Influence of Probiotic Microorganisms on Pre-Fermentation of Bran for Optimized Pizza Dough Preparation
 

Author: Marica Dasovic, MSc Biology

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Abstract

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This study explores the influence of probiotic microorganisms on the pre-fermentation of wheat Bran to optimize pizza dough preparation. Using Saccharomyces cerevisiae yeast combined with various fluids (including water, Yakult, Yakult with milk, and mozzarella water). Five test samples were evaluated over a 24-hour fermentation period. Samples containing probiotic-rich Yakult and Yakult with milk demonstrated accelerated fermentation, evidenced by earlier pH reduction, increased gas production, and enhanced microbial activity. These results highlight the beneficial role of probiotic bacteria such as Lactobacillus casei Shirota and Streptococcus thermophilus in promoting rapid and efficient bran fermentation. The findings suggest that incorporating probiotic microorganisms can improve fermentation performance and may lead to higher-quality, fiber-enriched pizza dough. Further, extra steps were taken to see real efficiency of the Yakult and Yakult with milk performing same study in less period of time. A follow-up test confirmed that these two samples were the most effective in promoting rapid pre-fermentation. While Sample 1 (water) provided the best visual and textural quality, Sample 5 (mozzarella water) showed a balanced profile in aroma and structure. This research highlights the potential of probiotic-enriched fluids to enhance yeast fermentation in bran-based dough, offering a foundation for future optimization of natural fermentation methods in functional baking.

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​Introduction​

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Yeast, especially Saccharomyces cerevisiae, plays a vital role in the baking industry due to its ability to ferment sugars into carbon dioxide and ethanol, enabling dough to rise and develop its characteristic texture and flavor. While yeast behavior is well understood in refined flour doughs, its performance in bran-rich dough remains underexplored. Bran, the outer layer of cereal grains, is nutritionally valuable but can inhibit yeast activity due to its high fiber content, limited available sugars, and its impact on gluten structure. This makes pre-fermentation – a process where yeast is activated and developed before final dough mixing –especially important when working with bran as a key ingredient.

Pre-fermentation success depends not only on the yeast itself but also on the environment in which fermentation begins. Liquids used in the preferment phase provide hydration and can influence microbial activity, nutrient availability, and fermentation speed. In this study, we explored how different liquid media—specifically water, milk, mozzarella water (the brine from fresh mozzarella cheese), and Yakult (a probiotic dairy drink) —affect yeast activity in a bran-based preferment.

All test groups used the same type and amount of Saccharomyces cerevisiae yeast and bran, allowing us to isolate the impact of the liquid component. Our interest extended beyond just yeast performance—we aimed to examine whether the bacterial cultures naturally present in milk, mozzarella water, or Yakult could enhance or inhibit fermentation, either by producing complementary byproducts or by affecting the microbial balance in the dough.

• Yakult contains the probiotic strain Lactobacillus casei Shirota, known for its resilience and ability to survive gastric acidity. This lactic acid bacterium can produce organic acids and enzymes that may influence yeast fermentation or compete with it.

• Milk may include Lactococcus lactis, Streptococcus thermophilus, and other naturally occurring lactic acid bacteria, particularly in raw or unpasteurized forms.

• Mozzarella water, depending on the source, can contain residual Lactobacillus and Streptococcus strains used in cheese production, along with salts and whey proteins that could influence fermentation speed and dough texture.

The core research question we sought to answer was: Which liquid medium—among water, milk, mozzarella water, and Yakult—provides the most effective conditions for pre-fermentation of bran using a constant yeast strain? We hypothesized that fluids rich in beneficial bacteria or nutrients (such as Yakult or mozzarella water) would enhance yeast fermentation more effectively than plain water.

The primary objective of this study was to determine which fluid supports the most rapid and optimal yeast activity in bran-based preferments. A secondary goal was to observe how bacterial interactions, especially from probiotic or fermented sources, might impact dough development, gas production, and potential flavor outcomes in pizza dough made with high-bran content.

