Why Room Temperature Fermentation Makes a Better Pizza Dough [Based on Science]

When it comes to long-fermented pizza dough, many people believe that cold fermentation is superior in terms of flavor development, resulting in deeper and more complex flavors. The goal of this article is to present why the opposite is true, and why, in terms of flavor development, long fermentation at room temperature is actually superior

Introduction

While this article is part of the fermentation series, it differs from the other articles in that it is more of an “opinion article” (supported by scientific evidence), with the main goal of expanding your perspective and opening your mind.

Most of the scientific review in this article is credited to Craig Lindberg, whose work has greatly contributed to its content.

In the article on fermentation basics, we learned about the two main processes that simultaneously occur in dough, directly and indirectly affecting flavor and aroma development:

1. The activity of yeast, bacteria, and the by-products they produce.
2. The activity of enzymes naturally present in the flour or produced by the organisms in the dough (yeast and bacteria).

Now, with that foundation, I will specifically focus on flavor development (the primary reason for long fermentation) in the context of room temperature fermentation versus cold fermentation. The following sections provide an overview of the impact of cold fermentation on each of the factors mentioned above, supported by scientific literature and relevant studies.

Temperature Effects on Dough Enzymatic Activity

Before discussing the impact of cold fermentation and room temperature fermentation on the dough, it is important to first understand enzymes and their role in the dough.

Dough Enzymes Basics

Enzymes, whether naturally occurring in the flour or produced by yeast and bacteria, play a significant role in the development of flavor and aroma in the dough. They achieve this through two main processes:

1. Breaking down starch into simple sugars by amylase enzymes.
2. Breaking down proteins (gluten) into amino acids and peptides by protease enzymes.

The activity of enzymes, like all chemical reactions, depends on temperature. For most enzymes, including those found in dough, an increase in temperature leads to increased activity and a corresponding acceleration in the chemical reactions they catalyze (up to the point where too high a temperature will cause the enzymes themselves to denature).

As a general rule, at room temperature (between 7-30°C/45-86°F), enzyme activity doubles for every 10°C increase in temperature. Similarly, a decrease of 10°C results in a twofold or greater decrease in enzyme activity. The extent of this change varies depending on the specific enzyme, with some exhibiting less than a twofold increase and others exceeding it. Nevertheless, the commonly accepted guideline is a doubling of activity with every 10°C increase in temperature.

The enzymes found in flour, particularly alpha-amylase, are at the upper limit of this range, meaning that their activity doubles and even increases further every 10°C.

Enzyme concentration is also a factor that influences their activity. The relationship is straightforward (and logical): greater enzyme concentration leads to stronger/more activity.

The enzymes responsible for breaking down starch into simple sugars are amylases, specifically alpha and beta amylase. Amylases play a crucial role in the dough as they break down starch into simple sugars, which the yeast and bacteria can then utilize to generate energy and produce the by-products we refer to as “fermentation”. Thus, amylases are considered the enzymes with the most significant function in the dough.

In practical terms, amylase enzymes provide food for yeast and bacteria. Without the breakdown of starch into simple sugars by amylases, none of the fermentation processes in dough – including rising, maturing, and flavor production – would occur. Therefore, the activity of amylase enzymes directly impacts the extent of fermentation and maturation in dough.

Enzymatic Activity in Cold Fermented Dough

Now that we understand the definition and function of enzymes in dough, let’s discuss the specific impact of cold fermentation on enzymatic activity in the dough.

The Activity of Alpha-Amylase Enzymes

So we know that enzymatic activity, which affects the processes the dough undergoes during fermentation, approximately doubles for every 10°C increase in temperature. To further illustrate this, please refer to the graph below from a study titled Alpha-Amylase I from Malted Barley – Physical Properties and Action Pattern on Amylose, which examined the activity of alpha-amylase (derived from malt) at different temperatures.

Pay attention to the markings in red and blue on the grids (my addition). At a temperature of 15°C, the activity of alpha-amylase is three times higher than its activity at a temperature of 4°C; And at a temperature of 30°C, the activity is 12 times higher compared to a temperature of 4°C, when the stability of the enzyme is still 100% (meaning that at this temperature, there is no decline in activity over time).

In practice, this means that the rate at which starch in the flour will be broken down into simple sugars will be 3-12 times higher when the dough ferments at room temperature compared to cold fermentation (depending on the specific temperatures). Accelerated breakdown of sugars means more food available for yeast, which means more active yeast and bacteria, and in short – faster dough fermentation and maturation.

