The Effect of Time on the Spoilage of Lactose

A comparison of the effect of time on the spoilage of lactose containing milk, and almond milk, by measuring transmission, through a change in arbitrary, over a period of 5 days.

1: Introduction

Within our society, more people than ever are following dairy-free diets due to a rise in self-diagnosed ‘lactose intolerance’. Lactose intolerance is the ‘inability to digest lactose, a component of milk’, which is a result of ‘the lack of an enzyme called lactase in the small intestine.’ (MedicineNet, 2016). Therefore, as someone who faces a personal struggle with lactose intolerance, I have always been intrigued to see the comparison of the spoilage of lactose containing milk (semi-skimmed), and non-lactose containing milk (almond). This is due to the observations that I have made in my own home regarding the spoilage of the two different types of milk, that have both been stored in the fridge over an extended period. For my Internal Assessment, I will be conducting an experiment, which will directly compare the transmission of light in both the lactose milk (semi skimmed) and lactose-free milk (almond), as they go on to spoil over a period of 5 days at 37 degrees Celsius.

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The spoilage of milk is a fermentation process (Lu, 2013), which requires the use of enzymes; enzymes are not only biological catalysts of chemical reactions, but they are also oxidising agents, which aid to the spoilage of milk. By leaving my two milk samples at the same temperature, I will be able to investigate whether lactose or the above enzymes will spoil the milk quicker. The reason I chose to investigate lactose as my independent variable is due to its important role in the spoilage of milk. Lactose is an enzyme that functions as an oxidising agent, which aids the spoilage of milk. Lactose spoils milk when it breaks down into two monosaccharides, glucose and galactose, which trigger the build-up of lactic acid, thus causing the sour taste of the milk (Y, 2017); this process is called bacterial fermentation.

The inspiration for my IA originated from an experiment conducted by Al-Holy (2008). This experiment consisted of the researchers using reflectance spectroscopy as a way of measuring the transmission of milk as it pasteurises over three set temperatures. However, in order for me to be able to adapt this experiment to make it my own, I will be changing their independent variable to a constant temperature, but using two different milk types, which will be lactose-free (almond) milk and lactose (semi-skimmed) milk. When researching, I found this experiment interesting due to its different approach. By reading this experiment, I was able to learn about spectrophotometry, and how beneficial it is within the food industry. This experiment explores the change in transmission of light as lactose milk spoils over a period, giving the researchers a clear indication of the change in arbitrary as it pasteurises. The results from this experiment showed that when lactose breaks down into glucose and galactose, the formation of lactic acid causes a change in the transmission of the milk, which they established through a change in arbitrary on the spectrophotometer. Therefore, when conducting my independent enquiry, I will be focussing on the difference in arbitrary when there is no lactose present, in order for me to be able to see if the enzymes in lactose-free milk (gelatinase, pepsin, gastric lipase and gastric amylase) will perform similarly to the lactose over a course of 5 days. Although, when I reviewed this experiment I found that it has limitations. For example, this experiment is that they used peculiar temperatures for their independent variable, which were 6 degrees, 21 degrees and 37 degrees (Celsius), therefore when applying this study to my own investigation; I cannot use their data to predict a trend in my own.

2: Investigation

2.1: Hypothesis

H₁: As time increases, the transmission of light (arbitrary) in lactose milk (semi-skimmed) will increase more than in lactose-free milk (almond), meaning that the lactose milk will spoil more than the lactose-free milk.

H₀: As time increases, the transmission of light (arbitrary) in lactose-free milk (almond) will increase more than in lactose milk (semi-skimmed), meaning that the lactose-free milk will spoil more than the lactose milk.

2.2: Background Knowledge

Casein and fat are the cause for the white colour complex that milk reflects, meaning that they reflect all transmissions of light. As milk progressively spoils, lactase enzymes such as lactobacilli break down in the lactose in milk to form glucose and galactose, which result in the production of Lactic Acid, a hygroscopic organic compound. Lactic Acid causes the casein molecules in the lactose milk to clump together thus forming curds. The yellow liquid ‘by-product’ of this reaction is called Whey, leaving very different physical properties such as lumps of casein and a separation of milk and whey. I expect to see very different physical properties when observing the spoilage of the lactose and lactose-free milks due to the lactose free milk being unable to form lactic acid, which is primarily the cause of curds within milks. In the lactose free milk, I do not expect to see any changes in the texture of the milk or any sour smells as the enzymes in the milk does not contain casein molecules.

