Relationship of Temperature to Fungal Growth on Bread

Bread makes up a substantial part of many people’s diets, and moldiness presents a considerable issue in regards to maintaining bread’s freshness. However, there’s more to the mold-fungus- growing on bread that meets the eye. Fungi are made up of filaments called hyphae, and the network of hyphae are known as mycelia. All fungi are heterotrophic and secrete hydrolytic enzymes that break down their surrounding material, subsequently absorbing the broken-down nutrients. Fungi enjoy feeding on sugars, and Rhizopus stolonifer in particular flourishes amongst sucrose and glucose.

The phyla Zygomycota contain species of fungi that decompose fruits, bread, etc. Rhizopus stolonifera, commonly known as black bread mold, is a prevalent mold seen growing on bread. Other genera of fungi that can contaminate bread are Alternaria, Aspergillus, Cladosporium, Mucor, and Penicillium (Vagelas, Gougoulias, Nedesca, and Liviu, 2011; Lohano, Sheikh, and Shahnawaz, 2010).

The contamination of bread itself occurs after the bread has been prepared, as any existing spores would either be damaged and/or destroyed during the baking process. Rather, contamination can happen anytime during transportation, storage, and packaging either from the air or from contact with surfaces (Vagelas, 2011). There have also been some cases of insects spreading spores as well, though it’s seen mainly in fruits (Amiri, 2011).

And while fungal contamination from surfaces can be reduced, it is more difficult to control the spores in the air. A study conducted on the quality of indoor air in university rooms in Poland looked at the concentration of bacterial and fungal in the air of nine university rooms (lecture room with ventilation, lecture room without ventilation, chemical laboratory, library, reading room, dean’s office, canteen, toilets, and corridor). The highest concentration of fungus in the student canteen in the morning was Rhizopus (58%) and Penicillium (19%), and in the afternoon, Cladosporium (46%) and Penicillium (24%). The same study was conducted the following year and in the same room, the dominating morning species were once again Rhizopus (33%) and Penicillium (30%). Penicillium (57%), and a tie for Cladosporium and Aspergillus (14%) in the afternoon. For reference, the CFU/m3 in the canteen averaged 1.7x103 for mornings and afternoons for both years (Stryjakowska et al., 2007). This study displays the prevalence of fungus in the air, especially in areas of high traffic, therefore demonstrating the likelihood of contamination of bread from contact with indoor air.

Using Rhizopus stolonifer as a demonstration, once contamination of bread occurs, hyphae begin to grow into and over the surface, secreting their digestive enzymes and feeding on the resulting nutrients. During times of low environmental stress, the zygomycetes will enter their asexual phase, where stalks of bulbed, black sporangia grow on the surface of the bread and release spores aerially. During times of stress, this fungus can engage in sexual reproduction using mycelia; sexual reproduction produces a zygosporangium that will remain dormant until the period of environmental stress passes at which point it go through meiosis and becomes a sporangium, thus recommencing the life cycle (Urry et al., 2016).

The role of artificial preservatives is also worth mentioning. Preservatives serve to slow down microbial growth while not having a meaningful effect on pH and composition, making them very popular across the world. Three common mold inhibitors used in bread are potassium sorbate, calcium propionate, and ethanol. Other mold inhibitors include sorbates, dimethyl fumarate, acetates, salt- though it alters flavor and processing too much to be effective, pasteurization, and freezing (Lohano et al., 2010). Bread that is rich in fibers has also shown to be more resistant to fungal growth than white bread (Vagelas et al., 2011).

The purpose of this experiment is to determine if there is a correlation between temperature and the formation of mold on the surface of bread, which will most likely consist of zygomycetes Rhizopus, specifically Rhizopus stolonifera, and Mucor, as well as possibly Penicillium. It was hypothesized that the bread samples kept at room temperature (~25℃) would have greater fungal growth than the samples kept in a refrigerated environment (~4℃). It is also important to note that the visual evidence of bread mold is when the fungus has entered its reproductive state, and therefore the presence of any fungi extended prior to visual evidence.

Methods

Ten slices of bread were taken from a single loaf of bread. Each slice was carefully measured and cut into 10x10 cm squares (note that when handling the bread, clean hands or sterile gloves were used to avoid cross contamination). The ten squares were allowed to sit undisturbed at room temperature for an hour, during which ten sandwich-sized plastic zip lock bags were collected and a 10x10cm (100cm2) grid pattern was drawn on the outside of all ten bags using a thin permanent marker. Five of the bags were labeled F1, F2, F3, F4, and F5 for the five refrigerated samples, and the other five were labeled R1, R2, R3, R4, and R5 for the room temperature samples.

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Once the hour had passed, a slice of bread was placed into each of the zip lock bags and sealed shut. These bags were not opened again for the entirety of the experiment and even afterwards. The zip lock bags were placed in their respective environment (F samples in fridge and R samples at room temperature). The refrigerated samples were placed in the back of the fridge, where they would remain undisturbed and received less light than upper shelves. The room temperature samples were placed in a location that matched those the refrigerated samples’ environment as closely as possible.