Although the study examined alpha-amylase derived from malt (which is the main source for the production of alpha-amylase used as a flour additive and dough improver), it is reasonable to assume that alpha-amylase from other sources (such as wheat, bacteria, or fungi) will behave similarly in terms of temperature effects, according to the general behavior of enzymes. For example, this study found that the activation energy of alpha-amylase from different sources is similar to that of alpha-amylase derived from malt.

The Activity of Protease Enzymes

What about the activity of another important group of enzymes in the dough – protease enzymes – whose role is to break down the proteins (gluten) in the dough, ultimately affecting the flavor of the dough?

I could not find specific studies on the activity of protease enzymes in dough at temperatures below 20°C/68°F. This area may not have been extensively researched as the activity of these enzymes at low temperatures is generally accepted to be very low.

However, while there is limited research on this topic, we can reasonably assume that the activity of protease enzymes, like that of α-amylase and enzymes in general, will be significantly lower at a temperature of 4°C/40°F. This assumption is logical since the purpose of a refrigerator is to slow down all processes in the dough, including enzyme activity, in order to prevent food spoilage.

For the sake of example, let’s consider a scenario where the activity of protease enzymes does not reduce in the fridge, but instead remains higher compared to other processes in the dough. In this case, more gluten would break down into amino acids, ultimately resulting in more flavor. However, this effect comes with a downside. The accelerated breakdown of gluten would also cause the dough to reach over-fermentation much, much faster.

In practice, the above assumption is incorrect, because cold fermentation actually allows for a much longer fermentation duration compared to room temperature fermentation. This means that the activity of protease enzymes becomes much slower during cold fermentation and remains in sync with the other processes in the dough.

If the activity of protease enzymes were higher in the fridge compared to other processes in the dough, a long cold fermentation would lead to excessive breakdown of gluten, resulting in an over-fermented dough that loses its elasticity and strength; However, this does not happen in practice.

In conclusion, cold fermentation does not prioritize the activity of protease enzymes, and does not contribute to the production of more flavors in this regard.

Conclusion: The Effect of Temperature on Enzymatic Activity

Based on the assumption that the activity of amylase and protease enzymes is crucial for flavor development in the dough, a significant decrease in their activity will result in a much slower flavor development.

Therefore, it can be concluded from the information provided that the fermentation processes in a cold fermented dough (4°C) occur at a rate 3-12 times slower compared to fermentation at room temperature (15-30°C). This slower rate of fermentation affects various processes, including flavor development.

Temperature Effects on Yeast and Bacteria Activity in Dough

The importance and impact of lactic acid bacteria (LAB) and their significant role in flavor development in dough, both directly and indirectly, were discussed in a separate article. When it comes to cold fermentation, it is widely believed that lower temperatures prioritize the activity of LAB over yeast, resulting in distinct flavors that can only be achieved through cold fermentation.

In this section, we will examine the validity of this claim by referring to relevant scientific literature as much as possible.

This section aims to address three key questions:

1. Can LAB function effectively at low temperatures?
2. Does the activity of LAB suffer less from low temperatures compared to yeast?
3. Do yeast and LAB compete for the same resources in the dough?

Can LAB Function Effectively at Low Temperatures?

The rationale for this section is straightforward. If the LAB function well or become more active at low temperatures compared to yeast, then cold fermentation will prioritize their activity over yeast. This, in turn, will allow for the creation of more acids and flavors.

I have personally not found any research that supports the idea that LAB found in doughs made with baker’s yeast perform well at low temperatures. Craig L., who conducted the original scientific review, also did not find any evidence to support this claim.

On the contrary, studies such as “Environmental stress responses in Lactobacillus: A review” have shown that when temperatures drop below 7°C, tested LAB strains, including those common in sourdough, cease to reproduce.

Another study titled “Glutathione Protects Lactobacillus sanfranciscensis against Freeze-Thawing, Freeze-Drying, and Cold Treatment” found that two strains of L. sanfranciscensis, one of the most common LAB strains in sourdough, begin to die almost immediately at low temperatures. After a week at 4°C, the death rate was almost 100%.

As mentioned earlier, there are no studies demonstrating that LAB found in sourdough or dough made with baker’s yeast, thrive at low temperatures. Most studies on LAB and low temperatures focus on food safety, meats, charcuterie, and fermented foods like kimchi or sauerkraut. The absence of research in the context of dough speaks volumes in itself.

A study titled “Behavior of Psychrotrophic Lactic Acid Bacteria Isolated from Spoiling Cooked Meat Products” did find strains of lactic acid bacteria that could reproduce at 4°C. However, these strains were not common LAB found in dough.

