Fermentation of yogurt, isolation and preservation of lactic bacteria

1. YOGURT
FERMENTATION

2. GROUP 1
Tran Huyen Trang
Bui Xuan Tu Bui Thi Tuoi
Ha Thi Loi
Le Thu Thuy Duong Le Long
2

3. Table of Contents
Reference
Results and explain
Implementation plan
Collecting and processing data
Steps of processing
Materials, chemicals, instruments and equipment
Objectives and Requirements
Overview
Introduction
3

4. - What exactly is yogurt ?
Yogurt is a food produced from
milk fermentation under the
influence of lactic bacteria.
Yogurt provides almost every
nutrient that your body needs. It is
especially high in calcium, B
vitamins and trace minerals.
Introduction
4

5. Yogurt provides probiotics, vitamins
and minerals, all of which may boost
immune health and prevent certain
illnesses.
Isolation and preservation of strains of
lactic bacteria is one of the prerequisites
for storing and developing good sources
in the future.
“
5

6. 2. Overview
2.1.Fermentation mechanism
Lactic acid fermentation is a metabolic process by
which carbohydrates are converted lactic acid.
It is an anaerobic fermentation reaction that
occurs in some bacteria, typically lactic bacteria.
6

7. Two types of lactic acid fermentation
Homofermentative
process
Homofermentative bacteria convert glucose to
lactate and use this reaction to perform substrate-
level phosphorylation to make ATP:
Glucose + 2 ADP + 2 P i → 2 lactate + 2 ATP
Heterofermentative
process
Heterofermentative bacteria produce less lactate
and less ATP, but produce other products
(ethanol, glycerin, CO2, H2O,...)
Glucose + ADP + P i → lactate + ethanol +
CO2 + ATP
7

8. The two bacteria used in yogurt production,
Lactobacillus bulgaricus and Streptococcus
thermophilus, help each other grow until they reach
a stable balance. Together, they transform the
lactose in milk into lactic acid, creating yogurt.
The suitable temperature for the growth of a
mixture of these two bacteria (ratio 1:1) is 43-45℃.
8

9. The changes in
fermentation
Protein in milk is
hydrolyzed to peptides
and amino acids,
creating the flavor for
the product.
Lactose is broken
down by the enzyme
galactosidase to form
glucose and galactose.
Glucose is
converted into
lactic acid by
lactic bacteria
The fermentation also
produces flavor compounds
such as acetaldehyde,
acetone, and di-acetyl.
9

10. 2.2. Lactic bacteria in yogurt
These are gram-positive bacteria, stick-shaped or
spherical, non-motile and do not form spores, aerotolerant
and acidophilic.
Lactobacillus bulgaricus Streptococcus thermophilus
10

11. Lactobacillus bulgaricus
• Optimum temperature range: 43 -
46°C in the high acidic condition.
• Low pH: around 5.2–5.6.
• Can produce a large amount of lactic
acid from lactose.
Streptococcus thermophilus
• Optimum temperatures range: 35 -
42 °C.
• The suitable pH is 6.6-6.8.
11

12. 2.3. Isolation and preservation
Lactic bacteria were isolated on MRS medium in petri plate
and incubated at 30ºC for 24 to 48h, adding CaCO3 to easy
identify the Lactic bacteria.
After isolation, reserving the bacteria in 4ºC.
12

13. 2.4. Identify the factors affecting the
yogurt fermentation
Time
Temperature
The ratio of varieties
Incubate the mixture in
30℃ and 25℃ to see the
differences.
Anneal the mixture for
30,60,90,120 minutes to
see the changes.
5% and 10% to see the
differences.
13

14. 2. Objectives and requirements
1.Objectives
Presenting the mechanism of fermentation, yogurt making
process and requirements for yogurt quality.
Understanding the role of lactic bacteria, factors affecting
the growth and development of them.
Knowing how to perform, set up experiment to isolate and
preserve the source of lactic bacteria
Finally, storing the bacteria at low temperature for a long
time to research in the future.
14

