The document discusses various methods for producing and preserving starter cultures for fermented dairy products. It describes traditional liquid culture production methods and highlights that they are time-consuming and risk contamination. It then outlines several improvements to culture production including concentrated, freeze-dried cultures and cryoprotected frozen cultures that allow for direct inoculation and overcome issues with traditional methods. The document also discusses factors that affect survival of freeze-dried cultures and outlines three main systems for bulk starter culture production: using simple techniques; mechanically protected tanks; and propagation in a phage-inhibitory medium.

2. • The fermentation process of any cultured dairy
product relies entirely on the purity and activity of
the starter culture, provided that the milk or growth
medium is free from any inhibitory agent (e.g.,
antibiotics or bacteriophage).
• The traditional method for the production of bulk
starter(Liquid culture) is time-consuming, requiring
skilled operators and may lead to contamination by
bacteriophage (which is one of the major hazards in
the industry), it is still widely used.
• Nevertheless, research work has been intensified in
this area to overcome various problems, and
developments in this field have focused on the
areas of starter preservation and concentration.

3. • An ideal starter culture must
• contain the maximum number of viable organisms,
• must be highly active under production conditions in the dairy, and
• must be free from contaminants.
• Provided that culture inoculation is carried out under aseptic
conditions and growth is initiated in a sterile medium, Foster
(1962 ) suggested that one of the following principles must be
adopted in order to maintain activity:
– Reducing and/or controlling the metabolic activity of the
microorganisms
– Separating the organisms from their waste products.
• The former principle is evident in refrigeration, while the
latter approach is mainly used during the concentration and/or
preservation of starters-that is, during the production of a
concentrated, active bulk starter, either in a continuous
fermentor or in a batch process, for direct to- vat inoculation
(DVI) of the milk

4. Methods of Preservation of Lactic Acid
Bacteria
• It is essential that starter cultures be preserved in order to maintain an
available stock of organisms, especially in the case of a starter failure.
• Also, successive subculturing can induce mutant strains that may alter the
overall behavior and general characteristics of the starter.
• Dairy cultures may be obtained from research establishments, educational
colleges, culture bank organizations, or commercial manufacturers, and
starter culture bacteria may be preserved by one of the following methods:
1. Liquid starter (mother, bulk)
2. Dried starter:
(a) spray-dried (unconcentrated),
(b) freeze-dried or lyophilized (unconcentrated), and
(c) concentrated freeze-dried.
3. Frozen starter:
(a) frozen at -20°C (unconcentrated),
(b) deep frozen at -40 to -80°C (concentrated), and
(c) ultra-low temperature freezing at -196°C in liquid nitrogen
(concentrated).

6. Production of industrial starter cultures

7. Liquid Starter Cultures
• This is the most popular
and widely used form in
which starter cultures
are handled in the dairy.
• Starters are normally
preserved in small
quantities, but to meet
the required volume for
any production line, a
scale-up system of
propagation is required.

8. • The working stock cultures are maintained in autoclaved (0.1
Mpa for 15 min) reconstituted, antibiotic-free, skimmed milk
powder [10-12g lOOg-1
solids non-fat (SNF)], with either
weekly or daily subculturing.
• Cheese starter cultures (L. lactis subsp. lactis and subsp.
cremoris, L. mesenteroides subsp. cremoris) can be propagated
up to 50 times without any fear of mutation, and the sterilized
medium is inoculated at a rate of 1 ml 100ml-1
and incubated
at 22°C or 30°C for 18 h or 6 h, respectively.
• However, that repeated subculturing of certain strains of
starter bacteria may lead to a loss of plasmid material that,
consequently, can affect the characteristics of the organism
(i.e., phage-resistant becomes phage sensitive).

