The Effects of Vitamins B6 and C on Yeast Growth By: Gianmarco Frezza Pittsburgh Central Catholic HS Grade 9.

The Effects of Vitamins B6 and C on Yeast Growth

By: Gianmarco FrezzaPittsburgh Central Catholic HS

Grade 9

Rationale

• Humans need vitamins B6 & C from their diets or as supplements

• Vitamin B6 RDA: 1.3 mg/day

• Vitamin C RDA males: 90 mg/day, females: 75 mg/day

• Does vitamin B6 or C affect the microbial flora of the body?

Microbial Flora

• Refers to microorganisms living in or on the body (gastrointestinal tract)

• Little is known about the association between humans and their flora

• Effects are mutualistic, parasitic, pathogenic, and commensal

• Perform functions beneficial to the host, including the manufacture of essential vitamins, and the prevention of colonization by undesirable microbes

• Human foods and supplements may have unintended effects on the flora populations and their functions

Saccharomyces cerevisiae

• A fungi• One of the most

intensively studied eukaryotic model organisms

• Commonly known as yeast

• Can be used as a model for human microflora

Vitamin B6 (Pyridoxine)

• Important for more than 100 enzymatic reactions involved in metabolism and brain development during pregnancy and infancy

• Helps the immune system function properly

Vitamin C (ascorbic acid)

• An antioxidant, protects cells from damage from free radicals

• Helps the immune system function properly• Growth and repair of tissues• Protects against cardiovascular disease• Prevents scurvy

Purpose

• To test if Vitamin B6 or C have an effect on S. cerevisiae growth individually or synergistically

Hypothesis

• Null: the vitamins WILL NOT have an significant effect on yeast growth individually or synergistically.

• Alternate: the vitamins WILL have a significant effect on yeast growth both individually and synergistically.

Materials• 36 sidearm flasks• Aluminum foil• Lab coat• Disposable rubber gloves• Micropipetters• Sterile micropipette tips• Sterile dilution fluid

(100mMKH2PO4, 100mMK2HPO4, 10mMMgSO4, 1mMNaCl)

• Macropippetter• Sterile macropipette tips• 3 Incubators

•1% stock solution vitamin B6•1% stock solution vitamin C•YEP media (1% yeast extract, 2% peptone, 2% glucose)•S. Cerevisiae•Klett Spectrophotometer

Procedure

1. 7.5 mL of YEP media was pipetted into each sterile sidearm flask.

2. Four replicates of the following combinations of variables were prepared as follows.

3. 0.5 mL of S. cerevisiae (KU=100) was pipetted into each flask.4. The flasks were covered with a sterilized aluminum foil cap.5. The 36 sidearm flasks were placed in incubators (32⁰C), and

readings were taken every half hour for six hours in a Klett spectrophotometer.

Mixtures by GroupsS.Cerevisiae(mL)

YEP media(mL)

Concentrations (B6, C) %

Vitamin C (mL)

Vitamin B6 (mL)

SDF (mL)

Group 1 0.5 7.5 [0%] B6 [0%] C 0 0 2

Group 2 0.5 7.5 [0%] B6 [0.01%] C

0.1 0 1.9

Group 3 0.5 7.5 [0%] B6 [0.01%] C

1 0 1

Group 4 0.5 7.5 [0.01%] B6 [0%] C

0 0.1 1.9

Group 5 0.5 7.5 [0.01%] B6 [0.01%] C

0.1 0.1 1.8

Group 6 0.5 7.5 [0.01] B6 [0.1] C 1 0.1 0.9

Group 7 0.5 7.5 [0.1%] B6 [0%] C 0 1 1

Group 8 0.5 7.5 [0.1] B6 [0.01] C 0.1 1 0.9

Group 9 0.5 7.5 [0.1%] B6 [0.1] C 1 1 0

Concentrations of the Vitamins within Groups

[0%] (0) B6 [0.01%] (L) B6

[0.1%] (H) B6

[0%] (0) C 00 L0 H0

[0.01%] (L) C 0L LL HL

[0.1%] (H) C 0H LH HH

Vitamin Effects on Yeast Growth

60 120 180 240 300 3600

20

40

60

80

100

120

140

Group 1 CGroup 2 0L Group 3 0HGroup 4 L0Group 5 LLGroup 6 LHGroup 7 H0Group 8 HLGroup 9 HH

Group 1 CGroup 2 0L Group 3 0HGroup 4 L0Group 5 LLGroup 6 LHGroup 7 H0Group 8 HLGroup 9 HH

Time (min.)

KU

L= [0.01%] H= [0.1%]B, C

Groups at 180 minutes

0% C 0.01% C 0.1% C0

20

40

60

80

100

120

0% B0.01% B0.1% B

Concentrations of vitamin C

Klett Units

Two factor ANOVA synergy P= 0.1162

Single Factor vitamin B P= 0.958538

Single factor vitamin C P= 0.972004

Groups at 360 minutes

0% C 0.01% C 0.1% C0

20

40

60

80

100

120

140

0% B0.01% B0.1% B

Concentrations of Vitamin C

Klett units

Two factor ANOVA synergy P= 0.1785

Single Factor vitamin B6 P= 0.000429

Single factor vitamin C P= 0.16963

Conclusion• The null hypothesis is accepted for all trials other than vitamin B6 at 360 minutes exposure.

• Vitamin B6 and C did not have a significant effect on yeast growth individually or synergistically.

Limitations and Extensions

Limitations• Only two

concentrations of the variables used

• Only growth was tested

• Six hour duration

Extensions•More concentrations

•Longer Duration

•Study survivorship

•More species

Resources• http://en.wikipedia.org/wiki/VitaminC

• www.ods.od.nih.gov/factsheets/VitaminC-HealthProfessional/

• http://en.wikipedia.org/wiki/VitaminB6

• www.ods.od.nih.gov/factsheets/VitaminB6-HealthProfessional/

• http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

• Mark Krotec, PTEI

• www.epa.gov/epawaste/nonhoz/municipal/

Raw Data at 180 minutes

Groups (Replicant 1) Klett units

Group 1 71

Group 2 73

Group 3 87

Group 4 73

Group 5 59

Group 6 41

Group 7 62

Group 8 79

Group 9 101

Groups (Replicant 2) Klett units

Group 1 70

Group 2 84

Group 3 80

Group 4 72

Group 5 78

Group 6 35

Group 7 57

Group 8 94

Group 9 100

Raw Data at 360 minutesGroups (Replicant 1)

Klett units

Group 1 128

Group 2 107

Group 3 127

Group 4 92

Group 5 105

Group 6 24

Group 7 118

Group 8 105

Group 9 113

Groups (Replicant 2) Klett units

Group 1 125

Group 2 108

Group 3 120

Group 4 94

Group 5 98

Group 6 25

Group 7 119

Group 8 111

Group 9 117

ANOVA at 180 minutes

ANOVA at 360 minutes