Maintenance and Preservation of Starters

The stock and mother cultures are propagated in the laboratory, while the feeder and bulk cultures are produced at the starter room of the dairy. An active bulk starter culture must have the following characteristics.

•  It must contain the maximum number of viable cells.
•  It must be free from any contaminants, e.g. coliforms or yeasts and moulds.
•  It must be active under processing conditions in the dairy and hence maintenance of the intermediate and other cultures is extremely important.

The mother and feeder cultures are grown in sterile media, mainly milk, under aseptic conditions and the activity of such cultures can be maintained by applying one of the following approaches. First, reducing or controlling the metabolic activity of the organisms by ordinary refrigeration; this is for short-term storage of a starter culture and it can be kept viable for up to a week. Second, concentration and separation of the organisms from their wastes, followed by re-suspension in a sterile medium and finally preservation by drying or freezing. The latter forms are used for extended storage of the starter bacteria and such cultures may be obtained from stock collections available in dairy research establishments, colleges or culture bank organizations, or from commercial starter manufacturers.

Methods of Starter Culture Preservation

It is essential that starter cultures are preserved in order to maintain an available stock of these microorganisms for the production of bulk starter and in the case of a starter failure, some typical preserved cultures could be used for direct-to-vat inoculation (DVI). Also, successive culture transfers or sub-culturing, can induce mutants which may alter the overall behaviour and general characteristics of the starter. Furthermore, in the case of mixed starter cultures, successive sub-culturing could alter the balance or ratio of S. thermophilus; L. delbrueckii subsp. bulgaricus; in “bio: starters the counts of Lactobacillus acidophilus and Bifidobacterium spp.will be altered.

In general, dairy starter cultures may be preserved by one of the following methods:

•  Liquid starter.
•  Dried starter: (a) un-concentrated (spray dried or freeze dried/ lyophilized; these methods are rather old and not used at the present time), and (b) concentrated freeze-dried.
•  Frozen starter: (a) frozen at –20oC (un-concentrated), (b) deep frozen at –40oC to –80oC (concentrated), and (c) ultra low temperature freezing at –196OC in liquid nitrogen (concentrated).

The main methods of starter culture preservation involve concentration of the bacteria,as well as various techniques of drying and freezing, and hence, the viability of a preserved culture may be dependent on:

•  the basic growth medium,
•  the presence of cryoprotective agents,
•  rapid removal of metabolic compounds, e.g. lactic acid and carbonyl compounds, the nature of the suspending medium (if employed),conditions of freezing and/ or drying.
•  Rate of thawing (deep frozen cultures),
•  Methods of concentrations.

The latter aspect, sometimes referred to as cell biomass concentration, is of great importance; the number of bacterial cells per unit weight or volume is measured by counting the number of colonies produced after serial dilution, on an agar medium and the results are recorded by colony forming units (cfu) ml-1 or g-1. However, the cell biomass can be concentrated using different systems.

i) Liquid starters: Starter cultures can be preserved in a liquid form using one of two different growth media. The first type is reconstituted skimmed milk powder (SMP) (10-12 g 100ml-1 SNF (solids-not-fat), which is free from antibiotics. The milk is sterilized by autoclaving at 121oC for 10-15 min, and a sample is incubated for a week at 30oC to check its sterility. After inoculation,the milk is incubated at 30oC for 16-18 hours or at 42oC for 3-4 hours. At the end of the incubation period, the clotted culture must be cooled immediately and it can then be stored for up to a week at ordinary refrigeration temperature (e.g. <10oC). Alternatively, cool, autoclaved milk may be inoculated with a starter culture and then stored under refrigeration for incubation whenever it is required. It is worthwhile to note that successive sub-culturing is labour intensive, expensive and can induce mutant strains; furthermore, trained personnel are required to perform such duties in the laboratory. A maximum limit of 15-20 sub-cultures is recommended for the yoghurt starter bacteria to safeguard the proper ratio between cocci and the rods, and to reduce the effect of mutation.A slightly extended preservation of liquid cultures (i.e. reserved stock culture)can be achieved using litmus milk.

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.

ii) Dried starters: An alternative method for the preservation of starter cultures is drying. The different drying methods used are:

•  Vacuum drying (old methods not used at present time)
•  Spray drying
•  Freeze drying or lyophilization (widely used in the laboratory)
•  Freeze drying of concentrated cultures (widely used commercially).

The main objectives behind these developments are first, to reduce the workload which is involved in maintaining liquid cultures, second, to improve the shelf life of the preserved cultures, and third, to facilitate the dispatch of cultures by post without any appreciable loss in their activity.

The drying process prior to 1950s was carried out under vacuum and the results were not encouraging (i.e. the preserved dried cultures contained only 1-2 per cent viable bacteria). To regain maximum activity several dried subculturing were required. In essence, this method of preservation consisted of taking an active liquid starter culture, adding lactose as a protective agent and calcium carbonate to neutralize the excess acid, followed by partial concentration of the mixture (i.e. removal of whey). The concentrated starters, which were by then in a granular form, were dried under vacuum.

iii) Frozen starters: Starter cultures can also be preserved in the frozen form and such cultures are produced by two different routes:

•  Deep or subzero freezing (-30 to –80oC).
•  Ultra low temperature freezing (-196oC) in liquid nitrogen.

 Sterile liquid milk freshly inoculated with an active starter cultures is deep frozen at–30 to –40oC to preserve the mother or feeder culture. Such frozen cultures can retain their activity for several months when stored at –40oC and this method of culture preservation became popular in the dairy industry because deep frozen cultures produced in centralized laboratories could be dispatched to a dairy in dry ice whenever required. These cultures are mainly packed in plastic containers and a typical example is the Astell-type plastic bottle.