2.3: Variables

Independent Variables: the type of milk. I will be using lactose milk (Semi-Skimmed) and lactose-free milk (Almond).

Dependent Variable: the transmissions of light (arbitrary) when a sample of milk is passed through a SpectroVis plus.

Controlled Variables:

The temperature of the environment was maintained at 37 degrees (the optimal temperature for enzymes) by a water bath.

The volume of water in the water bath was kept at 500ml so that the milk samples were kept submerged by the same amount each time.

Every time a sample was taken a different pipette was used, this was to prevent any cross-contamination of bacteria, and for my samples to be completely separate.

The time of day when the readings were taken was kept constant (8:00am); to ensure the time gap between recordings was precisely one day.

Every time a sample was taken, the same SpectroVis was used so that the samples could be compared. The SpectroVis measures the arbitrary of light (the percentage of light transmitted)

The same sized cuvettes were used to take the sample, so that the same volume of milk was placed through the GoDirect SpectroVis Plus.

The cuvettes were placed in the SpectroVis Plus for the same amount of time (1 minute 30 sec) in order for my readings to be taken after the same amount of time.

2.4: Preliminary Experiment

A preliminary experiment was conducted over a period of 2 days to evaluate if any alternations to the original methodology were necessary. The method used for the preliminary experiment was the same as that given in section 3.3, except for the fact that I used different time intervals varying by 30 seconds (±0.05 seconds), in order to find the optimum time to use. The value that I found to be the most successful in measuring concordant results was 1 minute 30 seconds.

3: Procedure

3.1: Apparatus

1)      1 Water Bath

2)      250ml Alpro’s Almond Milk

3)      250ml Semi-Skimmed Milk

4)      2 Conical flasks (250ml)

5)      2 orange bungs

6)      1 GoDirect SpectroVis plus

7)      2 pipettes

8)      2 measuring cylinders

9)      cuvettes

3.2: Photograph of set-up

A photograph taken by myself using an iPhone 5S, on 14/09/2018, that displays the Almond Milk and Semi-Skimmed Milk samples, as well as my water baths.

3.3: Methodology

1)      Fill the water bath carefully and set to 37°C

2)      Measure out 250ml (+/- 0.5ml) of Lactose Milk (Semi Skimmed) into a conical flask with a measuring cylinder, and firmly place an orange bung on the top.

3)      take an initial sample using a pipette and place it into the 1 GoDirect SpectroVis plus by squeezing 2 drops of the milk into the cuvettes.

4)      Repeat the above with the Lactose-Free Milk (Alpro’s Almond). Label each conical flask with correct name of ‘Lactose’ or ‘Lactose Free’

5)      Partially submerge ¾ the flasks into the water bath (one of each type of milk), and secure with the clamp stands.

6)      Take small samples of both using the pipettes and calculate the transmission of light (arbitrary) every 24 hours

7)      Repeat all of the above 5 times in order for you to gain 5 sets of results

8)      Calculate your range, mean change in arbitrary and compare your results for both types of milk.

3.4: Justification

The mentioned independent variable (the type of milk used – lactose-free (almond) milk or lactose (cow’s) milk) was chosen because the aim of my experiment is to measure and compare how quickly lactose milk spoils compared to lactose-free milk, in this case almond milk. I selected Almond Milk as the lactose-free milk in the experiment as it is readily available in a range of supermarkets and is widely used by the lactose intolerant / vegan population.

3.5: Risk Assessment

Safety Issues: due to the handling of hot water, glass wear and bacteria, I will ensure that I wear suitable items to protect me during the experiment, such as glasses, having my hair tied back, and wearing appropriate PPE such as gloves and a lab coat. Moreover, I will be keeping the milk contained in a sealed container due to allergies, intolerances and a general dislike to milk. Lastly, I will not be allowing people to touch, drink, or smell the milk prior, during and following my experiment to prevent cross-contamination and exposure to air.

