Pobre, pie no plantígrado, mal alineamiento severo, síntomas.

Considerable high cell concentration of cell samples at initial storage period demonstrated the ability of the strains used to withstand high temperature during freeze-drying. Thus, this preservation method is suitable for preparing dairy-based culture powders containing high numbers of viable cells of bacteria. Higher initial cell concentration was observed for O-type culture samples than LD-type samples, but was not significantly variable. Our results agree with Yao (2009), who reported low survival rates after freeze-drying of Leuconostoc strains compared with Lactococcus or Lactobacillus strains under the same conditions. Furthermore, variations in cell viability of each product at initial sampling time were assumed independent of the freeze-drying protocol used, especially the drying temperature.

Throughout storage, higher cell counts and acidification of freeze-dried lactic starters and P. camemberti cultures were observed at lower storage temperature, with storage conditions at 37°C showing the least stability. The loss of bacterial viability and higher inactivation of cells were associated with higher degree of discoloration, with brown colour being most pronounced among samples stored at 37°C for 5 months. As regards culture composition, significant decrease in cit+ _{and cit}- _{bacteria occurred} in all samples with increase of storage temperature. Findings of this study are in agreement with previous studies. Gyosheva et al. (1995) reported that S. thermophilus strains kept in freeze-dried state at 6°C were viable for 10 years, with no significant change in morphological, biochemical and technological characteristics. According to

127

Bullimore (1983), most starters of lactic cultures can be kept for 6 months at 4°C. Castro et al. (1995) reported a higher decrease in viability of Lb. bulgarius under 20°C than 5°C during storage. Wang et al. (2003) showed a difference of 20% in the survival rate of freeze-dried S. thermophilus stored at 4°C and 25°C. Viability of Pantoea agglomerans decreased by 0.5 logs after 90 d at 4 °C, compared to a decrease of 3 logs after 28 d at 25°C (Casta et al., 2002). Bruno and Shan (2003) demonstrated that temperature maintained at -18°C was ideal for the long-term storage of probiotic capsules to maximize viability of bifidobacteria. Storage at 20°C showed the highest decline in the viability of cells, whereas that at -18°C showed the least decrease. Higher storage temperature conditions also result in lower survival rates of bifidobacteria (Abe et al., 2009; Wirjantoro and Phianmongkhol, 2009) and E. faecium and Lb. plantarum (Strasser et al., 2009) in freeze-dried powder.

It is apparent from the results of this study and other studies that although frozen and refrigeration storage may be impractical from commercial point of view, it is however necessary for optimal culture viability of stored freeze-dried powders. The results of the high-temperature storage test also lead us to consider the effect of transportation temperature on the viability of product samples. Although there was accelerated decrease in viability of cultures stored at room temperature compared with their storage at frozen and refrigeration temperatures, it may also be feasible to store cells at ambient temperature due to the remaining high cell counts at end of storage above the recommended level. Storage at ambient temperatures can also remove the more costly requirements of refrigeration, energy and suitable package. However, the application of dairy cultures stored at high ambient temperature above 20°C should be limited and further work is also needed in this area.

The results showed that single cultures of L. subsp. lactis and mixed cultures of L. subsp. lactis and Leuconostoc exhibited tolerance at high temperatures. Compared to O-type starters, LD-type starters produce lactic acid at a slower rate, probably because Leuconostoc species are slow-acid producers. Leuconostoc spp. probably had longer lag time than L. subsp. lactis for repairing reversible cellular injuries prior to growth and acid production (Parente and Cogan, 2004). The shorter lag time needed for L. subsp. lactis was probably due to the better survival of the bacteria under stress of dehydration during freeze-drying and storage (Tamime and Robinson, 1999). Microbial inactivation is highly complex. The behaviour of lyophilized cultures during storage is speculated in here in order to explain the reason for loss of viability. Studies by Castro et al. (1995; 1996; 1997) reported the adverse effect of oxygen on survival of freeze-dried LAB during storage at high temperature, during which polyunsaturated fatty acids of cellular membrane were oxidised and showed a

