A.

Size distribution and morphology of powders

The size distribution of the powders is shown in Fig. 4 and Table 2. For both
strains, the size distribution of powders right shifted as the TS in the culture
media increased. This result also corresponded to the observation by
scanning electron microscopy, shown in Fig. 5.The powders dried from
culture media with high TS (30%) had a wider size distribution (Span > 2),
but with D0.5 larger than 10 m.The powders from 5% medium were finer,
with a narrow size distribution (Span between 1 and 1.5) peaking below 10
m. However, it was difficult to find the bacteria cells on the surface of
powders in the SEM observation, even for the powders obtained from the
medium with 5% TS. This indicated that the 5% TS sweet whey was already
sufficient for full microencapsulation of bacteria during spray drying.This
coincided with the results for spray dried powders of Lactobacillus
plantarum with trehalose as drying medium (Perdana et al., 2014).

B. Viability of probiotics during storage

The changes in bacteria population in powders were monitored
during storage at 4 °C for 120 days (Fig. 6). The viability of both L. casei
and P. freudenreichii strains in the powders dried from the culture media
with 20% ~ 40% TS remained constant, with a maximum log reduction of
0.89. After storage for 120 days, the L. casei population in the powders from
20% and 30% culture media remained the highest at around 7 × 108 CFU
g−1 , while the P. freudenreichii population was around 2 × 109 CFU g−1 .
Since the initial bacteria populations in the powders from 40% medium were
relatively low, the L. casei and P. freudenreichii populations were around 4
× 107 and 1 × 107 CFU g−1 after 120 days, respectively. In comparison, the
viable L. casei population in the powder from 5% medium decreased
significantly over the entire storage time, with a final population below 106
CFU g−1 at the end of storage (~2.8 log reduction at 120 days).
Moreover, the L. casei in the powder from 10% medium remained
unchanged during the first 90 days (viability loss < 1 log), but decreased
considerably after 90 days (~2.2 log reduction at 120 days). The P.
freudenreichii powders from the 5% and 10% culture media exhibited very
steady viability during the first 60 days. Although there was subsequently
reduction in viability after storage (~1.5 and 1 log reduction for 5% and
10%, respectively), the final viable bacteria population still remained above
108 CFU/g at the end of 120 days. The ability of P. freudenreichii to
accumulate polyphosphate, trehalose and glycogen as energy and carbon
storage compounds, as well as compatible solutes, might permit better
survival throughout a long-term storage (Boyaval et al., 1999; Cardoso,
Castro, Borges, & Santos, 2007; Thierry et al., 2011).
It was obvious that the two probiotic strains in the culture media with
lower TS (i.e. 5% to 10%) survived less well than those with higher TS
(20% to 40%). Three reasons can be proposed. First, although the bacteria
in powders from culture media with lowerTS were also covered by the
drying matrix, the lower solid content in these powders led to a thinner
encapsulating wall layer, which may not be effective enough to protect
bacteria from oxidation stress in the long-term storage (Elversson,
Millqvist-Fureby, Alderborn, & Elofsson, 2003; Perdana et al., 2014).
Second, the bacteria in the powders with lower TS had lower survival levels
after spray drying. This means that there may be more damaged bacteria in
these powders which might die as time goes on if they are not able to recover
from the damage (Ananta et al., 2005; Teixeira, Castro, Malcata, & Kirby,
1995).Third, the bacteria in culture media with higher TS were more tolerant
due to the cellular response triggered by the high osmolality during growth
(e.g. accumulation of compatible intracellular solutes) (Kets, Teunissen, &
Debont, 1996; Selmer-Olsen, Birkeland, & Sorhaug, 1999).The viability of
two probiotic strains in the powders, especially after long-term storage,
were still relatively low as compared to a part of commercially available
deep-frozen or freeze-dried cultures (1010 to 1011 CFU/g). In the future
work, the optimization on sweet whey medium, spray drying process or
 storage condition can be further carried out to obtain higher remaining
probiotic viability in the powders.

C. Conclusion
This is, to the best of our knowledge, the first report describing the
feasibility of simplifying the process of spray drying bacteria by utilizing a
double-used medium with increased total solid content. It was found that
culturing L. casei and P. freudenreichii in sweet whey with 20% ~ 30% TS
resulted in greater biomass production and sustained viability after spray
drying and following a long-term storage. The results of this study add to
the value of sweet whey and may indicate a new route to producing probiotic
powders with high levels of production efficiency and probiotic viability.
However, the mechanism of cellular adaptation to high osmolality and its
relationship with protection during spray drying and storage should now be
investigated at a molecular level. Moreover, the feasibility of scaling-up this
process should now be explored on an industrial scale.