According to the research, it took these bacteria one to two days to double their population at 4°C. In ideal conditions (between 30-45°C for LAB), bacteria can double their population every 20-30 minutes; Therefore, the doubling rate at low temperatures is approximately 100 times slower, indicating a significant decrease in their activity, even in cases where LAB were found to be relatively active at low temperatures.

Does the Activity of LAB Suffer Less From Low Temperatures Compared to Yeast?

Cold-loving bacteria, known as psychrophiles, thrive in cold environments ranging from -20°C to 20°C. While there are strains of LAB that are considered psychrophilic, these are mainly relevant to food preservation and the fermentation of meats and other foods, rather than LAB found in dough.

The majority of studies focusing on LAB in the context of dough (bread) concentrate on the optimal temperature range for their activity, which is typically between 20°C and 40°C. This is likely because the activity of LAB found in dough at low temperatures is significantly lower and not sufficiently interesting to warrant further research.

A study titled “Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation” examined the activity of two strains of L. sanfranciscensis (again, one of the most common LAB strains in bread dough, comprising between a third and 95% of the LAB composition in sourdoughs in domestic and industrial environments).

The study found that the activity of the two tested LAB strains decreases significantly with temperature. Additionally, their activity at low temperatures is at least comparable to the activity of the wild yeast in sourdough, specifically C. milleri, a strain of wild yeast commonly found in sourdough:

Although the study compared the LAB strains to wild yeast found in sourdough rather than baker’s yeast, it is reasonable to assume that the activity of baker’s yeast in dough is at least as high as, if not greater than, the activity of these wild yeast strains. This is because baker’s yeast is specifically chosen for its superior ability to leaven dough compared to other yeast strains.

In conclusion, this study, along with the studies mentioned in the previous section, disproves the claim that cold fermentation prioritizes the activity of lactic acid bacteria over yeast. The activity of lactic acid bacteria decreases with temperature, similar to the activity of yeast.

Do Yeast and LAB Compete for the Same Resources in Dough?

Both yeast and LAB “feed” off the same energy source – simple sugars. Theoretically, if the yeast consumes sugar and other resources at a much higher rate than LAB (so the yeast “steals” food from the bacteria), using cold fermentation would slow down yeast activity compared to LAB, resulting in more bacterial activity than yeast activity. But is this assumption correct?

A study titled “Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough” examined the interaction between LAB commonly found in sourdough and the yeast strain S. cerevisiae – the yeast strain used in baker’s yeast.

The study found that the maximum activity/reproduction rate of all tested LAB, except one strain, was not affected by the presence or competition with yeast. The researchers even noted that “Growth of lactic acid bacteria is believed to be promoted when co-cultured with yeast […] regardless the antagonism for the main carbon source”. (‘carbon source’ being sugars and other organic compounds).

Another study titled “The sourdough microflora: Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates” examined the interaction between baker’s yeast and LAB. The researchers found that the two LAB strains tested grew at a faster rate in the presence of yeast compared to an environment without yeast:

The graph shows that bacterial growth was lower in the absence of yeast (represented by squares) compared to its growth in the presence of baker’s yeast (represented by circles), and even lower compared to its growth in the presence of wild yeast (represented by triangles). In simpler terms, the growth of LAB was higher in the presence of both strains of yeast.

In summary, these two studies demonstrate that there are LAB strains common in dough that can successfully ‘compete’ with yeast for resources. While some studies show that the growth rates of certain LAB strains are negatively affected to some extent by the presence of yeast, I have not yet come across a study showing that yeast consumes MOST or ALL of the resources in the dough before the LAB have a chance to consume them.

Furthermore, as mentioned in the previous sections, both yeast and bacterial activity decline at low temperatures in a refrigerator, while the activity of the LAB declines even more. Therefore, even if there were competition for resources between the yeast and LAB, cold fermentation would not “solve” this matter, and would not cause more bacterial activity over yeast activity. In fact, it will be the other way around – cold fermentation will actually favor yeast activity.

LAB Concentration in Dough Made With Baker’s Yeast

In dough made with baker’s yeast, there are between 10,000-100,000 yeast cells for every single LAB bacterium. In sourdough, the ratio is reversed, with about 100 LAB bacteria for every single yeast cell.

In simpler terms, the concentration of LAB bacteria in dough made with baker’s yeast is VERY small; Therefore, it is necessary to allow the LAB enough time to work in order to achieve sufficient LAB activity in the dough. This is one of the main reasons for long fermentation – LAB activity is crucial because it is one of the major contributors to the flavor in the dough.