128

decrease in ratio of unsaturated and saturated fatty acids of cells. The change in lipid ratio can consequently lead to a disruption of membrane structure and fluidity. Under oxidizing conditions, Maillard reactions between nucleophilic groups in oxidized DNA and free carbonyl groups also susceptible to occur in presence of water, eventually lead to browning of cultures (Higl et al., 2007; Kurtmann et al., 2009a; Kurtmann et al., 2009c). Such carbonyl groups involved in reactions are mainly reducing fermented medium containing sugars in the concentrated bacterial suspension. As mentioned earlier, the peptidoglycan layer, a cell wall component of LAB, could also be hydrolysed during storage forming reducing carbonyl groups, which undergo condensation reactions with amino group of cell membrane protein, resulting in browning of freeze-dried cells. According to Garcia (2011), oxidation and browning process in freeze-dried bacteria may be inter-related processes. Reduction in cell viability at high storage temperature may also be ascribed to inactivation of

β-galactosidase within in the LAB cell membrane (Champagne et al., 1996; Mazzobre et al., 1997; Vasiljevic and Jelen, 2003). The lactose hydrolysing activity of

β-galactosidase in B. longum lost during storage at 20°C was about twice that observed at 4 and -20°C, while losses of viable cell counts at 20°C were approximately a hundred times greater than those at 4°C (Champagne et al., 1996).

Freeze-dried samples, with the exception of DSL-LL50A and DSL-30A, contained in each laminated sachet were made to 1 g from pure culture and lactose. Since lactose was the only compound incorporated in pure bacterial species of samples except for DSL-LL50A and DSL-30A, discrepancy of results in cell viability may be attributed to differences in physical state of lactose in samples. Several authors have reported on the stabilization effect of lactose in cultures due to their ability to form glassy states (Schebor et al., 1997; Schebor et al., 2000; Thomsen et al., 2005; Higl et al., 2007). As mentioned previously, microbial inactivation is derived from complex physico-chemical reactions. Principally, being a glassy state below Tg, the sugar-bacteria matrix has very high viscosity and low mobility (Roos, 2002; 2004), thus rates of both oxidative and Maillard reactions are limited. Crystallization of amorphous lactose is a spontaneous process, which is also very slow under conditions of high viscosity. A low reduction in β-galactosidase activity within cell membrane was also reported when cells were stored under glassy state (Champagne et al., 1996).

In this study, the addition of lactose in freeze-dried samples probably had low pronounced effect on bacterial stability; this aspect was not investigated. During storage, high cell inactivation was observed in all samples irrespective of the presence of lactose. Samples containing lactose as a major part of the freeze-drying matrix exhibited higher losses of viability in comparison to samples containing no

129

lactose. Higher viability loss was also observed in samples with higher concentration of lactose in their formulation. Considering that samples containing lower amounts of lactose had higher constituent species content in the formulation, it may reasonable to attribute higher stability of cells due to the higher concentration of bacteria. The results may be also associated with changes in Tg of culture-lactose matrix due to impact of aw during storage, especially when stored at high temperatures (Higl et al., 2007).

Although Tg is an important in understanding the stability of microorganisms, it cannot be used in isolation to characterize material as it is also water-dependent (Thomsen et al., 2005; Abe et al., 2009). Several previous studies demonstrated that Tg for freeze-dried cultures in lactose is strongly dependent on both aw and storage temperature (Thomsen et al., 2005; Kurtmann et al., 2009b) (Figure 38). For each of the investigated storage tempeatures, -18, 4, 20 and 37°C, a certain aw exists where the Tg equals the stroage tempeature, thus aw creates a border between the two physical states (glassy and non-glassy) of the sample at a specific storage temperature (Higl et al., 2007; Kurtmann et al., 2009b). Dry pure lactose has Tg of 101°C corresponding to aw of 0. The Tg was reported to be equal to the storage temperature (25°C) with aw of 0.395; at aw of 0.30, the storage temperature was 38°C (Thomsen et al., 2005). According to Thomsen et al. (2005), the aw limit for a glassy state of freeze-dried L. paracasei in a lactose matrix stored at 37°C was ≈0.17 aw, while for 20°C it was ≈0.29 aw.

