What is the effect of increasing concentrations of green tea extract (0.0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%) on the rate of fermentation of baker’s yeast (Saccharomyces cerevisiae) measured by the change in carbon dioxide concentration over ten minutes?

• A layer of foam developed after incubating all yeast cultures for 24 hours.

• Higher concentrations of green tea extract resulted in darker solutions.
• For all concentrations of green tea extract and the control, the fermentation process made the color of the solution slightly darker.
• During the fermentation process of green tea, bubbles were observed in the mixture.

Safety Concerns: Baker's yeast containing S. cerevisiae may irritate the skin and eyes upon contact. Yeast that is respiring naturally produces carbon dioxide, and under rare circumstances, an excessive amount of carbon dioxide can cause hypoxia. (2013) Lalley Biofuels and Distilled Spirits, As a result, the experiment needs to be carried out in a space that is adequately ventilated.

 

Environmental Concerns: The baker's yeast S. cerevisiae has killer toxins, which are glycosylated proteins that kill the target bacterium by attaching to particular surface receptors. (US EPA, 1997) Therefore, following the experiment, solutions containing the yeast cultures should be carefully disposed of away in approved waste receptacles.

The graph shows that the rate of fermentation over 10 minutes increases as the content of green tea extract is increased. Table 5 shows that the average carbon dioxide concentration changed at the slowest rate of 35.22 ppm/min for the control and at the quickest rate of 313.28 ppm/min for the 2.5% solution. Even though there is only a 2.5ppm/min difference between the 2.0% and 2.5% concentrations, there appears to be a plateau at higher concentrations. However, more research with larger concentrations of green tea extract should be done to identify a more distinct trend. The content of green tea extract and the rate of fermentation exhibit a strong positive association, as seen by the graph's high R² value of 0.972. The R² number, also known as the coefficient of determination, indicates how well the regression line fits a given set of data. The alternative hypothesis H₁ is supported by this, albeit further statistical analysis is required to prove this.

 

As the amount of green tea extract was increased, the rate of fermentation accelerated because green tea contains a lot of polyphenols like catechins and epigallocatechin gallate (EGCG). The redox-sensitive Yap1 transcription factor is activated by green tea extract, which causes the TRX2 gene to be induced, according to a study looking at the cellular TRX (thioredoxin) in S. cerevisiae. In 2005, Takutsume et al. By accelerating the decrease of TrxR (TRX reductase), which raises tolerance for a variety of lignocellulose-derived inhibitors including acetic acid and formic acid found in green tea, the TRX2 gene is activated. Cell survival under oxidative stress is increased by the simultaneous expression of TRX and TrxR. Better fermentative profiles with a shorter lag time and improved cell viability under oxidative stress were produced as a result. (2017) (Gao et al. High specific growth rates, which have also been demonstrated to be associated with enhanced fermentative capacities, promote cell survival. Van Hoek and others, 1997)

 

The standard deviation to mean ratio, which illustrates how the data points are spread in relation to the mean, is used to determine the coefficient of variation. The greatest calculated CV for the control is around 0.56, and all computed values are below 1. As a result, the distribution is thought to have a low variance. The graph's error bars stand in for the standard deviation. The fact that the error bars are so small indicates that there is not much variation in the data around the mean. The error bars for the 0.5% and 1.0% solutions hardly overlap, indicating that the difference in the rate of fermentation could not be very significant. Other concentrations do not overlap with one another, which shows that the difference might be substantial. Error bars, on the other hand, can only indicate whether there is a significant difference; statistical tests must be performed in order to reach a reliable result.

 

In order to ascertain the statistical significance of the outcomes seen from the various concentrations, an ANOVA (Analysis of Variance) was carried out. Table 6 shows that there is a significant difference between the various concentrations because the F value is more than the F critical value of 2.62 and the p-value is below 0.05 at 1.3919 10-17. As a result, the alternative hypothesis H₁ is accepted and the null hypothesis H0 is rejected.

