 2-Water Activity Experiment

Label flasks E through H. Add 80 mL of 40^oC water (neutral pH only) to each flask and dissolve the following amounts of sucrose in each:

Flask E-0 g (water only)

Flask F-5 g

Flask G-30 g

Flask H-50 g

Add 4 g of rapid-rise yeast to each solution and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

Group 3-pH Experiment

Label flasks I through L. Add 80 mL of tap water (neutral pH only) to each flask and add vinegar or ammonia to adjust the pH as shown below. Use pH paper to verify the pH.

Flask I-add vinegar to adjust the pH to 3.

Flask J-add vinegar to adjust the pH to 5.

Flask K-add vinegar or ammonia to adjust the pH to 7.

Flask L-add ammonia to adjust pH to 10.

Dissolve 5 g of sucrose in each flask and warm the solutions to 40^oC. Add 4 g of rapid-rise yeast to each solution and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

Group 4-Nutrient Experiment

Label flasks M through P. Add 80 mL of tap water (neutral pH only) at 40^oC to each flask and dissolve 5 g of each of the following sugars:

Flask M-fructose

Flask N-glucose

Flask 0-sucrose

Flask P-lactose

Add 4 g of rapid-rise yeast to each solution and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

All Groups‑Observations

1. After 15 minutes, record initial observations in the table provided for each test. Then make additional observations These observations should include a description of the fermentation activity and a measure of the amount of gas produced, either by measuring the actual volume of gas produced (see ancillary activities below) or by measuring the circumference of the balloon. To measure the circumference, wrap a string around the balloon at its widest point, then measure the length of the string.

2. Prepare bar graphs of balloon circumference (or cm³ of gas produced) against each of the following:

Temperature

Type of sugar

Water activity

TEACHING TIPS

· Warm water baths (40^oC and 80^oC) should be set up prior to the experiments for the placement of the flasks (except for the temperature experiment). If a commercial water bath is not available, a Styrofoam chest or bucket can be used. A pot of hot water will need to be available to add water as needed to maintain the water temperature in the water baths.

· There is a tendency for the flasks to float in the water bath during incubation. To prevent this, the students can physically hold down the flasks, use lead "donuts" around the neck of the flasks, tape the flasks to the side of the water bath, or add enough marbles to the flasks prior to water addition to keep the flasks from floating.

· Caution: Remind students to be careful when attaching balloons to the flasks. They must carefully tape the balloon securely to the flask to avoid any leaks. Also, they must remove the balloons very carefully, as the foam, which will rise into the balloons, will be expelled under great pressure on removal. It is suggested that the balloons remain attached to the flasks until the next class period to allow the CO₂ gas to escape overnight.

· It is important that the tap water be warmed to about 40^oC for each test prior to adding the sugar and yeast. Keep enough water available at this temperature for all tests.

· Students may use a lighted match to demonstrate that CO₂ is present in the balloons, as follows: Trap some of the gas from the flask into a test tube. (See ancillary activities below.) Place the lighted match into it. The match should be immediately extinguished. (Here is an opportunity to discuss the use of CO₂ in fire extinguishers).

· In the temperature experiment (Group 1), Flask C should show the best production of CO₂, as 40^oC is the ideal temperature for yeast growth. There should be little or no production of CO₂ in Flasks A, B, and D. Students should make observations regarding the fact that there is no foam in Flask D. They may be confused about the slight expansion of the balloon in Flask D. This is a result of air being heated inside the flask, which causes slight expansion of the balloon.

· In the water activity experiment (Group 2), Flask H should show little growth. In this solution, enough sugar is present to tie up the water molecules so that the yeast growth is inhibited. Flask E is the control in this test and should show no growth. Any inflation is due to air being heated inside the flask. As an additional experiment, have students try other sugar concentrations

· In the pH experiment (Group 3), there should be the most growth in flask K because the ideal pH for yeast growth is around 7. There should be little or no growth in flasks I, J, and L. As an additional experiment, have students try additional pH levels.

