INTERNATOINAL BACCALAUREATE BIOLOGY EXTENDED ESSAY (2019)

Research Question: -

Does the increasing concentration (%) of natural colorant anthocyanin (C15 H11 O+) obtained

from Red-cabbage (Brassica oleracea) and synthetic colorant allura-red (C18H14N2Na2O8S2)

affect the evolution of CO2 (ppm) during respiration of Saccharomyces cerevisiae?

Word count : 3956

Personal code : gzs420

Table of Contents

1. Introduction: - ............................................................................................................... 1

2. Background information................................................................................................... 3 2.2.1.PigmentAllura red : - ...................................................................................................................... 3 2.2.2.Natural food-colour- Red-cabbage (Brassica oleracea) ................................................................... 5

2.3. Respiration............................................................................................................................. 6 2.3.1 Saccharomyces cerevisiae (Yeast) .................................................................................................. 8

3. Hypothesis ........................................................................................................................ 9

3.1.1. Null hypothesis :- ................................................................................................................ 9

3.1.2 Alternate hypothesis :- ......................................................................................................... 9

4.1Variable ........................................................................................................................... 9

4.1.1. Independent variable .......................................................................................................... 9

4.1.2. Dependent variable ............................................................................................................ 9

4.1.3.Controlled variable .............................................................................................................. 9

5. Methodology .................................................................................................................. 10

5.1 The preliminary experiment.................................................................................................. 10

5.2. Preparation of different concentration of synthetic and natural food colours ................... 11

5.3 Yeast (Saccharomyces cerevisiae) Suspension preparation ................................................... 12

5.4 Exposure of Allura red and Anthocyanin food-colour on yeast: ........................................... 12

6. Data collection and graphs ............................................................................................. 13

6.1 Method of data collection ..................................................................................................... 13

7. Discussion and Result: .................................................................................................... 15

8. Conclusion ...................................................................................................................... 18

9. Limitations ...................................................................................................................... 19

10. Improvement ................................................................................................................ 19

11. Further studies:- ........................................................................................................... 20

12.Bibliography .................................................................................................................. 20

13. Glossary ........................................................................................................................ 22

14. Appendix....................................................................................................................... 23

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1. Introduction: - The basic amenities in one’s life are food, shelter and clothing. In many developing

countries it has become difficult to suffice the need of food for the entire population.

Different delicacies attract people with their looks. Colourful food affects our appetite.1 To

meet this need they use food colorants that enhance the look and flavour of the dishes. Food

companies and restaurants try to make their delicacies not only delicious but as attractive

as possible. In the old times food colours were obtained from plant parts like flower, bark

and root extract.2 This was time taking and expensive process hence people switched to

chemically prepared synthetic food colours.3 The question arises that are these colorants

(synthetic and natural) having any adverse effect on the human physiological processes?

According to International research and recommendation of Codex Committee on food

additives there is fixed permissible amount that must be used.4

A research article stating ‘Utilization of microbial bioassays for screening the possible

toxicity in regularly used food dyes’ by Ruchika Atri et al showed that even a very small

quantity of some colorants can lead to inhibition of the rate of respiration of Saccharomyces

cerevisiae. Many people love eating fancy food, in the developed nations most of the people

are dependent on the packaged food without knowing the amount of food additives used in

its preparation. Though they are aware of the ill effects, still they tend to eat the food rich

1Onlystaples. "Colors That Affect Your Appetite... – Onlystaples – Medium." Medium.com. Medium, 30 Nov. 2016. Web. 27 July 2018. <https://medium.com/@onlystaples/colors-that-affect-your-appetite-cfdb8fecbd72>. 2Delgado-Vargas, Francisco & Jiménez-Aparicio, Antonio & Paredes-Lopez, Octavio. (2000). Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics, Biosynthesis, Processing, and Stability. Critical reviews in food science and nutrition. 40. 173-289. 10.1080/10408690091189257. 3Rohrig, Brian. "The Chemistry of Food Colorings." American Chemical Society. N.p., Oct. 2015. Web. 27 July 2019. 4"Comment on the Codex Committee on Contaminants in Food 12th Session." International Rice Commission Newsletter Vol. 48. FAO of the UN, 26 Mar. 2018. Web. 28 Aug. 2018.

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in colorants either because of their interest or no option left because of their work

schedules.5

The research done by Centre for science in the Public Interest6 states that these food dyes

have side effects such allergic reaction, cancer, ADHD 7etc. Studying the effect of natural

and synthetic food dyes on Saccharomyces cerevisiae will help in making the society aware

of not only their deleterious effects but also the impacts they have on the physiology of an

organism. In this investigation Saccharomyces cerevisiae is used because it is a simple

eukaryote that has many physiological processes similar to humans and therefore, the finding

can be helpful to the human race. Keeping the above priorities in mind regarding the effect of

colorants the following research question was formed:

Does the increasing concentration (%) of natural colorant anthocyanin (C15 H11 O+)

obtained from Red-cabbage (Brassica oleracea) and synthetic colorant allura red

(C18H14N2Na2O8S2) affect the evolution of CO2 (ppm) during respiration of

Saccharomyces cerevisiae?

