THE CASE OF THE GOOD APPLE VS. THE BAD APPLE
PROBLEM:

You own a grocery store, and lately you've been having problems in your produce section. It appears your fresh carrots are turning bitter very rapidly and you are losing money. Having a background in Food Science, you decide you must solve the problem before you loose any more money.

BACKGROUND INFORMATION:

The Good Apple

Cancer is a deadly disease that has touched the lives of many people in the world today. The body receives numerous insults everyday from sources such as the sun, the diet, random mistakes in transcription, etc. These insults are usually repaired before mutation by two important pathways: the body's natural defense system and antioxidants consumed in the diet.

As the body ages the natural defense system becomes less effective and antioxidants in the diet become more important. A large segment of the American population is approaching the over 50 age bracket with the expectation of living well into their 80's. Research into antioxidant activity and other benefits of a healthy diet is becoming more and more important. It is much easier to prevent the onslaught of a major illness than to fight it after it has become well established.

The dietary defense system is set up as an interconnecting system. When a free radical is formed after an insult, an antioxidant neutralizes the free radical and itself becomes a free radical. [A free radical is a destructively reactive atom or molecule, in this case usually oxygen, with an unpaired electron in its outer shell. It is oxidized when another atom or molecule donates one electron. The donating atom or molecule is then one electron short and is a free radical.] The free radical is then passed down a pathway of various antioxidants (much like people passing sandbags) until it is excreted for the body. If even one of the critical antioxidants is missing due to a dietary deficiency, the system is less efficient or may fail to work altogether.

It is very important to have a constant supply of a variety of antioxidants from many different sources. Relying on antioxidant tablets may set up a problem where there are too many members of one link in the chain and too few of another. Because science has yet to identify and isolate all of the necessary antioxidant links, a varied diet of fruits and vegetables is the best alternative. An apple a day may indeed help keep the doctor away.

The Bad Apple

In addition to beneficial elements, plants may produce detrimental compounds as well. Isocoumarin from carrots is one example. Isocoumarin is a bitter tasting compound produced in carrots during times of stress. Isocoumarin has antifungal properties. Another compound of interest is ethylene gas, which is released by most fruits and vegetables during the ripening process. Rapidly ripening fruits, like apples and tomatoes, release greater amounts of ethylene. Bruising or damage to a fruit or vegetable increases the amount of ethylene released and speeds the ripening process. A chain reaction of ripening is why "one bad apple spoils the bunch."

When mold attacks a carrot growing in a field, the carrot begins to produce isocoumarin. This antifungal agent will stop the spread of the mold, but will cause the carrot to become impalatable to humans. Thus, it is very important for farmers to keep molds and yeast out of their field during a wet spell. They must also handle the carrots carefully during shipment. Rough treatment and scarring also triggers the formation of isocoumarin and the carrots will be bitter.

The production of isocoumarin in carrots is of special interest to baby food manufacturers. Babies will quickly reject a bitter product and the company loses profits and customers. Manufacturers like Gerber's have turned to the University of Arkansas Food Science Department to help solve a bitter carrot dilemma.

Problems caused during growth and transportation were solved and the carrots arrived at Gerber's sweet and crunchy. However, after storage in large walk-in coolers, the carrots became bitter. The coolers were being used to store a variety of other fruits and vegetables to be used in baby food products. Do you know what was happening to the carrots?

A third mechanism for the production of isocoumarin in carrots was soon discovered - the presence of ethylene gas. Rapidly ripening fruits, like apples, being stored in the same cooler were releasing ethylene and triggering the production of isocoumarin in the carrots. The company was losing a great deal of profits (up to $250,000/year in retail value) over a storage problem! Thereafter, the carrots were stored separately. This solution greatly reduced the bitter carrot dilemma.

A surprising aside to this problem is that even after separate storage, the carrots were slowly developing a bitter flavor. The carrot cooler and the apple cooler were located across the hall from each other. The process of opening and closing the cooler doors allowed passage of enough ethylene to measurably speed the production of isocoumarin. A level of 1 ppm of ethylene in the air is enough to stimulate the production of isocoumarin! [1 ppm (part per million) can be described as 1 tablespoon of salt dissolved in an Olympic sized swimming pool.]

