Starter Cultures – Preparation, Maintenance and Preservation

• The propagation and preparation of starting culture is one of the most vital processes in a plant’s life cycle.
• Since the quality of a starting directly affects the quality of a fermented product, the starter must be in good condition. For optimal performance and maintenance, milk should be purified by heating for one hour at 90 °C.
• The byproducts of milk’s thermal degradation serve as bacterial growth agents. This is likely one of the reasons why lactic acid bacteria grow faster in hot milk than in unheated milk.
• After a proper heat treatment, milk is chilled to between 22 and 25 degrees Celsius and inoculated with an adequate inoculum size. After inoculation, the culture is incubated at 22 to 25 degrees Celsius until coagulation occurs, and is then stored in the refrigerator.
• Additionally, a tiny portion of the culture is inoculated into a similar container containing sterilised milk for mother culture storage.
• The remaining culture is injected into the starter can or container that contains sterile milk. However, the purity of maternal culture is crucial.
• Inoculums are typically added at a rate of 1 percent from a culture containing roughly 0.8% acidity.
• The acidity level must not exceed 0.9% lactic acid. In the event of poor performance, including sluggish activity, evident abnormalities in behaviour, flavour, colour, and appearance, the starter should be removed and replaced with a new one.
• In order to create cultured dairy products efficiently, the purity and activity of the starter culture must be maintained by whatever methods necessary.
• The beginning culture must include the greatest number of live organisms and be highly active under the plant’s production conditions.
• To prepare fermented dairy products such as cheese, dahi, and yoghurt, etc. Frequently, starter cultures are maintained in milk heated to 90°C for one hour, and bulk starters can be cultured in milk heated to 90°C for thirty minutes.
• This heat treatment is sufficient to eliminate phages and any other bacterial vegetative cells.

Principles to maintain active tarter culture

• By decreasing the S.C.’s metabolic activity through chilling
• Through concentration and preservation, organisms can be separated from their waste products.

The preparation of cultures depends on a number of variables:

1. The choice of milk

• Diseases, such as mastitis, can alter the chemical composition of milk, rendering it inappropriate for the formation of starts in specific types of milk.
• Such milk may contain a high bacterial count, contaminate the starter, and cause off-flavors; therefore, it should not be used to prepare the starter.
• The selected milk must be from a healthy cow that is secreting normal, clean, and inhibitor-free milk.
• Also prohibited are milks with high lipolytic activity and low total solids.

2. Heat treatment of milk

• A minimum of 30 minutes at 71.1 degrees Celsius is required. Sometimes the use of sterile milk is advised, but it has a number of disadvantages, including the fact that more severe heating may hinder the culture.
• The culture that has created is soft and sloppy.
• Milk is steamed or boiled for 30 to 60 minutes. To minimise physicochemical changes in milk, it must be rapidly cooled to inoculation temperature after heat treatment.

3. Containers/Utensils

• For starting propagation, stainless steel, aluminium, and enamelled glasses are the ideal materials, with stainless steel containers being the most expensive.
• The ideal beginning vessel is a jar or cylindrical container that is at least twice as tall as its diameter and has a smooth, crevice-free surface to facilitate cleaning.
• After cleaning, the utensils must be steamed for at least 30 minutes at 100 degrees Celsius.

4. Amount of inoculum 

• The quantity of inoculum may have an effect on the performance of a starter, depending on time and temperature, individual culture features, and the condition of the culture at the time of inoculation.
• The optimal inoculum size for the preparation of strater is 0.5-2.0%.
• Some cultures may be injected at higher rates than others in mixed cultures if desired traits are to be maintained.

5. Aseptic culture transfer

• When transferring S.C., strict aseptic conditions must be maintained to prevent aerial contamination.
• A separate inoculation chamber should be fitted with a UV lamp and spraying equipment for detergents and sanitizers.
• It is required to sterilise inoculation needles, pipettes, spoons, and other equipment.

6. Time of inoculation

• After inoculation, the majority of cultures should be incubated between 21.1 and 30 degrees Celsius; the duration of incubation depends on the amount of the inoculum and the features of each culture.
• Normal inoculation time is between 14 and 16 hours when the inoculum size is 1.0%. Particularly during the summer, it is essential to maintain the ideal temperature.

7. Cooling

• After obtaining the desired level of S.C. growth, it must be rapidly cooled to cease its development so that it can be reused.
• Due to the cooling effect provided by refrigeration, mother cultures are often stored in the refrigerator until further usage.