As we have seen in the previous sections, cold fermentation not only does not contribute to the activity of lactic acid bacteria, but it actually slows it down, just as it slows down other processes in the dough. Consequently, in order to obtain sufficient activity of LAB in cold fermentation, the dough needs to be fermented for a much longer period compared to room temperature fermentation.

This is one of the reasons why cold fermentation usually lasts for at least 24 hours. Anything less than that will not result in enough LAB activity in the dough, which in turn limits the development of flavor.

Studies on Fermentation Temperature and Dough Flavor

So we now understand the theoretical impact of temperature on flavor development. But how do these factors actually affect the flavor in real-life situations, if at all?

Below, you will find two studies that explore the influence of fermentation temperature on the production of aromatic compounds in dough, which directly affect the overall flavor. The second study even includes a taste test conducted by a panel of professional tasters. While the studies focus on bread, they are also applicable to pizza dough, especially the first one that discusses the production of flavors in the crust – since pizza, unlike bread, is mostly crust.

Before we proceed, it is important to emphasize that scientific research requires resources, particularly financial funding. Most baking research is sponsored by large companies, specifically those involved in the large-scale production of baked goods. In this context, studies in this field primarily aim to explore new methods to maximize yield, baking properties, and functional characteristics, with flavor being a less significant aspect.

For this reason, currently, there are no studies – at least up until the time of writing – that directly examine the impact of long fermentation at room temperature compared to long fermentation at low temperatures in relation to flavor, and it is doubtful that such studies will ever materialize. In fact, there are very few studies in general that explore long fermentation in the context of flavor, regardless of temperature.

Study 1: Fermentation Temperature and Volatile Compounds in Bread Crust

The full study can be found here: Volatile compounds in whole meal bread crust: The effects of yeast level and fermentation temperature.

This study examined the impact of different fermentation temperatures on the production of aromatic compounds in the dough. Specifically, it focused on the concentration of these compounds in the crust (which refers to the outer part of the pizza or bread that undergoes browning). While the study also explored the effect of varying yeast concentrations, this aspect is less relevant to our purposes.

It is important to note that the study specifically focuses on the crust rather than the crumb. This distinction is highly significant because, unlike bread where the crumb makes up the majority, in the case of pizza, the crust comprises a large portion of the pizza, including the rim, bottom, and all the areas that undergo browning. In simpler terms: since pizza is mostly crust, the flavor of the crust has a significant impact on the overall taste of the pizza.

It’s also worth noting that the study used wholemeal bread as the type of bread for testing. Therefore, the results may not be directly applicable to pizza or white bread, but it is as close as possible, as studies on this subject are scarce.

Three fermentation temperatures were examined in the study: 8°C/46°F, 13°C/55°F, and 32°C/90°F. Additionally, three different concentrations of dry yeast were used: 2%, 4%, and 6%. It is worth noting that these yeast concentrations are significantly larger than what would typically be used for long fermentation.

All the doughs were prepared and baked using the same method, with the fermentation process considered complete when the dough reached a height of 8 cm. At this point, the dough was baked.

Following the baking process, the researchers analyzed the concentration of aromatic compounds in the crust of each bread.

Below is a table summarizing the fermentation temperatures and durations, as well as the yeast amounts used in each dough and bread:

As you can see, there is a significant difference in fermentation duration due to the “end point” of fermentation being set when the dough reaches 8 cm. For example, the dough containing 2% yeast fermented at 8°C for 20 hours, while the dough with the same amount of yeast fermented at 32°C for only two hours.

It is worth noting that the preparation method of the doughs (using water at 30°C/86°F and a long kneading time of 19 minutes in a bread machine) likely resulted in a VERY high final dough temperature. As a result, the fermentation process may have been accelerated and the dough may have taken longer to cool in the fridge. Unfortunately, the study does not provide any information on the final dough temperature.

For the doughs that underwent relatively short(er) fermentation periods at 8°C, most of the fermentation likely occurred at a temperature much higher than 8°C. It is doubtful that the dough even reached 8°C in such a short time, particularly for the dough fermented for 5 hours.

When dough is placed in the fridge, it takes a considerable amount of time, sometimes even 10 hours or more, to reach the actual refrigerator temperature. This is especially true when the final dough temperature is high, as was likely the case here.

In other words, the results for the short fermentation durations at 8°C are not very accurate or reliable, as it is probable that the dough actually fermented at a much higher temperature.

Additionally, it is important to acknowledge that the researchers’ chosen method of comparing the fermentation point of the doughs (based on height) is questionable. The fact that the dough physically rises and gains volume does not necessarily mean that it has matured and developed flavors. However, for the sake of this discussion, we will set aside this matter, as well as the issue of the final dough temperature and the absurdly high amounts of yeast used.