15. 2.Requirements
Perform a yogurt fermentation experiment and
determine the pH of yogurt based on the amount of
lactic acid formed through titration steps at different
times, different temperatures and ratio of varieties.
Defines a product's color, condition and favor.
Isolation and preservation of bacterial strains,
observing and determining morphology of lactic
strains.
15

16. 4. Materials, chemicals, instruments and equipments
Materials and chemicals
Condensed milk
Yogurt sample 1
Yogurt sample 2
Water, NaOH 0.1N,
Phenolphtalein 1%
MRS medium composition:
- Peptone ..............20.0 g
- Yeast extract ......5.0 g
- Meat extract…….10 g
- Glucose...............20.0 g
- Tween 80 ...............1.08 ml
- K2HPO4…………….2.0 g
- Sodium acetate........5.0 g
- Ammonium citrate ...2.0 g
- Magnesium sulfate...0.2 g
- Manganese sulfate...0.05 g
- CaCO3……………....5 g
pH: 6.5
16

17. Instruments and equipments
- Volumetric flask 1000ml and 200ml, flasks, tubes
- Thermostat tank, refrigerator, incubator box
- Thermometer, pH meter
- Burette
- Petri plates, pipette
17

18. 5. Steps of processing
Isolation of
Lactobacillus
Yogurt
fermentation
Purification of
Lactic
bacteria 18
Process of
preservation of
varieties

19. Sample 1
Sample 2
The ratio of
variety is
2.5%
The ratio of
variety is
5 %
Incubate in 30℃
Incubate in 25℃
Incubate in 30℃
Incubate in 25℃
Incubate in 30℃
The ratio of
variety is
2.5%
The ratio of
variety is
5%
19
Table 5.1.1: Design of yogurt Fermentation Experiment

20. 5.1. Yogurt fermentation
5.1.1. Preparation of fermentation
Dissolve 12 spoons of condensed milk in 800ml of warm water
(about 43°C), divide the mixture into four cups with same
proportions. Two cups are supplied breeding with 2.5% ratio
( group 1) and two cups are supplied with 5% ratio( group 2).
Take each cup of each group, incubate at 30℃ and room
temperature. Repeating with the second sample.
20

21. 5.1.2. Conducting titration.
30m 60m 90m 120m
After different incubation intervals: 30 minutes, 60
minutes, 90 minutes and 120 minutes conduct lethality
of the fermented yogurt solution to determine the content
of lactic acids born during the fermentation of yogurt.
21

22. 5.1.3. Conducting titration.
22
NaOH 0.1N
10 ml yogurt fermentation
5 µl phenolphthalein 0.1%

23. 5.2. Isolation of Lactobacillus bulgaricus and
Streptococus thermophilus from yogurt
Step 1: dilute samples to a ratio of
10−5
, 10−6
,10−7
.
Step 2: Use a pipette to transfer 0.1ml of
solution to the surface of the agar medium in a
petri dish.
Step 3: Dip the wiper head into the alcohol and
pass through the flame to disinfect.
23

24. Step 4: Use the wiper head to spread bacteria
evenly on the agar surface.
Step 5: Take the isolated disk Lactobacillus
bulgaricus and Streptococus thermophilus
cultured at a temperature of 30 degrees Celsius
for 48 hours.
24

25. ● Isolation of Lactobacillus bulgaricus and Streptococus thermophilus from
yogurtIsolation of Lactobacillus bulgaricus and Streptococus thermophilus from yogurt
25
Isolation of Lactobacillus bulgaricus and Streptococus thermophilus from yogurt

26. 5.3 Purification of Lactic bacteria with a ring
implant (3-phase streaked method on agar medium)
Step 1: Sterilize the implant with alcohol and burn over an open flame.
Step 2: Using an inoculum to take 1 round of suspension, streaking into
a zigzag line at an angle on the surface of the agar medium in the Petri
dish(1). Then sterilize the implant and let it cool.
Step 3: Continue to place the implant starting from region(1) to area(2).
Then sterilize the implant and let it cool.
Step 4: Continue to place the implant starting from region(2) to area(3).
26

