To what extent do the pHs 2.00, 3.00, 4.00, 5.00, 6.00, and 7.00 affect the growth of Lactobacillus found in soy yogurt, measured at 625nm by a spectrophotometer?

The gut microbiota is defined as those microorganisms that live in vertebrates' gastrointestinal tracts. In humans, the gut represents the main site of survival of the human microbiota composed of several microbial strains (Dahiya & Nigam, 2022). This part of the body is highly addressed because it inhibits the colonization of harmful bacteria, fortifies the layer of the internal mucosa, and strengthens the overall immune system (Xinzhou, Peng, & Xin, 2021) if a healthy balance between host bacteria and gut microorganisms is kept. Recent research has confirmed that the gut microbiome can be improved by the intake of functional foods, products with an active live population of probiotics, like dairy products (Dahiya & Nigam, 2022) containing lactic acid bacteria, such as Lactobacillus (National Center for Complementary and Integrative Health, 2019).

Bacterial survival is closely tied to pH levels. These microorganisms thrive best within a specific, optimal pH range. Any deviation from this range can result in less effective growth or even the death of the bacteria. Lactobacillus bacteria can tolerate acidic environments of pH 3.50-6.80 (LibreTexts, n.d.), with an optimum pH of 4.50-6.50 (Ślizewska & Chlebicz-Wójcik, 2020). Moreover, the pH levels along the human digestive tract vary from the mouth, where saliva is present, to the point of exit in the large intestine. Generally, gastric juices in the stomach have a pH range of 1.00-2.50, the small intestine maintains a pH of around 6.60, and the large intestine has a pH of approximately 7.00 (Evans, 1988).

What prompted my exploration was recognizing that bacteria have an optimum range where they grow more efficiently. This recalled the behavior of enzymes, with an optimal temperature and pH range at which they perform better as catalysts. If these conditions deviate from the norm, enzymes undergo denaturation, rendering them ineffective as catalysts. Knowing this, my previous belief in the health benefits of consuming probiotic yogurt was shattered. I couldn't comprehend how, considering the challenging journey the probiotics must undergo, being exposed to a wide range of pHs and specially the acidic environment of the stomach, they could survive and fulfill the promised contribution to the microbiota in the large intestine. This exploration aims to determine whether probiotics can indeed survive and thrive when they reach the large intestine. The potential outcome of this research carries significant real-world implications, especially in relation to the yogurt food industry and its claims of health benefits. Uncovering the growth rate of probiotics through the digestive system at different pHs could reveal whether these claims are accurate or potentially misleading.

This investigation seeks to determine whether altering pH levels would lead to the inhibition of the bacteria cultures of probiotics in soy yogurt by simulating the pHs found in the gut. To simulate the stomach and the wall of the large intestine, we will use nutrient agar plates containing different pHs to grow the cultures. The bacterial solution will be evenly spread along the surface and left to incubate overnight. Subsequently, three samples will be taken from each plate, and the spectrophotometer will be employed to measure the absorbance of Lactobacillus in each sample, at a wavelength of 625 nm. Higher absorbance values indicate a greater presence of bacterial colonies. Before the experiment, it is essential to carefully select, from the variety of available yogurts, one that exhibits the most efficient growth under overnight incubation conditions. As part of the preliminary work to this investigation, I examined various types of yogurts (soy yogurt, cow-milk yogurt, protein yogurt, and lactose-free yogurt) to determine which one prompted the highest growth of bacteria, and finally chose to work with soy yogurt.

A valid hypothesis would be that exposing the probiotic Lactobacillus to pH levels significantly deviating from the optimal range of 4.50-6.50 will lead to a decline in bacterial growth because enzymes will be denaturing. While, in pH conditions far from 3.50-6.80, bacterial growth is anticipated to be completely inhibited.

In this experiment, the independent variable is the different pH levels to which the probiotics are exposed. We will use pH levels of 2.00, 3.00, 4.00, 5.00, 6.00, and 7.00, achieved by adding hydrochloric acid (HCl) for acidification or sodium hydroxide (NaOH) for alkalization in the nutrient agar. To ensure a consistent pH throughout the agar, HCl and NaOH will be added and thoroughly mixed before the nutrient agar solidifies.

In this experiment, the dependent variable is the growth of probiotics such as Lactobacillus found in soy yogurt, as measured by a spectrophotometer at wavelength 625 nm. A spectrophotometer (Pasco wireless) is going to measure the light absorbed after it passes through a solution; a higher number of cells in the solution, the higher the absorbance because more light is scattered (Phillips, 2023).

• 5 g of cow milk yogurt
• 5 g of protein yogurt
• 5 g of lactose-free yogurt
• 5 g of soy yogurt
• 20 test tubes
• 70% ethanol
• 1 l of distilled water
• 66 g of sucrose
• 6 x 100 mL beakers (±0.5)
• 1.0 M HCL
• 1.0 M NaOH
• 12 g of nutrient agar
• 24 scoops of infant formula for 660 mL
• 18 agar plates
• 100 mL (±0.5) measuring cylinder
• 10 mL (±0.2) measuring cylinder
• Spatula
• Bunsen burner
• Matches
• Inoculation loop
• Refrigerator
• Incubator (±0.1°C)
• Steel test tube holder
• Pasco pH sensor, (±0.01) units
• Microwave
• Spectrophotometer (±0.001 AU)
• Latex gloves
• Electronic scale (±0.01 g)
• Bacterial metal spreader

As a first approach, we tried two methods to culture bacteria from four different probiotic yogurts (cow milk yogurt, protein yogurt, soy yogurt, and lactose-free yogurt) to select the most suitable options for our investigation. After using a streak method and diluting the yogurt in distilled water on separate plates, the most substantial growth (appendix table 6) was seen in the plates containing soy yogurt diluted in distilled water. Therefore, we have chosen this technique and yogurt for the experiment.