5Burger, Kyle S et al. "Assessing Food Appeal And Desire To Eat: The Effects Of Portion Size &

Energy Density." International Journal of Behavioral Nutrition and Physical Activity 8.1 (2011): 101.

Web. 27 Aug. 2018.

6"Food Dyes." Trans Fat | Center for Science in the Public Interest. Center for Sicence in Public Intrest, 30 July 2010. Web. 5 Aug. 2018. 7Wells, Katie, and Katie WellsKatie Wells. “Do Food Dyes Affect Behavior? |Wellness Mama.” Wellness

Mama®,Katie Wells Are Thank You Notes a Thing of the Past? They Shouldn't Be! Learn the Research-Backed

Ways Expressing Gratitude Not Only Feels Good for the Sender and the Receiver, but Also Benefits Physical

Health!, 8 June. 2018, wellnessmama.com/36873/do-food dyes-affect-behavior/.

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2. Background information 2.1.Colorants

Food colours are those substances that enhance the colour and the flavour of food which makes

the food look appealing. These food colours are categorised as natural and synthetic. The food

additives are added to attain the desired nutritional value, to modify the taste, also make it look

attractive. Nowadays colourful cakes and pastries set the trend to theme parties so people are

keen to use different colour in order to make the cake look more creative. The general effects

of the colorants are hyperactivity disorder in children which is generally caused by tartrazine,

brilliant-blue, sunset-yellow. However, colours like allura red have harmful effects like

genotoxicity, chromosomal aberration, reproductive toxicity, asthma and urticaria (skin

rashes)8. The amount of food dyes added is a matter of concern as many studies reveal a diluted

amount of colorants have negligible effects on body but more the concentration of the colorant

more deleterious effects could be noted. A study by Orlando et.al revealed that excessive

intake of phosphorous based food additives may disturb the mineral and bone metabolism

leading to a compromise in the bone health.9

2.2.1.PigmentAllura red : - Allura red is red coloured synthetic food dye which is chemically prepared and has the chemical

formula C18H14N2Na2O8S210. All the food colorants has a range of consumption for allura red

8Sezgin, Aybuke&Ayyıldız, Sibel. (2017). Food Additives: Colorants. 9Gutiérrez, Orlando M. et al. "Impact Of Phosphorus-Based Food Additives On Bone And Mineral

Metabolism." The Journal of Clinical Endocrinology & Metabolism 100.11 (2015): 4264-4271. Web.

19 June. 2018. 10Esmaeili S, et al. "Degradation Products Of The Artificial Azo Dye, Allura Red, Inhibit

Esterase Activity Of Carbonic Anhydrase II: A Basic In Vitro Study On The Food ... - Pubmed - NCBI ." Ncbi.nlm.nih.gov., 2016. Web. 18 Aug. 2018.

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it is 0.002 % by weight of pesticide formulation, above that they are harmful and can cause

severe health issues. It is commonly used in candies, cereals, confectionery, gelatine powder,

medicines and cosmetics. As its usage is permitted by Food and Drug Administration so

therefore a permissible amount is usually added to give red colour to the delicacies . The dye

allura red has many side effects like hyperactivity in children, swelling of mouth, cancer etc.11

It is a matter of concern to find out what percentage of this synthetic dye is permissible and

after what range will it be dangerous to consume. Allura red is now commonly used in many

bakery products and mouthwashes etc. It also has many adverse effects hence I chose to study

Allura Red to find to what extent it affects the human respiration.

Figure 1-Allura red ac12

11Wells, Katie, and Katie WellsKatie Wells. "Do Food Dyes Affect Behavior? | Wellness

Mama." Wellness Mama®. Katie Wells Are Thank You Notes a Thing of the Past? They

Shouldn't Be! Learn the Research-backed Ways

12"CID 5360805." Pubchem.ncbi.nlm.nih.gov. N. p., 2017. Web. 8 June. 2018.

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2.2.2.Natural food-colour- Red-cabbage (Brassica oleracea) The natural food colours are the ones that are obtained from fruits and vegetables for instance

red cabbage, pomegranate, blackberry and beetroot etc. are some fruits and vegetable that will

provide pigments like anthocyanin and beta vulgaris etc. Natural food colours are often noticed

to be safer than the synthetic ones, but as they are less stable to pH, temperature or light so they

tend to fade the colour because of which it makes the food decolorize easily and hence it is

inappropriate for the commercial use. Red cabbage contains a pigment called anthocyanin

which is used in this investigation and is a water soluble pigment that belongs to the Phenolic

group. This pigment is found in glycosylated form. The fruits and vegetables that range from

red to purple colour are the rich source of anthocyanin. Out of the anthocyanin pigments

cyanidin -3-glucoside is the most commonly found anthocyanin in the plants also in the red

cabbage.13 They were commonly used as traditional pigments in the past years. The colour and

stability of the anthocyanin depends on the temperature, light and ph. Under normal conditions

like low ph14 it appears red as the ph increases the colour changes from red to blue. Studies

have revealed that anthocyanins have antioxidant and anti -microbial activities, they have a

tendency to increase the visual and neurological health issues. Red cabbage also has health

benefits like prevention from cancer, controlling sugar levels, controls premature aging 6 etc.