27. Purification of Lactic bacteria with a ring implant
(3-phase streaked method on agar medium)
27

28. 5.4. Process of preservation of varieties in
inclined agar medium.
Step 1: Disinfect the head of the round implant.
Step 2: Bring the petri dish containing lactic
bacteria closer to the alcohol lamp flame and open
the lid of the disc for a sufficient amount to
perform the sampling.
Step 3: Use a sterilized round implant, cooled to
remove bacteria from petri dish.
28

29. - Step 4: Open the test tube button and leave the test tube
mouth facing the flame. Insert the implant into the protest
tube, move the head of the rod in the shape of a zigzag
gently on the jelly face from the bottom of the tube to the
top.
Step 5: Open the test tube mouth on the fire and cover the
button. Heat the implant, let it cool, label the culture tube.
Breeding in the suitable temperature.
29

30. 30

31. 6.Collecting and processing data
1. Collecting
and processing
data of yogurt
fermentation
- Determine the taste, color and status of the product
after the intervals of 30,60,90 and 120 minutes.
- After edging, determine the acid content generated
during milk fermentation according to the formula:
X % = V*K*100/P
In which:
X- lactic acid content(%)
K- the coefficient used for each acid( K= 0.009)
V- Volume NaOH 0.1 N(ml)
P- The weight or volume of sample
31

32. 2. Collection and
processing of
data of lactic
bacterial
isolation
- Observe how the proportion of colons at
dilution levels of 10−5, 10−6,107varies?
What's their color? Is it infected with
unwanted microorganisms?
32

33. 7. Implementation plan
Session 6
(09/04/2021)
Session 5
(05/04/2021)
Select
topics
&
discuss
topics.
Session 1
(22/03/2021)
Session 2
(26/03/2021)
Session 3
(29/03/2021)
Session 4
(02/04/2021)
Report on
the topic
outline.
Isolation
of lactic
bacteria
from 1
yogurt
sample.
Preparing
the MRS
medium
Fermentation
of one yogurt
sample
( sample 1)
Purification of
Lactic bacteria
with a ring
implant
Preserve seeds in
an agar slant
medium and
observe the
morphology of
bacteria
Fermentation of
remaining sample
(sample 2)
Report
on the
topic.
33

34. Table 8.1-1: The volume of NaOH solution after the titration at different
times, ratio supplied and temperature
- On 02/04/2021: Fermentation of one yogurt sample (sample 1 - TH true milk
yogurt). We have two tables:
8. Result and explanation
8.1 Fermentation
Table 8.1 – 2: The lactic acid content generated after different times, ratio
supplied and temperature.
34

35. - On 06/04/2021: Fermentation of remaining sample (sample 2 –
Vinamilk yogurt). We have a table:
Table 8.1 - 3: The volume of NaOH solution after the titration at different times, ratio supplied
and temperature
Table 8.1 – 4: The lactic acid content generated after different times ratio supplied and temperature.
35

36. EXPLANATION
Fermentation There does not appear to be a great difference in color, taste
or coagulation of the fermented yogurt solution because the
fermentation time is too short, and the temperature is not
suitable for woolen fermentation. The right temperature for
fermentation has been studied to be 40-50°C.
Titration Yogurt is the creamy texture solution, the plastic pipette is
difficult to attract and will be erroneous in successive
varieties. So, we should use grass pipettes
36

