Students will expand their knowledge of microbial growth and scientific food preservation methods to learn how honey can serve as an antibacterial agent. Students will learn how honey may be used as a preservative of milk in areas without access to electricity or refrigeration and how this preservation method relies on elements found specifically in honey that cannot be replicated with other sources of sugar. Grades 9-12

microbe: a microscopic living organism—e.g., bacterium, fungus, protozoan

preservative: a substance added to a product to destroy or inhibit multiplication of microorganisms

turbidity: a measure of relative clarity of a liquid

zone of inhibition: the area around an antimicrobial disc that contains no bacterial growth

Prior to this lesson High school students should be aware that food is at risk of spoiling because of microbial growth and contamination and that preservatives are present in the food we consume to prevent spoilage.  Students should be familiar with common ways to preserve food. This lesson expands students’ knowledge of preservation by exploring the use of honey as a preservative. Preservatives are an important topic for students to understand because without them we would not have the variety of available food products. 

Key STEM Ideas

All living organisms compete for food sources to fuel their energy needs. A variety of methods are used to protect and preserve our food from bacterial competition. Food preservation practices may include storage in an acidic environment (as with pickles or sauerkraut), refrigeration (many foods), removing moisture (as with dried meats or fruits), or a heavy concentration of sugar (as with fruit cake or Twinkies).

Connection to Agriculture

Honey bee societies like human societies have to manage logistics such as adequate space, shelter, and a safe, year-round food supply.  Bees rely on plants that flower during summer months to provide nectar, a carbohydrate and pollen, a protein source to last throughout the year.  Bees need to safely store their food for times when flowering plants are not available.

Honey is a food produced by bees that is antimicrobial and antibacterial under the right environmental conditions and can be used to our benefit as a preservative. This is especially important in areas of the world where refrigeration may not be available due to cost or lack of consistent electricity.

Honey may offer an alternative means of preserving a nutrient-rich food such as milk without refrigeration improving quality of life and health for people in these regions. Honey is a good preservative that allows us to maintain a nutritious food supply that the population demands. Milk and dairy products are an important part of a healthy diet providing an inexpensive source of protein, vitamins, and minerals.  Spoilage of milk is a major limitation in providing this valuable food source to people who do not have access to refrigeration.  

1. Project the Preservation Power of Honey PowerPoint. Show students slide 2 and have students review with a partner the following two questions:
   • Why does food spoil or go bad? (Microbial growth, usually from bacteria)
   • What conditions make this more likely to happen? (Unsanitary conditions, when food is kept in conditions favorable for bacterial growth (temperature, moisture, etc.) when food is not cooked thoroughly enough)
2. Give each student one copy of the Power of Honey student handout. Using page one of the handout and slide three of the PowerPoint, introduce the list of foods- beef jerky, bread, milk, pickles, raw steak, cucumbers, cupcakes, croutons, and Twinkies. 
3. As a class, discuss what makes food spoil. Have students work individually to predict how they believe these foods are preserved.
4. Discuss answers as a whole class and record effective food preservation strategies. Answers are provided in the teacher notes, however, students may come up with additional or creative food preservation techniques. Students should discuss removing water (dehydration), keeping out air (sealing), refrigeration, increasing acidity (adding vinegar), and adding preservatives.

Activity 1: Measuring antibacterial properties of honey

1. Using the paragraph on page one of the handout, introduce how bees gather their food from flowers during the spring, summer, and fall, but must protect this food for use during dearth periods.
2. Have students predict and write down which preservation methods they think bees might use.
3. Listen online to the audio clip “Why Honey Doesn’t Spoil.” Discuss if their predicted answers matched those in the audio clip. (Bees use dehydration by removing water from nectar. This could also be described as highly concentrating the sugars in honey. Students will not be introduced to it yet, but bees also keep out water by sealing honey in honeycomb cells with a wax capping and by adding an enzyme that produces hydrogen peroxide).
4. Review how to grow a colony of bacteria on agar in petri dishes (use slides 4-6 of the PowerPoint for visual support). Point out how antibiotic discs can be applied to a petri dish to inhibit bacterial growth (if the bacteria are susceptible to the antibiotic in the disc). Facilitate student discussion of how antibiotics might be tested in the lab for their efficacy against bacteria. (Measuring and comparing the zones of inhibition for different antibiotics is one way.)
5. Show students how to measure the zone of inhibition (slide 7 of PowerPoint). (Use a ruler; measure area with no bacterial growth including the disc.)
6. Divide students into pairs to complete "Part 1" of the worksheet. Students will measure zones of inhibition for each antibacterial disc and use their data to determine the relationship between concentration of honey and the size of the zone of inhibition.
7. Facilitate a whole class discussion of what students observed from the experiment. (The higher the honey concentration on the disc, the larger the zone of inhibition, and therefore the fewer bacteria were able to grow.)
8. Listen online to the audio clip “Honey: Food for Yeast or a Natural Preservative.”
9. Guide discussion on why bacteria doesn’t thrive in honey. (By the process of osmosis, the high concentration of sugar in honey forces the water out of any yeast or bacteria cells, drying them out and destroying them.)
10. Have students work in pairs again to predict if a concentrated sugar solution could be used as a more cost effective preservative. (Based on what they have observed and listened to so far, students will likely predict that sugar is a good substitute for honey as a preservative.) Have students provide evidence for their prediction.

