LECTURE TWO

STARTER CULTURE DEVELOPMENT AND MAINTENANCE

A starter culture can be defined as a microbial preparation of large number of

cells at least one microorganism that is added to a raw-material to produce a
fermented product by accelerating and steering its fermentation process.

A starter culture may also be described as a preparation containing high

numbers of viable microorganisms which may be added to bring about
desirable changes in a substrate.

It is the preparation of one or strains of microorganisms deliberately added to

foods to initiate fermentation.

Starter culture causes rapid acidification of the raw-material through the

production of organic acid, mainly lactic acid. Also, their production of acetic
acid, ethanol, aromatic compounds, exopolysaccharides and several enzymes is
of importance thereby enhancing shelf life and microbial safety, improve
texture and contribute to the pleasant sensory profile of the end product
Starter culture arc employed in the industries for fermentation process.

Fermentation has been used as a means of improving the keeping quality

of food for more than 6000 years. In the past fermentation arose directly from
naturally occurring microorganisms in the food as found in cassava
fermentation subsequently producers of fermented foods recognized that a
portion of a previously fermented food can be added to the food to give a better
result. This is called "back-slopping".

Fermentation of foods has the advantages of

i) Improving or extending the shelf life of foods

ii) It keeps the food safe and prevent wastage

iii) Improve sensory quality

iv) It reduces toxics e.g.undesirable food compounds in food

components

Starter cultures are used to accelerate the fermentation process

to standardize the process

to prevent batch to batch variation in product quality.

To make fermentation predictable and consistent

 to maintain the quality of the product

The major advantage is that the outcome of the fermentation process is

predictable and consistent.

MICROORGANISMS USED AS STARTER CULTURES

Microorganisms used is starter cultures include, yeast, moulds and bacteria

MOULDS

Many substances are produced commercially by moulds; perhaps the most

significant is the antibiotic penicillin and others which can be processed using
Penicillum notatum, Penicillum chrysogenum.

Enzymes e.g. amylase, invertase are also produced and purify from cultures of
moulds such as Aspergilius, Mucor and Rhizopus.

 Moulds like Penicillium roquefort [blue mould] and Penicillium

camembertic [white mouzld] are widely used in the production of cheese
and fermented sausages in European countries.

 Soysauce prepared from soy bean and wheat by a mixed culture

fermentation with Aspergillus oryzae.

 In Africa, Aspergillus oryzae has been used to produce miso from soy
bean and wheat) Tempe (prepared from soybean with Rhizopus
oligospous).

YEASTS

Yeasts play an important role in fermentation of alcoholic beverages as well as

dairy industries. Saccharomyes cerevisae strains are used in the fermentation
of beer and wine.

They can be characterized as bottom and top yeast respectively depending on

where they settle in the fermenting containers or tanks.

 Candida utilis are used in the production of microbial protein which are
used as animal food supplements (Single cell protein).

 Saccharomyces rouxii are used in the production of soy sauce which is

used as food condiment.

 Candida millcri are used in the production of sour French bread.

Saccharomyces cerevisae is used in the production of commercial alcohol

(ethanol) which is used as vitamin supplement.
 Dehydrated yeasts is used for bread making and small scale brewing.

LACTIC ACID BACTERIA

Lactic acid bacteria are the most widely used bacteria in the fermentation of
foods in industries.

This is because first they aid in the preservation of food by utilizing the
carbohydrate and converting it to lactic acid. The resulting drop in pH provides
protection against spoilage microorganisms.

Secondly they produce highly acceptable texture and flavour modification in

the food.

Thirdly, they are pro-biotic i.e. they have beneficial health effect on intestinal
microflora.

They are the starter culture most extensively used in diary industries but they
are also used for the fermentation of foods like meat to sausage, wine,
vegetable, sourdough.

Lactic acid bacteria produce solely lactic acid (homofermentative bacteria) or

lactic acid and other organic acids (heterofermentative bacteria).

LAB include cocci and bacilli and belong to 6 genera; Streptococcus,

Lactococcus, Pedicoccus, Leuconostoc , Bifidobacterium and Lactobacillus.

Lactic acid bacteria are often inhibiting to other microorganisms and this is the
basis of their ability to improve the keeping quantity of many fermented food
products. The heterolactics are more important than the homo-lactics in
producing flavour and aroma components such as acetyladhyde and diacetyl in
foods. In fact, lactic bacteria have been the subjects of intense interest with
respect to the production of inhibitory compounds including anti-microbial
peptides. bactcriocins.

TYPES OF STARTER CULTURE

There are three main types starter culture:

1. A pure single strain culture: This is also known as simple or defined

strain culture. This is a culture made up of just a pure strain of
microorganism. A single strain of microorganisms is used here giving rise to a
consistently good and uniform product. It can be called Axenic culture, due to
its cultivation from "a single pure strain of microorganisms e.g. Lactobacillus
acidophilus used in the fermentation of milk to give. Acidophilus milk. This is
type of culture is good because it eliminate day to day variation in food flavour,
but it is at risk of bactenophage attack. Contamination of this strain is quite
easy and attacks by bacteriophages are high except for strains that are
resistant.

2. Multiple strain culture: This is a blend or mixture of two more compatible

pure strains of microorganisms e.g. Lactobacillus bulgaricus and Streptococcus
thermophilus used to produce yoghurt.

3. Mixed strain culture or Compound Multiple Strain Culture: This is a culture

containing two or more naturally occurring microorganisms. It is a culture
containing two or more strains of the same species or different microorganisms
that are not antagonistic towards each otheri.e. each individual microorganism
do not produce or secret inhibitory substances that will adversely affect the
other e.g. a culture of a variety of materials:

CATEGORIZATION OF STARTER CULTURES

Starter culture may be grouped into the following categories on the basis
of their temperature range.

