The Effect of Different Incubation Temperature (20.0, 40.0, 60.0, 80.0, 100.0) of 10.

000
grams of Yogurt on the Lactic Acid Concentration (g/L3)

By Krishay Pradeep

Introduction:
When I was younger, my mother made yogurt in an Insta-Pot. She would use Dairyland
Milk with healthy bacteria to ferment and create delicious yogurt. She told me that yogurt
contained lactic acid which would help fuel muscle growth and assist different functions in the
human body such as immunity and glucose production. However, my mother also told me that an
excessive amount of lactic acid can cause health problems such as muscle weakness. This
sparked my curiosity for yogurt and the concentration of lactic acid; three years later from that
conversation, I had the idea to test the relationship between different incubation temperatures of
yogurt and the lactic acid concentration. Thanks to the lab equipment found in my school lab and
numerous kitchen tools, I can make this dream happen. The results of this lab will help me, and
other yogurt makers determine what incubation temperature have the healthiest amount of lactic
acid. Furthermore, it can help individuals like my mother who do not have accurate or precise
chemical tools, determine what temperature range results in perfect amount of lactic acid.
Based on research I believe the relationship between incubation temperatures and lactic
acid concentration will be inversely related because bacteria thrive in acidic conditions with
temperatures between 40°F and 140°F (4.44°C and 60°C) (USDA, n.d). These temperatures
will cause bacteria to thrive on lactose but increase temperatures will kill yogurt bacteria
colonies therefore decreasing the concentration of lactic acid. Therefore, increasing temperatures
lower the lactic acid concentration in yogurt.

Research Questions: How is Latic Acid concentration (g/L3) in 10 g of Yogurt affected by

different incubation temperatures (20.0, 40.0, 60.0, 80.0, 100.0) (± 0.5°C)?

Background Research:
Yogurt is a type of fermented milk containing healthy bacteria (or cultures) which are
popular in many cultures such as India or Canada (Mom, 2022). Yogurt is best made with
homogenized cow or goat milk as the cream has already been divided into small particles (Mom,
2022). Yogurt has a huge amount of protein and calcium which helps for different functions of
our body such as bone strength, increasing immunity throughout our body and protecting against
colon cancer (Smith, n.d). In terms of chemistry, yogurt is created when bacterium ferment
lactose, a milk sugar lactic acid (Smith, n.d). This lactic acid decreases the pH value of yogurt
(around 4) and correlates with the taste of the yogurt; more lactic acid contributes to more
sourness in the yogurt (Smith, n.d). Furthermore, the lactic acid shrinks the protein in the yogurt
(Smith, n.d), a process which is irreversible (Oxford IB Chemistry Course Companion, 2014).
 Lactic acid is a type of acid produced because of bacteria (specifically consuming
Lactobacillus bulgaricus bacterium) (Smith n.d). Latic acid was first synthesized by the chemist
Carl Wilhelm Scheele in 1780 and found to have had a pH level of 2.43 (Vendantu, n.d). The
chemical formula for lactic acid is C3H6O3 (Oxford IB Chemistry Course Companion, 2014).
Lactic Acid has a melting point of 530°C and a boiling point of 1220°C and therefore can
withstand high temperatures (Vendantu, n.d). Lactic acid is usually found as a yellowish syrup in
liquid form but in solid form it is found as a white powder (Vendantu, n.d). Lactic acid is
corrosive and to metals or cellular tissue and therefore can have health consequences if
consumed such as muscle fatigue (Vendantu, n.d).
Since yogurt is acidic because of lactic acid, it can be titrated with a base through an acid-
base titration. This method helps us find the concentration of an analyte (in this case lactic acid)
using the volume utilised of a titrant. Yogurt can be titrated with NaOH, a base with a pH level
of 13.0. This can create an acid-base reaction between lactic acid and NaOH which is shown
below.
C3H6O3 + NaOH → NaC3H5O3 + H2O

The equation is perfectly balanced, hence proving that NaOH is the best titrant to use for yogurt
titrations. To titrate yogurt properly, three drops of a pH indicator are dropped (phenolphthalein
indicator); if the yogurt turns faint pink during the titration, it means the titration has been
completed successfully. The concentration of lactic acid can be used using the volume of NaOH
used with these formulas: n = cv where n is the number of moles, c is the concentration and v is
the volumes and d = m/v, where d is the density, m is the mass and v is the volume.