Preparation of mother culture

The preparation of the mother culture is a crucial stage in the manufacture of bulk starter. The mother cultures are kept in poly ethylene bottles with a short neck that have been washed with detergent, sanitised over steam, and filled with 0.1% hypochlorite solution. The seals and caps are also autoclaved and stored in the hypochlorite solution. The advantage of this method is that contamination of the starter at any stage does not result in contamination at the subsequent stage. The preparation steps consist of the following:

1. The sterilised bottles containing three-quarters of the milk are heated to 90 degrees Celsius for one hour, cooled to 22 to 25 degrees Celsius, and sealed with an inverted top.
2. This milk is then aseptically inoculated with a starter whose purity is confirmed by a trusted supplier.
3. After inoculation, the culture is incubated at 22 to 25 degrees Celsius until coagulation occurs, and then refrigerated.
4. A small portion of the culture is re-inoculated at the appropriate time into a similar container containing sterilised milk for storage of the mother culture. The remaining culture is inoculated into the milk-filled starter pail or container.
5. The inoculum should be added at a concentration of 1% from a culture with an acidity of 0.8%.

Preparation of working cultures

• Working cultures and little mother cultures in bottles are identical.
• To avoid creating a huge number of these functional cultures for a series, a small number of large bottles might be used to inoculate the entire series.

Preparation of bulk starters

1. The used vessels are sanitised with steam and then filled with raw milk in bulk.
2. The milk in the container is steamed at 72-73 degrees Celsius for 45 minutes and then cooled to the incubation temperature.
3. The starter was then inoculated in the same manner as the mother culture and the vessel was rotated to incorporate the starter into the milk.
4. Under certain incubation conditions.

Continuous starter production

• This procedure is more inexpensive, convenient. Many cheese factories utilise up to 100,000 gallons of milk per day for cheese production, requiring between 500 and 1,000 gallons of starters each day.
• Such large quantities of starters necessitate numerous expensive pieces of equipment and significant cleaning and sterilising upkeep.
• If the culture is attacked by a phage, the entire mass and all equipments become contaminated, making the management of purity and activity crucial.

Preparation of master culture

• Litmus milk previously sterilised in glass bottles is put into polyethylene tubes for the creation of master cultures.
• The procedure of inoculation is identical to that of the mother culture; however, it is pasteurised or sterilised. These cultures can be maintained forever with careful monitoring and testing every three months.

Maintenance And Preservation Of Starter Cultures  

• Maintaining and preserving microbial cultures is a worthy endeavour. The fundamental idea of culture preservation is to maintain the morphological and physiological properties of the organism. In a laboratory, large collections of starting cultures are utilised for numerous purposes.
• Therefore, it is vital to carefully preserve them for future usage. The objective of microbial culture protection is to keep a microbial strain alive, uncontaminated, and without variation or transformation, as if it were unique.
• Numerous sorts of work demand immediate access to microorganisms. It can be inappropriate to acquire them from other sources or to seek to re-isolate them from their natural habitat.
• Sometimes the same isolate cannot be obtained again. Occasionally, repeated attempts to reisolate the same organism have failed.

Importance of maintenance and preservation

• Considerable effort has been given to discovering ways for preserving cultures in a healthy and stable state.
• Utilization of microbe cultures is crucial to microbiology’s productive routine.
• Authentic reference strains are necessary for comparing laboratory isolates, serving as control cultures in conventional methods of analysis, and employing in research and education.
• The significant increase in the number and scale of commercial fermentations has underlined the importance of maintaining microorganism collections, notably of production strains, test organisms, and related species.
• Additionally, industrially significant microbes are kept for use in various industrial processes.
• Microbiological research requires the maintenance of bacterial stock cultures to preserve their viability and biochemical or pathogenicity features as an indispensable component.
• The majority of microbiological laboratories require simple access to cultures that develop efficiently.
• Routinely, cultures are required daily for quality control, comparative testing, bioassay inoculum, and a variety of other applications.

Methods of starter cultures preservation

• The purpose of preservation techniques is to preserve the viability and genetic integrity of a culture by lowering the organism’s metabolic rate, hence extending the time between subcultures.
• To preserve starting culture microorganisms, a variety of preservation methods have been applied.
• The developed and employed techniques can be split into three categories: 1- Constant growth, 2-Dehydration, and 3-Frozen storage
• Factors that lengthen the duration between subcultures include manipulating growth circumstances by restricting carbon, nitrogen, and energy sources, reducing temperature, and preventing dehydration.
• Other ways for preserving microorganisms include air-drying, desiccation in or above a desiccant, and drying in a vacuum, either from a liquid or frozen condition.
• The term “frozen storage” refers to storing an organism at a temperature low enough to freeze it in order to slow or stop its metabolism and physical change.
• Age of the culture at the time of preservation as well as the use of the appropriate medium and cultivation method determine the success of the preservation.

