The Effect of Different Temperatures on the Growth of Bacteria in Milk

The Effect of Different Temperatures on the Growth of Bacteria in Milk. Research Question: To what degree does the initial increase in temperature (20° C,40° C,60° C,80° C,100° C.) of milk have an effect on the rate of growth of Bacteria (Lactococcus lactis) (measured by changes in pH levels) over a fixed period of time (24 and 48 hours)? Content 1. Introduction 2. Exploration 2.1 Background Information 2.2 Research Question 2.3 Aim 2.4 Hypothesis 2.5 Variables 3. Experiment Design 3.1 Apparatus 3.2 Methodology 3.3 Risk Assessment 4. Results 4.1 Qualitative Data 4.2 Quantitative Data 4.3 Graphical Data 5. Analysis 5.1 Sources of Error 5.1.1 Uncertainties 5.1.2 Random Error 5.2 Discussion of Results 6. Evaluation 6.1 Strengths 6.2 Limitations 7. Bibliography 1.Introduction: Consumption of milk has been a constituent of the human diet for centuries, and is consumed on a daily basis by majority of the world and due to its biodegradable nature, there have been many efforts to try and increase its shelf life and example of this being Pasteurisation. Pasteurisation of milk to increase its shelf life was first done by Louis Pasteur in in 1862, it is the process by which milk is treated with mild heat, usually less than 100 °C, to eliminate harmful pathogens which spread food borne diseases. Growing up in both England and India I was quick to realise that the milk that was purchased in India was usually brought to a boil after purchasing it to increase its shelf life despite it being industrially pasteurised, I concurred that this is possibly due to fluctuations of temperature during transportation where the cold chain i.e., the maintaining of a certain temperature to ensure quality and avoid spoilage[8]; was often not maintained thereby increasing the risk of quicker spoilage due to an overall warm climate. However, this was not the case in England possibly because of the comparatively colder climate which posed less of a risk of the milk getting spoilt and the conditions to avoid spoilage of milk are maintained. Since the optimum temperature range for most bacterial growth is 30 °C and 40°C [11] , I was curious to test at which temperature of bacterial growth would be highest. Therefore, I decided to perform this experiment to study the rate of growth of the bacteria in milk at varying temperatures. 2. Exploration: 2.1 Background Information. Bacterial Growth: A population of a certain type of bacteria can increase rapidly over a period of time this is due to their ability to reproduce exponentially, bacteria reproduce asexually under favorable conditions through the process of binary fission where the bacteria would replicate its singular DNA molecule and create a copy of the original chromosome after which the cell continues to grow forming a septum between the two DNA molecules. Eventually a distinct cell wall is formed around the two DNA molecules resulting in two different bacteria. Conditions which favour bacterial growth after pasteurization: Although pasteurization of milk kills almost all acid forming bacteria, except heat resistant ones such as Bacillus, Clostridium, Lactobacillus, Micrococcus and Streptococcus. These bacteria often grow at low temperatures and some are also psychotropic i.e., they grow optimally at 7 ° C. However, the bacteria which is the most common reason for milk fermentation is Lactic acid bacteria (Lactococcus lactis), which causes the sour taste and unpleasant odor in spoilt milk. Milk contamination: Milk is contaminated during the processes of milking, handling, storage, and other pre-processing activities. If we look into the microbiology of milk one of the types of bacteria present in milk are lactic acid bacteria this group of bacteria help in the fermentation of lactose to lactic acid, they are usually present in milk inherently and are also used in starter cultures in the production of dairy products such as yogurt. Some examples of lactic acid bacteria include Streptococcus lactis and Lactobacillus casein. Coliforms are another type of bacteria present in milk they are anerobic in nature and have an optimum growth