Anthocyanin from Red cabbage was chosen for this study as it is active colorant majorly used

in baking and cooking industries.

13Khoo, Hock Eng et al. "Anthocyanidins And Anthocyanins: Colored Pigments As Food,

Pharmaceutical Ingredients, And The Potential Health Benefits." Food & Nutrition Research 61.1 (2017): 1361779. Web. 16June. 2018. 14 Keller, Michael J. "Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method." Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method. College of Chemical & Life Sciences 1222 H.J. Patterson Hall (Bldg. 073) University of Maryland, 12 July 2011. Web. 07 June 2018. <http://www.terpconnect.umd.edu/~kellermi/Keller(2010)_ABLE.pdf>.

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Figure 2-Structure of Anthocyanin15

2.3. Respiration

Respiration is process of breaking down of glucose either in the presence or absence of oxygen.

When this happens in the presence of oxygen it is referred as aerobic respiration while in the

absence of oxygen it is called anaerobic respiration. Respiration is one of the characteristics of

all living beings it is divided into three pathways including glycolysis, kreb’s cycle and electron

transport chain (ETC). Glycolysis occurs in cytoplasm of the cell while kreb’s cycle and ETC

occurs in mitochondria. Glycolysis is the step common to both anaerobic and aerobic

respiration occurring in mitochondria. Yeast (Saccharomyces cerevisiae) which is used in this

investigation respires anaerobically and produces ethanol, CO2 and H2O as the end products.16

C6H12O6 cytoplasm Pyruvate 𝐴𝑛𝑎𝑒𝑟𝑜𝑏𝑖𝑐𝑟𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 Ethanol + CO2 + H2O

At times, in anaerobic-respiration, it is observed that if the conditions like substrate-type and

concentration along with presence of oxygen prevails, it may lead to mixed respiro-

15Khoo, Hock Eng et al. "Anthocyanidins And Anthocyanins: Colored Pigments As Food,

Pharmaceutical Ingredients, And The Potential Health Benefits." Food & Nutrition Research 61.1

(2017): 1361779. Web. 16 June. 2018. 16Weem, Minka Peeters. "Metabolisim, Cell Respiration and Photosynthesis." Biology 4 Edittion. Ed. Jenna Crierie. 4th ed. Melton: IBID, 2014. 232-37. Print.

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fermentative metabolism resulting in the ethanol collection.17 The aerobic production of

ethanol by yeast depends on the pathways like fermentation and respiration. More the glucose

level, higher will be the glycolytic rate and thereby, more generation of pyruvate decarboxylase

and as a result increased ethanol production.

Inhibitors and promoters of cellular respiration:CO2 is released as a result of oxidative

decarboxylation of pyruvate and this converts into the various compounds during the Kreb’s

cycle. Hence any change seen during the evolution of the CO2 is because of the steps involved

in the cellular respiration.

The rate of cellular respiration shoots up in the Saccharomyces cerevisiae with increase in

certain factors like sucrose or glucose concentration. With the increase in the sucrose

concentration the formation of pyruvate increases as well in the glycolysis process. A study

by Chiba M, et al. observed that the natural colorant is able to provide more sugar and therefore

faster and more production of ATP along with the CO2 emission occurs that facilitates the

growth rate of yeast. Hence more will be the production of the ATP showing greater

effectiveness in the yeast. On the contrary, rate of cellular respiration falls if the enzyme

pyruvate dehydrogenase is deficient as the rate of CO2production declines. Moreover the

chemicals like isocyanates, carbendazim, fungicidal chemicals and benomyl will inhibit both

the processes of respiration and fermentation making yeast less efficient in CO2 production.18

In a similar way, the investigation is going to explore the inhibitory effects of synthetic and

natural colorants on the cellular respiration in yeast.

17Otterstedt, Karin et al. "Switching The Mode Of Metabolism In The Yeast Saccharomyces

Cerevisiae." EMBO reports 5.5 (2004): 532-537. Web. 19 Aug. 2018. 18Chiba M, et al. "Inhibition Of Yeast Respiration And Fermentation By Benomyl, Carbendazim,

Isocyanates, And Other Fungicidal Chemicals. - Pubmed - NCBI ." Ncbi.nlm.nih.gov. N. p., 2018.

Web. 17June. 2018.