OBJECTIVE:

The student will observe the effects of ethylene gas produced by apples on the production of isocoumarin in of carrots.

MATERIALS:

  • Ziplock® bags
  • Apples
  • Carrots
  • Extension Exercise:

    Solutions of acids, bases and other chemicals can be very dangerous. They can cause severe chemical burns and destroy cell membranes. Contact with the skin and eyes must be prevented. Should contact occur, rinse the affected area with water for 15 minutes. If the contact involves the eyes, medical attention should be sought while the rinsing occurs.

    TIME:

    ½ of a class period need to prepare baggies. One week of storage. One class period to test results.

    PROCEDURES:

    Teachers: Divide the students into groups of four. There should be enough carrots in each treatment for each student to have two carrots per treatment. If doing the extension there should be three carrots per treatment.

    1. Store several carrots in a zipper lock plastic bag. Poke several holes in the zipper lock bag. Store 24 hours at room temperature then transfer to a refrigerator.
    2. Store an equal number of carrots from the same source along with an apple in a zipper lock bag. Poke several holes in the zipper lock bag. Store 24 hours at room temperature in a separate location from #1 then transfer to a separate refrigerator.
    3. Repeat 1 and 2 except store at room temperature the entire time. Be sure to store the two room temperature treatments (treatments 3 and 4) in different locations.

      One week later:

    4. Each member of the group should taste test one carrot from each of the four treatments. Do not peel the carrots.
    5. Drink water and eat a soda cracker between each taste of carrot. This will clean your palate of residual flavors.
    6. Taste carefully and discuss among the members of your group how the treatments should be ranked according to bitterness. Be sure to compare tops to tops or middles to middles. Do not compare the top of one carrot to the middle of another.
    7. Each student should still have one carrot from each treatment. Use these carrots to taste which region (top, middle or tip) has the bitterest flavor. Also try to taste test a piece of the peel.

    QUESTIONS:

    1. Why are the treatments stored separately?



    2. Why are holes punched in the bag?



    3. Which of the treatments gave the most bitter carrots? Why?



    4. Which part of the carrot is the most bitter?



    5. What is the best way to assure the consumption of the sweetest carrots?



    EXTENSION: QUANTITATIVE ANALYSIS OF ISOCOUMARIN

    Teachers: One carrot extra per treatment should be stored to do this activity.

    1. Each student in the group should grind an entire carrot from one treatment in a blender. Carefully weigh 8 g of the ground carrot into a 30-ml glass container with a tight fitting lid. Wash the blender between grinding each treatment to avoid contamination. All four treatments will be evaluated in the same manner.
    2. Add 15 ml of 0.5 N sodium hydroxide.
    3. Shake well every 5 minutes for a total of 20 minutes.
    4. Filter the ground carrot from the solution.
    5. Carefully measure 5 ml of filtrate into another 30-ml glass container with a solvent resistant lid.
    6. Add 5 ml of 2 N HCl and 10 ml of n-hexane.
    7. Agitate gently end over end for 1 minute.
    8. Remove the hexane layer.
    9. Sometimes the color compounds (alpha and beta-carotene) interfere with the measurement of isocoumarin. A further step removes these color compounds.
    10. Add an equal volume of 75% ethanol/water to the hexane layer in step 8.
    11. Agitate gently end over end for one minute.
    12. Remove the ethanol layer (the bottom, clear layer) and measure its absorbance at 267nm. The value of this reading is proportional to the amount of isocoumarin. A higher reading means more isocoumarin was present in the carrot.
    13. Repeat steps 5-13 for the remaining three Treatments.

    QUESTIONS:

    1. Why do you grind the entire carrot and then weigh out only 8 grams?



    2. Hexane and aqueous solution are immiscible. Which layer was on top? Why?



    3. Why is it so important to weigh and measure carefully (quantitatively)?



    FOR ADDITIONAL INFORMATION CONTACT:

    Dr. Luke Howard

    Steve Talcott, Research Specialist, Food Science Department, U of A - Fayetteville
    stalcot@comp.uark.edu

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