Fricano-Le 1

Introduction
Some 10,000 years ago, milk drinking became a dietary staple for humans first in
Afghanistan and Iran and later in Turkey and Africa. By the time cows arrived in the
Americas around 1600, the rest became history (Gipi). Humans likely first started
drinking milk for the taste, and in modern times have improved upon the original with
milk products such as cheese, ice cream, yogurt, and cottage cheese. For good reason toothe calcium that is necessary for skeletal growth, blood clotting, nerve impulse
transmission, and heartbeat regulation can be found in only one of two ways (Cheung). It
is either consumed in food products or stolen from existing bones. The latter is not an
ideal situation, so the consumption of quality milk and milk products is vital to a healthy
life. However, there are several obstacles to overcome in order to have the highest quality
milk.
Raw milk initially contains numerous bacteria strains such as Salmonella, E. coli,
and Listeria. In modern countries that raw milk is pasteurized which is the heating of the
milk in batches to an industry standard of 62.8 C for 30 minutes (Heat). This heating
process kills off harmful bacteria and gives milk a shelf life if refrigerated, as the
refrigeration slows bacteria regrowth. Pasteurized or not, if milk is left susceptible to
bacteria the acidity of milk will increase, making its taste sour and unpleasant at the very
least. The acidity increase will cause the milk to coagulate into solid pieces, but the
bacteria can cause life threatening food borne illnesses long before the coagulation
happens (Lu). The Center for Disease Control reports that unpasteurized milk is 150
times more likely to cause foodborne illness and results in 13 times more hospitalizations
than illnesses involving pasteurized dairy products (Dangers). Between 1993 and
2006, it was reported that over 1500 people became ill from consuming raw milk. The

Fricano-Le 2
most severe of these cases included miscarriage, kidney failure, and life-threatening
infections. Most milk cases still involved strong flu-like symptoms (Dangers). It is
essential then, that milk for human consumption is kept free from bacteria at all costs.
While pasteurization is the optimal method for bacteria suppression, it is not
always a feasible procedure in developing countries with limited industry and resources.
Alternative methods, such as the addition of the chemicals thiocyanate and hydrogen
peroxide to raw milk, stimulate the milks natural antibacterial mechanism and are a short
term solution for preservation. A small-scale, rural famer can potential increase
household income by almost 40% with the chemical treatment, factoring in the amount of
milk normally lost to spoilage (Lactoperoxidase). Another more natural solution is the
addition of antibacterial spices such as turmeric to the milk. This research experiment
will examine both methods and determine which method is the best way to preserve raw
milk without means of refrigeration or pasteurization.
In order to determine this, three solutions of milk were prepared. The first was
raw milk mixed with trace amounts of sodium thiocyanate and sodium percarbonate
(hydrogen peroxide source), or the chemically treated milk. The second was raw milk
mixed together with turmeric, or the naturally treated milk. The third solution was a
control solution consisting of just raw milk. Thirty test tubes were filled for each milk
solution, with a total of 90 test tubes. The pH level was measured as an indicator of the
spoilage level of the milk after 48 hours. Statistical tests were run to determine if there is
a significant difference between the three treatment methods. Through these procedures,
the researchers hoped that that a distinction between the chemical and natural methods
would be observed.

Fricano-Le 3
After all data was collected, an ANOVA test was run to determine if among the
three treatment types there was a significant difference in mean pH levels. From the
results of this test, a two-sample t-test was deemed appropriate to run comparing the
chemical and natural methods. A significant distinction between these two samples was
made known, accomplishing the objective of the experiment. The outcomes of this
research experiment are valuable as they demonstrated accessible methods to preserve
raw milk. The scientific community benefits as the knowledge of turmeric as a food
preservative has been expanded to raw milk. The experiment proved that by preserving
milk, even with just a common spice, reduces bacteria as compared to no treatment at all.
Research in the area of the chemical milk treatment (Lactoperoxidase system) is
expanded, as the time frame for milk preservation by this method was extended beyond
previously researched time frames. Overall, this research experiment was critical support
for research in the area of food science pertaining to alternate preservation methods.
With this research, society could benefit in numerous ways. By discovering a
difference between chemical and natural preservation methods, food preservation
companies could improve upon their current techniques. More effective and enduring
preservation treatments can be created by either increasing concentration of various
chemicals or by implementing a naturally-occurring ingredient into the treatment. This
research will create a direction for preservation developers to follow. Additionally, areas
in the world without access to chemical or mechanical methods can informally explore
food preservation through inexpensive, natural means by using similar procedures that
the researchers created. The researchers' preservation treatment procedures can be used as
a base for future preservation methods if similar effects were desired to be produced.

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Extending beyond the food industry, the field of medicine can use the data produced to
explore turmeric's further ability to inhibit bacterial growth and create possible turmericbased remedies.

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Review of Literature
Approximately one quarter of the world does not have access to electricity
(Kelly); individuals are left to despair in these hostile climates without a means to heat
their homes, light their workplaces, or refrigerate their foods. The lack of resources and
electricity to produce and work refrigerators or other machines can prove detrimental as
medicine and foods must be preserved at specific temperatures. Federal Centers for
Disease Control and Prevention claim 97% of food illnesses can be linked to improper
food handling such as preparation practices and temperature control (Pivarnik). As a
result, alternative methods for preservation are necessary practices to eliminate or reduce
food borne illnesses from spoiled foods. These methods can include mechanical,
chemical, and natural processes. The experiment will compare a chemical method to a
natural method of preserving a worldwide food staple, milk.
The measure of spoilage in milk is not something many would consider a
quantifiable measurement. If the milk has a strong odor, strong taste, or coagulation, it is
considered spoiled by the general population (Marshall). However, there is a noted
method to accurately measure the spoilage of milk. Acidity of milk correlates to spoilage
level, and thus can be measured by pH level (Lu).