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2.3.1 Saccharomyces cerevisiae (Yeast) Saccharomyces cerevisiae is a microbe that is a facultative anaerobe used to bake

confectionaries like bread, cakes, puffs etc. It is also used in the brewing industry for the

preparation of alcohol by fermentation process19. It respires anaerobically which results in

formation of CO2, water and ethanol. Saccharomyces cerevisiae is preferred as it resembles in

the genetic coding of Humans20 (Homo sapiens) and the rate of replication of Saccharomyces

cerevisiae is much faster than that of Homo sapiens (dividing once in every 2 hours). Hence,

using Saccharomyces cerevisiae makes it simpler for the scientist to replicate and form of

colonies faster to observe the effect of chemical and hence framing a conclusion at faster pace.

Because of this reason also it is used in this investigation. Saccharomyces cerevisiae baker’s

yeast uses the sugars (sucrose) and produce ethanol and carbon dioxide at both the anaerobic

and aerobic conditions.

2.4. CO2 sensor- During this investigation the rate of CO2 production (ppm) during anaerobic

respiration by yeast was measured using CO2 sensor. The calculation of the CO2 produced

would have been difficult without the sensor as there would have been inaccurate values

obtained. In order to get the authentic and accurate values the use of sensor was done. The

Vernier technology CO2 sensor was used in this investigation.

19 Pages, Biology. "Cellular Respiration." Elements and Atoms. Biology Pages, 2016. Web. 18 Aug.2018. <http://www.biology-pages.info/C/CellularRespiration.html>.

20Karathia, Hiren et al. "Saccharomyces Cerevisiae As A Model Organism: A Comparative

Study." PLoS ONE 6.2 (2011): e16015. Web. 18 June 2018.

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3. Hypothesis 3.1.1. Null hypothesis :-

There will be no significant change in the amount of CO2 collected by the CO2 sensor when

the concentration of either of the colours synthetic (allura red) and natural pigments

(anthocyanin) are increased.

3.1.2 Alternate hypothesis :-

In case of allura red the amount of CO2 collected by the respiration of yeast will decrease

as the concentration increases. But in the case of natural pigment anthocyanin the amount

of CO2 evolved will increase with the increasing concentration.

4.1Variable 4.1.1. Independent variable

The increasing concentration (0%,3%,6%,9%, 12%, 15%) of the solutions of allura red and

anthocyanin food colours are identified as the independent variable .The range of the

concentrations was selected by taking the running averages of the pre-lab. These concentrations

were perfect for observing the effect of both the colourants.

4.1.2. Dependent variable

Þ Amount of CO2 (ppm) produced during respiration by Saccharomyces cerevisiae.

4.1.3.Controlled variable

® The temperature for the yeast to ferment (40°C) was fixed as respiration is enzyme

dependent process using a water bath.

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® The time for the yeast to ferment was controlled using a stop watch as after 15

minutesCO2 evolved was measured. This ensured that the fermentation time is equal

for each trial.

5. Methodology 5.1 The preliminary experiment

Allura red powder the synthetic food additive and anthocyanin the natural food-colour

obtained from red-cabbage was selected for the investigation. Pure sample of Allura red

was purchased from a chemical shop and for the anthocyanin the red-cabbage was

dehydrated and crushed into powder. The red-cabbage was not boiled to obtain the extract

as anthocyanin is protein which might denature at high temperature. Then after obtaining

the pigments the solution was prepared in 5ml beaker. The solutions were made up of three

different concentrations to decide and select the range for concentrations to be used in the

main lab. Sucrose solution and yeast suspension were prepared and the CO2 sensor was

connected. The readings were recorded for three different solutions that were 0% that acted

as control, 6% and 15%. After surveying the nearby sweet shops and local ice-cream

makers this range was found appropriate for the investigation. In all the three set ups the

CO2 evolution was recorded. And finally the range selected for the main Lab was

0%,3%,6%,9%,12% and 15%.

Figure 3- Lab investigation of food colour on Saccharomyces respiration

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5.2. Preparation of different concentration of synthetic and natural food colours

To make the solutions of the food colours six beakers were taken and were labelled ‘1-

6 allura red’ and in the beaker labelled ‘1-allura red’ 0 gm of allura red and 100ml of

water measured using measuring cylinder was added that formed 0% solution, the

beaker labelled ‘2- allura red’ was added with 3 gm of allura red measured using a

weighing scale and 97 ml of water to form 3% allura red solution after dissolution the

solution was filtered using a filter funnel and filter paper as shown in figure 4. Similarly

by adding 6gm, 9gm, 12gm and 15gm of allura red in the beaker number 3,4,5 and 6-

allura red was measured and water was added to form 6%, 9%, 12% and 15% of allura

red solution. A similar procedure was carried out to form the solutions for the natural

food colorant anthocyanin in separate beakers labelled ‘1-6 anthocyanin’. The above

preparation is shown in the table below ( table- 1).

