TITLE: Food grade colorimetry of anthocyanins (A Youth Summer Camp Activity)
AUTHORS: Eric S McLamore, Geisianny AM Moreira, Diana Vanegas, dbahamo, Lisseth Casso-Hartmann, Lidadi Agbomi, Maria J Torres

[DESCRIPTION]
This protocol describes activities used in summer education camps for youth (14 years and older). The activities involve extraction and cryo-concentration of plant pigment (anthocyanin) from red cabbage. This protocol describes summer camp activities for encapsulation/spherification in alginate beads and other alternative methods for teaching basic principles related to cross linking and colorimetry.

 


[BEFORE_START]
Before starting
For most applications, it is recommended to heat extract the anthocyanin at least two days prior to events, allowing at least one day to freeze the extract.
If the anthocyanin is heat extracted during activities, ensure there is ample space between camp participants and cooking equipment. Induction cookware is advised to reduce footprint and limit heat exposure. It is also recommended to perform the heat extraction in a well-ventilated area as not all people enjoy the smell of cooked cabbage! 


 


[STEPS]
SECTION: Anthocyanin activities for camp
1. Step 1) Gather and prepare materials
Group participants into teams of 2-4 people (Fig 1).

 
Table 1 presents a planning table for a typical summer camp supporting 40 youth (ten teams of 4). For a camp of this size, 3-5 leaders are typically engaged to guide youth through activities.
 
 


 

The following items are required for heat extraction and cryo-concentration of anthocyanin:
66 Quart Stainless Steel pot for boiling cabbage
Freezer (0° F)
50mL Falcon tubes for freezing extract
Red cabbage (Brassica oleracea var. capitata, f, rubra) purchased from a local grocer
Optional: 500W Stainless Steel Immersion Blender

 

The following items are required for spherification:
Food grade calcium chloride OR food grade calcium lactose 
Food grade sodium alginate
Transfer pipettes
Small plastic tubs/dishes with lids (2 tubs per team) 
Plastic spoons (2 per team)

The following items are required for pH testing:
Sodium bicarbonate (baking soda) 
White distilled vinegar 
Small plastic tubs/dishes with lids (2 tubs per team) 
Plastic spoons (2 per team)
Optional: Color Name AR app
Optional: Colorimeter

SECTION: Anthocyanin activities for camp
2. Step 2) Heat Extract Anthocyanin
Using the vegetable cleaver, cut the cabbage head into quarters.
Chop leaves into small 1-2 inch wide strips (approximately 3-4 inches long).
Place Stainless Steel pot on cooking surface.
Place cabbage strips into pot.
Fill the pot with water until the cabbage is immersed (approximately 450 g of cabbage for each Liter of water). 
Next, heat the pot until the water boils (approximately 80°C).
Reduce heat to 75% of maximum level and cook for 120 minutes.
Check the pot every 30 minutes and stir to avoid burning material on base of pot.
Turn off heat (cooking equipment).
Cool down at room temperature for at least 20 minutes but no longer than 60 minutes
Carefully remove cabbage leaves from the purple liquid extract. 
If possible, a strainer should be used, and the liquid should be collected

 


SECTION: Anthocyanin activities for camp
3. Step 3) Cryo-Concentrate Anthocyanin 
Gather the 50mL Falcon tubes.
Carefully pour approximately 40mL of anthocyanin extract into each tube and then seal.


 

Freeze for at least 6 hours (or until a solid ice block forms in each tube).
After freezing solid, remove the block of frozen extract.
Place at room temperature for 10 min.
Remove the lid of the Falcon tube and carefully invert the tube to collect the dark purple syrup (known as “cryo-concentrate”).
Pour this dark syrup into a separate container. The cryo-concentrate may be combined together, but avoid adding any excess water.
Approximately 2-4 mL of cryoconcentrated pigment can be acquired for each 40mL vial.

