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IV. Resultados y discusión. Capítulo II 163
2.7 mmol/L KCl, pH 7.4) until they reached concentrations of 10 8 colony
forming units (cfu) per mL.
2.2.2 Experimental design for the almond milk fermentation
process
Amounts of glucose, fructose, inulin and starter inoculum were selected
as growth factor variables to obtain fermented almond milks. Central
Composite Design (CCD) with randomised Response Surface methodology
(RSM) was used to study how different combinations of growth factors
affect almond milk fermentation. Other authors also used RSM in the
development of probiotic products (Cruz et al., 2010; Gupta et al., 2010;
Liew et al., 2005; Shuhaimi et al., 2009; Stepheine et al., 2007; Yaakob et
al., 2012). Statistical analysis of the data was carried out by using an
orthogonal CCD 2 4 + star, which studied the effects of 4 factors in 31 runs.
Levels of glucose, fructose, inulin and starter inoculum were 1.5 to 3 g/100
mL for both glucose and fructose, 2 to 4 g/100 mL for inulin and 5 to 7
mL/100 mL for starter inoculum. These parameters were established by
taking previous fermentation studies with probiotics into account (Angelov
et al., 2006; De Souza-Oliveira et al., 2009; Franck, 2002). The variable
response was defined as the probiotic survival (cfu/mL) after fermentation
process.
Fermentation process of the 31 runs obtained in the design was carried
out by adding the corresponding starters (prepared by mixing in a
1:1volume ratio L. reuteri : S. thermophilus buffer suspensions) to the
formulated and pasteurised almond milk and incubating them at the optimal