Method of preservation

Two types of preservation techniques predominate:

1. Short-term preservation methods include serial transfer of organisms to fresh media, storage at low temperature, and maintenance of spores of spore-forming organisms in dry, sterile soil, etc.
2. Long-term preservation techniques are increasingly commonly utilised and involve freeze-drying or ultrafreezing in liquid nitrogen (-196°C). It is known that there is no preservation strategy universally effective for all microorganisms. Various groups of bacteria react differently to various preservation techniques. The techniques for preserving microorganisms, such as bacteria, viruses, fungi, and algae, take into account their unique biological features. The choice of preservation method is determined by the type of microorganism, the availability of equipment and qualified workers, and the desired level of preservation. The most prevalent techniques are discussed below.

1. Regular transfer of starter culture

• It is possible to preserve microbial starter cultures by regularly preparing new cultures from a previous stock culture.
• Through the arrangement of subcultures, the culture is maintained by alternating cycles of active growth and storage periods.
• The frequency of transmission differs from organism to organism. Numerous species are viable for weeks or months.
• After 24 hours of growth at 37 degrees Celsius, the slants can be stored at a low temperature for 20 to 30 days. The frequency of the beginning culture can be decreased if growing the organism on a minimally nutritive media slows its metabolism.
• Using the subculture approach, several aspects are examined when maintaining a microbial culture. The chosen temperature should favour moderate development rather than quick growth of the microorganisms.
• There is no danger of mutation when propagating cheese cultures (S. lactis, S. cremoris, L. cremoris) up to 50 times. And the sterilised medium is injected at 1% and incubated at 22 or 30 degrees Celsius for 18 or 6 hours, respectively.
• As a precaution against mutation, yoghurt S.C. are typically sub-cultured only 15 to 20 times, and incubation is conducted at 42C for 3 to 4 hours or 30C for 16 to 18 hours.
• The rate of cooling after incubation, the amount of acidity at the conclusion of the incubation period, and the duration of storage have an effect on the activity of the starter culture.
• The reserve stock-culture can be kept in liquid form, and inoculation media can be incubated for a brief period and stored under standard refrigeration conditions. Reactivation is required only every three months.

2.  Storage at the refrigerator

• Living starter cultures on a culture media can be preserved effectively in refrigerators or cold rooms (4oC). At this temperature, microbial metabolism significantly slows down.
• As a result, bacterial metabolism will become extremely sluggish, and less nutrients will be utilised.
• Before storing the cultures, they should be prepared using conventional procedures, with contamination avoided, and then sealed. In this procedure, sterilised vials containing 1 ml of agar medium are filled.
• Then, microorganisms are added to the set agar. The starter culture is incubated for 12 hours and then stored at 4 degrees Celsius.
• This procedure cannot be used for an extended period of time due to the accumulation of poisonous substances that are lethal to microorganisms.

3. Paraffin Method

• The simplest and most cost-effective approach for maintaining starting cultures for an extended period of time at room temperature is the paraffin method. In this technique, agar slants are infected and incubated until healthy growth is observed.
• They are then coated with sterile mineral oil to a depth of one centimetre above the slant surface’s tip. The layer of paraffin prevents the medium from dehydrating, and by maintaining an aerobic environment, the microorganisms remain dormant.
• Transfers are accomplished by withdrawing a loop containing the growth, touching the loop to the glass surface to drain excess oil, inoculating a new medium, and conserving the initial stock culture.
• The most frequently used oil is paraffin or Vaseline with a thickness of 1-2 cm. This approach decreases metabolic activity by reducing plant growth due to a decrease in oxygen tension.
• Additionally, cultures can be maintained by coating the agar slants with a layer of sterile mineral oil approximately half an inch above the slant surface.
• This approach preserves bacteria from the genera Azotobacter, Mycobacterium, and Bacillus with great cell viability.

4. Storage in soil

• Numerous genera of fungi, including as Fusarium, Penicillium, Alternaria, Rhizopus, and Aspergillus, among others, have proven productive for storage in sterile soil.
• Inoculating 1 ml of spore suspension into twice-autoclaved soil and incubation at room temperature for 5-10 days constitutes soil storage.
• This early period of growth helps the fungus to absorb the available nutrients before going dormant.
• The bottles are afterwards refrigerated. Few dirt particles sprayed on an adequate medium restores the culture.

5. Storage in Silica Gel

• At a low temperature, the bacteria and yeast can be preserved in silica gel powder for one to two years.
• Finely powdered, heat-sterilized, cooled silica powder is mixed with a thick solution of cells and stored at a low temperature in this procedure.
• The important rule of this technique is rapid drying at a low temperature, which prolongs the viability of the cell.

