TITLE: Dough Rise Assay
AUTHORS: Natalie Kruglyak, Erin Li, Chantle R Swichkow

[DESCRIPTION]
This protocol presents an accessible method for actively evaluating dough rise phenotypes in various yeast strains and yeast/bacteria combinations. As dough rise is a pivotal element in bread-making, this approach simplifies assessing the influence of genetic background on dough rise, particularly in non-food science laboratories and educational settings. The process begins by inoculating a sterile dough matrix with yeast and bacteria cultures in a graduated container. The graduations allow for accessible quantification of dough rise over 24 hours. The mixture's height is monitored over time, offering valuable insights into the dynamics of dough rise. This data allows for an active analysis of dough rise dynamics. This protocol is a valuable tool for actively assessing the leavening potential of yeast strains in a controlled and reproducible environment. It actively streamlines the selection of yeast strains that positively impact dough rise, thereby benefiting both the baking industry and sustainability goals through the potential reduction of the environmental impact of bread production.


[STEPS]
SECTION: Reducing Microbial Load by Autoclaving Flour
1. Combine unbleached All-Purpose flour and Stone Ground Whole Wheat flour in equal proportions.
SECTION: Reducing Microbial Load by Autoclaving Flour
2. Autoclave the flour for 20 minutes using the gravity cycle setting. 
SECTION: Reducing Microbial Load by Autoclaving Flour
3. Ensure the container is tightly covered, and store it at room temperature while maintaining cleanliness and hygiene.
SECTION: Streaking Yeast Cultures on YPD Agar Plates
4. Under sterile conditions, streak yeast to isolate single colonies onto YPD agar plates.
SECTION: Streaking Yeast Cultures on YPD Agar Plates
5. Repeat this process for all the yeast strains intended for use.
SECTION: Streaking Yeast Cultures on YPD Agar Plates
6. Incubate the plates at a temperature of 30°C for a period of two days or until visible colonies emerge.
SECTION: Streaking Yeast Cultures on YPD Agar Plates
7. Subsequently, wrap the plates with parafilm and transfer them to a storage environment at 4°C. This storage can be maintained for up to two weeks.
SECTION: Preparation of Yeast Overnight Cultures
8. Add 2 mL of synthetic sourdough medium into a culture tube
SECTION: Preparation of Yeast Overnight Cultures
9. Utilizing a wooden skewer, select a single colony from the streaked agar plate.
SECTION: Preparation of Yeast Overnight Cultures
10. Inoculate the sourdough media with the chosen yeast strain.
SECTION: Preparation of Yeast Overnight Cultures
11. Place the tubes in a shaking incubator set at 30°C for an overnight incubation.
SECTION: Preparation of Bacteria Overnight Cultures
12. Fill a 15 mL tube with 13 mL of the appropriate bacterial growth medium. L. Plantarum and L. Brevis grow in MRS, while L. Sanfranciscensis thrives in ATCC Medium: 0694 Sourdough Medium. 
SECTION: Preparation of Bacteria Overnight Cultures
13. Inoculate the media with bacteria from a glycerol aliquot.
SECTION: Preparation of Bacteria Overnight Cultures
14. Incubate the bacteria under static conditions, allowing them to grow overnight at 30°C. It's important to note that L. Sanfranciscensis may require an extended incubation period of two days or more to reach a sufficiently high concentration for use in the assay.
SECTION: Measurement of Optical Density (OD600)
15. Utilize a spectrophotometer to measure the optical density (OD600) of each yeast and bacteria overnight culture sample.
SECTION: Volume Calculation for 0.1 OD per Sample
16. Calculate the volume required to achieve an OD of 0.1 for each sample. Multiply the calculated volume by the number of replicates desired.
SECTION: Calculation of Water Addition
17. Calculate the amount of water needed to adjust the volume of a single culture to 500 µL.
SECTION: Combining Yeast and Bacteria
18. If working with a combination of yeast and bacteria, add the volumes required to reach an OD of 0.1 for each species.
SECTION: Combining Yeast and Bacteria
19. Calculate the remaining volume needed to reach a total volume of 500 µL.
SECTION: Preparation of Master Mix for Dough Samples
20. For each individual dough sample, allocate 2 grams of autoclaved flour and 1.5 grams of sterile water.
SECTION: Preparation of Master Mix for Dough Samples
21. Calculate the cumulative quantities needed for the entire set of samples, including an extra amount for contingencies. This total quantity ensures that all samples can be uniformly prepared.
SECTION: Mixing the Dough
22. Commence by measuring the designated amount of flour into a suitable vessel, such as a bowl or beaker.
SECTION: Mixing the Dough
23. Since heat-treating the flour may lead to clumping and dryness, it is advisable to break up the flour particles as needed before proceeding.
SECTION: Mixing the Dough
24. Gradually add the prescribed amount of sterile water to the flour.
SECTION: Mixing the Dough
25. Thoroughly mix the flour and water until a consistent and homogenous dough is achieved, ensuring uniformity in preparing the dough samples.
SECTION: Incorporating Cultures
26. Incorporate 500 µL of the previously prepared culture into each dough sample, ensuring that the cultures are evenly distributed within the dough mixture.
SECTION: Preparation of the Final Mixture
27. Add 3.5 grams of the 75% hydration dough mixture into each 15 mL Falcon tube.
SECTION: Preparation of the Final Mixture
28. Gently vortex the culture samples briefly to ensure uniformity.
SECTION: Preparation of the Final Mixture
29. In each Falcon tube, add 500 µL of the prepared inoculum.
SECTION: Preparation of the Final Mixture
30. Employ a clean wooden skewer to thoroughly mix the inoculum and the dough within each tube, ensuring a consistent distribution.
SECTION: Initial Measurement
31. Short spin the centrifuge the Falcon tubes to settle all the dough at the bottom.
SECTION: Initial Measurement
32. Measure the initial heights of the mixture within the tubes.
SECTION: Ongoing Height Measurements
33. At regular intervals, measure the height of the mixture within each tube using the gradations marked on the tube's side.
SECTION: Record Height Changes
34. Record and document the observed changes in height over time by subtracting the initial dough height from the height measured during subsequent intervals. This data will monitor and analyze the dynamics of the mixture over the specified duration.