The secondary objective of the study was to identify the most effective and efficient pre-fermentation strategy for bran-based pizza dough by comparing how different bacteria from fluids impact yeast activation and early fermentation performance. Ultimately, this research aims to improve the quality, consistency, and processing time of whole grain doughs for healthier and more practical pizza production.

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Materials and Methods

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This study was designed to evaluate the effect of different fluid bases on the pre-fermentation of bran using a consistent yeast strain. Five test samples were prepared using identical amounts of bran and dry yeast, with only the fluid component varying between samples (Table 1).

Bran: Odlums Wheat Bran, high in fiber (same amount used in all samples)

Dry baker’s yeast: (Saccharomyces cerevisiae, consistent brand and amount)

 

Fluid ingredients:

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• Filtered water (no added bacteria) Sample 1

• Yakult (contains Lactobacillus casei Shirota) Sample 2

• Yakult + water (no yeast) (contains Lactobacillus casei Shirota) Sample 3

• Yakult + milk (contains Lactobacillus casei Shirota, Lactococcus lactis, Streptococcus thermophilus) Sample 4

• Mozzarella water (may contain Lactobacillus delbrueckii, Streptococcus thermophilus, and other lactic acid bacteria from cheesemaking) Sample 5

 

 

 

 

 

 

Table 1: Ingredient quantities and bacterial sources used in the five test samples.

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For other important parameters, we recorded the pH levels of each sample both before and after fermentation to track acidity changes. Temperature measurements were taken twice daily (in the morning and at night), to monitor environmental conditions affecting fermentation. We also used microscopy not only to observe yeast and bacterial growth but to document the progression visually with images captured through the connected camera and laptop. These combined methods gave us a comprehensive understanding of the fermentation dynamics in each sample.
 

Preparation of Samples and Reagents

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All raw materials and reagents used in this study were sourced from local suppliers. Odlums Wheat Bran, known for its high fiber content, was used as the main substrate. Dry yeast (Saccharomyces cerevisiae) was obtained from a commercial bakery supply store. Fluids for pre-fermentation, including bottled water, Yakult probiotic drink, fresh milk, and mozzarella water (collected from fresh mozzarella packaging), were used as received without further treatment. All samples were prepared under controlled conditions to ensure consistency across test groups.

Sample Preparation:

• Five glass containers were cleaned, dried, and labeled from 1 to 5.

• The same amount of bran was added to each container.

• A consistent amount of dry yeast was added to all samples except Sample 3, which was a control without yeast to observe any spontaneous fermentation caused by Yakult bacteria (Lactobacillus casei Shirota) alone.
   

Fluid Addition:

• Sample 1: Bran + dry yeast + water

• Sample 2: Bran + dry yeast + Yakult

• Sample 3: Bran + Yakult + water (no yeast)

• Sample 4: Bran + dry yeast + Yakult + milk

• Sample 5: Bran + dry yeast + mozzarella water
   

All ingredients in each container were thoroughly mixed until a uniform paste-like consistency was achieved. Care was taken to keep ratios consistent in terms of the overall hydration level.

Samples were incubated at room temperature, which during the study period ranged from 24°C to 27°C (summertime conditions). Containers were loosely covered to prevent contamination while allowing gas to escape. Temperature was recorded twice daily (morning and night) using a thermometer. Fermentation activity was observed over a fixed period of time (e.g., 2 to 4 hours), and signs of yeast activity such as gas bubbles, rise, aroma, color and texture changes were recorded​​​​

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Observations and Measurements​​

Rise and gas production were visually assessed. Texture and consistency of the pre-fermented mixture were noted. Any differences in smell or visible microbial activity (particularly in Sample 3) were also documented.

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Results
 

The pre-fermentation process was conducted over a 24-hour period for all five test samples, each containing Odlums Wheat Bran and, except for Sample 3, the same amount of dry Saccharomyces cerevisiae. The primary variable among the samples was the type of fluid used: water (Sample 1), Yakult (Sample 2), Yakult + water without yeast (Sample 3), Yakult + milk (Sample 4), and mozzarella water (Sample 5). Room temperature during the test remained within a summertime range of 24–27°C and was recorded twice daily (morning and night).