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Figure 1. Common Items on a pH Scale

Figure 1 lists general items in correlation to their pH value and pH strip color an
indicator of the acidity of a substance in which the strip is dipped in. Using a pH strip is a
standard indicator for applications not requiring an exact measurement. The pH is the
concentration of hydrogen ions in a solution. A solution with a pH level below seven is
acidic; one with a level above seven is basic (Ophardt).
The pH of unspoiled milk is 6.7, which is almost neutral (Lu). A neutral solution
is considered neither acidic nor basic (Ophardt). Many bacteria strains thrive at this level,
but are controlled by refrigeration, pasteurization, or other methods. Pasteurization is the
heating of milk to kill bacteria that causes food borne illness such as Salmonella, E. coli,
and Listeria (Dangers). The milk is most commonly heated in batches to an industry
standard of 62.8C for 30 minutes (Heat). However, if the milk is left vulnerable to
bacteria, lactic acid builds up as lactose (a main component of milk) breaks down, and
once the pH level is below 4.6, the proteins coagulate, and the milk is considered spoiled
(Lu). Milk contains two main proteins: casein and whey. Milk is naturally negatively

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charged, and this allows the casein to be uniformly distributed in the liquid. When acid is
added to the milk until it reaches a pH of 4.6, the casein protein reaches its isoelectric
point and forms a precipitate as it can no longer spread evenly. This precipitate is the
solid chuck found in spoiled milk (Food).
Besides the practices of refrigeration and pasteurization, another method to
prevent milk spoilage is to chemically stimulate the milks natural preserving agent. The
lactoperoxidase system in milk is a natural antibacterial function in milk. Thought to exist
to protect the immune systems of calves, slight amounts of thiocyanate (SCN-) and
hydrogen peroxide (H2O2) in saliva activate this mechanism (Reiter). Sodium thiocyanate
(NaSCN) and sodium percarbonate (2Na2CO33H2O2 ) are the industry standard sources of
thiocyanate and hydrogen peroxide respectfully (Guidelines). Chemically speaking, the
lactoperoxidase system is a multi-stage chemical reaction in which the lactoperoxidase
protein and hydrogen peroxide allows for the oxidation of thiocyanate. As the reaction
occurs, the thiocyanate (SCN-) decomposes into hypothiocyanous acid (HOSCN). This
acid ionizes, and the hypothiocyanate ions (OSCN-) block metabolic and reproductive
growth in the bacteria. Any excess ions revert back to thiocyanate (SCN-) (Guidelines).
This reversible reaction is shown below (Lactoperoxidase).
( aq )+ H 2 O(l)
( aq ) + H 2 O 2 ( aq ) OSCN
SCN
Simply put, when hydrogen peroxide (H2O2), thiocyanate (SCN-), and the
lactoperoxidase protein are in simultaneous contact with the bacteria cell wall, the cell
walls are also permeated and the bacteria is killed (Mickelson).

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According to common practice for milk preservation illustrated in the Codex
Alimentarius, a standard book on milk products produced by the World Health
Organization, adding 14 mg of sodium thiocyanate (NaSCN) and 30 mg of sodium
percarbonate (2Na2CO33H2O2 ) per liter of milk is sufficient to activate the
lactoperoxidase system without adverse side effects to the consumers (Guidelines). The
molarity of these chemicals in the milk solution is comparable to the levels in human
saliva furthering the proof that there are no health effects to humans (Guan). Molarity, M,
is the number of moles, mol, over liters of solution, L.
M=

mol
L

While not used in the United States or other developed countries, this common practice
was applied in the experimental design of the current research to determine the process
for preparing the chemically preserved milk.
In many parts of the world where processed chemicals cannot be purchased or
access to refrigeration is not available due to economic or technical reasons, the only
option is nature. Recent studies have shown that spices are capable of effectively
suppressing bacteria growth over long periods of time. One of these many spices is
Curcuma longa, more commonly known as turmeric. Turmeric is a naturally occurring
popular spice in South Asia and the Middle East used as a food preservative and colorant
(Sinha). The spice has proven effective in reducing the numbers of common bacteria such
as Salmonella typhimurium, Pseudomonas aeruginosa, and E. coli (most commonly the
0157:H7 strain) in milk products, and decreases the spread of Staph. aureus, B. cereus,
and Listeria monocytogenes contamination during the storage period of the milk products

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(Moghadamtousi). Several experiments demonstrate turmerics power in this scope of
research.
In Antimicrobial Activity of Curcumin upon Pathogenic Microorganisms During
Manufacture and Storage of a Novel Style Cheese 'Karishcum , written by I.M Hosney,
researchers investigated the addition of curcumin to Karish cheese in regards to
antibacterial activity and taste alike. Karish is a soft , raw milk based cheese which is a
staple in Egyptian food culture. Karish cheese (35 samples) was obtained from a Cairo
marketplace to act as a control, and Karish cheese was prepared with fresh milk and
various concentrations of curcumin extract (0.2% ,0.3%, 0.6%, 0.8%). The fresh samples
were mixed with strains of Escherichia coli 0157:H7, Bacillus cereus, Listeria
monocytogenes, Staphylococcus aureus and Salmonella typhimurium, as these are the
most common bacteria found in raw milk. The zones of inhibition were measured, and it
was discovered that the zones increased as the concentration of curcumin increased. The
0.8% sample was effective against every bacteria strain except Listeria monocytogens. It
was noted that adding curcumin did not adversely affect taste, and instead improved it.
In Antibacterial and Antifungal Properties of Turmeric, written by Neha Tandon
Sinha, a high school student designed a procedure to investigate turmerics effectiveness
in suppressing bacteria and fungi in common foods such as strawberries, bread, and milk
in an accessible experiment. It was predicted that the turmeric would be capable of
suppressing the growth of bacteria and fungi when applied to the provisions. The solid
food types and milk were conducted separately by analyzing different response variables:
turmeric's antifungal effect was tested on the bread and strawberries, and its antibacterial
effect was tested on milk. In the separate setting, strawberries and bread were each