 

Optional: Cook a yummy and spicy meal!
For each head of cabbage, chop one full (large) onions, three bell peppers, one bunch of green onions
Obtain one small bag of carrots (optional to chop)
Heat olive oil in a large skillet over medium-high heat; add chopped onions, bell pepper, and green onions. 
Cook and stir vegetables until softened, about 5 minutes. 
For spicy: Mix in chopped Scotch bonnet pepper,
Add 2 sprigs fresh thyme, and salt as desired
Continue to cook for an additional minute
Stir boiled cabbage and carrots into mixture and cook for an additional 5 min in olive oil
Cover the skillet (or large wok) and reduce heat to medium-low. 
Allow to cook for 10 minutes.
While food is cooking, prepare a sauce with ¼ cup white vinegar and 2 tablespoons white vinegar. Stir well, and then add to skillet (or wok)
Cook, uncovered, stirring occasionally, about 2-3 more minutes. 
Discard Scotch bonnet pepper and thyme before serving.
 
 
 
SECTION: Anthocyanin activities for camp
4. Step 4) Prepare Alginate-Anthocyanin Solution
A total of five solutions should be prepared for this step (Fig 2). 
 


 

Solution 1: Prepare a 2% (w/v) sodium alginate solution.
Weigh 2 grams of sodium alginate powder and transfer it to a plastic tub.
Heat tap water (or bottled water)
Add 100 mL of hot water to the tub and stir vigorously until no clumps of alginate are present; this process can take up to 10 minutes of stirring.
Optional: Use an immersion blender to quickly dissolve the alginate in the water.

Solution 2: Prepare 1% (w/v) calcium chloride solution. Alternatively, a 1% solution of calcium lactose (w/v)
may be used.
Weigh 1 gram of calcium chloride (or calcium lactose) and transfer it to a plastic tub. 
Add 100 mL of water to the tub and stir until no powder is visible. 

Solution 3: Prepare a 2% (w/v) solution of sodium bicarbonate.
Weigh 1 gram of sodium bicarbonate and transfer to a plastic tub. 
Add 100 mL of water to the tub and stir until no powder is visible. 

Solution 4: Obtain approximately 4-5 mL of the cryo-concentrate solution from Step 3.

Solution 5: Obtain approximately 10mL of white vinegar.

SECTION: Anthocyanin activities for camp
5. Step 5) Spherification
Using a transfer pipette, add 1-2 mL of cabbage extract to a vial, and then add 5-7 mL of alginate solution (this ratio can be adjusted if desired, but no less than 1:1). 
Cap the vial and shake until well mixed.
Using a clean transfer pipette, drop small amounts of the alginate+pigment solution into the calcium chloride solution (or calcium lactose) (Fig 3)

 

Adjust the height of the pipette tip to alter bio-bead shape
After at least 1 min, use the plastic spoon to remove bio-beads from the calcium solution. 
Place beads in plastic weigh boats

 

SECTION: Anthocyanin activities for camp
6. Step 6) pH Testing 
Using the white vinegar or bicarbonate solution and a clean transfer pipette, expose add a drop of solution (Fig 4)
Wait approximately 1 minute.

 

Observe any color changes and record notes in laboratory handout or notebook provided. Participants should connect the color observed with the wavelength of visible light according to the electromagnetic spectrum (Fig 5)

 

If preferred, a cell phone app may be used to detect the color of the sample. For example, Color Name AR is a useful tool that is available for both iPhone and Android (as well as tablets).
 
 
 
 

Participants should attempt to reverse any color change(s) back and forth, if possible.
See Box for optional ideas that have been successful in various camp activities.

 

 


SECTION: Anthocyanin activities for camp
7. Step 7) Clean Up
Clean workstation/benchtop.
All powders, gels and liquid materials are food grade and may be either composted or disposed in the standard trash receptacle.
Used 50mL Falcon tubes and transfer pipettes may be reused after rinsing in warm water with soap.