37. 8.2. Isolation of Lactic bacteria
Appear 4 types of colonies in petri dish
• Type 1: Colonies are round, small in diameter,
milky white, with rounded edges and transparent
white, smooth and shiny colony surface, convex.
• Type 2: Colonies are round, small in diameter,
whole colonies are transparent white, with rounded
edges, with a smooth and shiny colony surface,
raised.
• Type 3: Colonies have irregular shapes, large
diameter, whole colonies are milky white, with lobed
edges, rough colony surface, flat.
• Type 4: Colonies are pink in color, round in shape,
small in diameter, with rounded edges, with a smooth
and shiny colony surface, convex.
- Colony density is the most. Petri dishes appear
many colonies pink. 37

38. - Appear 4 types of colonies like at
concentration of 10−5
- The density of colonies at this
concentration is less than that at
concentration of 10−5 . Appear
some areas pink.
38

39. - Appear 4 types of colonies like
at concentration of 10−5
- Colony density is the least,
many single colonies. Petri dish
appears only 1 pink colony
39

40.  Explanation about undesirable microorganisms:
• The isolated sample was re-used from the previous group.
• Undesirable microorganisms can contaminate the petri dish
by improper handling of our hands or tools.
• They can be other beneficial bacteria for our health that is
not Lactic bacteria.
40

41. 8.3. Purification of Lactic bacteria
41
Observe after 24h
The colonies of bacteria appear all 3
phages, do not have singer colonies.
The areas where Lactic bacteria
grow, CaCO3 is decomposed
Observe after 48h
The colonies of bacteria appear all 3
phages very clearly. CaCO3 is
decomposed very strong

42. 42
Observe after 24h
The colonies of bacteria appear all 2
phages, appearing a lot of single
colonies in central of petri dish. The
areas where Lactic bacteria grow,
CaCO3 is decomposed.
Observe after 48h
We easily observe the single colonies.
Almost CaCO3 is decomposed.

43. 43
Observe after 24h
All most colonies appear in first phage.
The areas where Lactic bacteria grow,
CaCO3 is decomposed
Observe after 48h
After 48h, the number of colonies
increases a lots.

44. 44
Observe after 24h
All most colonies appear in first phage.
The areas where Lactic bacteria grow,
CaCO3 is decomposed.
Observe after 48h
After 48h, the number of colonies
increases very clearly than at 24h.

45. 45
Observe after 24h
The colonies of bacteria appear all 2
phages. We easily observe the CaCO3
is decomposed
Observe after 48h
The colonies in second and third phage
grow up rapidly.

46. Explanation: The time factor effects the
number of colonies, Lactic bacteria increases
rapidly after 48h so they release more lactic
acid than at 24h to decompose CaCO3.
46

47. 8.4. Preservation of Lactic bacteria
Do not appear zigzag line. Test-tube
has a large amount of bacteria. The
colonies are milky white color.
47

48. Do not appear zigzag line. Test-tube has
a lot of single colonies that is spotlessly
white color.
48

49. Do not appear zigzag line. Test-tube has a
large amount of bacteria. The colonies are
milky white color.
49

50. Explanation: No zigzag appeared in all test tubes
because too many bacteria were taken during the
preservation operations.
50

51. The implementation process was not as planned because on
April 2, 2021 the medium was damaged (the agar was not
frozen), so it was not possible to purify Lactic bacteria. This
may be due to an uneven distribution of agar.
51

52. References
1. Fguiri, I. (2017). Isolation and Characterization of Lactic Acid
Bacteria Strains from Raw Camel Milk for Potential Use in the
Production of Yogurt. Food Science & Nutrition, 3(3), 1–8.
https://doi.org/10.24966/fsn-1076/100026
2. Giáo trình Thực tập vi sinh vật- NXB Nông nghiệp-2005
3. Giáo trình Thực hành công nghệ vi sinh- Trần Thị Hồng Hạnh
4. Giáo trình Thực hiện vi sinh vật chuyên ngành-NXB Nông nghiệp-
2007
5.https://vnuf.edu.vn/documents/454250/1795973/1.Nguyen%20Thi
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scbAwFErOveheblATNJ2Jk_fWCQSXPkacTSnMcQp9AIA
52

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