Bacterial Starter Culture - Definition, Types, Preparation and Applications

Bacterial starter culture is a prepared bacterial inoculum containing viable bacterial cells, used to start a new culture or a particular fermentation process. It may contain a single bacterial strain or a mixture of different strains.

In laboratories, a starter culture is used to inoculate fresh growth medium for further bacterial growth. Food fermentation has different requirements. Here, selected bacterial cultures are added to the raw materials to carry out the desired fermentation process. The selection of bacteria and their preparation depends on the intended application.

What Is a Bacterial Starter Culture?

Bacterial starter culture is a preparation of living bacterial cells used to initiate bacterial growth in a fresh culture medium or to start a specific fermentation process. It may consist of a single bacterial strain or a mixture of different strains. A small portion of this culture is transferred into a new medium or suitable substrate. This is referred to as an inoculum.

In microbiology laboratories, these cultures are used for further cultivation of bacteria. For food fermentation, selected bacterial cultures are added to the raw materials. Their selection and preparation differ according to the required fermentation process.

Not all starter cultures contain bacteria only. The broader term microbial starter culture also includes other microorganisms such as yeasts and molds, which may be used separately or along with bacteria.

Basic Characteristics of Bacterial Starter Cultures

Some of the basic characteristics of bacterial starter cultures are as follows-

Bacterial starter cultures contain viable bacterial cells that are capable of growing and multiplying when introduced into a suitable growth medium.
A starter culture may consist of a single bacterial strain or a mixture of two or more strains. The composition differs depending on the type of culture and its intended application.
A portion of the starter culture is used as an inoculum for initiating a new culture. The number of viable cells introduced can affect the initial growth of bacteria and the time required for the culture to develop.
The bacterial cells require suitable environmental conditions for their growth. These include the availability of nutrients, temperature, pH and oxygen requirements, which vary according to the bacterial species.
Starter cultures are selected according to the required metabolic activities. In food fermentation, some bacterial cultures produce organic acids, while others may contribute to the development of flavour and texture.
The purity of the culture depends on its application. Laboratory starter cultures are commonly prepared from isolated bacterial colonies to avoid contamination. Food starter cultures may contain selected combinations of bacterial strains.
Bacterial strains intended for food fermentation must be evaluated for their safety. The presence of pathogenic microorganisms and undesirable characteristics should be avoided.
During storage, the viability and required metabolic activity of the bacterial cells need to be maintained. Starter cultures may be available in liquid, frozen or freeze-dried forms, depending on their preparation and application.
The use of a standardized starter inoculum helps maintain similar initial culture conditions in laboratory experiments. In controlled food fermentation, selected starter cultures are used to obtain more consistent fermentation performance under specified processing conditions.

Types of Bacterial Starter Cultures

Bacterial starter cultures are of different types based on their growth temperature, number of bacterial strains, fermentation products, functions and method of preparation. The different types are described below.

1. Based on Growth Temperature

Based on their optimum growth temperature, bacterial starter cultures are classified into two types.

Mesophilic Starter Cultures- These are bacterial cultures that grow at moderate temperatures, generally around 20–32°C. Commonly used mesophilic starter bacteria include Lactococcus lactis and Leuconostoc species. They are mainly used in the production of Cheddar, Gouda and other cheeses prepared at relatively low temperatures.
Thermophilic Starter Cultures- These cultures grow at comparatively higher temperatures (37–45°C). The important members of this group are Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. They are used in the production of yoghurt and several types of high-temperature cheeses. In yoghurt production, a mixed culture of S. thermophilus and L. delbrueckii subsp. bulgaricus is commonly used.

2. Based on Strain Composition

According to the number and composition of bacterial strains, starter cultures are divided into the following types.