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soaked in water and turmeric was applied; the foods were set aside for several hours. The
quantity of fungus from a scale from one to five - with 5 being the highest - was
recorded. The latter part of the experiment consisted of applying turmeric to milk. Using
the methylene blue test, the student was able to determine the amount of time the milk
was preserved. The study concluded that turmeric elongated the quality of the milk,
partially prevented fungal growth of the bread, and had no significant effect on fungal
growth of the strawberries. In her experiment, the student concluded that turmeric had an
antimicrobial effect with milk, signifying turmeric's ability to prevent bacterial and fungal
growth in selective foods. By estimating the molarity of the turmeric-milk solution used
in the simplistic experiment, the molarity used in the current research was derived. The
experimental design in Sinhas experiment was made more scientifically conscious with
precise measurements and use of a control for the current research. While pH was the
dependent variable measured, the use of the methylene blue test was kept because it
offered a visual marker for the spoilage level of milk.
The methylene blue test is an effective visual marker because it will not affect the
chemical composition of any solution when added; therefore, any additional reactions in
milk will not be affected by the methylene blue. It acts simply as an indicator and does
nothing to preserve the milk. Methylene blue has been used for treatments of infections
and poisoning or in its dye component for diagnostics (McDonaugh). The test is more
commonly used as a dye that stains components such as cells, DNA, tissues, and bacteria
(McDonaugh). Milk containing living bacteria (spoiled-milk) will decolorize with
methylene blue. The enzymes associated with these living bacteria will cause the milk to

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go from blue to white due to an oxidation-reduction reaction in which the methylene blue
acts as the hydrogen receptor (Sinha).
A chemical reaction that involves a transfer of electrons, such as the methylene
blue test, can be classified as an oxidation-reduction reaction, or redox reaction These
reactions involve the combination of oxidation and reduction, where oxidation is used to
describe the addition of oxygen or the removal of hydrogen from a molecule and
reduction is used to describe the addition of hydrogen. In general terms, oxidation is the
loss of electrons and reduction is the gain of electrons (Helmenstine). The transfer of
electrons in methylene blue is apparent in Figure 2.

Figure 2. Structural Formula of Methylene Blue in Function of pH

The parent structure of methylene blue and its oxidation and reductions states are
illustrated above in Figure 2 (Mio). These oxidation and reduction states appear as the pH
of each solution reaches below 5.4 or above 6.0 by result of spoilage or a preservative
application. The part of the reaction in which there is an increase in oxidation state is
considered the oxidation half reaction. With methylene blue (C16H18ClN3S ) applied to

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milk, the oxygen (O2) present in the milk solution will oxidize and color the milk blue
(Blue Bottle). The reduction half reaction is the part of the reaction where there is a
decrease in oxidation state, apparent as the milk spoils and falls to a pH below 5.4. The
oxygen is taken from the milk solution due to bacteria metabolism; the milk will then
turn back to white: an indicator of milk spoilage. The diagram presents the addition of
hydrogen atoms and transfer of electrons as methylene blue becomes reduced. Each
arrow shows a progressive path of the methylene blue reduction half reaction. Methylene
blue discoloration is a completely separate chemical reaction that does not affect other
reactions occurring in solution (Blue Bottle).
There is definite evidence that utilizing the lactoperoxidase system is an effective
way to chemically preserve milk where refrigeration is not available. There is also
evidence that turmeric is also effective in preserving milk products from harmful bacteria.
While no formal research has been done comparing the two, it is likely that both
processes will demonstrate higher pH levels and thus less spoiled milk than no
preservation technique at all.

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Problem Statement
Problem Statement:
Will the natural preservation technique of curcumin (turmeric) be as effective as
the chemical preservation technique of the thiocyanate-hydrogen peroxide system in
delaying the spoilage of raw milk without refrigeration?
Hypothesis:
The 0.0014 % sodium thiocyanate (NaSCN) and 0.003% sodium percarbonate
(2Na2CO33H2O2 ) raw milk solution will have a higher pH level than the 7.5 % curcumin
(C21H20O6) raw milk solution.
Data Measured:
The dependent variable of the acidity (spoilage) of the raw milk was measured
with the pH scale. The color change of the milk was noted as a visual marker of
comparative spoilage of the different types of milk. The independent variables of
treatment type were the thiocyanate-hydrogen peroxide chemical method, curcumin
natural method, and no treatment. No treatment acted as a control to determine if the
treated milk spoilage was any different from that of untreated milk. The statistical
analysis used for this experiment was an ANOVA test between the three mean pH levels.
As the ANOVA test showed that the means are all different, a two-sample t-test was run

Fricano-Le 14
to determine if the difference between the mean pH level of chemically treated milk and
the mean pH level of naturally treated milk was significant.

Experimental Design
Materials:
14 mg Sodium Thiocyanate (NaSCN)

Pipet bulb

30 mg Sodium Percarbonate
(2Na2CO33H2O2)

(3) 2L Beakers

75g McCormick Turmeric Powder

(3) Liters Burnside Farms Raw Milk
Methylene Blue Dye
(3) Weighing dishes
(3) Test tube racks
(90) 30 mL Test tubes
(90) Test tube stoppers
(3) 25 mL pipets

1000 mL Beaker
(3) Glass Stir Rods
Electronic Scale (0.0001 g)
Logger Pro Labquest
Vernier pH probe (0.01)
Gloves
Kimwipes
Distilled water

Procedure:
***All milk solutions must be prepared within one hour of milking and are not for
consumption***
1. Randomize milk type used in each trial (See Appendix A).
2. Clean all glassware according to protocol described (See Appendix B).
3. While wearing gloves, measure out 30 mg sodium percarbonate, and 75 g
Turmeric in separate weighing dishes using the electronic scale. Set aside.
4. Prepare milk solutions according to procedures below.
Chemical Milk Preservation
5. Measure 14 mg sodium thiocyanate using a weighing dish and the electronic
scale.
6. Add 14 mg sodium thiocyanate to one liter of raw milk. Stir for one minute with
glass stir rod to evenly distribute the chemical.
7. Add 30 mg sodium percarbonate to the sodium thiocyanate milk solution. Stir for
two minutes to ensure that the hydrogen peroxide is evenly distributed. Set aside.
Natural Milk Preservation
8. Add 75g turmeric powder to separate one liter of raw milk. Stir for one minute to
evenly distribute spice.
General Procedure
9. Set up Labquest with pH probe (See Appendix C).
10. Measure initial pH level of the three milk solutions (chemical, natural, and
control)

11. Fill 30 test tubes each with 25 mL of desired milk solution (chemical, natural, and
control) for a total of 90 test tubes with a separate pipet for each type.
12. Add 6 drops Methylene Blue dye to each test tube. Seal each test tube with
stopper. Swirl test tube to distribute dye.
13. Set aside test tubes for 48 hours.
14. After 48 hours, measure pH of each milk sample in the randomly allocated order
using Labquest. Record pH measurement in data table. Rinse pH probe in
between trials with distilled water, and wipe probe with kimwipe. Observe
methylene blue discoloration and record in observation table.

