1. Comparing the Calcium Content of Various types of Milk
By: Rebecca Allen

2. Problem
Purpose: To determine how raw goat’s milk, pasteurized goat’s milk, raw cow’s milk, UHT 2% cow’s milk, 2% organic cow’s milk, 2% cow’s milk, whole cow’s milk, and skim cow’s milk compare in terms of calcium content by means of an EDTA titration.
Importance: Calcium is an essential mineral for a healthy body, and it is important for consumers to know which of the wide variety of types of milk on the market provides the greatest calcium content.

3. Background Information
Multiple studies have shown a greater content of calcium in raw milks as compared to pasteurized milk varieties. Also, studies have shown a greater content of calcium in goat milk varieties as compared to cow’s milk varieties.
Important Vocabulary:
Pasteurization
UHT
EDTA Titration
Organic Milk
Raw Milk
UHT - partial sterilization of food by heating it for a short time, around 1–2 seconds, at a temperature exceeding 135°C (275°F), which is the temperature required to kill spores in milk.
Pasteurization – A term describing milk that has been heat treated to destroy bacteria.
EDTA Titration – EDTA is used to generate a color change corresponding with calcium content in milk.
Organic Milk - is defined by the USDA as milk from cows that have been exclusively fed organic feed, have not been treated with synthetic hormones, are not given certain medications to treat sickness, and are held in pens with adequate space
Raw Milk – Milk that does not go antimicrobial treatment, pasteurization, homogenization, and is left unskimmed.

4. Background Information
Calcium is an essential mineral for…
Healthy bones and teeth
Regulation of metabolic processes
Lowering risk of colon cancer, heart disease, osteoporosis, and obesity
Prevention of preeclampsia, rickets, and strokes
Regulation of blood pressure
Improved premenstrual moods
Proper blood clotting

5. Hypothesis
If raw goat’s milk, pasteurized goat’s milk, raw cow’s milk, UHT 2% cow’s milk, 2% organic cow’s milk, 2% cow’s milk, whole cow’s milk, and skim cow’s milk are tested for calcium content, then raw goat’s milk will contain the highest quantity of calcium.

6. Controls and Experimental Variables
Independent Variable: Type of Milk
Dependent Variable: Calcium Content
Controls:
EDTA titration process used for all milk samples.
Milk products were tested within the expiration date.
Milk products were of the same brand when possible.
Each milk variety had its own set of labeled beakers and pipettes to eliminate contamination.
Each trial was compared to the color of the control sample respectively.
Extraneous Variables:
Different brands of milk
Different shelf life lengths
Added vitamins (can alter % digestion of calcium)
Retrieved from sources at different times
Different dairy farm locations and animal diets

7. Materials 800 mL NH₃ + NH₄Cl Buffer 1600 mL H₂O
Eriochrome Black T Solution
15 mL of each milk variety
EDTA Solution
48 Labeled Beakers
8 Pipettes and rubber bulb
2 Burettes and 2 Burette Clamps
Magnetic Stirring Objects

8. Procedure Label each of the 48 beakers with its appropriate title.
Prepare necessary solutions (Eriochrome Black T, EDTA, and Buffer to maintain pH > 7.0).
Rinse burettes with EDTA solution to remove prior residue.
Assemble burettes and magnetic stirrers.
Pipette 3 mL of milk into the corresponding labeled beaker (X Milk – Trial 1).
Add 40 mL of H₂O.
Add 20 mL of Buffer.
Add 10 drops Eriochrome Black T solution.
Magnetically stir.
Test pH – Color change only occurs at pH > 7.0
Titrate the mixture with the EDTA solution in the burette until a distinct color change from purple/magenta to blue.
Repeat this process for 5 trials of each of the 8 varieties of milk.