Independent Variable: The incubation temperatures (20.0, 40.0, 60.0, 80.0, 100.0) °C of 10
grams of yogurt. The instrument used to measure this variable is a mercury thermometer with an
uncertainty of (± 0.5°C). The range of this thermometer is between -20 °C and 120 °C

Dependent Variable: The Latic Acid concentration (g/L3) in the yogurt. This is measured and
found using titration methods with 1 M NaOH and multiple calculations

Controlled Variables:
Figure 1: Control Variables Table
What was controlled How it was Why it was
controlled controlled
1 The volume of water 300 mL of tap water If more than 300 mL
used for each were warmed at of tap water was used
different incubating different for each incubation
temperature of the temperatures (listed temperature interval,
yogurt above) and were then the water would
poured to surround overflow into the
the glass jars and yogurt, therefore
incubate the yogurt ruining the purity of
the yogurt. Vice
 versa, if there is less
than 300 mL of tap
water used for each
incubating
temperature, then the
water will not
incubate the yogurts
properly.
2 Temperature of the The milk was heated The milk is heated to
milk when heated to 180°F (82.2°C) 180°F to kill harmful
bacteria which could
be a health hazard to
us humans
(Steintrager, 2018).
These harmful
bacteria can overrun
yogurt bacterium and
therefore diminish the
latic acid
concentration in the
yogurt (Global News
TV Broadcast, 2021)
3 The concentration of 1 M of NaOH was Different
NaOH used used for each trial of concentrations for
each incubation NaOH would
temperature interval uncertainties to be
inconsistent and
would confuse
calculations for the
C3H6O3
4 The amount of milk 4 cups of 3.23% If more milk is added
and bacteria used Homogenous Milk and less bacteria is
and 3 tablespoons of used, there will be no
Lactobacillus proper fermentation
bulgaricus were used of yogurt and less
for each incubation lactic acid then I
temperature interval should get (Mom,
2021). If more
bacteria is used and
less milk is added,
the bacteria will be
crowded and run out
of lactose which will
(Smith, n.d) which
will affect the taste
and quality of the
 yogurt (Mom, 2021)

Materials (some uncertainties will be added later when lab work proceeds):
1) Phenolphthalein Indicator
2) One Funnel
3) Three Decimal Weighing Scale (± 0.001 g)
4) One Burette
5) One Burette Metal Stand
6) One 250 mL Beaker
7) 2000 mL of 1 M NaOH bottle (some of the NaOH will be extra)
8) One 100 mL Beaker
9) One 300 mL Beaker
10) One President Distilled Water Jug
11) One 50 mL beaker
12) One 10 mL Pipette
13) Lactobacillus bulgaricus packet
14) Three 3.25% Milk Jugs
15) Five Glass Jars
16) One Spoon
17) One Mercury Thermometer
18) One Insta Pot
19) One Plastic Tub
20) Some Paper Towels
21) One Cup measuring device
22) One tablespoon measuring device
23) One Plastic Cover

Safety Method:
When handling the hot Insta-Pot, wear heat-resistant gloves as it will prevent burns on
skin. Let the Insta-Pot cool down for the time listed in the procedure (see below). If there is hot
milk splattered in your face, immediately go the nearest sink, and wash with cold water and ask
someone to get first aid. Yogurt cultures used in this experiment have no dangers and can be
stored using a clip or thrown into the garbage (Mom, 2022). When using 1 M NaOH, make sure
to wear lab approved safety goggles, lab apron and gloves to prevent chemical spillage on hands
or eyes. If NaOH spills on skin or clothes, immediately wash area with cold water and get first
aid if necessary (ReAgent, 2008). Effects of NaOH on skin can include redness, cracking,
blisters, etc. (ScholAR Chemistry, 2009). Ingestion or inhalation can cause damage to organs
such as lungs or throat because of corrosive nature (ReAgent, 2008), so do not ingest or inhale
NaOH during the duration of this experiment. If NaOH gets in eye, rinse eye out immediately
and get the first aid to prevent blindness or damage to eyes (ReAgent, 2008). In conclusion,
NaOH can safely be disposed. Small amounts of 1 M NaOH can safely be disposed in a sink
(Mr. Bahia, 2022).

Procedure:
Making the Yogurt (this step can be done at your home): ^
1) Firstly, before creating the yogurt, gather these materials from stores, your fridge, your
pantry and/or your kitchen: one Insta Pot, one Plastic Tub, three 3.25% Milk Jugs,
Lactobacillus bulgaricus packet, one cup measuring device, one tablespoon measuring
device, five glass jars, 300 mL beaker, one mercury thermometer, some paper towels and
a sink or tap water source
2) Open the Insta-Pot up. Open one of the 3.25% Milk Jugs up and pour 4 cups of the 3.25%
Milk into the Insta-Pot using the one cup measuring device
3) After stirring, heat the milk of the Insta-Pot to 180°F (or 82.2°C) with a thermometer and
wait for 40 minutes
4) Then let the milk cool down, measuring a temperature of 43.9°C with a thermometer.
Open the Lactobacillus bulgaricus packet and add 4 tablespoons of Lactobacillus
bulgaricus to the Insta-Pot. Stir the contents of the Insta-Pot for 4 minutes.
5) Transport the milk into one of the glass jars. Put the glass jar into a plastic tub. Fill 300
mL of tap water into 300 mL beaker. Warm the water in the beaker to 20.0°C measuring
with a thermometer. Add this water to the plastic tub, pouring it around the glass jar
containing the yogurt. This will be the 20.0°C incubation temperature interval.
6) Add a plastic cover over the plastic tub and store in kitchen for 4 hours. Milk should be
curdled. Store in fridge after 4 hours of incubation
7) Repeat Steps 2-6 for each incubation temperature interval (40.0, 60.0, 80.0 and 100.0)
°C. Clean up all equipment when finished.