Table 1-To make the solutions take 6 beakers for each and were labelled ‘1-6 Allura red ’and

‘1-6 Anthocyanin’ shown in the following table :-

Beaker Concentration/ % Amt. of pigment (gm)

1 0 0

2 3 3

3 6 6

4 9 9

5 12 12

6 15 15

12

Figure 4- Different concentration of anthocyanin obtained from red cabbage.

5.3 Yeast (Saccharomyces cerevisiae) Suspension preparation

For yeast suspension preparation 10gm of baker's yeast was measured and was added

to a conical flask. 2gm of sugar (sucrose) was measured by the weigh-balance and

added to the same conical flask. 50ml of water was heated until 40℃ and then it was

added to the beaker and was stirred using a glass-rod. Then a clinch-film was used to

make the container airtight and create anaerobic conditions and avoid any contact with

oxygen. As respiration is a process that involves enzymes therefore, the temperature

was maintained using a water bath to avoid denaturation the enzyme. The conical flask

containing the yeast suspension was kept for 15minutes in the water bath at 40℃.

5.4 Exposure of Allura red and Anthocyanin food-colour on yeast:

To obtain the data from Allura red the3ml of suspension obtained using a dropper and was

added to a 100 ml beaker. In the same beaker 3ml of Allura red measured by the measuring

cylinder was added. Then using a clinch wrap, the beaker was sealed to prevent the oxygen

from going in (figure 5). It was kept for 15min at room-temperature and after that the record

of the carbon-dioxide production was noted. In the same way the preparation for anthocyanin

was also done.

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Figure 5- Different concentration of Allura red with Saccharomyces.

6. Data collection and graphs 6.1 Method of data collection

After the exposure of 15minutesof the yeast suspension to the solution of different

concentrations (0%, 3%, 6%, 9%, 12%, 15%) of both synthetic colorant Allura red and

anthocyanin were transferred one by one to the conical flask. a carbon-dioxide sensor and

logger pro software were then attached to the flask for the CO2collection which was measured

in parts per million (Vernier technology) ( shown in figure 6). Mean-value obtained for carbon-

dioxide production was recorded in the table prepared. In order to conclude accurately 10 trials

for each concentration of both the additives were conducted (in table 2 and 3). Later the average

was calculated and the standard deviation and percentage difference and affectivity were

calculated. For the statistical confirmation of the data the t-test and Pearson correlation test

were conducted.

Figure 6 : Setup for data collection

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6.2. Processed data table

Table: 2 Effect of Allura red on Saccharomyces respiration. Concentration/

% Carbon dioxide(ppm)

Average SD 1 2 3 4 5 6 7 8 9 10

0% 3467.0 3677.0 3589.0 3468.0 3345.0 3247.0 4564.0 3567.0 4536.0 3536.0 3699.6 464.5

3% 2950.0 3927.0 3179.0 3965.0 3496.0 3988.0 2929.0 1965.0 3836.0 3458.0 3369.3 638.4

6% 3195.0 3195.0 3317.0 2458.0 2615.0 3075.0 3073.0 3805.0 3359.0 3958.0 3205.0 459.9

9% 2340.0 3606.0 2430.0 3439.0 1760.0 1501.0 3286.0 3469.0 3393.0 3525.0 2874.9 794.5

12% 1699.0 1758.0 2458.0 1976.0 2004.0 2589.0 2615.0 1986.0 1974.0 1965.0 2102.4 330.3

15% 1148.0 1258.0 1148.0 2830.0 2828.0 1298.0 2846.0 1179.0 2456.0 2816.0 1980.7 825.3

Table:3 Effect of Anthocyanin on Saccharomyces respiration.

Concentration/% Carbon dioxide/ppm Average SD

1 2 3 4 5 6 7 8 9 10 0% 3467.0 3677.0 3589.0 3468.0 3345.0 3247.0 4564.0 3567.0 4536.0 3536.0 3699.6 464.5 3% 2934.0 2383.0 3908.0 4100.0 4115.0 4288.0 4156.0 5779.0 3049.0 3063.0 3777.5 962.8 6% 3009.0 10492.0 5909.0 3242.0 4132.0 4190.0 3680.0 3150.0 3160.0 3501.0 4446.5 2289.7 9% 4051.0 3764.0 3796.0 3380.0 4276.0 5788.0 4910.0 4057.0 6589.0 5988.0 4659.9 1100.0 12% 2660.0 4555.0 5548.0 5648.0 5978.0 4184.0 4555.0 4502.0 5295.0 5791.0 4871.6 1000.6 15% 3230.0 4137.0 3357.0 4681.0 7197.0 7100.0 7186.0 7265.0 4634.0 5292.0 5407.9 1645.9

Table: 4 Percentage difference in average carbon dioxide evolution(ppm) by Saccharomyces in various concentration of food colorant to control/%