Single-Strain Starter Cultures- A single-strain starter culture consists of only one selected bacterial strain. It is isolated and maintained as a pure culture. These cultures are used where specific metabolic properties of a particular bacterial strain are required during fermentation.
Multiple-Strain or Defined Mixed Starter Cultures- These contain two or more selected strains of bacteria whose identities are already known. The bacterial strains may belong to the same species or different species. Different strains are mixed together based on their required properties, such as acid production, flavour development and resistance to bacteriophages. Each strain may carry out different functions during the fermentation process.
Undefined Mixed Starter Cultures- These are mixed bacterial cultures whose complete strain composition is not known. The composition of the bacterial population may change during repeated propagation. Natural whey starters used in the production of certain traditional Italian cheeses are examples of this type. The fermented whey obtained from a previous batch is used for inoculating fresh milk. This method is referred to as “back-slopping”.

3. Based on Fermentation Products

There are two major types of bacterial starter cultures based on the end products formed during sugar fermentation.

Homofermentative Starter Cultures- These bacteria ferment the available sugars and produce mainly lactic acid. They are the primary acid-producing bacteria commonly employed in dairy fermentation. The important examples are Lactococcus lactis and Streptococcus thermophilus.
Heterofermentative Starter Cultures- Unlike homofermentative bacteria, these cultures produce different fermentation products along with lactic acid. The products include carbon dioxide (CO₂), ethanol or acetic acid. Leuconostoc species are examples of this group. They are often added along with other acid-producing starter bacteria for the development of flavour and other specific characteristics of fermented foods.

4. Based on Their Functions

Based on their functions during fermentation, starter cultures are classified into primary and secondary starter cultures.

Primary Starter Cultures- The primary function of these bacteria is to produce acid during fermentation. They ferment the available sugars into lactic acid. In dairy fermentation, commonly used primary starter bacteria are Lactococcus lactis, Streptococcus thermophilus and selected lactobacilli.
Secondary or Adjunct Starter Cultures- These are bacterial cultures added along with the primary starter for performing additional functions. They are mostly used during cheese production and ripening, where they help in the development of characteristic flavour and texture. Selected strains of Lactobacillus helveticus and other non-starter lactic acid bacteria are used as adjunct cultures.

5. Based on Preparation and Inoculation Method

Bacterial starter cultures may also be classified according to their method of preparation and their addition to the fermentation medium. They are of two types.

Bulk Starter Cultures– Bulk starter cultures are prepared by growing selected bacterial cultures in a suitable nutrient medium before their addition to the main fermentation batch. Initially, a small quantity of stock culture is transferred into a suitable medium, where it is grown to prepare the mother culture. This culture is further propagated to obtain the required quantity of bulk starter for fermentation.
Direct-Vat-Set (DVS) Starter Cultures– These are concentrated bacterial cultures prepared commercially in frozen or freeze-dried forms. They are added directly into the fermentation vat without any prior propagation. There is no need to prepare separate mother and bulk starter cultures at the production plant. This method also reduces the chances of bacteriophage contamination associated with repeated starter propagation.

Functions of Bacterial Starter Cultures

Bacterial starter cultures are used in food fermentation for acid production, flavour development, preservation and other biochemical activities. The important functions of bacterial starter cultures are as follows-