Study Results and Discussion

The researchers concluded that fermenting at a higher temperature resulted in the formation of more aromatic compounds in the crust.

It is important to note that the presence of aromatic compounds in the dough does not necessarily mean there will be an actual impact on flavor. Each aromatic compound has a minimum concentration threshold, known as the odor detection threshold, which determines whether we can perceive it through smell and taste.

In simpler terms, even if a specific aromatic compound is present in the dough, if its concentration falls below the odor detection threshold, it will have no effect on the actual flavor. Therefore, just because the study discovered an increase in the number of aromatic compounds under certain conditions, it does not automatically translate into noticeable flavor during a real taste test. To assess this, a tasting panel is necessary, as was performed in the next study we will discuss.

In addition to the limitations and issues with the study discussed in the previous section (regarding dough preparation and comparison), these factors render this study unreliable in terms of determining the effect of fermentation temperature on flavor development.

However, I chose to discuss it as an example for the following reasons:

1. The objective findings of this study indicate that fermenting at a higher temperature results in more aromatic compounds in the crust (which is particularly relevant since pizza primarily consists of crust).
2. This is one of the few studies that have explored flavor development in dough under different fermentation temperatures.

Therefore, at the very least, this study does not disprove the assumption that cold fermentation offers no advantages over room temperature fermentation in terms of flavor development.

Study 2: Fermentation Temperature and French Bread Flavor

The full study can be found here: Effect of fermentation conditions of bread dough on the sensory and nutritional properties of French bread.

This study investigated the impact of different fermentation conditions (duration and temperature) on the nutritional values and flavor properties of sourdough bread and bread made with baker’s yeast. Our focus will be on the bread made with baker’s yeast.

What sets this study apart is the inclusion of a professional taste panel consisting of trained tasters with experience in food research tasting. These experts actually sampled the bread, providing valuable insights into the real-life flavor effects of fermentation time and temperature.

Two doughs were prepared:

1. Dough fermented for a total of 2.5 hours at 23C/73F.
2. Dough fermented for 2.5 hours at 23C/73F, with an additional 24 hours at 6C/43F.

The graph below (taken from the study) illustrates the preparation procedure for each dough.

Following the baking process, each bread underwent flavor analysis, measuring indicators such as aroma formation and taste testing conducted by the taste panel.

Study Results and Discussion

The findings of the study are quite surprising (at least to me).

For the dough made with baker’s yeast, the taste panel found no significant difference in flavor between the two breads. Essentially, this means that the additional 24 hours of fermentation at 6C did not contribute to the flavor or texture of the baked bread! The graph below illustrates the results of the taste test (sensory profile) of the two breads, as rated by the taste panel:

As you can see, while there were certain aspects in which the dough that underwent an extra cold fermentation was slightly “better”, according to the study, the panel of tasters ultimately concluded that there was no significant difference in flavor between the two breads.

It is worth noting that the dough fermented at room temperature was only fermented for two and a half hours. A longer fermentation time at 23C (5 hours or more) would likely have surpassed the flavor of the cold-fermented dough.

In conclusion, this study also demonstrates that cold fermentation does not offer any advantage in terms of flavor development over room temperature fermentation.

Concluding Remarks

The main point I recommend taking from this article is that cold fermented dough takes 3-12 times longer to reach the same level of flavor development compared to dough fermented at room temperature (depending on the room temperature).

To put it simply, flavors develop much faster in dough fermented at room temperature than in cold fermentation. In terms of flavor development, as a rough generalization, dough fermented for 5 hours at a temperature of 25C/77F is equivalent to dough fermented for 40 hours at a temperature of 4C/40F.

It may sound strange, but once you try fermenting dough at room temperature, I promise you won’t look back (and many people I’ve personally accompanied can attest to this).

It’s important to note that the by-products of fermentation change depending on the temperature, including the aromatic compounds formed in the dough and other by-products that affect flavor.

Cold fermentation will inevitably result in a different flavor profile (for better or worse), so even if we reach the same theoretical level of flavor development through cold or room temperature fermentation, the flavor created in the dough will be different.

For example, if a 4-hour room temperature fermentation creates a theoretical 50 “flavor units”, then to achieve the same 50 “flavor units” in cold fermentation, the dough would need to ferment for 24 hours (these numbers are arbitrary and for demonstration purposes only).

In this theoretical scenario, although both doughs contain the same ‘amount’ of flavor, the flavor profile would be different depending on the fermentation temperature.

Beyond this, cold fermentation does not offer any added value in terms of flavor development. If you want to maximize flavors in the dough, room temperature fermentation is the way to go.

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