· In the nutrient experiment (Group 4), Flask M should have the fastest‑expanding balloon, since fructose is a simple sugar and therefore requires fewer steps to hydrolyze it to alcohol and CO₂. Flasks N and O will produce expanded balloons that are almost equal in size; however, since glucose is a simpler sugar than sucrose, the balloon on flask N may expand faster. There should be no expansion in Flask P, since baker's yeast does not produce the enzyme lactase needed to hydrolyze lactose during fermentation.

QUESTIONS & ANSWERS

1. What observations did you make about the flasks prior to the addition of the yeast?

Ans. Most sugar is dissolved, though some undissolved sugar may remain visible.

2. Which flasks showed the greatest yeast growth, i.e., most production of CO₂ gas?

Ans. Answers may vary, but should generally be: Flask C in test 1; Flask G in test 2; Flask K in test 3; and Flask M in test 4.

3. Did the contents of the flasks look the same at the end of the test time? Why or why not?

Ans. No, there should be a good deal of foaming present in many of the flasks. These changes are due to fermentation's having taken place to yield CO₂ and alcohol.

4. Knowing what you have learned about yeast "food," do you think yeast will hydrolyze gelatin or fat?

Ans. No. Yeast will not ferment fat or protein. The only nutrients yeast uses for energy are sugars and starches.

5. Which were the most favorable conditions for growth?

Ans. The most favorable conditions were 40^oC (slightly warm temperature), neutral pH (pH 7), and the presence of sucrose and fructose.

6. Which were the least favorable conditions for growth?

Ans. The least favorable conditions were too high or low pH, too hot or cold temperature, too much or too little sugar, and the presence of lactose.

ANCILLARY ACTIVITIES

1. To quantify the amount of CO₂ produced, a flask containing the test solutions may be stoppered with a glass tube extending from the stopper, as shown in Figure 1. Rubber or plastic tubing is connected to the glass rod on the flask and then placed inside a 250-mL graduated cylinder which has been filled with water and turned upside down in a water bath. As the CO₂ is expelled from the flask, it travels through the tubing and into the graduated cylinder. The water in the cylinder is displaced into the water bath at the same rate as the gas is expelled.

2. In place of the flasks and cylinders, 15-mL graduated conical centrifuge tubes with caps (Corning #25319) can be used. For further information, see "Fermentation, Respiration, and Enzyme Specificity: A Simple Device and Key Experiments with Yeast," by L. Reinking, J. Reinking, and K. Miller, The American Biology Teacher, March 1994, pp. 164-168.)

3. Incubate at different temperature.

4. Replace sugar with 5 g of cooking oil, corn starch, or gelatin. These variations will illustrate that yeast is unable to utilize protein or fat.

5. Grind one Lactaid or Dairy Ease tablet into a flask containing 5 g of lactose and 80 mL of water. Incubate at 40⁰C for 5 minutes. After the addition of the yeast, fermentation will occur because the Lactaid or Dairy Ease has broken down (hydrolyzed) the lactose into a mixture of glucose and galactose. While yeasts are unable to utilize the disaccharide lactose, they are able to ferment the monosaccharides glucose and fructose. This would be an ideal time to discuss lactose intolerance in humans.

DATA TABLE

Test/Flask	Conditions	Fermentation observed	Gas produced *
Temperature A	Sucrose + Ice bath	none	little or none
B	Sucrose + Room temperature	some initially	some
C	Sucrose + 40^oC	the most	the most
D	Sucrose + 80^oC	None	slight
Water activity E	40^oC + No sucrose	None	little or none
F	40^oC + 5g sucrose	some initially	some
G	40^oC + 30g sucrose	the most	the most
H	40^oC + 50g sucrose	Little	little

* Balloon circumference in cm or volume of gas produced in cm³

DATA TABLE

Test/Flask	Conditions	Fermentation observed	Gas produced *
pH I	40^oC + pH 3	none	little or none
J	40^oC + pH 5	some initially	some
K	40^oC + pH 7	the most	the most
L	40^oC + pH 10	none	little or none
Nutrient M	40^oC + Fructose	the most	the most
N	40^oC + Glucose	some	some
O	40^oC + Sucrose	some	some
P	40^oC + Lactose	none	little or none

* Balloon circumference in cm or volume of gas produced in cm³

STUDENT ACTIVITY GUIDE

What Affects Yeast Growth?