Initiation of Fermentation- Starter cultures are added to the food material to initiate the fermentation process. The selected bacteria multiply and start utilizing the available nutrients. It helps in controlling the fermentation rate and producing fermented foods with uniform quality in different batches.
Production of Organic Acids- The main function of lactic acid bacterial starter cultures is the production of lactic acid from the available carbohydrates. In milk fermentation, lactose is broken down by the bacteria and converted into lactic acid. As the acid accumulates, the pH of the medium decreases. In yoghurt production, this decrease in pH causes coagulation of milk proteins, forming the characteristic gel-like structure.
Preservation of Food- During fermentation, the production of organic acids lowers the pH of the food material, which inhibits the growth of many spoilage and pathogenic microorganisms. Some bacterial strains also produce antimicrobial compounds called “bacteriocins”. The starter bacteria compete with other microorganisms for nutrients, limiting their growth in the fermentation medium.
Flavour and Aroma Development- Different metabolic compounds are produced by starter bacteria during fermentation, such as acetaldehyde, diacetyl and organic acids. These compounds are responsible for the characteristic flavour and aroma of fermented food products. In cheese, the bacterial enzymes also break down proteins into peptides and amino acids, which are further metabolized to produce different flavour compounds during ripening.
Texture Development- Some starter bacteria produce extracellular polysaccharides, also called exopolysaccharides (EPS), which increase the viscosity and water-holding capacity of fermented foods. EPS-producing bacterial cultures are used in yoghurt production to increase thickness and reduce the separation of whey.
Cheese Ripening- Starter bacteria take part in different biochemical reactions during cheese ripening. The proteolytic enzymes produced by these bacteria break down milk proteins into smaller peptides and free amino acids. Some bacterial cells undergo autolysis during ripening. This releases intracellular enzymes which further take part in the breakdown of different food components.
Improvement of Nutritional Properties- Certain starter bacteria are capable of synthesizing vitamins during fermentation. They also produce enzymes such as β-galactosidase, which breaks down lactose into glucose and galactose in fermented dairy products. Some selected strains break down food proteins and release different peptides.
Gas Production- Certain heterofermentative starter bacteria, such as Leuconostoc species, produce carbon dioxide (CO₂) along with lactic acid and other fermentation products. The CO₂ produced during fermentation helps in the formation of small openings or “eyes” in certain types of cheese.

Characteristics of a Good Bacterial Starter Culture

A good bacterial starter culture must possess certain characteristics to carry out the required fermentation process and produce the desired food product. The selection of starter bacteria is based on their growth, metabolic activities, safety and stability under the processing conditions. Some of the important characteristics are as follows-

Rapid Growth- The selected bacterial culture must grow rapidly in the fermentation medium and utilize the available nutrients. It should become active within a short period after inoculation. In lactic acid fermentation, a high rate of acid production is required to lower the pH within the desired fermentation time.
Suitable Temperature Requirements- Starter bacteria should grow and remain metabolically active at the temperature used during fermentation. The temperature requirement varies with the type of culture. Mesophilic cultures are used at moderate temperatures, whereas thermophilic cultures are selected for fermentation at higher temperatures.
Safe and Non-pathogenic- The culture must be safe for food production. It should not produce toxins or possess harmful virulence properties. The bacterial strains must be properly identified and evaluated for their safety, including the presence of transferable antibiotic resistance genes. Production of harmful amounts of biogenic amines is also an undesirable characteristic.
Resistance to Bacteriophages- Bacteriophages are bacterial viruses that infect and destroy susceptible starter bacteria. A good starter culture, especially in dairy fermentation, should possess resistance to bacteriophages commonly present in the processing environment. Phage infection may slow down acid production or cause complete fermentation failure.
Production of Desirable Flavour and Aroma- The selected bacteria should produce the required flavour and aroma compounds during fermentation. They must not develop undesirable flavour, excessive bitterness or other unwanted sensory characteristics. The flavour-producing ability depends on the bacterial strain and the food material used.
Genetic and Metabolic Stability- The culture must retain its desired fermentation properties during repeated propagation and industrial production. A sudden change in acid-producing ability or loss of important metabolic activities is undesirable. The selected strain should give reproducible results under similar fermentation conditions.
Tolerance to Fermentation Conditions- During fermentation, the bacteria may be exposed to increasing acidity, changes in temperature and different concentrations of salt. The culture should tolerate the conditions present in the particular food material. Acid tolerance is especially important for bacteria used in lactic acid fermentation.
Compatibility with Other Starter Bacteria- In mixed starter cultures, the different bacterial strains must be compatible with each other. The metabolic activities of one strain should not unnecessarily inhibit the growth of the other required strains. Some bacteria may also support the growth of other members by producing essential nutrients or growth-promoting compounds.
Production of Required Enzymes and Metabolites- Starter bacteria are selected for specific biochemical activities depending on the product. Proteolytic activity is required in several dairy fermentations. Certain strains produce exopolysaccharides (EPS), which are useful for the development of texture and viscosity in fermented milk products.
Good Storage Stability- The bacterial cultures should retain their viability and fermentation activity during storage. They must be able to recover their metabolic activities after preservation, particularly in frozen or freeze-dried starter preparations.
Free from Contamination- Starter preparations must be free from unwanted microorganisms. Contaminated cultures may introduce spoilage organisms into the fermentation medium and affect the quality of the food product. A sufficient number of viable starter bacteria should be present at the time of inoculation.