MESOPHILIC:

These kinds of starter culture are cultivated and yield best within the
temperature range of approximately 200C - 300C Examples of such are:

- Lactobacillus spp and Lactococcus lactic subsp. Cremoris are used for
aroma and acid production during butter milk production

THERMOPHILIC

These starter cultures grow best at temperature above 45 0C.e.g yoghurt

fermentation Non fat or low fat milk is pasteurized ,cooled to 43c or lower and
inoculated with 1;1 ratio of Streptocococcus thermophilus and Lactobicillus
delbrueckii subsp bulgaricus (L.bulgaricus) . S.thermophillus grows more rapidly
at first and render the milk anaerobic and weakly acidic. L.bugaricus then
acidifies the milk more.acting together the two species ferment almost all the
lactose to lactic acid and flavor the yoghurt with diacetyl (S.thermophilus and
acetaldehyde (L.bulgaricus) other examples are L.casei , L.plantarum
MANUFACTURING AND DEVELOPMENT OF STARTER CULTURES

For the development a good starter culture system, research is normally

focused on the biochemical and genetic characteristics of the desired and
undesired properties of a potential starter culture.

The desired properties include phage resistance,

growth at low temperature,

highly active formation of favorable compounds which contribute to the

desired aroma, favour texture

survival during processing.

Ability to grow rapidly on a suitable substrate.

Environmental condition required for maximal growth must be simple, easy

cultivation in large quantity.

MAINTENANCE OF STARTER CULTURE

Once a desired microorganism has been developed and it's ready for use
in stability fermentation, strain and presentation are of concern. A variety of
culture preparation techniques may be used to maintain desired cultures.

1. Periodic transfer; Variables of periodic transfer to new media include

transfer frequency, medium used and the holding temperature.

2. Mineral oil slant: A stock culture is grown on a slant and covered with
sterilized mineral oil. The slant can be stored at refrigerator temperature.

3. Storing under refrigeration: Minimal medium, distilled water or water

agar washed cultures are stored under refrigeration. This culture can be
viable for 3 to 5 months.

4. Freezing in a growth medium.

5. Drying - Cultures are dried on sterile soil or on sterile filter paper. This
can be stored in a dessicator at refrigerator temperature by freezing

6. Freeze drying Lyophilozation: Water is removed from the culture by

sublimation. Very effective up to about 30 years.

7. Ultra freezing: Ultra freezing in liquid nitrogen. Cultures of fastidious

microorganisms have been preserved for more than 15 years.
 LECTURE THREE

MEDIA FORMULATION WITH RAW MATERIALS FROM LOCAL SOURCES

AND ITS ECONOMICS

All microorganism need energy, carbon, nitrogen, phosphorous and sulphur

and various minerals or trace elements. Precise composition of a satisfactory
medium depends on the species of microorganisms to be cultured because it
influences the economic competitiveness of a particular process or product. In
industries raw material input at a particular time determines the cost of a
product. It is important to use raw materials from local sources, these
materials are very cheap and very easy to obtain. Frequently low cost crude
materials are used as source of carbon, nitrogen, etc. Raw materials from
Agricultural sources (crops and residues) are the most important sources of
carbon used in media preparation. Industrial wastes are also used to prepare
media in industries. Materials like -straws, molasses, whey are examples of raw
materials used in industries. Waste materials both from industrial and
agricultural sources are important for economic and environmental reasons.
This is because many by-products from industries especially food industries
are of low economic value arid are often discharged into waterways creating
serious environmental problems.

Molasses is a by-product from the sugar industry. It contains about 50% of

sugar. It is the most easily available for use in many developing countries and
it is readily utilized by many microorganisms because it contains assimilable
sugars and some essential trace clements. However, it is relatively poor in
nitrogen content. Waste from pineapple is another very important source from
food industry. Cassava is also a very good substrate for the production of
methanol and single cell protein.

Most of these substrate are very rich in carbon, however, for certain
microorganisms. This substrate must be enriched with trace elements. The
best source of nitrogen enriched media is yeast.

The table below shows some major component of growth media used in
industrial processes.
NUTRITIONAL REQUIREMENT RAW MATERIAL/SOURCES

Carbon: Molasses, Whey, Grains, corncobs, Barley

Nitrogen: Corn-steep Liquor, Soybean meal Groundnut, Oat flour, Whey

Vitamins: Crude preparation of Plants, animals Products

Iron/trace elements: Crude inorganic chemicals

Buffers: Chalk, Crude carbonate fertilizers

Antiform Agents: Higher alcohols, silicones, natural esters, lard and vegetable
oils.

WATER: This is a dominant component of media in which microorganisms

grow. In respect of volume, water is the most important raw material in many
industrial processes. The quality and quantity of the water used in highly
relevant because its pH affects microbial growth and the products of biological
processes.

Frequently, the carbon source is added continually as a continuous feed. This

is important so that the microorganisms will not have excess substrate that
can cause undesirable metabolic waste products to accumulate.

Also, the level and balance of mineral especially iron and growth factors
can be very critical in medium formulation. Biotin by biosynthetic seen
controls product accumulation in many fermentation. The medium must also
be designed so that Carbon, N2, Phosphorus and a specific growth factor will
become limiting causes a shifting from the growth of M.O. to the products of
desired metabolites.

Sterilization of the medium is very important and must achieve a

maximum kill/destruction of the contaminating microorganisms with
minimum temperature damage to the medium components

Once a medium is developed, physical environment for microbial

functioning must be well defined or developed. This often involves precise
control of agitation, cooling, pH changes, oxygenation. Phosphate buffers can
be used to control pH which also functioning at the same time as a source of
phosphorous.