6. Storage by freezing

• Freezing is a common method for storing microorganisms. Therefore, thick bacterial suspensions can be frozen at -30°C. In general, the longer a culture will remain viable, the colder the storage temperature.
• By lowering the temperature, metabolic rates are reduced, and in the extreme instance of storage in liquid nitrogen at -196oC, they are thought to be eliminated.
• The freezing and thawing of cells is a well-known method for destroying them. In addition, as water is evacuated after freezing as ice, electrolytes become increasingly concentrated in unfrozen water, which may also be damaging since electrolyte concentrations outside cells become significantly different from concentrations inside cells, resulting in osmotic stress.
• Cultures can be adequately preserved when frozen in the presence of a cry protectant, which decreases ice crystal damage.
• As cryoprotectants, glycerol or dimethylsulphoxide (DMSO) are often utilised. Adding 15%(v/v) glycerol to a culture and storing it at -20oC or -80oC in a freezer is the simplest technique to preserve a culture.
• In glycerol stored at -40oC in a freezer, cultures can be maintained for several years. Approximately 2 cc of glycerol solution is added to the agar slant culture in this method.
• Shaking can emulsify the culture. The emulsion is then transferred to ampoules containing 5 ml of culture per ampoule.
• These ampoules are immersed in a combination of industrial methylated spirit and carbon dioxide then promptly frozen to -70 degrees Celsius. The ampoules are then extracted and placed directly in a -40oC deep freezer for use of the stock cultures.
• The ampoules are placed in a 45oC water bath for a few seconds before being utilised for plate cultures.
• The use of freezing in liquid nitrogen at -196oC is the optimum method, and its benefits include: convenience, culture dependability, increased flexibility, improved phage control, and potential quality enhancements.
• However, liquid nitrogen storage has many disadvantages, including the expense of the equipment and regular liquid nitrogen supplies, the risk of explosion, the loss of a large number of cultures if careful monitoring of liquid nitrogen levels is not performed, and the possibility of liquid nitrogen contamination in the storage container.

7. Storage by drying methods

• Some bacterial strains can be kept by drying them from a liquid state as opposed to freezing them.
• Several methods for drying bacterial suspensions for preservation purposes have been developed, which are useful for laboratories that cannot afford the expensive equipment required for storing at very low temperatures or for freeze drying, or for laboratories that rarely perform preservation of cultures.
• A number of the following drying processes offer an alternative option for culture preservation.
• The creation of these techniques aims to eliminate the labour required to maintain liquid stock cultures. It also permits the activity-preserving shipment of dried cultures by mail.

Vacuum drying

• was the standard procedure. The method involves combining a liquid culture with lactose and then neutralising excess acid with calcium carbonate.
• By separating the whey from the mixture, granules are produced, which are then dried under vacuum.
• The dried starters contain only 1-2% live bacteria and may necessitate multiple sub-culturings before returning to full activity.

Spray drying

• Spray-drying bacteria offers a greater production scale, cheaper energy costs, and a sustainable method.
• This is also a viable method for microencapsulating bacteria within a variety of protective matrices to improve their resistance to storage, technical, and digestive challenges.
• When utilising spray drying, higher survival rates are possible. This system has not been utilised in the marketplace.

8. Freeze Drying (Lyophilization)

• This procedure involves placing the microbial suspension in tiny vials. By spinning the vial in a mixture of dry ice (solid carbon dioxide) and alcohol or acetone at RIo C, a thin coating is frozen over the interior surface.
• Instantaneously, the vials are linked to a high vacuum line. The organism is dried while still frozen. Finally, the ampules are vacuum-sealed using a tiny flame.
• The freeze-drying process can damage the bacterial cell membrane, although some chemicals can mitigate the harm.
• Freeze-dried starter cultures are primarily utilised as inoculants for the growth of mother cultures.
• For direct inoculation of the bulk starters, large amounts and maybe an extended incubation period are necessary.
• Lyophilization needs the suspension of bacteria in a medium that maintains its viability through freezing, water removal, and subsequent storage. 

Quality control of the preserved starter culture

• Regardless of the approach adopted for the protection and maintenance of mechanically essential life forms, it is essential to assess the quality of the rescued animal population.
• Regardless of the strategy utilised for the preservation and maintenance of industrially significant beginning culture organisms, it is necessary to inspect the quality of the organism stocks that have been conserved.
• Each lot of recently preserved cultures is routinely examined to ensure their quality. A single colony was transferred to a shake-flask to assure the growth of a certain type of bacterium; additional shake-flask subcultures were employed to prepare an enormous quantity of vials.
• Few vials are checked to determine the purity, viability, and production of cultures. If samples fail any of these tests, the entire batch will be discarded. By utilising such a quality-control system, stock cultures can be maintained and utilised with certainty.