Water activity has a higher deteriorative effect/impact on cell inactivation at higher storage temperature (Higl et al., 2007; Kurtmann et al., 2009b; Passot et al., 2012). The effect of aw on culture viability loss and their contribution to accelerate rates of deteriorative reactions has been widely reported (Wang et al., 2003; Thomsen et al., 2005; Higl et al., 2007; Abe et al., 2009; Kurtmann et al., 2009a; Kurtmann et al., 2009b; Kurtmann et al., 2009c; Passot et al., 2012). Castro et al. (1995) reported the effect of RH on lipid oxidation profile; at high RH, lipid oxidation occurred more rapidly while cell viability markedly decreased. High aw of the cellular system could also accelerate Maillard reactions (Kurtmann et al., 2009a; Kurtmann et al., 2009c). This reflects faster browning of freeze-dried samples and loss of viability observed in this study as a consequence of Maillard reaction after high storage temperature. Moreover, water is produced by Maillard reaction (Zamora and Hidalgo, 2011). Thus, lactose crystallization may also be triggered due to an increase of released water into the matrix; this probably explains the occasional caking observed on some cultures by end of storage at high temperature [Figures 4 and 8, Appendix 2.4]. For pure amorphous lactose, crystallization was observed at aw>0.40 at 20°C, aw>0.32 at 30°C and aw>0.31 at 38°C (Roos, 2002; Thomsen et al., 2005). The lactose hydrolysing

130

activity of β-galactosidase was critically dependent on the aw at which the lyophilized bacteria was kept (Vasiljevic and Jelen, 2003). High aw was correlated to significant

β-galactosidase activity loss.

Figure 38. (a) aw-temperature state diagram of amorphous lactose (Thomsen et al., 2005). The

gray area indicates a transition zone separating stable amorphous lactose from unstable non-glassy lactose. The dotted line shows the borderline, where the storage temperature is identical to the Tg. (b) aw-temperature state diagrams of Lb. acidophilus freeze-dried in sucrose

or lactose matrix (Kurtmann et al., 2009b). (○) lactose matrix, (܆) sucrose matrix.

The preceeding discussion highlights the need to consider the storage condition of freeze-dried culture samples with regards to temperature and aw. Stability of dried LAB during storage has been shown to be optimal at 0.1-0.2 aw zone with 4% moisture content (Champagne et al., 1996; Simpson et al., 2005). In this study, aw of samples was not standardized throughout storage and the actual atmosphere in sample bags was also not measured. Initial aw of cultures were found to have a significant impact on the storage stability of cultures (Andersen et al., 1999). The initial aw may be different between samples as conditions for freeze-drying procedure may not be similar as they are obtained from different suppliers. The high storage temperature (37°C) used in this experiment may have amplified the response to aw in samples (Kurtmann et al., 2009b; Higl et al., 2007); and the real aw of samples in laminated package at each temperature may be different. Although cultures were packed in laminated sachets with low oxygen transmission and flushed with nitrogen. However, aw of cultures may still increase during storage due to the uptake of moisture from storage environment which influence by permeability of packaging material (Wirjantoro and Phianmongkhol, 2009). The initial aw of Lb. paracasei ssp.

paracasei in a lactose matrix after freeze-drying process was 0.12; after storage at 20°C for 25 d, the aw of samples increased to 0.23 (Higl et al., 2007). In order to achieve the Tg level required for prolonged storage life and stability of cultures, low temperatures have to be compensated for the increased water activity throughout storage.

131

It was probably because Tg could not be regarded as an absolute threshold of bacterial stability during storage, although the rate of deterioration reactions were markedly reduced (Schebor et al., 2000; Higl et al., 2007). The glassy state, as reported in various studies (Higl et al., 2007; Kurtmann et al., 2009b; Passot et al., 2012), does not prevent bacterial inactivation. However, formation of a glassy state during storage may be of practical importance to the producer/user of freeze-dried bacteria cultures. The inactivation of bacteria below Tg also suggests that good survival of bacterial cells at conventional frozen temperature may not solely depend on a sugar protectant.

132

5 SCREENING AND SELECTION OF STARTER AND