Effects of Commercial Protein Powders on Microbial Models

Harry Suver

Grade 11

Central Catholic High School

Gram Staining

• Invented by Hans Christian Gram• Is a method of differentiating bacterial species into

two groups, Gram-positive and Gram-negative• Gram-positive bacteria have generally a single

membrane (monoderm) surrounded by a thick peptidoglycan

• Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes

• When stained, a Gram-positive bacteria results in a blue/violet color while Gram-negative results in light red/pink

Gram(-) vs. Gram(+)

Gram-negative Gram-positive

Cell walls contains an extra layer of lipopolysaccharides for extra protection.

Outer membrane protects bacteria from several antibiotics.

Most pathogenic bacteria in humans are Gram (+) organisms.

Simple cell wall.

Some antibiotics work against the formation of the cell wall.

Staphylococcus Epidermidis

• Staph. species consisting of harmless skin-dwelling micro-organisms

• Species of Staph. are commonly used in microbiological experiments because of variety of strain types within each species –each strain has different features and toxins

• Gram-positive bacteria

• Most strains are not pathogenic

Escherichia coli

• Major cause of food-borne illness

• Consumption of undercooked meats

• Consumption of contaminated vegetables or unpasteurized milk

• Also resides in fecal matter• Most studied bacteria in the

world and very commonly used in the lab

• Resides in the digestive tract of animals

• Not all strains of E.coli can result in sickness or disease

• Is a Gram-negative bacteria

Whey Protein

• GNC Pro Performance 100% Whey Protein

• Whey protein is a mixture of globular proteins isolated from whey, the liquid material created as a by-product of cheese production.

• Whey protein is commonly marketed and ingested as a dietary supplement, and various health claims have been attributed to it in the alternative medicine community.

Soy Protein

• GNC Super Foods 100% Soy Protein

• protein isolated from the soybean.

• Made from soybean meal that had been dehulled and defatted

Purpose

• The purpose of this experiment was to see if protein powder had significant effects on the growth of microbial models

Hypothesis

• Null Hypothesis- The protein powder will not have a significant effect the growth of the bacteria

• Alternate Hypothesis- The protein powder will have a significant effect on the bacteria and will cause more colonies to grow than the control groups

Materials

• E.coli• Staph. E.• GNC pro performance

Whey Protein• GNC Soy Protein• LB media (.5% yeast

extract, 1% tryptone, 1% sodium chloride)

• Agar plates• Spreader bars• Bunsen Burner• Vortex

• Incubator• Micropipettes • Sterile pipette tips• Sterile dilution fluid

([SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2, 100mM NaCl)

• Sterile test tubes• Test tube rack• Ethanol• Matches

Procedure

1. Bacteria (E.coli and Staph. E.) were grown overnight in sterile LB Media.2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL.4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL.

5. Solutions were made up in sterile test tubes using sterile fluid and variable.6. 0.1mL. Of the cell cultures were added to the solutions which brought them to a total volume of 10mL.7. Solutions were pipetted onto LB plates using a micro-pipette.8. Spreader bars were sterilized and used to spread solutions on the plates.9. Plates were put in an incubator set at 37 degrees Celsius for 24 hours.10. Colonies were counted, data was recorded, results were analyzed.

Concentrations in TubesCONTROL 5% 10%

STERILEFLUID

9.9 mL 7.9 mL 7.9 mL

VARIABLE 0.0 mL 2.0 mL(of 25% stock)

2.0 mL(of 50% stock)

BACTERIA 0.1 mL 0.1 mL 0.1 mL

TOTALVOLUME

10 mL 10 mL 10 mL

5 replicates for each group

Stat Analysis

ANOVA

• Shows if there is any variations between the mean of 2 or more variable groups

Dunnetts Test

• Shows the variation between a control group and an experimental groups

Alpha=.05 T crit= 3.03

125

130

135

140

145

150

155

160

165

170

Control 5% 10%

Staph with Soy Protein

P-value = 0.037219 = Significant

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Dunnett's Test(Staph w/ Soy)

Concentration T-value Significance

5% 1.207 Not Significant

10% 3.34 Significant

0

20

40

60

80

100

120

140

160

180

Control 5% 10%

Staph with Whey ProteinP-value = .116984 = Not Significant

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Dunnett’s Test (Staph w/Whey)

Concentration T-value Significance

5% -0.99281 Not Significant

10% 1.2264 Not Significant

0

50

100

150

200

250

300

350

400

450

Control 5% 10%

E.Coli with Soy Protein

P-value = 0.003653 = Significant

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Dunnetts Test (E.coli w/ Soy)

Concentration T-value Significance

5% 3.2442 Significant

10% 4.4968 Significant

270

280

290

300

310

320

330

340

350

Control 5% 10%

E.Coli with Whey Protein

P-value = 0.016782 = Significant

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Dunnetts Test (E.coli w/ Whey)

Concentration T-value Significance

5% -0.5841 Not Significant

10% 3.8646 Significant

Conclusion

• The Null Hypothesis can be partially rejected, and the Alternate Hypothesis can be partially accepted.

• Soy protein had significant effects on both bacterial models at all concentrations and caused more colonies to grow.

• Whey protein did not have significant effects on either bacterial model at any concentration.

• While on some occasions the 5% groups had less colonies than the control groups, they had colonies that were larger in size

Limitations Continuations

• Did not have access to any other model organisms.

• Lag time between counting of colonies

• Experiment could be expanded to test protein powder on stem cells or algae.

• Could compare protein powder’s effectiveness to growth hormones and natural animal proteins.

ANOVA (Staph w/ soy)

SUMMARYGroups Count Sum Average Variance

Column 1 5 700 140 62.5Column 2 5 725 145 62.5Column 3 5 761 152.2 3.7

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 376.1333 2 188.0667 4.383838 0.037219 3.885294Within Groups 514.8 12 42.9

Total 890.9333 14

ANOVA (Staph w/ whey)SUMMARY

Groups Count Sum Average VarianceColumn 1 5 700 140 20.5Column 2 5 615 123 1678Column 3 5 809 161.8 501.2

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 3782.8 2 1891.4 2.579534 0.116984 3.885294Within Groups 8798.8 12 733.2333

Total 12581.6 14

ANOVA (E.coli w/ soy)SUMMARY

Groups Count Sum Average VarianceColumn 1 5 1530 306 348.5Column 2 5 1685 337 698Column 3 5 1918 383.6 1417.3

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 15257.2 2 7628.6 9.288822 0.003653 3.885294Within Groups 9855.2 12 821.2667

Total 25112.4 14

ANOVA (E.coli w/ whey)SUMMARY

Groups Count Sum Average VarianceColumn 1 5 1530 306 305Column 2 5 1480 296 247Column 3 5 1690 338 680.5

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 4813.333 2 2406.667 5.858012 0.016782 3.885294Within Groups 4930 12 410.8333

Total 9743.333 14

References

• CentralCatholichs.com/biology

• http://www.gnc.com/home/index.jsp