In this experiment milk was fermented into Kefir, then a series of tests were used to confirm that fermentation actually occurred. The tests used to confirm the fermentation were a gas production test, pH test, Gram stain, and turbidity test. The results showed during the conversion of milk to Kefir there was gas produced, a decrease in pH, more bacteria present, and an increase in absorbency. The results proved that fermentation occurred with three positive confirmatory results from the gas production, pH, and turbidity tests, while the Gram stain test acted as a negative confirmatory test. Introduction:
The main goal of this experiment was to successfully ferment milk into a yogurt-like product called Kefir. The fermentation is accomplished with a symbiotic combination of different bacteria and yeasts. The main type of bacteria found in the fermentation of milk to Kefir is Lactobacillus. Lactobacilli ferment the lactose found in milk and produces lactic acid, contributing to the sour taste, characteristic of Kefir. The fermented product, Kefir, is not know only for its taste, but more so for the many health benefits if can have for the body.
Experimental Procedures
The first step in this experiment was to make the fermented kefir product as well as a control. Two bottles were filled with milk; kefir grains were added to the first bottle to act as the experimental sample, and the second bottle, which contained only milk, was used as the control sample. A rubber glove was placed on the top of each bottle to act as a gas collector, and both bottle were allowed to sit for twenty-four hours.
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Litmus milk is a complex medium that can potentially distinguish many species of bacteria. Litmus milk has several components that can be metabolized: lactose (milk sugar); casein (milk protein); and litmus (a pH indicator). If lactose is fermented, the solution should turn pink. If gas is produced during fermentation, you may be able to see bubbles or cracks in the milky medium. If lactose is not ...
The first test used to confirm the presence of fermentation was to test for the presence of Carbon dioxide. This was carried out by checking the gas collectors, on the top of each bottle, for the presence of carbon dioxide gas. The second test performed used indicator paper to determine the pH of both samples. Two pieces of indicator paper were dipped in each of the samples, and the color change was recorded. The third test performed was a gram staining of both samples to determine the presence of bacteria (“Gram Stain Protocols.” ASM Microbelibrary).
The fourth test performed was a turbidity test to determine the absorbance of each sample. Five microliters of each sample was placed in a test tube and diluted with five hundred microliters of water. The test tubes were then placed in a spectrophotometer and the absorbance was recorded.
Results:
Gas Production
Samples
Result
Kefir
Positive
Milk
Negative
pH Test
Samples
pH
Kefir
4.5
Milk
7
Gram-Stain
Samples
Results
Kefir
Bacteria present
Milk
Bacteria present
Absorbance
Sample
Result
Kefir
Milk
Gas Production Observations:
The Kefir sample had an inflated gas collector
The milk sample had a deflated gas collector
(Figure 1)
pH Observations:
The indicator paper dipped in the Kefir had an orange color, correlating with a pH of 4.5 The indicator paper dipped in the milk had a yellow color, correlating with a pH of 7 (Figure 2)
Gram-stain Observations:
The presence of bacteria was observed in the Kefir sample.
The presence of bacteria was observed in the milk sample, but was a lower in amount than that of the Kefir sample.
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Turbidity Observations:
The Kefir sample was observed to have a higher absorbency compared to the milk sample. The milk sample was observed to have a lower absorbency compared to the Kefir sample.
Discussion:
The gas production test showed the Kefir sample produced gas while the milk sample did not. This was interpreted as an indication of fermentation, because carbon dioxide is a direct product of fermentation.
The pH test showed that the kefir sample was more acidic than the milk sample, which is exactly what was expected. This confirms the presence of fermentation due to the bacteria producing several different acids when fermenting the carbohydrates in milk. The pH of the milk was neutral as expected.
The turbidity test showed the Kefir sample to have a higher absorbance than the milk sample. This went exactly how we expected, and is used as confirmation of fermentation since the fermented product would have more bacteria present.
The Gram stain test showed that there were bacteria present in both the Kefir and milk samples, which was not an expected result. Before conducting the experiment, milk was believed to contain no bacteria due to the pasteurization process. Upon further investigation the group came to the realization that bacteria was present in milk due to the ubiquitous nature of bacteria. This is the test that is considered the negative confirmatory test.
Acknowledgements:
This experiment was funded by the selfless donations of Nick Fiore and Michael Ahrens, with additional funding from the University of Kansas. M.A., N.F., G.H., and M.C. designed and performed the experiment. M.A. wrote the report. No conflicts of interest were reported.
References
1) Ahrens, Micheal L. Figure 1. 2014. Micheal Ahren’s Phone, Lawrence, KS.
2) Cullom, Melissa. Figure 2. 2014. Melissa Cullom’s Phone, Lawrence, KS. 3) Smith, Ann C., and Marise A. Hussey. “Gram Stain Protocols.” Gram Stain Protocols. ASM Microbelibrary, 30 Sept. 2005. Web. 23 Oct. 2014.