temperature 37 ° C. they are responsible for the rapid spoilage of milk; they are able to ferment lactose to produce acid and gas. Escherichia coli is an example of a coliform. Relationship between the spoilage of milk and pH: The pH of unspoiled milk is around 6.7 (Michael Lu, 2013). Acidity tends to increase as the milk spoils; therefore, acidity can be used to measure the quality of milk and to what degree it’s been spoilt. Acidity in dairy products can be measured by measuring the hydrogen ion concentration or pH which indicates the strength of the acid. At normal pH levels, the main protein in milk is casein, remains evenly dispersed. (Michael Lu, 2013). As the pH starts to decrease the protein coagulates sue to the acid produced from fermentation. There are a few devices that exist nowadays which can measure the acidity levels of milk, pH electrodes and titrators quickly and accurately measure pH levels of milk and are often used by manufacturers as a means of quality control. Raw milk has high moisture, an almost neutral pH and is rich in nutrients all which make it an excellent medium for microorganisms and in turn making it prone to spoilage. Milk turns sour in both odor and taste as a result of spoilage which leads to the production of small amounts of propanoic acids by lactic acid bacteria which in turn lowers the pH and inhibits the growth of other microorganisms. 2.2 Research question: To what degree does the initial increase in temperature (20° C,40° C,60° C,80° C,100° C.) of milk have an effect on the rate of growth of bacteria in milk over a fixed period of time? 2.3 Aim: To investigate the effects of initial heating of fresh milk over a fixed duration of time. To understand which temperature is most favorable to bacterial growth. 2.4 Hypothesis: Since the optimum temperature for Lactococcus lactis (LAB) to grow between is 30-40 ° C [11]. The recorded pH of the milk corresponding to these temperatures would be the lowest indicating higher rates of bacterial growth. 2.5 Variables Independent variable: Temperatures at which milk is heated. Dependent variable: pH of skimmed milk after 24 hours and 48 hours. Controlled variables: Variable Method of Control Volume(cm ) of milk heated and Each sample of milk was 10cm3 and measured poured into each petri dish using a 25cm3 measuring cylinder Medium in which bacterial growth was All samples of milk were observed in petri dishes observed and measured of the same diameter(90mm). Method of heating the milk. All milk samples were heated using a hot water 3 bath to ensure even distribution of heat. Temperature of the room where petri All of the samples were placed in the same room dishes where placed after their initial heat treatment. Type of Milk used. All of the milk was sampled from the same carton i.e., skimmed milk. Table 1 3. Experiment Design 3.1 Apparatus: 1) 2) 3) 4) 5) Fresh milk* Petri dishes (16) Hot water baths Beakers (5) pH meter 6) 25cm3 measuring cylinder. 7) Thermometer. 8) Labeling stickers 9) Pencil Health and safety equipment: 1) Lab coat 2) Laboratory goggles *Note: For the purpose of this experiment Almarai’s low fat cows’ milk was used 3.2 Methodology: 1) Take 5 samples of fresh milk which are 10cm3 each 2) Pour each sample in to a beaker in order to be heated. 3) Heat each of the samples of milk to 20,40,60 80, 100 ° C respectively using the hot water bath. 4) Use a thermometer to verify the temperatures. 5) Label each petri dish according to its respective temperature. 6) Once the required temperature is obtained pour the heated milk into its respective petri dish 7) Observe the bacterial growth over the course of 48 hours within the petri dishes. 8) Measure the pH of each sample, using a pH meter dipping the electrode in distilled water after each use to avoid “confusing” the meter, every 24 hours. 9) Observe the qualitative changes in the milk after each 24- hour period and record them. 10) Record other physical changes within the petri dishes. 11) Repeat steps 1-11 for two more trials. Image showing arrangement of the milk samples, on 25/11/2020 Control experiment: A sample of the milk was left unheated and kept in the refrigerator for 48 hours and its pH was measured every 24 hours. 