PREPARATION OF DAIRY CROPS AND YOGURT

1. INTRODUCTION

Although there are numerous types of fermented milk, only yogurt has achieved
international diffusion. Its popularity is due to various facts, such as its pleasant and
aromatic flavor or its fame as a healthy food, but perhaps the most important thing is its
creamy consistency, which makes it an ideal vehicle for various types of fruits.

Yogurt is a semi-liquid, fermented dairy product, considered a healthy food. It is made with
whole or skim milk, cooked and concentrated by evaporation. Fermentation is achieved by
adding to it cultures of two bacteria, Lactobacillus bulgaricus and Streptococcus
thermophilus. New batches of yogurt can be obtained by adding a portion of the previous
batch to the concentrated milk.
 2. OBJECTIVES

2.1. GENERAL OBJECTIVE

 Follow the fundamental steps for the preparation of lactic cultures and yogurt

2.2. SPECIFIC OBJECTIVES

 Prepare lactic cultures and yogurt.

 Determine the behavior of acidity at regular time intervals.
 Inoculate and sow in broth and MRS agar.
 Determine the coccus – bacillus ratio in a sample of commercial yogurt.
 3. CONCEPTUAL FRAMEWORK

Fermented dairy products are characterized by the fact that they obtain their acid
character and typical texture by undergoing lactic fermentation combined with aroma
production. Different species and combinations of species of acidifying lactic bacteria
ferment a part of the lactose and sometimes the added sucrose to lactic acid and, to a
lesser extent, also to other organic acids and aromatic substances. Fermentation also
causes coagulation of proteins, which also undergo a certain degree of unfolding.

Yogurt is fermented milk made by adding a yogurt culture to pasteurized milk, sometimes
homogenized, and normalized in fat content, or fresh skimmed milk, which may contain
stabilizing products. To increase the natural proportion of dry extract, the milk is
concentrated by evaporation or milk powder or concentrated milk is added by ultrafiltration.

With the yogurt culture, lactic bacteria are added to the milk that will determine
acidification and fermentation. The quality of the final product depends strongly on the
quality of the crop, and for this reason the utmost importance must be given to the
composition and preparation of the crop.

Crop composition.
Yogurt cultures must contain, as microorganisms, exclusively the following thermophilic
bacterial species:

Streptococcus thermophilus
Lactobacillus bulgaricus.

Observed under the microscope, it is seen that S. thermophilus grows forming pairs
(diplococci) or medium-long chains of spherical or ellipsoid cells with an approximate
diameter of 0.7 – 0.9µm. L. bulgaricus has a relatively long rod shape of 0.2 – 0.4 µm wide
with a tendency to form ribbons.
The optimal growth temperatures are 37 – 42ºC and 42 – 45ºC for S. thermophilus and L.
bulgaricus , respectively.

Microscopy also allows determining the quantitative ratio of cocci/bacilli. S. thermophilus

and L. bulgaricus must be, in the prepared culture (and therefore also in the yogurt), in a
quantitative ratio of approximately 1:1 to 1:5.

This relationship is essentially established based on three factors:

- Amount inoculated.
- Incubation temperature.
- Incubation time.

After sowing, the ratio is modified several times during the course of incubation to regain
the initial value at the end of the process. The origin of this is found in relationships of true
symbiosis.

Symbiosis is the vital association that is established between different organisms and that
benefits all participants. In this case S. thermophilus and L. bulgaricus . In this symbiosis it
is S. thermophilus which initiates lactic fermentation and which develops very intensely up
to a pH of 5.5. The acidity, the consumption of oxygen and the release of volatile
substances, for example the formic acid it produces, create the ideal conditions for the
development of L. bulgaricus .

The proteolytic activity, that is, the release of amino acids, of the lactobacilli stimulates, in
turn, the growth and acidifying activity of the streptococci. Lactobacilli separately develop a
lipolytic activity, by which fatty acids are released and also produce acetaldehyde
(ethanal), thus becoming the main producers of yogurt aroma.

The quality of a culture is analyzed, apart from the cocci/bacilli ratio, by the absolute
number of germs. The starter capacity of the crop, that is, its acid production capacity, is
another criterion to assess its quality.
Crop preparation
The preparation of the culture must be carried out under strict aseptic conditions to avoid
contamination by yeasts, molds and bacteriophages. Culture maintenance should be done
under laboratory conditions and preparation should be done in a room separate from the
production rooms that has adequate facilities.