Activity 2: Comparing sugar to honey

1. Ask students to read the information found on page three of their handout to familiarize themselves with the sugar experiment setup explained in "Part 2" of the worksheet.
2. Divide students into pairs to measure zones of inhibition for each antibacterial disc and use their data to determine the relationship between concentration of sugar and the size of the zone of inhibition. (There is no effect of the concentration of sugar on the growth of bacteria.)
3. Ask students to compare the effectiveness of honey to a concentrated solution of table sugar and provide evidence to support their choice. (Honey was an effective antibacterial, however, concentrated sugar was not effective at inhibiting bacterial growth.)
4. Discuss if students thought this would be the case. Why or why not? (Based on information presented so far, this should be surprising to students. Honey must be more than simply a source of concentrated sugar.)
5. Read and discuss as a class how nectar is ripened into honey from the student worksheet and have students predict if there is anything special about honey.
6. Have students discuss why honey and concentrated sugar might have had different effects on bacterial growth. Examine the honey ripening diagram. Have students note that hydrogen peroxide and acid are produced by the addition of an enzyme, glucose oxidase.
   • What do we use hydrogen peroxide for? (Cleaning bacteria from wounds)
   • How have we used acid in food preservation? (Vinegar is used to pickle foods to prevent bacterial growth.)
7. Have students predict which components found in honey may have inhibited bacterial growth on their worksheet.

Activity 3: Can honey inhibit the growth of bacteria in food products?

1. Lead a discussion to review what has been learned about honey and how we might use what we have learned about the antibacterial properties of honey to benefit society (slide 8 of the PPT). (Answers may vary. Honey could be used as a wound dressing in hospitals or as a food preservative in areas without access to other means of food preservation such as refrigeration.)
2. Have students read part 3 of the student worksheet about an experiment testing honey as a preservative for milk. Discuss the following (slide 9 of PowerPoint):
   • What is turbidity? (Turbidity is a measure of how clear a liquid is.)
   • How does measuring turbidity help us to determine bacteria growth in milk? (Turbidity is an indicator of bacterial presence; the cloudier a liquid is, the higher the turbidity, the more bacterial is assumed to be present.)
3. Instruct students to calculate the percent difference in turbidity of milk with honey compared to milk without honey as a sign of inhibition of bacterial growth. Use this data as evidence when answering the two follow-up questions.
4. Facilitate class discussion about the feasibility of using honey as an effective preservative for milk. Have students make a list of benefits and challenges. (Potential benefits include availability of an inexpensive, protein-rich food source in areas with no refrigeration, better bone and teeth health, access to calories in areas dealing with malnutrition; potential challenges include cost of adding honey, change in flavor of milk, consumer acceptance of this new method for preservation.)
5. Using the class discussion as a starting point, have students complete the final question on the worksheet on their own. Encourage students to think critically about this issue.

Concept Elaboration and Evaluation

• Many areas throughout the world suffer from hunger due to an inability to properly preserve and store their food.
• Honey is produced by bees and can be used to preserve foods.
• Methods of food preservation include dehydration, sealing, refrigeration, increasing acidity, or adding a preservative such as honey.

1. https://glorybee.com/content/honey-facts-nutrition
2. http://nchfp.uga.edu/publications/nchfp/factsheets/food_pres_hist.html
3. Background info on honey from The Book of Honey by Eva Crane

• Burke Morrow: Lincoln East High School, Lincoln, NE
• Erin Ingram: University of Nebraska-Lincoln, IANR Science Literacy Initiative, National Center for Agricultural Literacy

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