Diagram:

Turmeric

pH Probe

Test
Tubes

Sodium
Thiocyanate

Sodium
Percarbonate

Methylene
Blue

Pipet
Bulb

Raw Milk

Labquest

Figure 3. Materials and Experimental Setup

Figure 3 shows the materials used in the experiment and the setup. The test tubes
are numbered in the rack. See Appendix C for a more detailed setup of Labquest screen.

Data and Observations

Table 1
Natural Treatment Trials
Trial
Type
pH
1
Natural
5.39
2
Natural
5.6
3
Natural
5.62
4
Natural
5.67
5
Natural
5.61
6
Natural
5.74
7
Natural
5.83
8
Natural
5.75
9
Natural
5.7
10
Natural
5.58
11
Natural
5.33
12
Natural
5.67
13
Natural
5.54
14
Natural
5.49
15
Natural
5.8
Average
S. Deviation

Trial
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Type
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural

pH
5.35
5.44
5.97
5.42
5.48
5.68
5.38
5.65
2.12
5.63
5.57
5.72
5.52
5.63
5.8
5.49
0.65

Table 1 shows the pH levels measured for the natural treatment trials along with
the average and standard deviation of the pH levels. The standard deviation is higher than

the other groups, indicating more variability within the sample than the other groups.
However, it is still relatively low, indicating little variability. See Table 5 for a discussion
of the obvious outlier of trial 24.

Table 2
Chemical Treatment Trials
Type
Trial
pH
Chemica
31
l
5.04
Chemica
32
l
5.06
Chemica
33
l
4.88
Chemica
34
l
4.92
Chemica
35
l
5.30
Chemica
36
l
6.04
Chemica
37
l
5.12
Chemica
38
l
4.81
Chemica
39
l
4.99
Chemica
40
l
4.74
Chemica
41
l
5.09
Chemica
42
l
4.70

Trial
46
47
48
49
50
51
52
53
54
55
56
57

Type
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l
Chemica
l

pH
5.05
4.83
4.96
4.90
4.91
5.46
4.96
4.75
4.66
4.70
5.04
5.14

Chemica
43
l
Chemica
44
l
Chemica
45
l
Average
S. Deviation

5.43

58

4.63

59

4.96

60

Chemica
l
Chemica
l
Chemica
l

4.79
4.75
5.21
4.99
0.29

Table 2 shows the pH levels measured for the chemical treatment trials along with
the average and standard deviation of the pH levels. The standard deviation is low,
indicating little variability within the sample.

Table 3
Control Treatment Trials
Trial
61
62
63
64
65

Type
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l

pH

Tria
l

4.75

76

4.82

77

4.70

78

4.57

79

5.34

80

Type
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l

pH
4.65
4.44
4.83
4.79
4.32

Contro
66
l
Contro
67
l
Contro
68
l
Contro
69
l
Contro
70
l
Contro
71
l
Contro
72
l
Contro
73
l
Contro
74
l
Contro
75
l
Average
S. Deviation

4.70

81

5.48

82

4.84

83

4.79

84

4.67

85

4.49

86

4.82

87

4.48

88

4.77

89

4.53

90

Contro
l
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l
Contro
l

4.61
4.92
4.77
4.58
4.40
4.82
4.72
4.71
4.86
4.41
4.72
0.24

Table 3 shows the pH levels measured for the control treatment trials along with
the average and standard deviation of the pH levels. The standard deviation is low,
indicating little variability within the sample.
Table 4
Original pH Levels
Type
Original pH
Natural
6.93
Chemical
6.95
Control
6.93
Table 4 shows the original pH values of the three milk types. The pH was
measured immediately after the milk solution was made. The original pH was measured
to prove that there was milk spoilage, and to note a baseline pH for the milk itself.

Table 5
Natural Treatment Observations
Trial
Number

Observation

Trial
Number

Observation

1-3

No noticeable color
change. pH consistent
with other trials.

19-20

No noticeable color
change. pH consistent
with other trials.

21

Milk solution spilled

during transport. pH
still consistent

5-7
8

10
11-14
15
16-17
18

Stopper popped off at

unknown time. pH still
consistent with other
trials
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent

22
23

24

25
26-28
29
30

No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent
Milk solution spilled
during transport.
Lowest pH level
recorded, measured
several times. Lowest
level of milk solution
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent.
No noticeable color
change. pH consistent
with other trials.
Stopper popped off at
unknown time. pH still
consistent

Table 5 shows the observations for the trials performed with the natural milk solution.
No trends were apparent during the duration of trials. A general observation for the natural
trials was that the stoppers were placed much too tightly on some test tubes, and this allowed
for the stoppers to dislodge. The scientific reasoning for this occurrence is further discussed
in the conclusion. It was also noted the pipet bulb was an inaccurate measuring tool as it
would release either too much or too little milk solution at a time. Two cases of spillage

during the transport of the milk also greatly reduced the amount of milk solution (trials 21
and 24). These flaws did not appear to affect pH except in the case of trial 24.

Table 6
Chemical Treatment Observations
Trial Number

Observation

31-35

Definite color separation.

Creamy solid on top turned
white, rest of milk blue. pH
levels consistent with rest of
trials

36

Definite color separation, but

significantly darker blue
color. Highest pH measured

37-60

Definite color separation.

Creamy solid on top turned
white, rest of milk blue. pH
levels consistent with rest of
trials

Table 6 shows the observations for the trials performed with the chemical milk
solution. The pH levels measured were largely consistent, and no apparent trends over the
course of trials were recognized.

Table 7
Control Treatment Observations
Trial Number

Observation

Trial Number

61-64

Complete color change to

white. pH levels consistent
with rest of trials.