Titrating the Yogurt with NaOH (this step should be done at your school lab): ^^
1) Before titrating, gather these materials from your school lab: phenolphthalein indicator,
one funnel, one burette, one burette stand, one three decimal scale, one 250 mL beaker,
one 2000 mL of 1 M NaOH, one spoon, one 100 mL beaker, one 50 mL beaker, one
President’s Choice Distilled Water jug, one 10 mL pipette, five glass jars containing the
yogurt at different incubation temperatures and some paper towels
2) Pour the distilled water from the President’s Choice jug into the 50 mL beaker to the 30
mL marker. Using the 10 mL pipette, add some distilled water to the 100- and 250-mL
beaker. Swirl the beakers and pour into the nearest sink. This will remove any unwanted
chemicals in the beakers that could cause chemical reactions.
3) Calibrate the scale with the 250 mL beaker on it. Add 10.000 g of the 20°C incubated
yogurt into the 250 mL beaker using the scale to measure.
4) Grab the burette and insert into the burette stand near the edge of a sink. Then using the
10 mL pipette, collect the remaining 20 mL of distilled water and run it in the burette.
Release the distilled water into the nearest sink.
5) Add the funnel to the burette. Prepare the NaOH solution by adding 100 mL of NaOH
from the 2000 mL of 1 M NaOH bottle. Using the 10 mL pipette, add 5 mL of NaOH to
the burette and let it run through the sink. The previous and this step will help remove
unwanted chemicals and acclimatize the burette to the NaOH solution. Then after
emptying the burette, close the burette and it to the zero line with the remaining NaOH
(may need to add more NaOH to 100 mL beaker)
6) Move burette to the table and let the burette hover over the 250 mL beaker containing
yogurt. Make sure the burette is in the close setting to prevent accidental spillages
 7) Add four drops of phenolphthalein indicator into the yogurt beaker and swish it around.
Then open the burette and let the NaOH gently spill into the yogurt beaker. Watch for a
faint pink colour. If the yogurt beaker turns faint pink, close the burette, and record the
volume of the remaining NaOH solution on the burette.
8) Repeat Steps 2-7 for each incubation temperature interval (40.0, 60.0, 80.0 and 100.0).
These steps should be done for five trials for each incubation temperature interval

^ The first part of the procedure was adapted from my mom’s yogurt recipe (very delicious
indeed). The only difference being the multiple yogurts incubating at different temperatures
intervals (listed above) that were used in this experiment

^^ The second part of the procedure was adapted by Courtney Simons. The differences in this
procedure were the mass of the yogurt used (10.000 grams instead of 15.000 grams), there was
no cleaning of lab equipment with distilled water and the procedure by Courtney Simons does
not include multiple trials and yogurts at different incubation temperature intervals.

References
 Bylikin, S., Horner, G., Murphy, B., & Tarcey, D. (2014). Chemistry: Course companion.
Oxford University Press. 

ReAgent. (2008). NAN3 material safety data sheet chemical name ... - unil.ch. SAFETY
DATA SHEET SODIUM HYDROXIDE 1M (1N). Retrieved March 19, 2022, from
https://www.unil.ch/files/live/sites/cig/files/FAQ/Safety/PDF/MSDS/sodium
%20azide_MSDS.pdf 
ScholAR Chemistry. (2009). Material Safety Data Sheet - mccsd.net. Sodium Hydroxide
Solution, 1.0M. Retrieved March 19, 2022, from
https://www.mccsd.net/cms/lib/NY02208580/Centricity/Shared/Material%20Safety%20Data
%20Sheets%20_MSDS_/MSDS%20Sheets_Hydrochloric_Acid_Solution_0_1M_338_15.pdf 

Simons, C. (2022, February 3). How to determine acidity in yogurt. Food Science

Toolbox. Retrieved March 18, 2022, from https://cwsimons.com/determination-of-acidity-in-
yogurt-lab/ 

Smith, D. K. (n.d.). Yogurt made simple - OSU extension service. Yogurt Made Simple.
Retrieved March 20, 2022, from
https://extension.oregonstate.edu/sites/default/files/documents/8836/fs173emakingyogurt.pdf

Steintrager, M. O. (2018, May 16). How to make homemade yogurt step by step.

Epicurious. Retrieved March 20, 2022, from https://www.epicurious.com/expert-advice/how-to-
make-homemade-yogurt-step-by-step-article 

USDA. (2022, February 22). At what temperature do bacteria grow the

fastest? ASKUSDA. Retrieved March 20, 2022, from https://ask.usda.gov/s/article/At-what-
temperature-do-bacteria-grow-the-fastest

Vedantu. (2022, March 16). Lactic a\Acid. VEDANTU. Retrieved March 20, 2022, from
https://www.vedantu.com/chemistry/lactic-acid