The samples were checked on at noon every three days. The refrigerated samples were taken out one by one and the readings were taken as swiftly as possible while still maintaining the accuracy of the reading in order to reduce abrupt temperature changes. The data was collected through matching up the bread with the zip lock grid and counting the number of squares with visible fungus growth. The rule of thumb for incompletely filled squares was to round up if the coverage exceeded more than half a square, and otherwise rounding down. The number of squares filled could easily be converted into percentages, as the grid was already out of 100.

After the 21 days had passed and all data had been properly collected (eight data entries for both temperatures should have been made at this point) as well as final observations done, all ten samples were gathered and disposed of properly. Calculations were later made using the results, the most important of which was the statistical t-test.

Results

The average growth of the refrigerated sample on day 21 was 74.400% coverage. The average growth of the room temperature sample on day 21 was 87.200% coverage. The standard deviation for refrigerated samples and room temperature was 3.323%and 4.445%, respectively. A two-tailed t-test was conducted to determine if the refrigerated samples and the room temperature had a difference in growth rates. There was a significant difference between refrigerated samples and room temperature samples (T Refrigerated, Room Temperature = 4.613, T crit 12,0.05 = 2.776).

Discussion

Based on the results, it is safe to say the experiment went as expected. The hypothesis outlined at the onset of this experiment predicted that the room temperature samples of bread would have a higher rate of fungal growth than the refrigerated samples. The results supported the hypothesis and were statistically significant with a p-value of less than 0.05; this statistical significance allows for the elimination of the null hypothesis, demonstrating that the results were not due to chance, and also points to the possibility of a 5% chance of error.

In relation to other studies, a study conducted on bread enriched with fibers versus white bread using two species of Penicillium fungi (Penicillium digitatum and Penicillium italicum) at different temperatures garnered similar results. The bread was separated into two environments at different temperatures: one at 19℃ and the other at 25℃ for seven days. The researchers found that the amount of spores on both types of bread kept at 25℃ to be greater than the bread kept at 19℃. The study concluded by stating that fungus develops in environments with a temperature of 19-25℃ with 80% humidity for the reasoning that spores germinate well in these conditions and the hyphae they form is successful at penetrating the bread (Vagelas et al., 2010).

Another study looked at the effect of nutrient status, pH, temperature and water potential on germination and growth of Rhizopus stonolifer and Gilbertella persicaria, two zygomycetes. Focusing on their results of the culture growths in relation to temperature, they found that fungal germination was severely slowed at temperatures 5-10℃ and growth ceased entirely when temperatures dropped below 10℃. Growth was found to be most successful at 20℃ and germination at this temperature had a recorded 100% rate with the right water potential. Interestingly, the growth rate for both species of fungi decreased at 30℃ (Amiri et al., 2011).

From this point, these results can be used as an aid to further the effort of bread preservation. This experiment showed that cooler temperatures are effective at inhibiting fungal growth, but are not entirely successful; there was still a 74% growth rate on the 21st day. Perhaps this experiment could be repeated with frozen bread as a variable. The second study listed above mentioned a cessation of mycelial growth in temperatures less than 10℃, after all (Amiri et al., 2011). The possible vulnerability of contamination or a burst of fungal growth from dormant species upon thawing should also be looked into as well as any alterations to taste or composition of the bread. Decreasing humidity could also be another short-term inhibitor (Vagelas et al., 2011).

Despite all this, artificial preservatives do seem to be the popular route due to its ease of access and high rate of success. A combination of storing bread at refrigerated temperatures and adding preservatives could be another possibility to be looked at. Perhaps there are certain preservatives that function more successfully at lower temperatures relative to others so that the two techniques can be used conjointly. Overall however, storing bread in a refrigerator has proven to decrease fungal growth, and is an easy, accessible option (to many people) for an extended shelf life of bread.

References

1. Amiri, A., Chai, W., & Schnabel, G. (2011). EFFECT OF NUTRIENT STATUS, pH, TEMPERATURE AND WATER POTENTIAL ON GERMINATION AND GROWTH OF RHIZOPUS STOLONIFERAND GILBERTELLA PERSICARIA. Journal of Plant Pathology, 93(3), 603-612.
2. Bio II Lab Manual (year). The Moldy Bread Lab – Fungus Among Us. Nova Southeastern University Biology Department.
3. Lohano, D. K., Sheikh, S. A., & Shahnawaz, M. (2010). Effect of Chemical Preservatives on the Shelf Life of Bread at Various Temperatures. Pakistan Journal of Nutrition, 9(3), 279-283. Retrieved 2010.
4. Stryjakowska-Sekulska, M., Piotraszewska-Pająk, A., Szyszka, A., Nowicki, M., & Filipiak, M. (2007). Microbiological Quality of Indoor Air in University Rooms. Polish Journal of Environmental Studies, 16(4), 623-632.
5. Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Reece, J. B. (2016). Campbell Biology (Eleventh ed.). New York: Pearson.
6. Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Orr, R. B. (2019). Campbell Biology In Focus (Third ed.). Pearson.
7. Vagelas, I., Gougoulias, N., Nedesca, E., & Liviu, G. (2011). BREAD CONTAMINATION WITH FUNGUS. Carpathian Journal of Food Science and Technology, 3, 1-6.