72

65

Extremely light blue tint to

entire test tube. pH slightly
higher than rest of trials

73-83

66

Complete color change to

white. pH levels consistent
with rest of trials.

84

67

68-71

Extremely light blue tint to

entire test tube. pH slightly
higher than rest of trials
Complete color change to
white. pH levels consistent
with rest of trials.

85-86

Observation
Color pocket of blue at
bottom of test tube, rest of
milk white. pH consistent
with rest of trials
Complete color change to
white. pH levels consistent
with rest of trials.
Color pocket of blue on side
of test tube, rest of milk
white. pH consistent with rest
of trials
Complete color change to
white. pH levels consistent
with rest of trials.

Table 7 shows the observations for the trials performed with the control milk solution.
No trends appeared over the course of trials.
24
hours

48
hours

Figure 4. Turmeric Solution Before and After

Figure 4 shows the turmeric milk solution after 24 and 48 hours. The stoppers
were removed in the picture due to the picture being taken after pH measuring on the
second day.

24
hours

48
hours

Figure 5. Chemical Solution Before and After

Figure 5 shows the chemical milk solution after 24 and 48 hours. The stoppers
were removed in the picture due to the picture being taken after pH measuring on the
second day.

24
hours

48
hours

Figure 6. Control Solution Before and After

Figure 6 shows the control milk solution after 24 and 48 hours. The stoppers were
removed in the picture due to the picture being taken after pH measuring on the second
day.

Data Analysis and Interpretation

The data collected for this experiment was reliable due to control, randomization,
and replication. Having a control reduced confounding because if any lurking variables
existed, they affected all the groups equally. It can also be a standard to compare to any
treatment groups. This experiment ran trials with raw milk to see if any change in
preservation type made a significant difference in the pH level of milk. If there was a
significant change in pH, the comparison to the control of raw milk (without treatment)
would allow the researchers to conclude if it was due to any treatment as opposed to no
treatment at all and then further investigate what treatment type reduced the spoilage of
raw milk the most. Randomization reduces bias because each unit is equally likely to get
picked. Thirty trials of each treatment group (natural, chemical, and control) were
completed to ensure the datas validity. By replicating the combinations of the trials, it
was ensured that the observed results were less likely to be affected by random chance.
All test tubes were numbered 1 through 90, with 1 through 30 being designated to the
natural group, 31 through 60 to the chemical group, and 61 through 90 to the control
group. Each trial was given a random number generated from the TI-Nspire
randomization function and was tested in the order of its random number value. The
random order ensured that no patterns developed over time due to human fatigue or
equipment within any one treatment type. The randomly allocated preservation types
allowed an even distribution of the treatments throughout the course of the experiment.
Replication allowed for valid conclusions to be drawn from the data, as the results can be
attributed to an actual difference and not a rare occurrence. It was a basic assumption of
the statistical treatment used to analyze the data.
In order to assess the data collected, it first had to be graphed (see Figure 1).

Figure. 7 Box Plots of Data Collected

Figure 7 shows the box plots created for the natural, chemical, and control trials.
Points of interest have been labeled. The trials for the chemical and natural group appear
to be skewed slightly to the right, and the trials for the control group appear to be left
skewed. All treatment types have one or more indicated outliers. Because of this, the data
was graphed again without outliers before any discussion of overlap began (see Figure 8).

Figure 8. Box Plots of Data Collected Without Outliers

Figure 8 shows the box plots created for the natural, chemical, and control trials
without the outliers. Points of interest have been labeled. The trials for the natural and
chemical group appear to be slightly right skewed, and the trials for the control group
appear to be slightly left skewed. The chemical trials appear to have the greatest spread in
the range, indicating some variability. The chemical group distribution overlaps the
control and natural distributions; however the distribution of the natural and control
group do not overlap each other at all. The natural group has the largest median, and the
control group has the smallest. It is likely that there is a significant difference between the
three means; a statistical test was run to confirm this.
An ANOVA test was used for this experiment to compare the means of the three
independent populations. It tested how far apart samples means are with regards to the
variation within the samples.
An ANOVA test has three main assumptions. Generalized assumptions were made
specific to the situation. The first assumption was that there are three independent simple
random samples for each of the three populations. Due to the randomization completed

during trials, it was reasonable to say that the data collected was a simple random sample
for each preservation type. Each treatment was distinct in its chemistry, so the
preservation types were separate populations. The second assumption was that each
population had a normal distribution. The data appeared to be normally distributed, but
even so, each sample had thirty data points. By the Central Limit Theorem, the sampling
distribution was assumed to be normal. However, there were some outliers in each
sample. The test was run with and then again without these outliers. In order to maintain
that the data was still normally distributed with outliers removed, normal probability
plots were created (See Figures 9, 10, and 11).

Figure 9. Normal Probability Plot for Natural Group

Figure 9 shows the normal probability plot for the natural group without outliers.
The data is primarily in a linear pattern, indicating it is normally distributed.

Figure 10. Normal Probability Plot for Chemical Group

Figure 10 shows the normal probability plot for the chemical group without
outliers. The data is primarily in a linear pattern, indicating it is normally distributed.

Figure 11. Normal Probability Plot for Control Group

Figure 11 shows the normal probability plot for the control group without outliers.
The data is in a linear pattern, indicating it is normally distributed.
The third assumption for an ANOVA test was that all populations have the same
standard deviation, whose value is unknown. A rule of thumb was that this assumption
would be met when the largest sample standard deviation was no more than twice as big
as the smallest standard deviation. This assumption was met with the outliers removed
(see Appendix D for table of all means, standard deviations, sample sizes, and
calculations for ANOVA test).

All ANOVA tests have the same set of hypotheses. The null hypothesis, Ho, was
that the means are all equal to one another, and the alternative hypothesis, Ha, was that
not all means are equal as shown below.
H o : n=ch =c
H a : not all n , ch c are equal
This null hypothesis was tested to see if a difference in the pH of milk occurred across the
three treatments types.
The test statistic, F, was calculated by dividing the variation among sample means
between each population, or mean square group (MSG), by the variation among
individuals in all samples of each population, or mean square error (MSE) as shown
below.
F=

MSG
MSE

A sample calculation of the F-statistic, along with formulas and calculations for MSG and
MSE can be found in Appendix D. Two ANOVA test were run with (see Figure 12) and
without the outliers (see Figure 13).