3.3 Risk Assessment Apparatus Hot water bath Hazard Involves electricity and hot water. Prevention Make sure there is no contact between the water and the electric socket and precaution must be taken whilst handling the hot water. Table 2 Environmental and ethical concerns Disposal of the milk samples must be done carefully by placing them into a plastic bag and throwing them away. No particular environmental concerns however contamination of humans or plants in proximity must be considered. 4.Results 4.1 Qualitative Data After 24 hours no noticeable physical or visual changes are noticed However, it was observed that after 48 hours the milk in the petri dishes of 20 and 40° C respectively showed visible discoloration showing a slight brown tinge. Furthermore, it was also observed that after 48 hours the milk showed slight curdling* had a mild sour odor. In the sample heated to 60° C there was mild discoloration as the milk had turned off-white and there was no visible curdling*. In the petri dishes where milk had been heated to 80° C and 100° C there was neither discoloration nor curdling*. *As the pH of milk drops, casein molecules in milk attract each other and float in a solution of translucent whey. 4.2 Quantitative Data Temperature +/- 1° C. pH after 24 hours Trial 1 Trial 2 20° C. 6.56 6.55 40° C. 6.43 60° C. 80° C. 100° C. Mean pH* +/- 0.01 pH after 48 hours Mean pH* +/- 0.01 Trial 3 6.56 6.56 Trial 1 6.46 Trial 2 6.38 Trial 3 6.41 6.41 6.44 6.43 6.43 6.31 6.35 6.37 6.34 6.60 6.59 6.62 6.60 6.48 6.48 6.49 6.48 6.68 6.68 6.68 6.68 6.58 6.60 6.61 6.59 6.72 6.70 7.73 6.72 6.60 6.59 6.59 6.59 *Values taken to 2 significant figures. Table 3. Note: The initial pH of the milk was measured using the pH electrode and had a pH of 6.76 and the control had a pH of 6.75 on both days indicating only a 0.1 decrease in pH. Tables showing the differences between the initial pH of the milk and that after 48 hours for each variable. (all values taken to 2 s.f) Trial number Initial pH after Difference Trial number Initial pH after Difference pH of milk 48 hours pH of milk 48 hours 1 2 6.76 6.76 6.46 6.38 0.30 0.38 1 6.76 6.35 0.41 3 6.76 6.41 0.35 2 6.76 6.37 0.39 6.41 0.34 3 6.76 6.34 0.42 6.34 0.40 Mean Table 4 (Temperature= 20° C) Mean Table 5 (Temperature= 40° C) Trial number Initial pH of milk pH after 48 hours Difference 1 6.76 6.48 0.28 2 6.76 6.48 0.28 3 6.76 6.49 0.27 6.48 0.28 Mean Trial number Initial pH of milk pH after 48 hours Difference 1 6.76 6.58 0.18 2 6.76 6.60 0.16 3 6.76 6.61 0.15 6.61 0.16 Table 6 (Temperature =60° C) Table 7 (Temperature= 80° C) Trial number Initial pH of milk pH after 48 hours Difference 1 2 6.76 6.76 6.60 6.59 0.16 0.17 3 6.76 6.59 0.17 6.59 0.17 Mean Table 8 (Temperature= 100° C) 4.3 Graphical Data pH vs Temperature Recorded pH of the milk Recorded pH of the milk 6.65 6.6 6.55 6.5 6.45 6.4 6.35 6.3 6.25 6.65 6.6 6.55 6.5 6.45 6.4 6.35 6.3 6.25 6.2 20° C. 6.2 20° C. 40° C. 60° C. Temperature 80° C. 100° C. Table showing the pH of milk after 24 hours 40° C. 60° C. 80° C. Temperature Table showing the pH of milk after 48 hours 100° C. Mean difference in pH after 24 hours Graph showing the mean difference in pH for different temepratures from 0-48 hours. Difference in pH vs temperature. 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 20 40 60 80 100 120 Temperature 5. Analysis 5.1 Sources of Error 5.1.1 Uncertainties Percentage uncertainty = (margin of uncertainty/ value measured )100% Apparatus Used Margin of Uncertainty Total Percentage Uncertainty. Thermometer ±1° C. 11.41% 3 25 cm measuring cylinder The pH electrode (Percentage uncertainty pH calculated for trial 1 for variable 20° C.) 3 ±0.1 cm ±0.01. (calculated by adding the percentage uncertainty for each variable.) 5.00% 0.15% Table 9. 