Ethical Issues: from an ethical prospective, another reason why I will be containing the lactose milk in a clearly labelled container is to make it clear for people who are Vegan; therefore, they would not go near the lactose milk, or mistake lactose milk for non-lactose milk.

Environmental Issue: to protect the environment around me, I will be sealing the milk throughout the experiment in a conical flask with an orange bung covering the top, thus sealing it and preventing cross-contamination with the air and local environment. This will not only prevent the spreading of bacteria, but will also prevent the smell from spreading during the course of the experiment.

4: Raw Data

Raw Data Table 1: A table showing how the transmission of light in samples of Lactose and Lactose-Free Milk varies over 5 days in a water bath. (±0.05 arbitrary)

Raw Data Table 2: A table showing how the transmission of light in samples of Lactose and Lactose-Free Milk varies over 5 days in a water bath. (±0.05 arbitrary)

Raw Data Table 3: A table showing how the transmission of light in samples of Lactose and Lactose-Free Milk varies over 5 days in a water bath. (±0.05 arbitrary)

Raw Data Table 4: A table showing how the transmission of light in samples of Lactose and Lactose-Free Milk varies over 5 days in a water bath. (±0.05 arbitrary)

Raw Data Table 5: A table showing how the transmission of light in samples of Lactose and Lactose-Free varies over 5 days in a water bath. (±0.05 arbitrary)

I have decided to have 5 repeats as it will allow me to gain a reliable set of data as I will be able to easily spot and disregard anomalies, as well as making my mean more reliable as more values makes the mean closer to the true value.

5: Processed Data

Processed Data Table 1: A table showing how the mean transmission of light in samples of Lactose and Lactose-Free Milk varies across 5 days (±0.05 arbitrary)

Table of Means

Calculation of average transmission of light: $\frac{\sum \mathit{ transmission\; of\; light\; on\; each\; day\; for\; each\; repeat}}{\mathit{Number\; of\; repeats}}$

The average transmission of light will be rounded to two decimal places [2.d.p] because the uncertainty of the transmission of light is ±0.05g. This uncertainty is because ± 0.05 is half of the smallest reading on my apparatus. .

Processed Data Table 2: A Table showing what the mean and standard deviation in samples of Almond Milk and Cow’s Milk are at both 24 hours and 96 hours

5.1 Statistical Test

In order to determine if there is a significant difference between the rates at which the milks spoil, I will perform a Student’s T Test. This statistical test will compare the means and standard deviations of the two sets of data to confirm if any differences are due to the manipulated variable (type of milk) or due to chance. I will be using my means and standard deviations after 96 hours as this set of data will show how the milk’s were affected after a significant period. The equation used is:

Where:    

• x1 is the mean of sample 1           
• s1 is the standard deviation of sample 1             
• n1 is the sample size of sample 1                
• x2 is the mean of sample 2                
• s2 is the standard deviation of sample 2                
• n2 is the sample size in sample 2

Result:

t = $\frac{(895.06–478.04)}{\sqrt{\frac{(4.98)²}{5}+\frac{(8.82)²}{5}}}$

t = 23.45

Graph 1: A graph showing a constant temperature affects the transmission of light (arbitrary) in lactose (semi-skimmed) milk and lactose-free (almond) milk over a period (days)
 

The errors bars were set to the maximum error per reading, which was found to be 5% [rounded to 3 significant figures (s.f)].

Calculation for maximum percentage error

maxiumum = $\frac{\mathit{uncetainty\; of\; apparatus\; used\; to\; measure\; dependent\; variable}}{\mathit{lowest\; dependent\; variable\; value }}*100\%$

= $\frac{0.005}{378.9}*100\%$

     = 0.011% (rounded to 3. s. f)

The error bars of the graph represent standard error on each data point and the line of regression indicates a strong positive correlation between transmission of light in almond milk and cows milk, over a period of time. The size of the error bars indicate that the early readings had a lower chance of error, increasing the accuractness of the data that I obtained during my experiment.

4.2: Notes and Qualitative Observations

1) The Cow’s Milk (lactose) showed signs of spoilage earlier than the Almond Milk (lactose-free)

2) The Cow’s Milk (lactose) produced a foul smell, whereas the almond milk carried the same ‘nutty’ smell throughout the experiment.