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pH Measurements and Early Fermentation Activity

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pH measurements taken before and after fermentation revealed notable changes across all samples. Samples 2 and 4, which contained Yakult and Yakult + milk, respectively showed the most significant drop in pH, indicating earlier and more active microbial fermentation.

As shown in Figure 1, these two samples experienced a faster acidification process compared to the others. This early drop in pH was accompanied by clear signs of fermentation (such as visible rise, gas production, and textural changes), appearing sooner than in the remaining samples (Figure 1).

This suggests that the presence of Lactobacillus casei Shirota (from Yakult), and additionally Lactococcus lactis and Streptococcus thermophilus (from milk), enhanced the metabolic environment for yeast activity and promoted rapid fermentation. As a result, Samples 2 and 4 were identified as the most efficient in supporting fast, effective pre-fermentation.

To confirm these observations, a follow-up experiment was conducted focusing exclusively on these two samples, with the goal of further evaluating and optimizing the fermentation process.





 

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Figure 1: pH levels before and after 24-hour fermentation in all five samples​​

The bar chart shows the initial pH ("Before") and the pH after 24 hours of fermentation ("After") for each sample. The X-axis lists the five samples: Water, Yakult (L. casei), Yakult + Water (No yeast), Yakult + Milk (L. casei, L. lactis, S. thermophilus), and Mozzarella Water (L. delbrueckii, S. thermophilus). The Y-axis represents the pH level. Two bars per sample compare pH changes, showing how fermentation affected acidity. Samples containing probiotic bacteria (Yakult variants and Mozzarella Water) show a significant decrease in pH, indicating acid production during fermentation, while Water remained stable.

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​Visual and Physical Changes in Samples

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Visual and sensory observations were recorded throughout the fermentation period. Before-and-after images were taken for all samples to evaluate changes in color, texture, gas formation, and aroma.

Samples 2 and 4 demonstrated the fastest and most noticeable fermentation activity. They showed significant volume expansion and gas bubble formation, clear signs of active fermentation. Sample 2 (Yakult) exhibited strong yeast activity with abundant gas production and a pronounced rise, although its color was paler compared to others. Notably, Sample 2 also emitted a strong alcoholic smell, typical of ethanol production during yeast fermentation under anaerobic conditions (Figure 2).





 

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Figure 2: Observation of Pre-fermented Bran Samples 2, 4 and 5 During Fermentation

This image shows three pre-fermented bran samples under daylight conditions at 25 °C. Each beaker is labeled with its sample number and fermentation temperature.

Samples 2 and 4 (left and center), covered with transparent film, show visible gas build up beneath the plastic wrap and bubbling on the surface, indicating active fermentation and gas production. This suggests vigorous microbial activity, most likely due to the presence of Lactobacillus casei Shirota (Yakult in Sample 2) and the combination of L. casei, Lactococcus lactis, and Streptococcus thermophilus (Yakult + milk in Sample 4).

Sample 1 (right) is shown with a pH meter inserted, reflecting the monitoring of acidification. The surface appears more liquid and less active compared to Samples 2 and 4, indicating milder fermentation activity.

These observations visually support the data on gas production and pH change, highlighting the stronger fermentation performance of bacterial combinations in Samples 2 and 4.

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Sample 4 (Yakult + milk) also produced rapid fermentation but resulted in a looser texture and a more acidic, yogurt-like aroma, likely due to the combined presence of Lactobacillus casei Shirota, Lactococcus lactis, and Streptococcus thermophilus.

Sample 1 (water) displayed the most visually appealing color and structure, with a warm golden-brown tone and a balanced, slightly firm texture. Sample 5 (mozzarella water) showed good fermentation, offering a favorable texture and subtle dairy aroma (Figure 3).

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Figure 3: Before and after images of Samples; illustrating visible rise, texture changes, and color variation.