Preparation of Bacterial Starter Cultures

The preparation of bacterial starter culture involves the growth and propagation of selected bacteria in a suitable nutrient medium under controlled conditions. In conventional dairy starter preparation, the bacterial culture is propagated from the stock culture to mother culture and subsequently into intermediate and bulk starter cultures, depending on the quantity required for fermentation.

The different steps involved in the preparation are as follows-

1. Selection of Bacterial Culture

The first step is to select the suitable bacterial strain or mixed culture based on the type of food product to be fermented.

The selected culture must possess the required properties like acid production, growth rate and other metabolic activities necessary for the fermentation process.

2. Preparation of Culture Medium

A suitable nutrient medium is prepared for the growth of selected bacteria. The composition of the medium depends on the nutritional requirements of the culture.

In dairy starter preparation, skim milk or reconstituted skim milk is commonly employed. The medium must contain sufficient nutrients and should be free from inhibitory substances, such as antibiotic residues and residual cleaning chemicals.

3. Sterilization and Cooling of Medium

Before inoculation, the prepared culture medium is subjected to a suitable heat treatment to eliminate unwanted microorganisms.

For dairy starter cultures, skim milk is usually subjected to high-temperature heat treatment.

After the treatment, the medium is cooled to the required inoculation temperature, which varies according to the bacterial culture selected.

All the culture transfer vessels and other equipment are properly cleaned and sterilized before use.

4. Activation of Stock Culture

The selected bacterial culture is taken from the preserved stock. It may be available in frozen or freeze-dried form.

For cultures that require activation, a small quantity of the preserved stock is transferred into a suitable sterile medium and subjected to incubation under the required growth conditions. During this process, the preserved bacterial cells recover their metabolic activities before further propagation.

5. Preparation of Mother Culture

It is the initial propagation stage where an active bacterial culture is prepared from the selected stock culture. This culture is referred to as the “mother culture”.

A small amount of active stock culture is aseptically transferred into the previously prepared culture medium.

The inoculated medium is then incubated at a temperature suitable for the selected bacterial strain.

During incubation, the bacterial cells multiply and carry out their metabolic activities. Incubation is continued until the required bacterial growth and fermentation activity are obtained.

The incubation temperature depends on whether the culture is mesophilic or thermophilic. Different strains also require different incubation periods depending on their growth and the desired acidity.

6. Examination of Mother Culture

Before further propagation, the prepared mother culture is examined for its purity and fermentation activity.

The examination includes checking bacterial growth, acid production, pH and other required culture characteristics. Cultures showing abnormal appearance, contamination or poor fermentation activity are rejected.

7. Preparation of Intermediate Culture

When a large quantity of starter is required, the mother culture is further propagated to prepare an intermediate culture.

A portion of the prepared mother culture is transferred into a fresh, previously treated nutrient medium. The medium is then subjected to incubation under suitable conditions where the bacterial cells multiply, increasing the bacterial population.

The quantity of intermediate culture prepared depends on the final amount of starter required. This step may be omitted in small-scale starter preparation.