Concentration/%

Percentage difference

Allura Anthocyanin

0% 0.00 0.00 3% -8.93 2.11 6% -13.37 20.19 9% -22.29 25.96

12% -43.17 31.68 15% -46.46 46.18

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Graph: 1 Relationship between respiration (%) and increasing concentrations of allura-red

and anthocyanin

7. Discussion and Result: In this exploration, different concentration of two distinct colorants allura red and anthocyanin

were chosen. Solutions were made separately to see the effect of increasing concentrations

(0%, 3%, 6%, 9%, 12% and 15%) of these colorants on the release of CO2 (ppm) by yeast

during respiration, this helped to comprehend the effect of the colorants on Homo sapiens as

Saccharomyces cerevisiae has many physiological processes similar to them. The experiment

certified that the increasing concentration of the natural food pigment anthocyanin extracted

from Red-cabbage is better on yeast’s respiration than the artificial colorant Allura red which

was able to clearly answer the research question in finding the effect of food colorants on the

rate of respiration in yeast. The increasing percentage of anthocyanin increased the volume of

CO2 produced ( table 3 ) which showed that the respiration is better with this natural colorant.

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In red-cabbage powder the amount of sugar was about 3.32g which would have act as a

substrate for respiration and would have increased the rate of respiration leading to increased

carbon-dioxide production. On the contrary the increasing concentration of Allura red (0%,

3%, 6%, 9%, 12% and 15%) showed the deleterious effects on the respiration of yeast there

by reducing volume of CO2 produced because of absence of any sugar content that would have

supported the process of respiration.

Many research papers recommend that diluted amount of synthetic colorants are permissible

as stated by Prithviraj et al in one of their papers “evaluation of toxicity of synthetic food

colours on human, normal flora and yeast.” 21Whereas a research paper by Abdellah A.

Dafallah et al “22physiological effects of some artificial and natural food-colouring on young

male albino rats” says synthetic food colours decrease the high density lipoprotein cholesterol

and increase the low density lipoprotein whereas there was no such effect seen with natural

colorants. All these studies are undoubtedly in synchronisation with my research question,

which clearly is reflected with the data collected that increasing concentrations of synthetic

colorant like allura red from 0% to 15 % declines the CO2 production from 3699.60 ppm to

1980.7 ppm showing a strong negative correlation between the two (table 2) the rate of CO2

production was maximum at control that is 0% and the value was 3699.60ppm, at 6 % it was

3205ppm it further decreased to 1980.7pmm at highest concentration that is 15% this clearly

21Sadar, Prithviraj. "Evaluation of Toxicity of Synthetic Food Colors on Human Normal Flora and Yeast." UGC

Approved Journal: International Journal of Health Sciences and Research. International Journal of Health Sciences and Research, 8 Aug. 2017. Web. 30 Aug. 2018.

<http://www.ijhsr.org/IJHSR_Vol.7_Issue.8_Aug2017/IJHSR_Abstract.017.html>.

22Hashem, Mohamed & Atta, Attia & S Arbid, Mahmoud & Nada, Somaia&Mouneir, Samar

& Asaad, Gihanfarag. (2011). Toxicological impact of Amaranth, Sunset Yellow and

Curcumin as food coloring agents in albino rats. J. Pak. Med. St.. 1. 43-51.

17

show that yeast was unable to respire at concentrations of allura red.A comparison between

data collected for allura red ( synthetic additive ) and anthocyanin (natural additive) was

able to prove that anthocyanin showed a strong positive correlation as from 0% to 15% as the

CO2 production increased from 3699.60ppm to 5407.90 ppm ( table 3) at control again CO2

production was minimum that is 3699.60 ppm but at 6% CO2 production rose to 4446.5ppm

with its highest value at 15 %which was 5407.9ppm of 2 . The data in the above tables

suggest that the synthetic additives have more drastic effects on the metabolic processes like

fermentation. The synthetic dye might have affected the fermentative pathway and decreased

the production of carbon-dioxide unlike the natural colorant anthocyanin. The yeast solution

was prepared for different concentration (0%, 3%, 6%, 9%, 12% and 15%)and was added in

the flask for measuring the carbon-dioxide production. It was noted that some oxygen was

present in the flask as the complete removal of oxygen was not possible and therefore the

yeast was performing a mixed respiro-fermentative pathway, hence the mitochondrial

respiration was also involved during this process. The food-colour allura red inhibited the

mitochondrial respiration with the support of succinate or alpha keto-glutarate

as it was unable to couple the state III respiration which is ADP stimulated.23 Moreover

higher the concentrations of the sugar or substrate, greater are the chances for the yeast to

changes its form of respiration( anaerobic to mixed respiration).24 The data obtained was

clearly stating that with increasing concentration of anthocyanin the sugar content increased

as well this lead to more sugar availability and therefore more CO2 evolution. From table 2 it

can be observed a negative co-relation of the CO2 evolution and increasing concentration of

23Reyes FG, et al. "Effect Of Organic Synthetic Food colours On Mitochondrial Respiration. - Pubmed

- NCBI ." Ncbi.nlm.nih.gov. N. p., 2018. Web. 18 Aug. 2018. 24Keller, Michael J. "Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method." Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method. College of Chemical & Life Sciences 1222 H.J. Patterson Hall (Bldg. 073) University of Maryland, 12 July 2011. Web. 07 June 2018. <http://www.terpconnect.umd.edu/~kellermi/Keller(2010)_ABLE.pdf>.