(A) Pre-fermented bran fermentation: Bran samples were soaked and inoculated with different microbial sources to initiate wild yeast and lactic acid bacterial fermentation. From left to right:

Sample 1 – Filtered water (control, no added bacteria)

Sample 2 – Yakult (contains Lactobacillus casei Shirota)

Sample 3 – Yakult + filtered water (no yeast, contains L. casei Shirota)

Sample 4 – Yakult + milk (contains L. casei Shirota, Lactococcus lactis, Streptococcus thermophilus)

Sample 5 – Mozzarella water (may contain Lactobacillus delbrueckii, S. thermophilus, and other LAB from cheesemaking)

(B) Dough formation: After fermentation, each pre-fermented bran sample was used to leave the whole wheat dough. The doughs show differing textures and rise, reflecting the varying fermentation activities of the microbial communities.

(C) Final baked sourdough loaves: Baked loaves prepared from each dough in (B). Visual differences in crust, rise, and shape demonstrate the impact of different bacterial inoculants on leavening performance and bread quality.​

Note: Although this experiment aimed to evaluate pre-fermented bran as a leavening agent, the microbial activity observed was not strong enough to fully ferment the dough. Therefore, commercial yeast was added in the final dough to ensure proper rise and baking.

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Overall, Samples 2 and 4 proved to be the most active in terms of fermentation speed and microbial activity, while Sample 1 showed excellent visual qualities and texture. Sample 5 offered a well-balanced profile in terms of both its structure and very nice smell. These findings provide useful insights into selecting effective fluid ingredients for pre-fermenting bran-based pizza dough (Table 2).

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​Table 2: Summary of physicochemical changes and observable fermentation characteristics in five dough samples prepared with varying fluid ingredients.

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The pre-fermentation period was set for 24 hours for all test samples; however, accelerated fermentation was observed in Samples 2 and 4, where signs of active fermentation appeared much earlier than in the other samples. This suggests that the fluid components in these samples (Yakult in Sample 2 and Yakult combined with milk in Sample 4), enhanced the fermentation rate, resulting in faster microbial activity during the pre-fermentation process. These findings indicate that Samples 2 and 4 were the most effective in promoting rapid and efficient pre-fermentation, and consequently, a follow-up experiment was conducted focusing exclusively on these two samples to further evaluate and optimize the pre-fermentation process.

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Discussion

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This study aimed to determine the most effective fluid ingredient for pre-fermenting bran in order to optimize the preparation of pizza dough. The results clearly show that the type of fluid used in the pre-fermentation mixture had a significant impact on the speed and quality of fermentation. Among the five test samples, Samples 2 (Yakult) and 4 (Yakult + milk) demonstrated the most rapid and vigorous fermentation, with earlier signs of microbial activity, greater gas production, and more pronounced pH reduction compared to the other samples. These outcomes suggest that the presence of probiotic bacteria, particularly Lactobacillus casei Shirota (found in Yakult), and additional strains such as Lactococcus lactis and Streptococcus thermophilus (from milk), played a synergistic role in enhancing yeast fermentation.

The strong alcohol aroma detected in Sample 2 further supports the evidence of accelerated yeast metabolism, as ethanol is a primary byproduct of Saccharomyces cerevisiae fermenting sugars anaerobically. Microscopic observations confirmed active yeast budding and dense microbial presence in Samples 2 and 4 at earlier time points, aligning with their rapid fermentation behavior. These findings support existing knowledge that lactic acid bacteria can improve dough fermentation by acidifying the environment and stimulating yeast performance.

While Sample 2 achieved the fastest and most intense fermentation, Sample 1 (water) showed superior visual qualities, including a warm golden color and balanced texture, despite its slower fermentation rate. Sample 5 (mozzarella water) also performed well in terms of texture and had a pleasant dairy aroma, likely due to the presence of Lactobacillus delbrueckii and Streptococcus thermophilus. These observations suggest that while bacterial stimulation enhances speed, the choice of fluid also affects sensory and structural qualities that are important in dough development.

A follow-up experiment focusing on Samples 2 and 4 was conducted to further evaluate their effectiveness. The results reinforced their status as the most efficient in terms of fermentation speed, making them strong candidates for future optimization. However, texture and aroma should also be considered when selecting fermentation agents for food products like pizza dough.