9. • The yogurt starter cultures (S. thermophilus and L.
delbrueckii subsp. bulgaricus) are normally subcultured only
15-20 times as as safeguard against imbalance and to retain
the ratio of cocci: rods as 1 : 1 .
• Incubation is carried out at 42°C for 3-4h, or at 30°C for 16-
18h using 2 or 1ml 1OOml-L
inocula, respectively.
• Starter culture activity is affected by
– the rate of cooling after incubation,
– level of acidity at the end of the incubation period, and
– the temperature and duration of storage.
• Cooling is important to control the metabolic activity of the
starter; in practice, however, a warm starter (freshly
incubated and uncooled) is sometimes used in cheese
factories and, to some extent, in the yogurt industry.

10. Dried Starter Cultures
• The preservation of starter cultures by drying is an
alternative method for culture retention.
• The development of such processes seeks to overcome the
work involved in maintaining liquid stock cultures.
• It also facilitates the dispatch of dried cultures by post
without any loss in activity.
• The different methods used are as follows:
1. Vacuum- and spray-drying (i.e., old methods not used
at present time
2. Lyophilization or freeze-drying (this method is widely
used in laboratories
3. Freeze-drying of concentrated cultures (widely used
commercially).

12. Factors affecting the survival rate of freeze-dried
dairy starter cultures
1. Growth Phase: Starter cultures are less sensitive to
freezing and drying if the cells are harvested toward
the latter part of the exponential phase with the
exception of L. delbrueckii subsp. bulgaricus and
Lactococcus lactis subsp. cremoris, where the cells
are harvested in the early stages of the stationary
phase.
2. pH of Media: Media in a pH range of 5-6 are more
favorable to high survival rates, and neutralization of
the growth and the suspending medium is essential.
3. Moisture Content: The moisture content of the dried
culture must be less than 3g/100g.

13. 4. Type of Packaging: Vacuum or modified
atmosphere packaging of the dried cultures is
highly recommended because the preserved
organisms are sensitive to oxygen.
• However, the most popular type of packaging
material for dried cultures is the glass vial,
followed by the laminated, aluminum foil sachet.
5. Freezing Temp.-Freezing cultures at -20 to -30°C
and drying at temperatures between -10 arid
30°C results in high bacterial activity of the dried
culture
6. Storage Temp.: Dried cultures stored at 5-10°C
showed higher rates of survival during prolonged
storage than did those stored at room
temperature.

14. 7. Presence of Carbonyl Compounds: Carbonyl compounds, such
as pyruvate and diacetyl, which can react with the amino
groups within the preserved cells, can accelerate their death.
• Hence, it is recommended that these compounds should be
separated from the harvested cells.
9. Use of cryoprotective agents: For the long-term preservation of
freeze-dried cultures, the suspending medium must be fortified
with non reducing sugars, amino acids, and/or semi carbazid
10. Rehydration temperature can affect the leakage of cellular
ribonucleotides from damaged cells.
• The optimum rehydration temperature of LAB is recommended to
be 20°C.

15. • Starter cultures preserved by freeze-drying tend
to have a prolonged lag phase, and they are
mainly used as inoculants for the propagation of
mother cultures ( System 1).
• Larger quantities are needed for direct
inoculation of the bulk starter, and an extended
incubation time may be required .
• Developments in the last few years have made it
feasible to produce concentrated, freeze-dried
cultures (CFDC) for direct inoculation of the
bulk starter vessel or for DVI of milk for the
manufacture of cheese and other fermented dairy
products ( Systems 2 and 3, respectively).

16. Frozen Starter Cultures
• Starter cultures can also be preserved in the frozen
form, and such cultures are produced by two
different routes:
1. Deep or subzero freezing (-20 to -8O'C).
2. Ultra-low-temperature freezing at -196°C in liquid
nitrogen.
• Sterile milk freshly inoculated with an active starter
culture is deep frozen at -30 to -40°C for preservation as a
mother or feeder intermediate culture.
• Such frozen cultures can retain their activity for several
months when stored at -40°C.
• Such cultures have now been replaced by the concentrated,
frozen type for direct inoculation of bulk starter tanks or
DVI of milk for the manufacture of cheese or fermented
milks (Systems 2 and 3, respectively