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allura red. This was statistically proved by performing Pearson correlation test between

%respiration and increasing concentration of both allura-red and anthocyanin, and the value

of R for allura red was -0.9757 that showed the relationship between the concentration and

CO2 evolution is a strong negative correlation. The value for R in case of anthocyanin was

0.9811 and it clearly depicts that there exist a strong positive correlation between

%respiration the and anthocyanin concentration (table shown in appendix A and B).

Moreover from the graph-1 it can be seen that anthocyanin was more affective on the

evolution of the CO2 as the values of different concentrations clearly indicates that. At all the

concentrations, anthocyanin was more affective except as at the control value (0%) where the

CO2 was the same but as the concentration kept on increasing the anthocyanin acted better

then synthetic dye allura red. Graph-1 clearly depicts that a positive correlation occurs

between the increasing concentrations of anthocyanin and a negative correlation occurs

between allura–red and respiration rate in terms of CO2 evolution seen with the help of

trendline equation for both the additives anthocyanin and allura red (𝑦 = 9.2966𝑥 +

88.482,𝑦 = −9.8269+ 112.02).

For allura red t-value is 5.73972 and p-value is 0.000019whereas the t value is -3.15882 and

p values is 0.005432for anthocyanin. It is clear from the result of the t test since the t> cv

(2.101) we reject null hypothesis and accept alternative for allura red whereas t< cv for

anthocyanin that clearly indicates that null hypothesis is accepted and alternative rejected

Though from the data collected the alternate hypothesis was appearing to be true for

anthocyanin as well but this wasn’t explained statistically. It showed there are 5% chances for

the alternate hypothesis to hold true but statistically the null hypothesis is accepted.

8. Conclusion The above exploration with the research question: Does the increasing concentration (%)of

colorant anthocyanin (C15 H11 O+ ) from Red-cabbage (Brassica oleracea) and synthetic

19

colorant allura red (C18H14N2Na2O8S2) affect the evolution of CO2 (ppm)during

respiration of Saccharomyces cerevisiae , aided to conclude that synthetic food pigment

Allura red show inhibitory effect on Saccharomyces cerevisiae as it reduces the rate of

respiration thereby decreasing the CO2 production whereas the data acquired through the

experiment proves that the anthocyanin pigment in Red-cabbage enhances the respiration in

yeast there by releasing large amount of CO2 and hence becomes more efficient in respiration

and energy output. Hence it is easy to comprehend the effects of the colorants on humans as

yeast has profusion of genetic similarities with the Homo sapiens.

9. Limitations It was difficult to obtain the pigment anthocyanin in its purified form. Therefore there would

have some flaws in the experiment as there might be the presence of some other substances

that would have affected the rate of CO2 production. It is difficult to control the life processes

of the microbes and it was true in the case of yeast as well, the respiration of Saccharomyces

cerevisiae could be modified but it could not be controlled as the process of respiration depends

on the organisms. The accuracy of the data is not acceptable for other synthetic food colours

that are red in colour due to different chemical composition in varied types of food colours like

Carnosine, Red2G, Erythrosine etc. the collected data for Red-cabbage cannot be accepted for

other natural red pigments obtained from pomegranate, beet root etc as they have varied

concentrations of the sugars and different pigments and the results could not have been much

reliable.

10. Improvement It is important to get the anthocyanin in its purest form to make any authenticated conclusion.

Some laboratory procedure like column assays were needed to extract the anthocyanin from

red-cabbage as explained by Marcelo Fonseca Xavier in his paper ‘Extraction of red-cabbage

anthocyanin: optimization of the operation condition of column process’ which was beyond

20

the scope of the school laboratory.25 It is difficult to control the metabolic processes in the

living organisms like yeast as a little variation in any of the substrate will lead to change in the

rate of respiration.

11. Further studies:- Quantity of substance (substrate) used up can be calculated both after and before the respiration

of yeast to deduce the impact of natural and artificial colorants. The investigation can even

include the solution of both: artificial and synthetic food colours, for the result it can be

compared with the control solution, and analysis of the effect could be obtained. Also by

keeping the proportion of the colorants equal the accuracy of the result might increase, this

could lead to improvised comparison bagged with explanation of the significance of the two

food colours on respiration, as there will some inhibiting and promoting parts present in the

solution which will give better results, hence the results could be analysed more effectively.

12.Bibliography •

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25Fonseca Xavier, Marcelo & Lopes, Toni &Quadri, Mara & Bastos Quadri, Marintho. (2008). Extraction of red-cabbage anthocyanins: Optimization of the operation conditions of the column process. Brazilian Archives of Biology and Technology - BRAZ ARCH BIOL TECHNOL. 51. 10.1590/S1516-89132008000100018.