Figure 12. ANOVA Test with Outliers

Figure 12 shows a screenshot from the TI-Nspire program computing the results
of the ANOVA test done with outliers. The calculated F-statistic is 23.94, with a
corresponding p-value of 5.21 10-9 (see Appendix D for formulas and sample
calculations). The null hypothesis was rejected because the p-value of 5.21 10-9 was
significantly less than the alpha level of 0.05, the assumed significance level. Variations
in sample means this extreme would occur close to 0% of the time, if the null hypothesis
was true. This means there is a significant difference among the different preservation
treatments.

Figure 13. ANOVA Test without Outliers

Figure 13 shows the results of the ANOVA test done without outliers. The
calculated F-statistic was 202.33, with a corresponding p-value of 1.22 x 10-32 (see
Appendix D for formulas and sample calculations). The null hypothesis was rejected
because the p-value of 1.22 x 10-32 was significantly lower than the alpha level of 0.05.
Variations in sample means this extreme would occur about approximately 0% of the
time, assuming the null hypothesis was true. There was a significant difference among the
different preservation types. In running the test without outliers, the sample means were
much more accurate. This would explain why the null hypothesis was rejected at a greater
degree than with the outliers included. To test the difference between the natural and
chemical treatment groups, a two sample t-test was run.
To run a two-sample t test, the following equation is used, where n represents the
natural treatment and c represents the chemical treatment:

t=

In this equation,

x n

x n x c

s 2n s 2c
+
nn nc

is the sample mean from the natural group and

x c

is the sample

mean from the chemical group. The sample standard deviations from the two groups are

respectively

sn

and

sc

along with the respective sample sizes,

nn

and

nc

. The

t value calculated represents the number of standard deviations above or below zero, no
difference, on a t distribution that the difference between these two samples means lie.
See Appendix D for sample calculation of a two-sample t test.
The following hypotheses on the data set with and without outliers tested are:
H o :n = c
H a : n c
The null hypothesis,

Ho

, states that there is no difference in the mean pH levels

between the natural and chemical group,

hypothesis,

Ha

and

respectfully. The alternative

, states that there is a difference in the mean pH levels, as the

experimental results differed from the original hypothesis.

The assumptions for this test are met as each group is a sample from a distinct
population, the responses in each group are independent of each other, and the sample
sizes for both groups have normal distributions by the Central Limit Theorem and the
normal probability plots.

Figure 14. Two Sample t-test with Outliers

Figure 14 shows the results of the two-sample t test for the two groups (natural
and chemical) with outliers. The test produced a t value of approximately 3.79 and pvalue of almost zero.

Figure 15. Probability Distribution with Outliers

Figure 15 shows the shaded probability distribution for the test run with outliers.
Running the test produces a p-value of approximately zero. Therefore, the null hypothesis

was rejected; there is convincing evidence that there is a significant difference in the pH
level of milk when given the two different treatments. There is almost no chance of
getting this extreme of a difference in average pH by chance alone, if the null hypothesis
is true, that is, if there is no difference in the pH of milk when exposed to the two
treatments.

Figure 16. Two Sample t-test without Outliers

Figure 16 shows the results of the two-sample t test for the two groups (natural
and chemical) without outliers. The test produced a t value of approximately 13.09 and Pvalue of almost zero.

Figure 17. Probability Distribution without Outliers

Figure 17 shows the shaded probability distribution for the test run without
outliers. Running the test produces a P-value of approximately zero. Therefore, the null

hypothesis was rejected; there is convincing evidence that there is a significant difference
in the pH level of milk when given the two different treatments. There is almost no
chance of getting this extreme of a difference in average pH by chance alone, if the null
hypothesis is true, that is, if there is no difference in the pH of milk when exposed to the
two treatments.
Conclusion
This research attempted to ascertain the best method of non-mechanical
preservation for raw milk. In order to determine this, pH of spoiled milk when treated by
the natural (turmeric) method, the chemical (Lactoperoxidase system) method, and no
treatment were compared. The pH is a direct indication of the spoilage of the milk as
bacteria growth creates buildup of lactic acid (Lu). The pH of unspoiled milk is around
6.7, and spoiled milk with complete coagulation is around 4.6 (Food). It was predicted
that the chemical method would reduce the spoilage of milk the most since it is used in
industry as an alternative to pasteurization. However, the data did not support this
hypothesis. The hypothesis was rejected because there was significant evidence
illustrating that the natural method had the most normal pH.
From the ANOVA test, it was found that there was a significant difference
between the means of each sample with a p-value of 5.2110-9, illustrating that applying a
preservation treatment to raw milk produced a significant difference in pH compared to
no treatment at all. In other words, there was approximately a 0% chance of getting
results this extreme due to chance alone assuming all preservation types were equal.
Contrary to what was predicted, the raw milk treated by the chemical had a mean pH
(4.99) very close to that of the control sample (4.72), indicating that the chemical method

had little effect on the change of pH; when compared to the natural group, it was found
that there was a significant difference in pH with a p-value of 4.9910-4 with the natural
sample having a higher mean (5.49) than that of the chemical group (4.99). Again, there
was approximately 0% chance of getting a result this extreme due to chance alone if the
natural and chemical treatments had an equal effect.
While the chemical method presented in this research is a common, effective
method used to preserve milk, it is not designed to preserve the quality of milk over an
extended period of time. The addition of thiocyanate and hydrogen peroxide to raw milk
activates the milks natural antibacterial system, the Lactoperoxidase system, by
increasing the natural thiocyanate concentration. At 15 C, the system will preserve raw
milk up to approximately 24 hours ("What is Lactoperoxidase?"). This system allows
milk producers a sufficient amount of time to transport milk to a location suitable for
processing. As the experiment was conducted in extrapolated conditions - room
temperature (22 C) for a period of 48 hours - the Lactoperoxidase system could not
sustain the quality of the raw milk. Chemically speaking, the Lactoperoxidase system is a
multi-stage chemical reaction. As the reaction occurs, the thiocyanate (SCN-) in the form
of sodium thiocyanate (NaSCN) decomposes into hypothiocyanous acid (HOSCN). This
acid ionizes, and the hypothiocyanate ions (OSCN-) block metabolic and reproductive
growth in the bacteria. Any excess ions revert back to thiocyanate (SCN-) The oxidation
of the thiocyanate does not occur to a great extent naturally, so increasing the
concentration of thiocyanate and initiating the reaction with hydrogen peroxide in the
form of sodium percarbonate allows the reaction to operate to its full extent.
(Guidelines). At a neutral pH (as unspoiled milk is), this reaction is not stable and is