Green tea concentrations range from 0% to 5% to 1.0% to 2.5% (g/100cm³). This will be determined by making a stock solution with 2.5% green tea extract and diluting it again until it is 0.5%. The 0.0% solution is created exclusively using water.

• Pour 20 ml of the yeast culture and 20 ml of the green tea solution into a beaker.
• Set up a water bath set at 30°C and put the beaker containing the mixture in until it reaches the desired temperature.
• Pour the contents of the beaker into a plastic bottle.
• Cut a small hole in a glove and cover the top of the CO₂ sensor to cover the holes.
• Connect the CO₂ gas sensor to CH1 of the data logger.
• After calibration, set the sampling rate at 1 sample per minute.
• Insert the CO₂ gas sensor into the fermentation chamber.
• Immediately start the data collection after the sensor has been placed.
• After 5 minutes, record the data using the table of readings.
• Repeat steps 3-8 with the rest of the solutions with different concentrations. For the control, add 20 ml of distilled water in place of the green tea solution.
• Repeat the same procedure 4 times for each of the different concentrations and the control.

• Heat the beakers containing the green tea on a Bunsen burner until fully dissolved

C. Measuring carbon dioxide concentration during the fermentation process

Green tea extract content does not impact the rate of fermentation, according to H_0.

 

Null. Green tea extract concentration influences the rate of fermentation, according to H₁ Alternative.

Overnight, yeast cultures are prepared for fermentation. Different concentrations of green tea solutions are created. In order to track the rate of change in carbon dioxide concentration during a 10-minute period, the culture and green tea are combined, and the initial carbon dioxide concentration is then established.

 

A. Preparing yeast cultures for fermentation

 

• Peptone, malt extract, baker's yeast, and glucose should all be measured out to equal masses (1.25g) using an electronic balance (on separate weighing boats).
• To create the yeast culture, combine the contents of all the weighing boats with 500ml of DI water.
• Incubate the yeast culture for 24 hours at 25 °C

Rate of change in carbon dioxide concentration per minute

 

 = \(\frac{change\ carbon\ dioxide\ concentration}{total\ time\ in\ minutes}\)

 

= \(\frac{299}{10}\) = 29.90 ppm/min

 

 the average rate of change in carbon dioxide concentration per minute:

 

= \(\frac{sum\ of\ \ change\ in\ CO_{2\ }concemtration}{total\ number\ of\ trails}\)

 

For average carbon dioxide concentration produced for control

 

= \(\frac{29.9+70.3+24.8+27.6+23.5}{5}\) = 35.22ppm/min

 

Standard deviation

 

σ = \(\sqrt{\frac{\Sigma(x_i-\mu)^2}{N}}\)

 

𝜎 – population standard deviation

x_i – each value from the population

𝜇 – the population's average

𝑁 – number of trials

 

standard deviation for control

 

σ = \(\sqrt{\frac{(29.9-35.22)^2+(70.3-35.22)^2+(24.8-35.22)^2+(27.6-35.22)^2+(23.5-35.22)^2}{5}}\)

 

= 19.77

 

Coefficient of Variation

 

CV = \(\ \frac{\sigma}{\mu}\)

 

𝜎 – population standard deviation

𝜇 – the population's average

 

Coefficient of Variation for control:

 

CV = \(\ \frac{19.76681563}{35.22}\) = 0.5612

One of the most popular drinks consumed worldwide is tea. Black tea, oolong tea, and green tea are just a few of the numerous varieties of tea that can be made from the same plant, Camellia sinesis. When I visited Kyoto, my parents' former coworker took us to a traditional green tea house where I had the chance to taste green tea in its most natural state. Japan is highly known for its tea culture, and I was fortunate enough to experience it for myself. After experiencing green tea of the highest caliber in Japan, I was intrigued to learn how to improve the flavor of less expensive teas without using flavorings. I observed that recent studies have indicated that the yeast fermentation of green tea can result in a higher taste quality since the procedure enhances specific scents, giving it a more fruity and floral taste. 2020 (Wang et al.)