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• Dafallah, Abdellah A. "PHYSIOLOGICAL EFFECTS OF SOME ARTIFICIAL AND NATURAL FOOD COLORING ON YOUNG MALE ALBINO RATS." PHYSIOLOGICAL EFFECTS O. Journal of Food Technology Research, Feb. 2015. Web. 20 Aug. 2018. <http://www.conscientiabeam.com/pdf-files/agr/58/JFTR-2015-2(2)-21-32.pdf>.

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• Gallik, Stephen. "Exercise 5. Determination of the Anthocyanin Concentration in Table Wines and Fruit Juices Using Visible Light Spectrophotometry." Exercise 2 - CellBiologyOLM. N.p., 4 Oct. 2012. Web. 07 Feb. 2018. <http://stevegallik.org/cellbiologyolm_Ex01_P06.html>.

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• Hashem, Mohamed & Atta, Attia & S Arbid, Mahmoud & Nada, Somaia&Mouneir, Samar & Asaad, Gihanfarag. (2011). Toxicological impact of Amaranth, Sunset Yellow and Curcumin as food coloring agents in albino rats. J. Pak. Med. St.. 1. 43-51.

• Hewitson, John, and Charles Hills. "How Does Sugar Affect Yeast Growth?" Welcome. Science and Plants for School, 11 July 2005. Web. 20 Nov. 2018. <http://www.saps.org.uk/saps-associates/browse-q-and-a/169-q-a-a-how-does-sugar-affect-yeast-growth>.

• Keller, Michael J. "Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method." Manipulation of Yeast Respiration Using Acetic Acid to Demonstrate the Scientific Method. College of Chemical & Life Sciences 1222 H.J. Patterson Hall (Bldg. 073) University of Maryland, 12 July 2011. Web. 07 June 2018. <http://www.terpconnect.umd.edu/~kellermi/Keller(2010)_ABLE.pdf>.

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13. Glossary a) Anaerobic – pertain to cause by the absence of oxygen

b) Anti -microbial-destructive to or inhibiting the growth of microorganisms.

c) Amenities –any feature that provides comfort, convenience, or pleasure:

d) alpha keto-glutarate- Alpha-ketoglutaric acid is a substance found naturally in the

human body.

e) Deleterious– Injurious to health

f) Eukaryotes- any organism having as its fundamental structural unit a cell type that contains

specializedorganelles in the cytoplasm, a membrane-

bound nucleus enclosing genetic material organizedinto chromosomes, and an elaborate syste

m of division by mitosis or meiosis, characteristic of all life forms

g) Hypothesis- a proposition assumed as a premise in an argument.

23

h) Inhibition- something that inhibits; constraint.

i) lipoprotein-any of the class of proteins that contain a lipid combined with a simple

protein.

j) Metabolic processes- The organic process in a cell that are necessary for life

k) Pesticide- a substance used for destroying insects or other organisms harmful to

cultivated plants or to animals.

l) physiological processes -The functions of living organisms and their parts, and the

physical and chemical factors and processes involved

m) substrate - A substrate is a molecule acted upon by an enzyme. A substrate is loaded

into the active site of the enzyme, or the place that allows weak bonds to be formed

between the two molecules

n) suspension– A substrate is a molecule acted upon by an enzyme. A substrate is loaded

into the active site of the enzyme, or the place that allows weak bonds to be formed

between the two molecules

14. Appendix Appendix A

Allura red

Concentration/% % difference

% Respiration

0% 0.00 100.00 3% -8.93 91.07 6% -13.37 86.63 9% -22.29 77.71

12% -43.17 56.83 15% -46.46 53.54

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Appendix B Anthocyanin

Concentration/% %

Difference Respiration/

%

0% 0.00 100.00 3% 2.11 102.11 6% 20.19 120.19 9% 25.96 125.96

12% 31.68 131.68 15% 46.18 146.18

Sample calculation

a) Allura red average of 0%

For allura red on 0% conc.

Formula : ?@ABCDEF.FG?@ABCD

Example : HIJJ

3467.0 + 3677.0 + 3589.0 + 3468.0 + 3345.0 + 3237.0 + 4564.0 + 3567.0 + 4536.0 + 5356.010 = 3699.6

b) Allura red Standard deviation 0%

Formula: 𝜎 = OP(QRQ̅)T

E Example: 𝜎 = OP(JRH)T

J

𝜎 = OP(UVWX.YRUWZZ.W)T

HY= 464.5

c) Allura red % diffrence

Formula: (GBEACR[FE?@FC)[FE?@FC

× 100 Example: (URH)H

× 100

For allura red on 3% conc.

(3369.3RUWZZ.W)UWZZ.W

× 100= -8.93

d) Allura red % respiration

Formula: (%diffrence – 100) Example: (3-100)

For allura red in 3%conc.

(-8.93 -100) =91.07

25