1. Disinfect the workplace with 70% ethanol to minimize contamination.
2. Sterilize eighteen Petri dishes with 70% ethanol.
3. Label the Petri dishes, pH 2.00, 3.00, 4.00, 5.00, 6.00, and 7.00 for the soy yogurt's bacteria, that grew the most.
4. To prepare the agar for each pH (3 plates), mix 110 ml of distilled water with 4 scoops of infant formula in a beaker. Add 2 g of nutrient agar and 11 g of sucrose to the solution.
5. Use the pH meter to confirm that they are at the desired pH. Add 1.0 M hydrochloric acid to lower the pH to the desired ones.
6. Heat it in the microwave until completely dissolved.
7. Pour each pH agar solution into three Petri dishes. A total of 18 petri dishes will be filled with agar. Let it solidify.
8. Spread the previously collected soy bacteria (now in a 0.2 ml distilled water solution) into the agar plates. Add three drops to each agar plate.
9. Use the sterilized metal spreader to spread the bacteria evenly on the agar's surface.
10. Incubate the agar plates at 28°C overnight.

1. After the exposure period of 12 h, remove the agar plates from the incubator.
2. Cut with the edge of a test tube three equal size pieces of agar from each agar plate. For each pH we have three plates, so nine samples in total for each pH.
3. Put each piece in a different sterilized beaker with 3 mL of distilled water.
4. Remove the agar leaving the bacteria in the water.
5. Use a spectrophotometer to measure the absorbance at 625 nm of each solution, which indirectly measures the amount of probiotic bacterial growth after exposure to different pH.

Qualitative: Petri dishes containing cultures with pH levels of 5.00 and 6.00 exhibited a consistently large, yellow culture at the center of the plates. Plates with pH levels of 3.00, 4.00, and 7.00 displayed a less dense, uniform culture on the surface of the agar. No visible change was observed in plates with a pH of 2.00. Furthermore, pH 5.00 plates displayed the presence of white dots.

The average of this data will be calculated for each pH to facilitate the identification of patterns. The equation for the mean is the following:

Where:

\[ \bar{x}=\frac{\sum_{i=1}^{n} x_{i}}{n} \quad \begin{aligned} & \bar{x}: \text{ the mean } \\ & \\ & n: \text{ number of trials } \\ & \\ & i: \text{ the index } \end{aligned} \]

Calculating the mean for pH 3.00:

\[ \bar{x}=\frac{0.235+0.219+0.171+0.234+0.238+0.224+0.234+0.235+0.172}{9}=0.218 \]

The population's standard deviation will also be calculated to incorporate error bars on the average graph. A greater deviation indicates lower precision in the data. The equation for the standard deviation is the following:

Where:

\[ \sigma=\sqrt{\frac{\sum(x_{i}-𝜇)^{2}}{n}} \]

σ: the standard deviation
μ: the mean
n: the number of data points
x_i: each of the values of the data

Calculating the standard deviation for pH 3.00:

\[ \sigma=\sqrt{\frac{\sum(x_{i}-0.218)^{2}}{9}}=0.027 \]

The chart illustrates a consistent rise in Lactobacillus absorbance as pH increases. The curve starts at pH 2.00 with an almost inhibited growth of the bacteria. Due to the acidic conditions, a tiny fraction of the bacteria could survive to divide and increase the population. From there, the graph follows a pattern of growth that persists until it reaches a peak absorbance at pH 5.00, after which the absorbance begins to decrease. From there on, there is a progressive and slow decline in absorbance as the pH increases. The absorbance at pH 6.00 closely approximates the absorbance at pH 5.00 but diverges significantly from pH 7.00, which indicates the steady and notable decline starts at pH 6.00. The best pH to cultivate Lactobacillus, based on this information, is between 5.00 and 6.00. This was overly expected as the optimal pH range of Lactobacillus is said to lay between 4.50 and 6.50 (Śliżewska & Chlebicz-Wójcik, 2020).

There is a considerable degree of uncertainty in the results, as indicated by the significant scatter represented in the length of the error bars, derived from the standard deviation of the collected data. A likely reason for this could be attributed to the procedure itself. When immersing the agar pieces into distilled water to detach the bacteria grown on the surface, some may have remained attached. This limitation is attributed to the limited materials available in the school.

A T-test was conducted to assess whether a significant difference existed between pH levels, given the considerable overlap of error bars for each pH. A 5% significance level was adopted, assuming the hypothesis is supported by data with an expected error of a 5%. With 16 as degrees of freedom, the critical value was established at 1.746. Acceptance of the alternative hypothesis occurs when values surpass 1.746, indicating a significant difference between the samples.