12. Calculations EDTA⁻⁴ + Ca2+  CaEDTA⁻2 1:1 mole ratio of EDTA : Ca
Molarity of EDTA = 0.01 mol/L
Atomic Weight of Ca = g
Sample Calculation:

14. Type of Milk
Trial Number
Initial EDTA Burette Reading (mL)
Final EDTA Burette Reading (mL)
Volume EDTA (mL)
Raw Goat 1
0.45
12.55
12.10 2
13.81
28.20
14.39 3
31.70
41.47
9.77 4
16.51
28.23
11.72 5
40.13
11.90
Pasteurized Goat
8.16
16.35
8.19
19.98
37.74
17.76
1.30
14.70
13.40
27.79
13.09
40.64
12.85
Raw Cow
17.60
27.97
10.37
18.40
31.52
13.12
39.51
11.54
50.00
10.49
40.90
9.39
UHT 2% Cow
12.61
23.60
10.99
0.92
12.48
38.79
15.14
26.91
13.51
40.94
14.03
2% Cow
3.52
18.01
14.49
0.83
16.40
15.57
32.10
14.09
30.35
44.40
14.05
17.00
31.69
14.69
Whole Cow
1.00
16.05
15.05
18.35
33.90
15.55
28.94
12.89
46.27
12.37
42.60
13.66
Skim Cow
2.99
15.00
12.01
2.75
14.50
11.75
25.70
10.70
14.45
25.65
11.15
36.11
10.46
Organic Cow
25.50
40.38
14.88
49.89
13.78
5.97
19.78
5.48
20.70
15.22
7.18
20.30

15. Average Amount of Calcium (mg)
Type of Milk
Average Amount of Calcium (mg)
Standard Deviation
Raw Goat’s Milk
8.385
Pasteurized Goat’s Milk
7.455
Raw Cow’s Milk
4.401
UHT 2% Cow’s Milk
5.303
2% Cow’s Milk
5.844
0.6167
Whole Cow’s Milk
5.571
Skim Cow’s Milk
4.493
0.6629
Organic Cow’s Milk
5.675

16. Statistical Analysis
One Way Analysis of Variance (ANOVA) – Compares the means of each group
Fisher F-Value was calculated for the data:
Fisher F-Value corresponds to P-Value of significance.
P < = Less than a million to one chance that results were not significant.
In summation, each of the 8 groups were significantly different from each other, in terms of calcium content.

18. Analysis Possible explanations for the results:
Pasteurization process and UHT processing.
Partial removal / full removal of fat content in 2% cow’s milk and skim cow’s milk
Calcium content diminishes with freshness
Feed quality/characteristics
Lactation cycle stage
Seasonal changes in calcium content
Daily fluctuation

19. Analysis Possible errors:
Milk products were not purchased directly from the manufacturer in some cases, so superior freshness could not be insured.
Milk products were purchased from different sources.
One cannot be sure of the amount of variability allowed on either side of the “best by” date, despite all milk products being well within this estimate.
One cannot be sure of the lactation cycle stage of the goats from which the goat milk products were produced.
One cannot be sure of the specific weather conditions of each farm from which milk products were produced at the time of milking.

20. Conclusion
The milk variety shown to have the highest calcium content: raw goat’s milk (8.385 mg Ca)
The milk variety shown to have the lowest calcium content: raw cow’s milk (4.401 mg Ca)
The hypothesis is accepted because the data showed raw goat’s milk to have the highest calcium content of all varieties of milk tested.
In conclusion, the consumer may be better off purchasing raw goat’s milk as compared to other varieties of milk from a nutritional standpoint.

21. Recommendations
Purchase milk varieties from the same manufacturer to rule out variables dealing with weather conditions and location.
Purchase directly from the manufacturer to insure superior freshness.
Test milk varieties for calcium content immediately after production and processing to insure superior freshness.
Determine lactation cycle stage of goats used for milk production prior to milking.

22. Extensions
Comparing calcium content of alternative milks, such as soy, almond, and rice milk.
Exploring the effects of farm location and climate on calcium content of milk.
Exploring the effects of diet on cow and goat milk varieties.
Comparing the calcium content of various type of cheeses, yogurts, or other dairy products.
Investigating the impact of age and lactation cycle stage of animals on calcium content of milk.
Determining which variety milk has the most humanly digestible calcium content.

