Dough Rise Assay

Natalie Kruglyak; Erin Li; Chantle R Swichkow

Oct 30, 2023

Dough Rise Assay

DOI

dx.doi.org/10.17504/protocols.io.n2bvj3z2wlk5/v1

Natalie Kruglyak¹,
Erin Li²,
Chantle R Swichkow³

¹Geffen Academy at UCLA;
²Choate Rosemary Hall;
³University of California, Los Angeles

Chantle R Swichkow

University of California, Los Angeles

DOI: dx.doi.org/10.17504/protocols.io.n2bvj3z2wlk5/v1

Protocol Citation: Natalie Kruglyak, Erin Li, Chantle R Swichkow 2023. Dough Rise Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3z2wlk5/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: In development

We are still developing and optimizing this protocol

Created: October 30, 2023

Last Modified: October 30, 2023

Protocol Integer ID: 90115

Keywords: sourdough, yeast, fermentation

Abstract

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.

Materials

Reagents
YPD media agar
Synthetic sourdough medium
ATCC Medium: 0694 Sourdough medium
MRS media
All-purpose flour
Whole-wheat flour
Distilled water

Equipment
15 mL centrifuge tubes with gradation
Sterilized toothpicks or wooden dowels
30°C incubator
30°C shaker
Spectrophotometer
Autoclave
Centrifuge

Recipes
Yeast Peptone Dextrose Media (YPD) - 1L
20 g Bactopeptone
10 g yeast extract
20 g glucose
pH 5.0 (HCl)
2% agar (for plates)

Synthetic sourdough medium - 1L
24 g wheat peptone
0.2 g Magnesium sulfate
0.05 g Manganese sulfate
4 g Monopotassium phosphate
4 g Dipotassium phosphate
1 mL Tween 80
15 g Glucose
35 g Maltose
0.2 mg Cobalamine
0.2 mg Nicotinamide
0.2 mg Folic acid
0.2 mg Pantothenic acid
0.2 mg Pyridoxal-phosphate
0.2 mg Thiamine
pH 4.5 (Citric acid)

ATCC Medium: 0694 Sourdough Medium - 1L
20g Maltose
3g Yeast extract
15 mL Liquid yeast extract
0.3g Tween 80
6g Trypticase
pH 5.6 (20% Lactic acid)

deMan, Rogosa, and Sharpe broth (MRS) - 1L
10 g Proteose Peptone No. 3
10 g Beef extract
5 g Yeast extract
20 g Dextrose
1 g Polysorbate 80
2 g Ammonium Citrate
5 g Sodium Acetate
0.1 g Magnesium Sulfate
0.05 g Manganese Sulfate
2 g Dipotassium Phosphate

Reducing Microbial Load by Autoclaving Flour

Combine unbleached All-Purpose flour and Stone Ground Whole Wheat flour in equal proportions.

Autoclave the flour for 20 minutes using the gravity cycle setting. 

Ensure the container is tightly covered, and store it at room temperature while maintaining cleanliness and hygiene.

Streaking Yeast Cultures on YPD Agar Plates

Under sterile conditions, streak yeast to isolate single colonies onto YPD agar plates.

Repeat this process for all the yeast strains intended for use.

Incubate the plates at a temperature of 30°C for a period of two days or until visible colonies emerge.

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.

Preparation of Yeast Overnight Cultures

Add 2 mL of synthetic sourdough medium into a culture tube

Utilizing a wooden skewer, select a single colony from the streaked agar plate.

Inoculate the sourdough media with the chosen yeast strain.

Place the tubes in a shaking incubator set at 30°C for an overnight incubation.

Preparation of Bacteria Overnight Cultures

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. 

Inoculate the media with bacteria from a glycerol aliquot.

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.

Measurement of Optical Density (OD600)

Utilize a spectrophotometer to measure the optical density (OD600) of each yeast and bacteria overnight culture sample.

Volume Calculation for 0.1 OD per Sample

Calculate the volume required to achieve an OD of 0.1 for each sample. Multiply the calculated volume by the number of replicates desired.

Calculation of Water Addition

Calculate the amount of water needed to adjust the volume of a single culture to 500 µL.

Combining Yeast and Bacteria

If working with a combination of yeast and bacteria, add the volumes required to reach an OD of 0.1 for each species.

Calculate the remaining volume needed to reach a total volume of 500 µL.

Preparation of Master Mix for Dough Samples

For each individual dough sample, allocate 2 grams of autoclaved flour and 1.5 grams of sterile water.

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.

Mixing the Dough

Commence by measuring the designated amount of flour into a suitable vessel, such as a bowl or beaker.

Since heat-treating the flour may lead to clumping and dryness, it is advisable to break up the flour particles as needed before proceeding.

Gradually add the prescribed amount of sterile water to the flour.

Thoroughly mix the flour and water until a consistent and homogenous dough is achieved, ensuring uniformity in preparing the dough samples.

Incorporating Cultures

Incorporate 500 µL of the previously prepared culture into each dough sample, ensuring that the cultures are evenly distributed within the dough mixture.

Preparation of the Final Mixture

Add 3.5 grams of the 75% hydration dough mixture into each 15 mL Falcon tube.

Gently vortex the culture samples briefly to ensure uniformity.

In each Falcon tube, add 500 µL of the prepared inoculum.

Employ a clean wooden skewer to thoroughly mix the inoculum and the dough within each tube, ensuring a consistent distribution.

Initial Measurement

Short spin the centrifuge the Falcon tubes to settle all the dough at the bottom.

Measure the initial heights of the mixture within the tubes.

Ongoing Height Measurements

At regular intervals, measure the height of the mixture within each tube using the gradations marked on the tube's side.

Record Height Changes

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.

Protocol references

1.	Landis, E. A. et al. The diversity and function of sourdough starter microbiomes. Elife 10, (2021).
2.	Bigey, F. et al. Evidence for Two Main Domestication Trajectories in Saccharomyces cerevisiae Linked to Distinct Bread-Making Processes. Curr. Biol. 31, 722–732.e5 (2021).

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