mainly temperature dependent, meaning it will happen faster at higher temperatures, and
slower at lower temperatures (Guidelines). Having the milk at room temperature will
drive the reaction to completion much faster than if it was refrigerated. Once the reaction
is complete, there are no more ions available to block bacteria growth. The only way to
extend the widow of time in which the reaction occurs to procure a more long term
method is to decrease temperature. This is why the lactoperoxidase system is not a
substitution for refrigeration.
Spices have been long known for their antimicrobial effect and preservation
power. What makes turmeric an especially powerful preserving agent is its active
ingredient, curcumin. Curcumin exhibits a broad spectrum of inhibitory effects against all
food-borne organisms and has been used for centuries to preserve foods over extended
periods of time (Y et. al). A 0.8% curcumin concentration in solution is sufficient to
preserve raw milk products like karish cheese against bacteria (Hosney). It is noted that
curcumin is not very soluble. The natural group's test tubes appeared to be over-saturated,
with solid turmeric settling on the bottom of the test tubes. This oversaturation may have
well caused the large difference in pH between the natural and chemical group. With
oversaturation, the pH reading of the naturally preserved milk mixture was abnormally
high compared to the chemically preserved mixture. Conventional turmeric powder has
an average curcumin concentration of 3.14% which is much higher than the solution that
Hosneys karish experiment utilized (Tayyem). The turmeric made approximately 7% of
the natural treatment milk solution. Hosneys experiment showed a correlation between
increasing the curcumin concentration and observing increased bacteria growth

suppression. It follows then that having a large concentration of turmeric in the milk
solution would be very effective in suppressing bacterial growth.
Overall, the results of this research agree with and support existing evidence that
both the chemical and natural treatment is effective in suppressing bacteria growth in raw
milk. However, no other published experiments exist comparing the two systems. More
data is required to make a more conclusive determination that one treatment option is
better than the other, as the quantity of each treatment differed.
Minor errors were encountered during the experiment. The turmeric did not fully
dissolve into the raw milk and settled at the bottom of the test tube. The milk solution
was not tasted, so it is unknown if the level of turmeric in the milk had adverse effects on
taste. It was assumed that the milk solution would be useful for sauces, but if the turmeric
content is indeed too high, the natural solution has no practical value outside of specific
recipes. The strong turmeric content might not be a favorable taste for the conventional
drinking of milk. Additionally, overnight, several of the turmeric group's test tube
stoppers dislodged from the test tubes. The common bacteria (E.coli, Listeria,
Salmonella, etc.) found in raw milk are all facultative anaerobic bacteria, meaning they
function aerobically and require oxygen when oxygen is present (Lowy). Regardless of
aerobic or anaerobic (not requiring oxygen) bacterial respiration, gases are produced as
the bacteria grow (Jurtshuk). If certain stoppers were initially wedged too tightly into the
test tubes, the buildup of gasses would increase the pressure within the test tubes. The
increased pressure would dislodge those stoppers with a tighter seal than those with a less
tight seal. Once dislodged, the tubes were left to the open air for an unknown amount of
time. The presence of oxygen may have increased the rate of the milk spoilage, as it

provided more oxygen for the aerobic bacteria to multiply. It was also impossible to keep
the environment of the milk truly consistent for the entirety of the experiment. The
mixtures of each group were created at an outside source and were then transported to the
school setting. The school temperature fluctuated over the two days of testing. A decrease
in temperature would have refrigerated and preserved the milk longer, whereas an
increase in temperature would have accelerated the milk decomposition.
A multitude of steps could be enforced in order to improve the procedures and the
experimental design to reduce experimental error and bias. Larger test tubes could be
used with the same amount of milk solution to decrease the pressure of the gases
produced. A larger space for the buildup of gases reduces the overall pressure. This way
all stoppers would remain secure, and the lurking variable of open air exposure due to
dislodged test tubes would be decreased significantly. The temperature setting of the
experiment could be kept constant, reducing the lurking variable of temperature
preserving or decomposing the milk.
In future experiments, one could test the preservation ability of various spices
available around the world. Turmeric is primarily grown in India and Asia; extending to
different areas in the world and their natural resources would prove beneficial on a global
scale, creating a far-extending application. Testing the limits of the lactoperoxidase
system by increasing the sodium percabonate and sodium thiocyanate concentrations
would allow the researchers to see if the system can be improved as it currently does not
last very long. The optimal conditions capacity could be increased if a means to drive the
reaction or stop it could be discovered. Through the use of bacteria cultures, it could also
be determined which strains of bacteria the lactoperoxidase system and turmeric are most

effective against. The effects of the chemical treatment could also be investigated as a
supplementary method to pasteurization instead of a replacement. The experiment should
also be explored in different temperature settings as countries without access to electricity
may have environments with extreme temperatures.
The practical elements of this research are numerous. Governments and public
health agencies world-wide can use the results from this research to better protect their
people against food-borne illnesses. Developing countries can use this experiment as a
basis to fall back on natural treatment when chemical and mechanical methods are simply
not available. Accessible refrigeration, pasteurization, or necessary chemicals may be too
expensive or remote for certain communities to consistently use. If no other preservation
method is useable, these third world communities can use a spice like turmeric to kill the
bacteria in their milk. Researchers can investigate ways to further the effectiveness of the
chemical method presented in the experiment. First world households can even use the
presence of spices such as turmeric to supplement other precautions such as refrigeration
in the quest to further preserve dairy products. Even with modern pasteurization
practices, the spoilage of milk will occur eventually with time. Adding antibacterial
spices could keep milk and milk products at optimal freshness for the average consumer.