 

Aspergillus niger is a fungus that is fermented to create the bioactive components that give teas like Pu-erh their characteristic flavor. After reading Wang's paper, I was curious to find out more about how varying concentrations might impact how quickly fermentation occurs. Since green tea experiences less oxidation than other types of tea, such as black tea, it keeps a greater portion of the original flavor of the tea plant. The baker's yeast Saccharomyces cerevisiae was selected as the model organism for this study because of its quick fermentation rates and low nutritional needs, which make it simple to manipulate in a lab setting. (2018) (Villarreal-Soto et al.) This prompted the investigation into the impact of increasing green tea extract concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%) on the baker's yeast (Saccharomyces cerevisiae) rate of fermentation as determined by the change in carbon dioxide concentration over ten minutes.

B. Preparation of different concentrations of green tea

• Grind tea leaves into a powder using a mortar and pestle.
• Measure 2.5g of green tea leaf powder and combine it with 100 ml of DI water in a beaker.
• Carry out a dilution according to the table below

A concentrated type of green tea, green tea extract has the same quantity of beneficial compounds as a typical cup of green tea. Since it is a well-known source of antioxidants, it has also been linked to a number of health advantages, including bettering heart, liver, and brain health. By preventing free radicals from causing cell damage, the antioxidants in green tea extract can help lower oxidative stress. Additionally, studies have demonstrated that it is an effective weight-loss tool. by Gunnars in 2020 The human small intestine's ability to absorb glucose has been demonstrated to be suppressed by the catechins found in tea. 2019 (Ueda-Wakagi)

 

When glucose is subjected to anaerobic respiration, ethanol and carbon dioxide are produced as a result of yeast fermentation. The process of generating energy without the use of oxygen is known as anaerobic respiration. 2020 (Macias) This procedure is crucial for making bread because it promotes rising and the formation of air pockets in the baked good. The carbon dioxide expands considerably more during baking. (2007) Shimiya et al. In the process of brewing beer, glucose is broken down into alcohol and carbon dioxide to create another use for yeast fermentation.

 

Glycolysis, the initial stage in the regulated breakdown of carbohydrates like glucose, kicks off the fermentation process. A hexose sugar (6 carbons) is converted into two molecules of pyruvate during the process of glycolysis, which takes place in the cytoplasm of the cell (3 carbons). Glycolysis involves four primary processes: photophosphorylation, lysis, oxidation, and ATP synthesis. Hexose sugar is phosphorylated by two ATP molecules to produce hexose bisphosphate during photophosphorylation. The molecule becomes more reactive as a result of this action, which also stops cell-exiting diffusion. Hexose bisphosphate, a six-carbon sugar, is divided into two triose phosphates during the lysis process (3 carbon sugars). Each of the 3C sugars loses a hydrogen atom during oxidation, which reduces NAD+ to NADH (+H). One NADH molecule is created by each sugar, for a total of two molecules of NADH. When some of the energy from the sugar intermediates is used to directly synthesize ATP, the substrate level is formed. Since the glucose is not further oxidized in the absence of oxygen, no new units of ATP are created. The two pyruvates are transformed into two carbon dioxide and two acetaldehyde in the cytosol. As a result of the two NADH molecules reducing the two acetaldehydes, two moles of ethanol are transformed back into NADH from NAD+. To continue producing some ATP in the absence of oxygen, the products can be changed back into pyruvate. The available supplies of NAD+ are rapidly exhausted during anaerobic respiration, which halts further glycolysis. (2011) Aranda et al. The formulas for yeast fermentation are as follows:

 

C₆H₁₂O_{6 (aq)}  2C₂H₅OH_(aq) + 2CO_{2 (aq)}

 

Glucose →Ethanol + Carbon Dioxide 

Using a CO₂ sensor at 1-minute intervals, the amount of carbon dioxide will b monitored over a period of 10 minutes to determine the rate of fermentation.