23. Resources
Reykdal, Olafur, and Ken Lee. “Soluble, Dialyzable, and Ionic Calcium in Raw and Processed Skim Milk, Whole Milk, and Spinach.” Journal of Food Science 56.3 (2006) : Web. 29 Oct Pettifor, John M. “Nutritional Rickets: Deficiency of Vitamin D, Calcium, or Both?” The American Journal of Clinical Nutrition 80.6 (2004) : n.p. Web. 29 Oct Ott, Christopher. “The Surprising Benefits of Calcium.” Natural Health Jan.- Feb. (2002) : 55. Web. 28 Oct Krebs, Nancy F. “Optimizing Bone Health and Calcium Intakes of Infants, Children, and Adolescents.” Pediatrics (2006) : n.p. Web. 29 Oct “Building Strong Bones: Calcium Information for Health Care Providers.” U.S. Department Of Health and Human Services – National Institute of Child Health and Human Development (2006) : 1-4. Web. 28 Oct Baron, JA, M. Beach, J.S. Mandel, et al. “Calcium Supplements for the Prevention of Colorectal Adenomas.” New England Journal of Medicine (1999) : Web. 27 Oct

24. Resources
Bucher, HC, G.H. Guyatt, R.J. Cook, et al. “Effect of Calcium Supplementation on Pre-Induced Hypertension and Preeclampsia.” Journal of the American Medical Association 275 (1996) : Web. 29 Oct “Calcium 101: How It Does a Body Good.” The Cleveland Clinic Foundation Web. 28 Oct University of Granada. "Goats' Milk Is More Beneficial To Health Than Cows' Milk, Study Suggests." Science Daily 31 July January 2010 < /releases/2007/07/ htm>. "Standard Deviation Calculator." Easy Calculation.com. HIOX India, n.d. Web. 21 Feb <http//:easycalculation.com/statistics/standard-deviation.php>. Tóth, Á., M. Borbély, and Z. Gyori. "Examination of protein and calcium content of consumer milks and breakfast drinks." CABI Abstract Database. Tejgazdaság, n.d. Web. 21 Feb < "Raw Milk Vs. Pasteurized Milk." A Campaign for Real (Raw) Milk. Farm-to- Consumer Legal Defense Fund, n.d. Web. 21 Feb <

25. Resources
Gillis, Erin . "The Effect of Heat Treatment on the Nutritional Value of Milk." Faculty Articles. California State University - Los Angeles, 1 Sept Web. 19 Feb <
"Goat Dairy Foods." Dairy Research & Information Center. UC Davis - DRINC Program, n.d. Web. 26 Feb <
Kluiber, RW. "EDTA Titration for Ca+2 in milk.." Milk: Calcium and Nutrition. Rutgers University, 18 Nov Web. 20 Feb <
Nolte, Molly. "Info on Raw Goat Milk & Pasteurization." Fias Co Farm. N.p., 18 Jan Web. 26 Feb <
Shelton, Herbert. "Destructive Effects Of Pasteurization."ChestofBooks.com. N.p., n.d. Web. 20 Feb < Orthotrophy/Destructive-Effects-Of-Pasteurization.html>.
"The Michigan Fresh Unprocessed Whole Milk Workgroup: Benefits and Values." Michigan Food & Farming Systems - MIFFS. N.p., 23 Oct Web. 26 Feb <
"Varieties of Milk." The Dairy Council. N.p., n.d. Web. 20 Feb < of Form

26. Acknowledgements
Special thanks to Mrs. Joan Bechtel for all of her assistance in the lab during this experiment. It would not have been possible without her expertise and guidance.
Thank you to my parents for their continued support of everything I endeavor to achieve.
This project would not have been possible without the help of PJAS and CASEF sponsors Mr. Jason Sibbach and Mr. Jason Ambler.
Thank you to all the professionals who make PJAS and CASEF possible for students to learn and compete in these competitions.