8. Preparation of Bulk Starter Culture

It is the stage where a larger quantity of active bacterial culture is prepared for the main fermentation process.

The active intermediate culture or other suitable starter inoculum is transferred into a larger volume of prepared culture medium. The inoculated medium is incubated under the required conditions, allowing the bacteria to multiply and utilize the available nutrients.

During this process, the temperature, pH and fermentation activity are monitored. Excessive accumulation of lactic acid may inhibit further bacterial growth in lactic acid bacterial cultures. To regulate acid production, some industrial processes employ buffered culture media or controlled neutralization.

Incubation is continued until the required quantity and activity of bulk starter culture are obtained.

9. Cooling and Storage

After obtaining the required fermentation activity, the prepared starter is cooled down to slow further bacterial growth and acid production.

The cooled culture is maintained under specified storage conditions until use. The storage temperature and duration depend on the bacterial culture and its method of preparation.

10. Quality Testing and Inoculation

The prepared starter culture is tested before being used for the main fermentation process.

The culture is examined for its microbial purity, viability and required fermentation activity.

After quality testing, the active starter is transferred into the main fermentation medium in the required quantity.

The inoculation rate is selected based on the bacterial strain, type of food material and fermentation conditions.

Note: In the case of commercial direct-vat-set (DVS) starter cultures, the concentrated bacterial preparation is directly added to the fermentation medium. These cultures are available in frozen or freeze-dried forms and do not require the preparation of mother, intermediate or bulk starter cultures at the production plant.

Evaluation of Bacterial Starter Cultures

Evaluation of bacterial starter cultures involves different microbiological, biochemical and technological tests to determine their suitability for food fermentation. The selected culture is examined for its purity, growth, acid production, metabolic activities and performance under the required fermentation conditions.

The following are the important parameters used for the evaluation of bacterial starter cultures.

Purity of Culture- The bacterial starter culture is examined for the presence of unwanted microorganisms. Microscopic examination and cultivation on suitable microbiological media are employed to detect contamination. For defined starter cultures, the identity of the selected bacterial strains is also confirmed using appropriate identification methods.
Viable Cell Count- It is used to determine the number of viable bacterial cells present in the starter culture. The culture is subjected to serial dilution and plated on a suitable growth medium. After incubation, the colonies are counted and expressed as colony-forming units per millilitre (CFU/mL) or per gram (CFU/g).
Acid-Producing Activity- One of the major parameters in evaluating lactic acid bacterial starter cultures. The culture is inoculated into a suitable fermentation medium and incubated at the required temperature. During this process, the pH is measured at different time intervals to determine the rate of acid production. Titratable acidity is also measured to estimate the total acid produced. A slow decrease in pH may indicate poor starter activity.
Growth and Temperature Tolerance- The growth of the selected bacteria is examined under different temperatures and other conditions associated with the fermentation process. Some starter bacteria are mesophilic, whereas others require higher temperatures. Salt tolerance and the ability to grow under increasing acidity are also tested where required.
Enzymatic Activity- Starter bacteria possess different enzymes involved in the breakdown of food components. The important activities examined include proteolysis and lipolysis, depending on the type of fermented product. Proteolytic activity involves the breakdown of proteins into smaller peptides and amino acids. Lipolytic activity is measured to determine the ability of the selected bacteria to break down fats.
Flavour and Aroma Production- The starter culture is tested for its ability to produce the required flavour and aroma during fermentation. Certain bacteria produce compounds such as acetaldehyde and diacetyl. The fermented product is also examined for the presence of undesirable flavour and aroma.
Texture Development- Some bacterial strains are examined for the production of exopolysaccharides (EPS). In yoghurt preparation, the viscosity, water-holding capacity and separation of whey (syneresis) are measured to evaluate the textural characteristics of the fermented product.
Antimicrobial Activity- The selected bacteria may produce antimicrobial compounds that inhibit the growth of certain undesirable microorganisms. The activity can be examined against selected indicator organisms using appropriate inhibition assays. The inhibitory effect of organic acids and other metabolites must be distinguished from the activity of bacteriocins.
Safety Evaluation- The selected bacterial strains are examined for undesirable characteristics before being used in food fermentation. Tests may include antibiotic susceptibility, haemolytic activity, production of biogenic amines and the presence of virulence factors. Acquired antibiotic resistance genes that can be transferred to other bacteria are of particular concern.
Resistance to Bacteriophages- Bacteriophages infect susceptible starter bacteria and may cause slow or incomplete fermentation. The sensitivity of starter strains to relevant bacteriophages is examined using plaque assays, spot tests or activity tests. Changes in acid production may also be monitored during phage challenge tests.
Storage Stability- The ability of the bacterial culture to retain its viability and fermentation activity during storage is evaluated. Frozen and freeze-dried preparations are examined after storage and subsequent activation. The loss of viable cells and changes in acid-producing activity are recorded.
Performance During Fermentation- The selected culture is tested in the actual food material under the required processing conditions. In mixed starter cultures, the compatibility and combined activities of different bacterial strains are also examined. Fermentation time, acidification and the required characteristics of the final product are recorded.

