The Effects of Vitamins B6 and C on Yeast Growth By: Gianmarco Frezza Pittsburgh 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 1% stock solution vitamin B6 1% stock solution vitamin C YEP media (1% yeast extract, 2% peptone, 2% glucose) S. Cerevisiae Klett Spectrophotometer 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 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 (32C), and readings were taken every half hour for six hours in a Klett spectrophotometer. Mixtures by Groups S.Cerevisiae (mL) YEP
Concentrations media (B6, C) % (mL) 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 0.1 0 1.9 Group 3 0.5 7.5 1 0 1 Group 4 0.5 7.5
0 0.1 1.9 Group 5 0.5 7.5 0.1 0.1 1.8 Group 6 0.5 7.5 [0%] B6 [0.01%] C [0%] B6 [0.01%] C
[0.01%] B6 [0%] C [0.01%] B6 [0.01%] C [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 140 120 Group 1 C Group 12C0L Group Group230L Group 0H Group 3 0H Group 4 L0 Group 4 L0 Group 5 LL Group Group 56LL Group LH 6 LH Group Group 77H0 H0 Group Group 88HL
HL Group 99HH Group HH 100 KU 80 60 40 20 0 60 120 180 240 Time (min.) 300 360
L= [0.01%] H= [0.1%] B, C Single factor vitamin C P= 0.972004 Groups at 180 minutes Single Factor vitamin B P= 0.958538 Two factor ANOVA synergy P= 0.1162 120 100 80 Klett Units 0% B 0.01% B 0.1% B 60 40
20 0 0% C 0.01% C 0.1% C Concentrations of vitamin C Single factor vitamin C P= 0.16963 Groups at 360 minutes Single Factor vitamin B6 P= 0.000429 Two factor ANOVA synergy P= 0.1785 140 120 100 Klett
units 80 0% B 0.01% B 0.1% B 60 40 20 0 0% C 0.01% C 0.1% C Concentrations of Vitamin C 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
Groups (Replicant 2) Klett units Group 1 71 Group 1 70 Group 2 73 Group 2 84 Group 3 87 Group 3 80 Group 4
73 Group 4 72 Group 5 59 Group 5 78 Group 6 41 Group 6 35 Group 7 62 Group 7
57 Group 8 79 Group 8 94 Group 9 101 Group 9 100 Raw Data at 360 minutes Groups (Replicant 1) Group 1 Klett units Groups (Replicant 2)
Klett units 128 Group 1 125 Group 2 107 Group 2 108 Group 3 127 Group 3 120 Group 4 92
Group 4 94 Group 5 105 Group 5 98 Group 6 24 Group 6 25 Group 7 118 Group 7 119
Group 8 105 Group 9 113 Group 8 111 Group 9 117 ANOVA at 180 minutes ANOVA at 360 minutes
