Preparation of Modern Starter Cultures
On an industrial scale, starter cultures are mainly prepared by batch fermentation, since this process is simpler and more convenient than continuous fermentation. Since in the latter undesirable microbial contaminations as well as bacteriophage infections may occur, this process needs a much more complex equipment to assure aseptic conditions throughout each run.
The growth medium is usually formulated on the nutritional requirements of the spe- cies/strain to be developed and balanced between ingredients costs and ability to support the biomass growth in terms of high number of cells in the final batch.
During fermentation temperature, pH, oxygen presence, type of neutralizer, agitation type, speed and frequency are the main process parameters that can affect the growth and the activity of lactic acid bacteria. But also the optimum cooling/harvesting time to the growth curve has to be considered. Streptococcus thermophilus cultures lose their activity fast if cooling and harvesting is postponed when reaching the stationary phase, whereas lactobacilli can be harvested at the stationary phase without activity loss.
Nutritional requirements for optimal growth are very important: the culture medium must contain sources of sugars, protein, nonprotein nitrogen (NPN), mineral salts, vitamins, and other micro-nutrients. Vitamins requirements may vary from species to species: Lactococci require niacine (PP), panthotenic acid (B5), riboflavin (B2) and pyridoxine; Lactobacilli require calcium panthotenate (B5), niacin (PP), and riboflavin (B2). Some lactobacilli, such as Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus acidophilus, require cobalamin (B12) in addition.
After fermentation, the biomass is concentrated by membranes and/or centrifugation to harvest the cells from the culture medium.
Centrifugation is mostly used in industrial operations when favorable conditions occur such as low viscosity of fermentation media, size, and arrangements of the cells to be harvested. Temperature during centrifugation is usually kept low (+5 to +15°C). The pH of cell concentrate is very important during freeze-drying, because it can improve or damage the cell wall. The optimum pH for lactobacilli concentrate is 5.4 to 5.8, whereas S. thermophilus cultures can lose their activity if the pH of concentrate is below
6.2 to 6.6.
After cell harvesting, cryoprotectans are added to improve their survival and protect their activity during freezing, frozen storage, and freeze-drying. The composition of cryoprotectans is different for cocci and lactobacilli and can vary among species. Freezing can affect the activity of cultures also strain-dependently. To formulate the cryoprotectant several main ingredients may be used: lactose, sucrose, milk, monosodium glutamate, ascorbic acid, other vitamins, salts.
The most efficient method to preserve cells is fast freezing in liquid nitrogen; in this way, it is possible to generate pellets that are easy to use as a frozen concentrate or for further freeze-drying.
The shelf-life depends on the form of the cultures: Frozen concentrated cultures are usually stored at -40°C or lower for 12 to 24 months without problem of loss of vitality and activity.
Freeze-dried products are usually stored at + 4°-6°C but also at -20°C and -40°C.