Common Factors Affecting Starter Culture Performance

Temperature- The activity of a starter depends on the temperature used for fermentation. At low temperature, acid production may become slow. The suitable range differs with the culture and the food being fermented.
pH- Lactic acid produced by the bacteria lowers the pH of the food. As acidity increases, some strains continue to grow while others show reduced activity.
Salt affects starter growth. A strain used in a salted food must be able to grow at the salt concentration present there. The response is not same for all strains.
Food material and nutrients- Fermentable sugars and other nutrients must be available to the culture. These differ in milk, vegetables and cereal dough. In sourdough, even the flour used affects the stability of the starter.
A suitable amount of starter is added so that the selected bacteria can establish in the food. In olive fermentation, its success also depends upon the olive variety and the processing method.
Other microorganisms- Microbes already present in the raw material may compete with an added starter. Sometimes the added culture fails to remain dominant. Yeasts, however, can help the growth of lactic acid bacteria in certain mixed fermentations.
Bacteriophages- These viruses infect the starter bacteria. In dairy products, phage infection may cause slow or incomplete fermentation.
Antibiotic residues in milk can inhibit the culture. Sanitizer residues are another cause of starter failure in dairy processing.
Fermentation time and storage conditions- The period of fermentation affects the activity and composition of a starter culture. Its performance is also influenced by the conditions in which the food is stored.

Applications of Bacterial Starter Cultures

Yogurt and fermented milk- Starter bacteria ferment lactose into lactic acid. The pH falls, milk turns sour and becomes thick. Yogurt is prepared using Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus together.
Cheese production- Cultures are added to produce acid. During ripening, some strains break down milk proteins and fats, giving the cheese its flavour and aroma. Different cheese types require different cultures.
Fermented meat- In fermented sausages, added sugars are fermented by lactic acid bacteria. The pH comes down. Selected cultures can restrict unwanted microorganisms; some bacterial starters also take part in colour and flavour development during ripening.
Fermented vegetables- Starter cultures may be used in sauerkraut, pickled cucumbers, olives and kimchi. Lactic acid is produced. Many traditional vegetable fermentations are carried out without an added starter, by spontaneous fermentation.
Sourdough and cereal foods- Lactic acid bacteria and yeasts occur together in sourdough. The bacteria bring about acidification and help in flavour formation. Yeasts help the dough rise. Selected bacterial cultures are also used for some fermented cereal foods and beverages.
Food preservation- Acids and antimicrobial substances (such as bacteriocins) produced by some selected strains can hold back spoilage organisms. It depends on the strain, the food and fermentation conditions. A starter culture cannot make a heavily contaminated raw material safe.

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Bacterial Starter Culture – Definition, Types, Preparation and Applications