3) Both Milk’s produced clumps of spoiled milk, which were thick in texture.

4) The Cow’s milk’s (lactose) naturally occuring emulsifiers broke down very quickly, in comparison to the Almond Milk’s (lactose-free), which broke down into a clear liquid in an extended time.

5: Evaluation

5.1: Conclusion

From the results of this experiment, I can conclude that the transmission of light (arbitrary) in Lactose Milk is greater than the transmission of light (arbitrary) in Lactose-Free Milk. Therefore the null hypothesis that I presented in section 2.1 is rejected and my initial hypothesis (H₁) is accepted. I have displayed this through my observations, results, statistical tests and my graph, which all follow my experiment. The use of my t-test and standard deviations indicate that there is a strong correlation between the differences in spoilage in these two milks. This supports my hypothesis as my t-test shows that there is a significant difference in the ways the milks respond to being left at a set temperature over time. As part of my experiment, I have also proven that the transmission of light in lactose milk is larger than in lactose-free milk when passed through a spectrometer (SpectroVis Plus), as in lactose milk there is a larger amount of suspended particles thus meaning that there are more bacteria particles and other metabolites. The role of the bacteria in milk is to break down Lactose into Glucose and Galactose, this is due to lactose-free almond milk not containing lactose, and therefore the enzymes in the lactose-free almond milk are less penetrable than in regular lactose milk, meaning that they take longer to break down.

This is evident in my averages, more specifically the mean transmission of lactose-free almond milk and the lactose semi-skimmed milk after being exposed to a consistent temperature for 96 hours (processed data table 2) as at 24 hours, the average transmission of the lactose milk was 381.2 arbitrary units, which was less than that of the lactose-free milk(381.98 arbitrary units). However, after 96 hours had passed, the average transmission of lactose milk was significantly higher than that of the lactose-free milk, with the difference between the two being 387.02 arbitrary, thus supporting my statistical tests.

5.2: Strengths

A strength of my experiment is that it has a low systematic error due to my low level of uncertainty (±0.05nm). Moreover, my maximum percentage error was very low (0.011%) in comparison to the large levels of transmission that I faced throughout my experiment. Overall, my figures were very strong as they showed a strong correlation through my standard deviations and statistics test; the t-test.

Another strength of my experiment is that I maintained the temperature at which the milk was kept in by using a water bath. This ensured that there was no fluctuation in the temperature, as I know from previous knowledge that temperature is a factor affecting enzyme performance, which could have affected my data significantly.

Lastly, the small error bars on my graph indicate that I had a low chance of an error occuring, which systematicallt increases the certainty and accuracy of my collected data over the course of my experiment.

5.3: Weaknesses

A weakness of my experiment is that my experiment is soley focussed on the short term effect of time on the transmission, focussing majorly on the change in the number of suspended particles in each of the milks, which changes as the milk’s go on to spoil. Therefore, this limits the extend to which my experiment is applicable in regards to my real life situation as I only looked at the effect over a short period of 5 days in order for me to obey my institutions health and safety concerns regarding the bacteria in the milk’s.

5.4: Extensions

Following on from my point above; if I was able to adapt and repeat this experiment, I would change the temperature at which the milk’s are stored, meaning that I could observe and take readings on the changes present for a much longer period of time, which would allow me to be able to apply my findings back to my real life situation of milk being left on the side in a building. 

Bibliography

• Al-Holy, 2008. Monitoring quality loss of pasteurized skim milk using visible and short wavelength near-infrared spectroscopy and multivariate analysis.. [Online]
  Available at: https://www.ncbi.nlm.nih.gov/pubmed/18292250
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• Lu, M., 2013. Milk Spoilage: Methods and Practices of Detecting Milk. [Online]
  Available at: https://file.scirp.org/pdf/FNS_2013070515384782.pdf
  [Accessed 12 October 2018].
• MedicineNet, 2016. Medical Definition of Lactose Intolerance. [Online]
  Available at: https://www.medicinenet.com/script/main/art.asp?articlekey=6205
  [Accessed 14 September 2018].
• S, P., 2017. Spectrophotometric Color Measurement Assures Milk Quality and Safety. [Online]
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