17. • Freezing cultures in liquid nitrogen has made
possible the DVI of milk for cheese and yogurt
production, or direct inoculation of the bulk starter
( Systems 2 and 3).
• The advantages of this approach are as follows:
• convenience,
• culture reliability,
• improved daily performance and strain balance,
• greater flexibility,
• better control of phage, and
• possible improvement in quality.
• However, the disadvantages are as follows:
• difficulties in providing liquid nitrogen facilities,
• higher cost,
• greater dependence on starter suppliers, and
• apportioning of responsibility in case of starter failure

18. PRODUCTION SYSTEMS FOR BULK
STARTER CULTURES
• The production of a bulk starter culture-that is, the culture used
directly in the fermentation process-necessitates several stages
of subculturing in order to meet the quantity required, or direct
inoculation of the bulk starter tank using concentrated cultures
(systems 1 and 2)
• The most important aspect of starter production is the
preparation of the growth medium, along with the protection of
the culture from phage attack. The methods used may be
divided into the following systems:
1. the use of simple microbiological techniques;
2. the use of mechanically protected tanks; and
3. the propagation of the starter culture in a special medium that is
inhibitory to phage.
• The mechanically protected approach is widely used in the
United Kingdom, Australia, and New Zealand, whereas the
latter system is popular in the United States

20. 1. Simple Microbiological Techniques
• In this system, the
equipment/materials
are basically
laboratory utensils
and a starter tank,
which may be of a
simple design-that is,
batch pasteurizer,
incubator.

21. 2. Mechanically-Protected Systems
• Different techniques have been developed in the dairy
industry for the production of starter cultures in
mechanically protected systems; and in these systems,
the following aspects are important:
1. the processing of the growth medium and starter growth
take place in a completely enclosed tank; and
2. the inoculation of the starter takes place through a
barrier which prevents the entry of unclean air.
Some examples of mechanically protected systems follow.

22. A. The Lewis System
• The technique consists of using reusable polythene bottles (115- and 850-g
capacity) for mother and feeder cultures, respectively.
• These bottles are fitted with Astell rubber seals, and the growth medium (i.e.,
10-12g 1OOg-1
reconstituted, antibiotic-free, skimmed milk powder) is
sterilized in the same bottles.
• The starter culture transfers are carried out by means of two-way
hypodermic needles, and the overall technique is illustrated schematically in
Figure.
Schematic illustration of the Lewis
system for starter culture transfers.
A, mother culture;
B, feeder intermediate culture;
C, bulk culture;
D, detail of needle assembly
1, tap;
2, Astell rubber seal;
3, hypochlorite solution

23. The Jones System
• The other protected
method is the Jones
system and, in this
case, the tank is not a
pressurized starter
culture vessel.
• During the heating or
cooling of the milk, air
leaves or enters the
starter vessel; that is,
the tank is not
pressurized.

24. • In the Lewis system, the starter culture transfer
from one container to another relies on squeezing
the polythene bottle to eject the culture.
• However, the Tetra Pak system (Bylund, 1995)
uses filter-sterilized air, under pressure, for
transferring the culture.
• The mother and feeded intermediate cultures are
prepared in a special unit called a “Viscubator,”
and the capacity of these containers is 0.5 and 20
liters, respectively.
Tetrapack System

25. Use of DVS cultures
• During the past 25 years,
cheesemakers around the world
have become aware of the
advantages offered by the direct
addition of frozen or freeze-dried
concentrated culture to the cheese
vat.
• In the production of Cheddar,
pizza, Cottage and white brine
cheeses such as Feta, DVS
cultures are well established.
• The use of DVS is estimated to be
20% in Germany, 65% in the
United Kingdom and 25% in the
United States, and to be a total of
30% of the total global cheese
milk set.

26. Advantages of DVS
1. The risk of phage attack and of accumulation of
bacteriophage in the dairy is reduced due to the
elimination of the bulk starter propagation.
2. DVS cultures are put through an extensive quality
control regimen before dispatch, assuring that the
cultures have the required activity and
bacteriological quality.
3. Mixtures of strains can be predetermined to ensure
consistent acid and flavour production, and
blending of mesophilic, thermophilic and possibly
probiotic strains into one starter culture can be
done to give customised cultures with specific
properties.