Incubation is a discontinuous process which manages time/temperature relationships to

provide optimal conditions for the growth of microorganisms. The incubation process
begins with the inoculum of the ferments. It is characterized by causing, in the lactic
fermentation process, the coagulation of milk casein. The acid coagulation of casein, that
is, the formation at the isoelectric point approximately at a pH of 4.65 of a yogurt gel, is a
sign that marks the end of incubation and fermentation. This gel must have a
homogeneous curdled appearance.

The cocci/bacilli ratio, and therefore the development of acidification, can be regulated by
fundamentally modifying the incubation temperature.

Temperatures < 43ºC → Predominance of coconuts.

Temperatures > 43ºC → Predominance of lactobacilli.

The Normalization of the dry extract is another very important process for the production
of yogurt, since by increasing amounts of 1 – 3% the proportion of lean dry extract of the
initial milk, the typical consistency is obtained. To normalize the dry extract, a
discontinuous preparation of the previous solution is usually carried out, in which the
powdered milk obtained by spraying in hot milk is dissolved in a heatable container.

There are selective media for the growth of lactic acid bacteria. One of the most used is
MRS (Man, Rogosa and Sharpe) broth and agar. These media are used for the
enrichment, cultivation and isolation of all Lactobacillus species, including species such as
L. brevis and L. fermenti difficult to cultivate.

MRS culture media contain polysorbate, magnesium acetate and manganese, which are
special growth factors for Lactobacillus as well as a rich and nutritious base. This type of
medium has very little selectivity, so Pediococcus and Leuconostoc species can grow. For
a better study or isolation, the material under study is incorporated into the MRS broth for
enrichment and subsequently seeded on MRS agar by exhaustion or by the pouring onto
plates procedure.

In the MRS broth, microorganisms grow such as: L. acidophilus, L. bulgaricus, L. Casei, L.
fermentum, E. coli and Pseudomona aeruginosa; while when sowing on MRS agar, growth
can be affected by E. coli and P. vulgaris which are not inhibited at all in this environment.
 4. PROCEDURE

4.1. DAIRY CULTIVATION

Reconstitute 200 ml milk powder

Sterilize 95ºC × 15 min.

In serological bath

Cool At 42ºC

Inoculate 3% diet yogurt

Incubate 43ºC × 2 and a half

hours

Measure Every ½ hr.

acidity 10 ml culture holder
with 0.1N NaOH
Lay off When the milk is like
jelly

Shake

Refrigerate
4.2. YOGHURT

Whole milk 1 Lt.

Increase At 3%
solids With powdered milk

Add sugar At 8%

Sterilize 95ºC × 15 min.

In serological bath

Cool

Inoculate 3% diet yogurt

Incubate 43ºC × 3 hours

Shake

Refrigerate

Add fruits
4.3 COCONUT - BACILLUS RELATIONSHIP

yogurt sample

Make smears

Pin up

Gram stain

Observe under
100x
microscope
objective

4.4. SOWING IN BROTH AND MRS AGAR

Sample 1ml commercial

yogurt

Inoculate
MRS Broth

Incubate
36ºC × 24hr.

Shake
Take inoculum

Sow
MRS agar

Incubate
36ºC × 24hr.
 5. RESULTS

5.1. DAIRY CULTIVATION

200 ml of lactic culture were prepared from a powdered milk solution to which 3% of
dietary yogurt was inoculated, which corresponded to an amount of 6 ml. And they were
kept in incubation for 3 hours until a type of clot formed.

5.1.1. Grams of milk: necessary to prepare 200 ml of 10% culture

g. of Milk = Total volume × desired %

100 – 3% humidity

 g. of Milk = 200ml × 10%

100 – 3%
 g. of Milk = 20.62 g

5.1.2. Total water volume:

V T H 2 O = Total volume – Grams of milk

 V T H 2 O = 200 – 20.62
 V T H 2 O = 179.38 ml.

5.1.3. Acidity percentage:

% Acidity = ml NaOH × 0.1N × 100

10ml sample
% Acidity = ml NaOH/ ml

The determination of the percentage of acidity was carried out by titration with 0.1N NaOH
every half hour obtaining the following results:
 Table 1. Percentage of acidity over time
Time (min.) 30 60 90 120 150 180
% Acidity 2.1 2.5 2.9 4.0 5.7 6.3

Therefore a graph of % Acidity vs. Time would be as follows:

5.2. YOGHURT

1000 ml of yogurt was prepared from normalized and sweetened milk.