ACTIVITY OBJECTIVE

In this activity, you will (1) determine what factors affect the growth of yeast cells during fermentation, and (2) observe that one by-product of the fermentation process is carbon dioxide (CO₂).

MATERIALS REQUIRED

Safety goggles

125-mL Erlenmeyer flasks or small (8-oz) glass soft-drink bottles

Balloons, 7.8-cm (7-inch) size

Table sugar (sucrose)

Fructose, lactose, and glucose from science supply catalog or health food store

pH paper

Wax pencil or marker

Masking tape

Large bottle or 16 packages of rapid-rise yeast

Vinegar

Ammonia

Clock or stopwatch

Warm water bath (40^oC and 80^oC)

Triple-beam balances or scales

100-mL graduated cylinders

Eyedropper

Thermometers

BACKGROUND INFORMATION

Yeasts are microscopic plants that exist naturally on the surface of the Earth. They are noted for their ability to ferment carbohydrates to produce various food products, including bread, beer, wine, and cheese. During fermentation, yeast cells convert complex sugars into simple sugars, which are further hydrolyzed into CO₂ and ethyl alcohol (ethanol). Yeast growth is affected by several factors, including temperature, pH, and nutrient content.

EXPERIMENTAL PROCEDURE

Group I-Temperature Experiment

Label flasks A through D. Add 80 mL of tap water (neutral pH only) to each flask and place the

flasks in the following conditions:

Flask A-in ice bath.

Flask B-at room temperature.

Flask C-in 40^oC water bath.

Flask D-in 80^oC water bath.

Dissolve 5 g of sucrose in each flask. Add 4 g of rapid-rise yeast to each flask and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

Group 2-Water Activity Experiment

Label flasks E through H. Add 80 mL of 40^oC water (neutral pH only) to each flask and dissolve the following amounts of sucrose in each:

Flask E-0 g (water only)

Flask F-5 g

Flask G-30 g

Flask H-50 g

Add 4 g of rapid-rise yeast to each solution and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

Group 3-pH Experiment

Label flasks I through L. Add 80 mL of tap water (neutral pH only) to each flask and add vinegar or ammonia to adjust the pH as shown below. Use pH paper to verify the pH.

Flask I-add vinegar to adjust the pH to 3.

Flask J-add vinegar to adjust the pH to 5.

Flask K-add vinegar or ammonia to adjust the pH to 7.

Flask L-add ammonia to adjust the pH to 10.

Group 4-Nutrient Experiment

Label flasks M through P. Add 80 ml, of tap water (neutral pH only) at 40⁰C to each flask and dissolve 5 g of each of the following sugars:

Flask M-fructose

Flask N-glucose

Flask O-sucrose

Flask P-lactose

Add 4 g of rapid-rise yeast to each solution and stir. Then place a balloon on each flask and seal it securely with masking tape. Periodically stir the contents by spinning the flask slowly.

All Groups-Observations

1. After 15 minutes, record initial observations in the table provided for each test. Then make additional observations at 10-minute intervals, and final observations. These observations should include a description of the fermentation activity and a measure of the amount of gas produced, either by measuring the actual volume of gas produced (see ancillary activities below) or by measuring the circumference of the balloon. To measure the circumference, wrap a string around the balloon at its widest point, then measure the length of the string.

2. Prepare bar graphs of balloon circumference (or cm³of gas produced) against each of the following:

Temperature

Type of sugar

Water activity

QUESTIONS

1. Which were the most favorable conditions for growth?

2. Which were the least favorable conditions for growth?

3. What observations did you make about the flasks prior to the addition of the yeast?

4. Which flasks showed the greatest yeast growth, i.e., most production of CO₂ gas?

5. Did the contents of the flasks look and smell the same at the end of the test time? Why?

6. Knowing what you have learned about yeast "food," do you think yeast will hydrolyze gelatin or fat?

Chapter 3

What Affects Yeast Growth?