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IV. Resultados y discusión. Capítulo III 255
and AOAC 958.48, respectively) (Horwitz, 2000). Sugars and ashes
contents were obtained following the protocols suggested by Matissek et al.
(1998). All the determinations were done in triplicate.
2.3 Preparation of fermented products
2.3.1 Inoculum preparation
Two different probiotic bacteria, Lactobacillus reuteri ATCC 55730
(further on R) (Biogaia, Stockholm, Sweeden) and Lactobacillus rhamnosus
ATCC 53103, most known as Lactobacillus rhamnosus GG (further on G)
(LGC Standards S.L.U., Barcelona, Spain) were used to obtain almond
fermented milks either alone or as a mixed starter culture with Streptococcus
thermophilus CECT 986 (further on T) (CECT, Paterna (Valencia), Spain)
by using a ratio 1:1.
Bacteria were activated and propagated from their frozen forms (stored
in 40 g/100 mL glycerol at -80 ºC), by transferring each one to its selective
broth until optimal bacterial growth was obtained. The selective broths were
MRS (Scharlab; Barcelona, Spain) for both probiotic lactobacilli and M17
(DifcoTM; New Jersey; USA) for T. Incubation conditions were 37 ºC/24 h/
anaerobically for both R and G and 42 ºC /24 h/aerobically for T.
For the starter inoculumm, strains were independently incubated in their
broths for 24 h and then centrifuged at 10,000 rpm-10 min at 4 ºC;
supernatant was discarded. Immediately afterwards, the bacteria were
resuspended in PBS-1x buffer (10 mmol/L phosphate, 137 mmol/L NaCl,
2.7 mmol/L KCl, pH 7.4) until they reached concentrations of 10 8 cfu/mL.