This study has some limitations. The test was conducted once under uncontrolled room temperature conditions (24–27°C), which may have influenced microbial activity. Additionally, microscopic analysis was observational rather than quantitative, and fermentation volume was not precisely measured. Future studies could improve reliability by using controlled environments, repeating the experiment with larger sample sizes, and measuring CO₂ output or rise volume more accurately.

In conclusion, the data indicates that the combination of bran with Yakult or Yakult and milk significantly enhances the speed and efficiency of pre-fermentation. These findings could contribute to developing a faster, more efficient dough fermentation method, especially in fiber-rich bran-based recipes. Further exploration of combinations that balance speed, structure, and sensory quality is recommended.

 

Conclusion

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This study investigated the effect of different fluid ingredients on the pre-fermentation of bran using dry Saccharomyces cerevisiae yeast. Key findings revealed that Samples 2 (Yakult) and 4 (Yakult + milk) showed significantly faster and more active fermentation compared to other samples, as evidenced by earlier pH drops, stronger gas production, and increased microbial activity observed under the microscope. Sample 2 demonstrated the most intense fermentation, while Sample 1 (water) resulted in the best texture and color, and Sample 5 (mozzarella water) provided a balanced outcome in both aroma and structure.

The research is significant as it identifies probiotic fluids, particularly Yakult and Yakult with milk, as effective agents for accelerating and enhancing the pre-fermentation of bran a key component in high-fiber pizza dough. These findings offer practical value for improving dough preparation in both home and commercial baking, where fermentation time and consistency are critical.

Future research should focus on optimizing combinations of probiotic fluids and yeast to balance fermentation speed with dough quality. More controlled testing conditions, quantitative CO₂ and rise measurements, and expanded trials with different types of bran or flour could help develop standardized, scalable pre-fermentation methods for fiber-rich dough products.

 

References

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Journal Articles:

1. Lin, M.-T., Hu, C.-C., Lin, T.-Y., & Yu, R.-C. (2022). Influence of Lactobacillus (LAB) Fermentation on the Enhancement of Branched Chain Amino Acids and Antioxidant Properties in Bran among Wheat By-Products. Fermentation, 8(12), 732. https://doi.org/10.3390/fermentation8120732

2. Arendt, E. K., Ryan, L. A. M., & Dal Bello, F. (2007). Impact of sourdough on the texture of bread. Food Microbiology, 24(2), 165–174. https://doi.org/10.1016/j.fm.2006.07.011

3. De Vuyst, L., & Leroy, F. (2007). Functional role of lactic acid bacteria in sourdough bread ecosystems. Food Microbiology, 24(2), 120–127. https://doi.org/10.1016/j.fm.2006.07.006

4. Corsetti, A., & Settanni, L. (2007). Lactic acid bacteria in sourdough fermentation. Food Research International, 40(5), 539–558. https://doi.org/10.1016/j.foodres.2006.11.001

5. Gobbetti, M., De Angelis, M., Di Cagno, R., Minervini, F., & Limitone, A. (2007). Sourdough lactic acid bacteria: Exploration of non-starter microorganisms and their metabolic role in bread making. Food Microbiology, 24(2), 151–160. https://doi.org/10.1016/j.fm.2006.07.001

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1. Hammes, W. P., & Hertel, C. (2009). The genera Lactobacillus and Carnobacterium. In E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt, & F. Thompson (Eds.), The Prokaryotes (pp. 320–403). Springer. https://doi.org/10.1007/978-3-540-69736-7_13

2. Cauvain, S. P. (2015). Technology of Breadmaking (3rd ed.). Springer. https://doi.org/10.1007/978-1-4899-7637-7

3. Hui, Y. H. (Ed.). (2006). Handbook of Food Science, Technology, and Engineering (Vol. 1). CRC Press.

4. Wood, B. J. B. (1998). Microbiology of Fermented Foods (2nd ed.). Springer. https://doi.org/10.1007/978-1-4615-4197-6