5.2.1. Soluble solids

To increase the soluble solids to 3%, powdered milk was added.

g. of Milk = Total volume × desired %

100 – 3% humidity

 g. of Milk = 1000ml × 3%
100 – 3%
 g. of Milk = 30.93 g

5.2.2. 8% sugar
An amount of sugar was added to the normalized milk until it reached a concentration of
8%.

g. sugar = Total volume × desired %

100 – 3% humidity

 g. sugar = 1000ml × 8%
100 – 3%
 g. Sugar = 82.47 g

5.3. COCO-BACILLUS RELATIONSHIP

–
Chains of fuchsia-colored Gram cocci (streptococci) very close together and elongated
Gram + diplobacilli of violet color were observed in a 5:1 ratio. (See annex A)

5.4. SOWING IN BROTH AND MRS AGAR

Cream-colored sediments were observed in the MRS broth, and the liquid was dark
orange in color.

On the MRS agar, well-formed colonies were observed that followed the seeding line.
Colonies of spherical, circular shape, with convex elevation, entire edge and semi-
transparent cream color. (See annex B)
 6. ANALYSIS OF RESULTS

6.1. DAIRY CULTIVATION

In lactic culture, the formation of clots indicates the fermentation of lactose by acidifying
lactic acid bacteria, producing lactic acid and, at the same time, coagulating the proteins of
the reconstituted milk. This leads to determining optimal growth by microorganisms.

The increase in the percentage of acidity is inherent to the growth of acid-producing

bacteria, therefore, the graph of % acidity vs. Time would be analogous to a graph of
bacterial growth vs. Time.

Initially the microorganisms adapt in the dormancy phase, later they begin to duplicate in a
logarithmic phase consuming the nutrients from the medium, producing the respective
acids characteristic of this type of fermentation which change the acidity and pH of the
medium, affecting the speed of growth. As time passes, nutrients decrease and
metabolites increase until reaching a stationary phase in which the number of
microorganisms remains constant.

6.2. YOGHURT
The sensory characteristics obtained at the end of incubation in the prepared product were
typical of a yogurt. The characteristic yogurt aroma was obtained by the production of
acetaldehyde by Lactobacillus . The sour taste and texture were a product of the
fermentation of lactose and part of the sucrose by the lactic acid bacteria present in the
yogurt. Finally, the characteristic viscosity was produced by the coagulation of the proteins
carried out after said fermentation.

6.3. COCONUT – BACILLUS RELATIONSHIP

The bacteria observed correspond to:

Fuchsia, spherical Gram bacteria –  Streptococcus thermophilus

Violet bacteria, Gram + bacillary  Lactobacillus bulgaricus
The observed ratio was 5:1 for Streptococcus thermophilus and Lactobacillus bulgaricus ,
respectively, indicating adequate growth and symbiosis between the two microorganisms.

6.4. SOWING IN BROTH AND MRS AGAR

In the MRS media, optimal growth of Lactobacillus bulgaricus contained in commercial
yogurt can be seen.
 7. RESEARCH

 What problem does it represent to sometimes find yeast in a high percentage

in products such as kumis and yogurt?
 8. CONCLUSIONS

 Yogurt is a dairy product obtained by the symbiosis between microorganisms:

Streptococcus thermophilus and Lactobacillus bulgaricus.

 The sensory characteristics of yogurt are obtained as a product of the fermentation of

lactose and sucrose by these lactic acid bacteria.

 The acidity changes proportionally to the growth of lactic acid fermenting bacteria.

 MRS medium is used for the enrichment and isolation of Lactobacillus.

 BIBLIOGRAPHY

 SPREER, Edgar. Industrial lactology. Ed. Acribia, SA 2nd edition. Zaragoza (Spain).
1991.

 TAMIME, A. and ROBINSON, R. YOGURT: Science and technology. Ed. Acribia,

SA Zaragoza (Spain). 1991.

 BALTIMORE BIOLOGICAL LABORATORY, INC. Culture media, materials and

instruments for microbiology laboratories. BBL Publishing Fourth edition. Baltimore,
Maryland. (USA). 1959.

 ENCARTA Encyclopedia 2000. Microsoft Corporation 1993 – 1999.

 ANNEX A.
RESULT OF THE MICROSCOPE OBSERVATION WITH GRAM STAIN
 ANNEX B.
RESULT OF BACTERIAL GROWTH IN MRS MEDIUM

MRS BROTH

MRS AGAR