Probiotics are commonly found in chilled short shelf-life foods, such as fermented milk drinks and yoghurts, but consumer demand for healthy foods in a variety of formats is increasing. This is why there is a growing interest in including viable probiotics in dried foods with a long- term ambient shelf-life, and in sufficient numbers (108 CFU per serving or 106 to 108 CFU per
gram of food) to provide a health benefit. A cost-efficient process (from growth to storage) would further help in expanding the market and promoting the usage of microorganisms for wider range of applications that could benefit consumers.
Following this line, the purpose of this research was to maximizing viability of L. casei 431 during fermentation, fluidized bed drying and ambient storage. This was achieved by investigating various parameters (Table 9.1.1) at various stages from growth to storage with the objective of finding best combination for maximizing the cell viability during laboratory and scale-up preservation of bacteria.
Table 9.1.1- Various parameters studied at each processing step to maximize L. casei 431l viability during drying and ambient storage
Processing step Parameters studied
1 Fermentation Media composition, pre stress by acid and osmotic (NaCl) stress
2 Harvesting Harvesting Time/growth phase (starvation stress) and harvesting method (laboratory and industrial scale)
3 Mixing Mixing technique, carrier water activity
4 Drying Drying time and temperature, initial solids/moisture before drying
5 Storage Storage temperature
Large amount of information was available discussing the effects of various growth medium components and growth conditions on viable cell count and other growth properties such as OD and biomass of various lactobacilli strains. But most of these studies were performed
under pH controlled conditions in a bioreactor. Many of these studies included comparison of batch, fed-batch and continuous bioreactor in terms of cell growth. But very few studies have investigated increasing cell growth properties of lactobacilli under uncontrolled pH conditions and moreover the focus of these studies was to increase cell biomass or OD rather than viable cell count. Furthermore bulk of the attention has been given to the preservation of lactobacilli cells either through freeze drying or spray drying with very few studies describing the drying and storage stability of cells dried using a fluidized bed (FB). Also, in these studies, poor stability of FB dried lactobacilli during drying as well storage at temperatures above refrigeration temperatures has been reported. Maintenance of probiotic viability is not only important during drying but also during the shelf-life of powder. This is the main determinant of commercial success of a product.
9.2 Conclusions
A probiotic strain Lactobacillus paracasei subsp. paracasei L.casei 431 used in this study was found to grow well in MRS medium. During uncontrolled pH fermentation, pH of the growth medium dropped to ~4.0 over a period of 10-14 h and thereby preventing further multiplication of bacterial cells. The maximum cell density obtained in MRS media under these conditions was 9.3 log cfu ml-1 media. Supplementing MRS with glucose or nitrogen extract did not increase viable cell density significantly. However, concentration of nitrogen source (in the form of yeast extract) was found highly significant for L.casei 431 biomass suggesting a partial uncoupling between the increase in cell number and the biomass production, as they were affected by the independent variables in a different way. When pH during fermentation was controlled at 6.5 in a bioreactor, maximum cell density still remained 9.3-9.5 log cfu ml-1. Yet again glucose supplementation did not affect the cell count probably due to substrate inhibition. However, fed-batch technique did increase the viable count to above 10 log cfu ml-1. For a commercial product, FB drying was chosen as an economical alternative to freeze drying. FB drying needs a carrier for bacterial cells and so whole milk powder (WMP) was chosen as a carrier for this work, but other carrier might offer more protection. During drying in a fluidized bed, pH controlled cells were found to suffer huge losses during drying and subsequent storage compared to pH uncontrolled cells. The reason for this loss was not related to osmotic shock caused during mixing of cells (wet) with WMP as the water activity of the WMP showed little effect on viability and the loss was also unrelated to presence of lactose crystals. The most probable reason could be due to the build up of resistance through starvation and acid stress in cells grown in uncontrolled pH conditions. The drying tests showed that the initial moisture content of harvested cells was a critical factor – if too high powder is difficult to dry
and drying and storage losses are high. Also, drying temperature should not be too high but drying time should be sufficient enough to dry powders to desirable final moisture content (~4 %) required for long term shelf-stability of dried bacteria.
For dried powders containing bacteria, the level of moisture appeared to be a critical factor. But, final moisture content highly depends on other factors such as drying temperature, drying time, initial moisture content before drying, harvesting techniques and type of protective carriers used. Thus, optimization of all these parameters in combination of final moisture content in dried powders is essential for stability of probiotic bacteria under ambient conditions. Furthermore, storage environment and type of packaging are important for long shelf-life for probiotics. An efficient packaging which could provide barrier to oxygen, light, heat and moisture would be beneficial. It is important to not overlook the necessity of inducing desiccation tolerance during the growth of the micro-organisms, and not just relying on protective agents within the drying matrix. It has clearly been demonstrated that cells need to be conditioned for desiccation tolerance, e.g. stationary phase cells in acidic growth conditions. Thus, each step ranging from growth to storage of cells could strongly influence viability of the cultures.
Some generic conclusions can be drawn from this study which may be useful in maximizing cell survival during drying and ambient storage:
Growth media should be high in nitrogen content.
Cell growth should be under uncontrolled pH conditions (acid stress).
Harvesting of cells should be done during early to mid-stationary phase (starvation and acid stress).
Total solids in harvested cells should be high.
Mixing of cells with the protective carrier should be slow. Initial moisture content of the mix before drying should be low
Maximum inlet drying air temperature of FB drier should not exceed 50 ˚C Relative humidity of drying air should be low
Drying time should be aimed to get final water activity of ≤ 0.2 and moisture content close to 4 % in dried culture
Packaging should be air tight and temperature of storage should not exceed 25 ˚C in order to ensure high viability of a commercial probiotic and starter culture.