Acknowledgements
Rick Lutz
Matthew Mio

Appendix A: Randomizing Using the TI-Nspire CX

In order to help insure good data, it is necessary to randomize the order that the
trials are done in. The trials received a random integer 1-90 for the order in which they
were performed in. 1-30 were allocated for the natural group, 31-60 for the chemical
group, and 61-90 for the control group.
Procedures for Randomizing:
1.

Turn on calculator

2.

On the home page, select new document and then a calculator page.

3.

Select menu, probability, random, integer.

4.

Type 1, 90, 90 in the parenthesis and press enter. This order is the order in
which trials will be performed.

5.

If there are any repeated numbers, copy the function and generate more integers
until the desired number of integers with no repeats is reached.

Appendix B: Glassware Cleaning Protocol

Materials:
2000 mL beaker
Hot Plate
Tongs
Thermal Gloves
Digital thermometer (0.01C)
80% ethanol solution

Procedure:
1.

Spray all test tube racks, lab surfaces, and thermal gloves down with ethanol
solution

2.

Fill beaker to 1400 mL mark with water.

3.

Turn hot plate on high.

4.

While wearing thermal gloves, monitor temperature of boiling water using

thermometer until the temperature reaches 98 C.

5.

Once the temperature is at least 98C, completely submerge all test tubes and
stoppers one at a time in boiling water using tongs. Once submerged, place test
tube or stopper on clean test tube rack to dry.

Appendix C: Using the Logger Pro

Before data collection with the Logger Pro can begin it must be setup first.
Setup and Usage Procedure:
1.

Turn on the Logger Pro by pressing the power button

2.

Choose New from the File menu

3.

Plug in pH probe to Logger Pro

4.

On the Meter Screen, select measuring mode as Selected Entry.

5.

To start data collection, press the green arrow button

6.

To save a measured pH level, tap the Keep button.

7.

Record the pH level for that trial. To examine a data point, tap that point.

8.

Save data by tapping the File Cabinet icon.

Figure 18. Labquest Screen

Figure 18 shows the screen of the Labquest with the pH probe connected. The pH
of tap water is being measured as a reference.

Appendix D: Sample Calculations

This appendix shows the calculations of the ANOVA test and the two sample t-test
used to analyze the data in this paper.
Table 8
ANOVA Test Values with Outliers
Treatmen
t Type
Natural
Chemical
Control

ni
30
30
30

XXi

si

5.49
4.99
4.72

0.65
0.29
0.24

Table 8 shows the values used to calculate all aspects of the ANOVA statistic with
outliers included.

Table 9
ANOVA Test Values without Outliers
Treatmen
t Type
Natural
Chemical
Control

ni
29
29
28

XXi

si

5.61
4.96
4.67

0.16
0.22
0.16

Table 9 shows the values used to calculate all aspects of the ANOVA statistic with
outliers excluded.

An ANOVA statistic, F, was calculated using the formula below in which the
mean square group was divided by the mean square error.

F=

MSG
MSE

Where MSG was the mean square group and MSE was the mean square error. MSG was
found using the formula below.

MSG=

n1 ( x 1 x )2+ n2 ( x2 x )2 +n3 ( x 3 x )2
I 1

In this formula, nz equaled the number of samples in group z, xxz was the average of group
z. I was the number of groups being compared. xx is

x =

n1 x 1 +n2 x2 +n 3 x 3
N

Where N was the total number of terms in all groups and the other variables were the
same as before. MSE, or the mean square error is given by

MSE=

( n11 ) s 12+ ( n21 ) s22 + ( n3 1 ) s 32

N I

In this formula, nz equaled the number of samples in group z, sz was the standard
deviation of group z, N was the total number of samples, and I was the number of groups
the samples were in. Figures 19 through 23 show sample calculations of these numbers,
up to the F-statistic.

x =

n1 x 1 +n2 x2 +n 3 x 3
N

x =

( 30 ) 5.49+ ( 30 ) 4.99+ ( 30 ) 4.72

90

x = 5.07

Figure 19. Sample Calculation of xx.

Figure 19 shows a sample calculation of xx, which was then used to calculate the
mean square group, or MSG.

MSG=

n1 ( x 1 x )2+ n2 ( x2 x )2 +n3 ( x 3 x )2
I 1

MSG=

30(5.495.07) +30 (4.995.07) +30 (4.725.07)

31

MSG=4.58

Figure 20. Sample Calculation of MSG.

Figure 20 shows a sample calculation of MSG, or mean square group.

MSE=

( n11 ) s 12+ ( n21 ) s22 + ( n3 1 ) s 32

N I

( 301 ) 0.652+ (301 ) 0.292+ ( 301 ) 0.242

MSE=
903
MSE=0.19

Figure 21. Sample Calculation of MSE.

Figure 21 shows a sample calculation of mean square error, or MSE, the second
part of finding the test statistic of an ANOVA test.

F=

MSG
MSE

4.58
0.19

F=24.35
Figure 22. Calculation of the F-statistic.
Figure 22 shows the calculation of the F-statistic, which corresponded to a Pvalue with degrees of freedom equal to the un-simplified
DF=

I1
N1

Where DF was the degrees of freedom, I was the number of groups, and N was the total
number of trials.

DF=

I1
N1

DF=

31
901

DF=

2
89

Figure 23. Sample Calculation of the Degrees of Freedom.

Figure 23 shows a sample calculation of the degrees of freedom.

A two-sample t test statistic, t, is calculated by dividing the difference between the

sample means, xx1 and xx2, by the square root of the squared sample standard deviations, s1
and s2, over the sample size, n1 and n2.

t=

( x 1 x 2 )

s1 s2
+
n1 n2

See Figure 24 for a sample calculation.

t=

t=

( x 1 x 2 )

s1 s2
+
n1 n2

( 4.994.72)

0.292 0.24 2
+
30
30

t=3.93

Figure 24. Sample Calculation of t- Statistic

Figure 24 shows the calculation of the t-Statistic which corresponds to a p-value.

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