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1. Introduction
I have always loved drinking milk, and it is found in my daily eating routine. My favorite
way of drinking milk is to drink it raw, but occasionally I drink milk with either Quice(syrup),
fruits, or Roohafza(syrup). Milk, a nutrient-rich food produced by the mammary glands of
mammals, is drunk by many. Milk has many sound effects on the human body such as:
strengthening your bones by providing nutritional values of calcium, phosphorus, B vitamins,
potassium, and vitamin D [1]. However, milk can also damage the human body if drunk when
spoiled. Drinking milk when spoiled could lead to effects such as abdominal cramps, vomiting,
and diarrhea.
Spoiled milk is not necessarily bad. Spoiled milk is often used to make sweets, salad
dressings, cheese, and to bake. An example of how spoiled milk is used in my daily life is
through the use of Rasmalai. Rasmalai has been a special dessert regulating the dinner table since
I was young and is what prompted me to carry out this investigation. The Pakistani dessert,
Rasmalai, is made through the use of spoiled milk. Spoiled milk is used in Rasmalai since
spoiled milk acts as a leavening agent.
2. Exploration
2.1: Research Question: How do varying temperatures (5°C, 10°C, 15°C, 24°C, 45°C ) affect
the rate of milk spoilage (3.25% fat) over 72 hours, measured by the change in pH?
2.2: Variables:
Dependent Variable:
- Rate of milk spoilage measured by the change in pH
Independent Variable:
- Temperatures
Controlled Variable:
Variable Identification of Reason for controlling the variable +
Variable How to control the variable
Light Intensity The intensity of light is kept the same, as the intensity of light could cause a change in
Controlled temperature. The light bulb or sun could potentially increase the temperature and therefore change
Variable the ideal temperature. The only exceptions will be the samples placed in 45°C; a light bulb is used
to increase the temperature, but the rest of the samples will be provided with no light.
pH probe Instead of using pH strips to take measurements, pH probes will be used throughout the
experiment. This will help with the precision of the results.
Volume of milk The volume of milk placed within the sample will also be kept constant. This is to make sure the
experiment is set fairly. If a sample were to have a higher volume of milk, it can be presumed that
the experiment was not fair as that higher volume of milk might have more bacteria production
and fermentation.
Number of trials The number of trials will also be kept constant to minimize errors and also to have conclusive
evidence of the results at the end of the experiment. Not only that, but the number of trials is kept
constant to ensure the fairness of the experiment.
Source of milk The source of milk will be bought from the same company (Almarai), at the same time, with the
, same expiry, and from the same store. This is to ensure that the milk is as close to being similar as
it possibly could be. This is done to minimize any mistakes as bacteria production and
fermentation could play a role if not held constant.
Equipment used Identical beakers were used throughout the experiment to ensure that every milk sample contained
the same amount of milk or close to the same amount of milk. Not only that but the same type of
thermometer was also used to have consistent temperature readings throughout the experiment.
2.3: Background Information:
85% of the world’s milk production comes from cow species, followed by goats, ewes,
mares, donkeys, camels, sheep, and buffaloes. One thing in common is that milk in all species
contains a percentage of proteins, salt, water, lipids, minerals, and carbohydrates [4]. More
specifically, milk contains carbohydrates in the form of lactose, proteins in the form of casein,
and lipids in the form of butterfat. Additionally, for this experiment, cow’s milk will be used.
Cow’s milk has the protein casein and also the protein whey, which mainly includes
α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) [3]. Understanding the composition of milk is
the key to understanding the nature of milk spoilage. To be more precise, it is the bacteria in the
milk that causes the milk to spoil.
The production of lactic acid is the key to milk spoilage. Milk contains significant
amounts of lactose sugar, which bacteria uses to ferment into lactic acid. Milk spoilage occurs
from the metabolism of lactose, nitrogenous compounds (proteins, amino acids, urea, ammonia,
etc), unsaturated fatty acids and triglyceride, which produces lactic acid [3].To be more specific,
lactic acid bacteria (LAB) are responsible for the fermentation of
lactose to lactic acid or lactate. The most common LAB in milk
includes Lactococcus, Lactobacillus, Leuconostoc,
Streptococcus and Enterococcus [5].
Lactic acid bacteria, firstly, breaks down lactose into
glucose and galactose with the help of the enzyme called lactase.
This is done in order to use the glucose for cellular respiration.
The glucose goes through the anaerobic glycolysis reaction to
produce lactic acid or lactate (shown to the right). It goes
through the anaerobic process as the LAB does not use oxygen.
The pH of milk is approximately 6.7 pH. Spoiled milk
has a pH of 4.0 - 5.0 and this is due to lactic acid production [6].
Since lactic acid is an acid, it will decrease the overall pH of the milk. As the pH decreases, more
lactic acid is produced, and more of the milk is spoiled. Hence, pH is used as a measure to detect
and analyze milk spoilage for this experiment.
Methods that are commonly used to lower the rate of lactic acid fermentation, in order to
stop milk spoilage, include refrigeration, salting, dehydration, and pasteurization. The
experiment will aim to investigate temperature as a method to observe the rate of spoilage in
milk. The temperatures used were -4°C, 5°C, 15°C, 24°C, and 45°C. These temperatures were
selected due to their impacts on bacteria. -4°C and 5°C were chosen as these temperatures have
no bacteria growth. 15°C and 24°C were chosen as these temperatures are usually temperatures
, at which bacteria are normally found. 45°C was chosen because at 45°C, the bacteria growth is
most efficient. One thing to note is that the protein, whey and casein, denature at a temperature
of about 70°C. Thus proteins and amino acids, responsible for lactic acid fermentation, will most
likely not denature at 45°C as 45°C is not a high enough temperature for protein denature [8].
2.4: Hypothesis:
H1 = There is a significant difference between the change in pH between the different
temperature conditions.
H0 = There is no significant difference between the change in pH between the different
temperature conditions.
This experiment expects that there will be significant difference between the change in
pH between the different temperature conditions. The experiment hypothesizes that the higher
the temperature the quicker the milk spoilage, and vice versa, the lower the temperature the
slower the milk spoilage. The highest temperature, 45°C, will have the quickest rate of milk
spoilage within 72 hours and will have the most significant difference in pH due to higher
increased production of lactic acid. This is because the enzymes - proteases, lactase and catalase
- in Lactic Acid Bacteria, responsible for the fermentation reaction, are working faster due to
being at a higher temperature. The 24°C samples will go through a significant change in pH, but
not as significant as the 45°C samples. The 15°C samples are expected to go through some
significant differences, but not much. The 24°C The 5°C and -4°C samples are expected to go
through a non-significant difference. The lower temperatures such as 5°C and -4°C will go
through a slow rate of fermentation reaction due to decreased rate of enzyme productivity. The
graph below shows the rate of change for each temperature, which shows that the 45°C will go
through the most change.
Graph 1: Predicted rate of change in pH for each temperature
2.5: Risk assessment:
Risk Risk Management
Use of electronic equipment pH probes have vast electricity. Special care will be used while using the probe - making sure liquid
and glassware products are not near the pH probe.
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