5.1.2 Random errors: - Although the milk samples were kept in the same room over the course of the experiment minor temperature fluctuations in the room may have had an effect on the growth of bacteria. - Another source of error could be the residue on the pH electrode from the previous sample, even though the electrode was dipped in distilled water which acted as a buffer to avoid confusion between samples there may or may not have been an effect on the readings recorded. Although the milk was poured into the Petri dishes after reaching the temperature there could potentially be variations in the time taken to pour the milk into its petri dish after heating it. 5.3 Discussion of Results. After conducting this experiment, a general trend was found which was consistent over the course of the 48-hours; the trend found was that the pH shows a decrease initially from variables 20° C to 40° C indicating an increase in bacterial growth however the pH then increases from temperature 60° C onwards indicating a lower rate of rate bacterial growth with increasing temperatures. There is also a notable decrease in pH from the 24- hour period to the 48 -hour period however the trend shown in graphs 1 and 2 remain consistent. Another observation made was the pH levels recorded in the temperatures 80° C and 100° C were almost equal after the 48- hour period. The fact that acidity was highest in samples heated to 40° C can be attributed to the fact that the optimal growth of bacteria is 37° C and this variable was the closest to that value. The second highest acidity level was that of the samples heated to 20° C as possible because of the fact that bacteria were not affected or killed by this temperature. Next were 60° C, 80° C and 100° C in that order specifically. Although the sample heated to 100° C was the most effective in inhibiting bacterial growth on day 1 the results on day show that both 80° C and 100° C were equally as effective. 6.Evaluation 6.1 Conclusion. It can be deduced that the highest rate of bacterial growth was in the milk samples heated to 40° C followed by that of 20° C. the lowest rate of growth was in the samples heated to 100° C indicating that this temperature was the most effective in inhibiting bacterial growth followed by that of 80° C. The bacterial growth in the samples heated to 60° C could be due to the presence of thermophiles whose optimum growth range is from 55° C to 65° C. Overall the results substantiated the hypothesis provide above. In conclusion Pasteurisation of milk is effective in inhibiting bacterial growth and increasing shelf life of milk to a certain extent. 6.2 Strengths: -Reliable results were obtained since three trials were carried out for each variable. - The trends exhibited were consistent through the course of the experiment and the values didn’t vary majorly. - Five variables of temperature were used to effectively understand how heating milk to varying temperature affect the rate of bacterial growth. - controlled variables mentioned in Table 1 ensured there was no variations that could potentially affect the results. - Each milk sample was heated in the same way and put into petri dishes of equal size and type. - All petri dishes were taken from a new pack therefore there was a low chance of external bacterial interference. - Research was done on the relationship between pH and bacterial growth in milk before the experiment was carried out to optimize understanding. - Uncertainty of the measuring cylinder used to measure the volume of milk did not exceed ±0.1 cm3 and the percentage uncertainty did not exceed 5%. 6.3 Limitations - It was expected that the difference of pH would be greater however the use of low- fat milk instead of full- fat milk may have inhibited bacterial growth therefore reducing the amount of acid produced. Due to time constraints increased number of trials were not performed to further increase the reliability of the experiment. There were various random errors as mentioned in section 5.1.2, which could have had an effect on the reliability of the results. Percentage uncertainty of all the temperatures was 11.41% which is relatively high in comparison to the measuring cylinder. Although the petri dishes were obtained from new packages, they were not sterilized with an antiseptic before conducting the experiment; there may have been surface bacteria present in the petri dishes conducting the experiment which may have affected reliability of the results obtained. Only one brand of milk was investigated for this exploration.