The studies of the behavior, intrinsic characteristics, and adaptive capacity to a food matrix of the bacterial strains are critical steps for developing functional foods with probiotics. It is known that many LAB strains possess the metabolic tools to degrade oligosaccharides through α-galactosidase activity (Silvestroni et al. 2002) and are capable of adapting their metabolism and growing in soy-based products (Aguirre et al. 2014; Farnworth et al. 2007; Granato et al. 2012). The LABs might present different metabolic activities, evidencing differences in their nucleotide sequence described by technologies that have the power to rapidly reveal important genetic detail for the LAB involved in these fermentations, as reported by Bergsveinson et al. (2017). In this sense, in this work, it was observed a satisfactory growth of L. paracasei BGP1 and L. plantarum CIDCA 8327 in the soy-based formula. In particular, the strain L. plantarum CIDCA 8327 was previously characterized as a potential probiotic strain, and it was demonstrated that it can ferment sucrose, fructose, and raffinose (Garrote et al. 2001; Golowczyc et al. 2008). The temperature is a factor that affects the bacterial growth rate in different matrices, and for both LAB analyzed in the present work, the bacterial growth in the soy-based formula at 37°C was significantly higher concerning the growth observed at 42°C. Moreover, lower pH and higher titratable acidity was found in the soy-based formula fermented at 37°C with respect to that obtained at 42°C. Other authors achieved viable levels between 108 and 109 CFU/mL and pH values near 4 in soymilk fermented at 42 ºC with different probiotic cultures (Mishra and Mishra 2013). Wang et al. (2002) reported pH values ranging from 6.13 to 4.19 and titratable acidity between 0.09 and 0.25% in soymilk fermented at 37°C for 48 h with culture combinations of bifidobacterias and lactic acid bacteria. Moreover, other authors informed lactic acid concentrations of 0.38–0.39% for soy beverages containing cane juice fermented 12 h at 41°C with L. johnsonii NCC533 and L. rhamnosus ATCC 53103 (Farnworth et al. 2007) and between 0.25 and 0.67%, for soymilk fermented with kefir grains which contains a symbiotic association of bacteria and yeast, at 37°C (dos Santos et al. 2019). The variability in the lactic acid content as a metabolic product detected in the fermented beverages is dependent on the metabolic and adaptive characteristics of each particular strain or starter.
In another hand, many authors have described that soymilk fermentation with different LAB cultures improves the product´s flavor and texture (Donkor et al. 2007; Granato et al. 2012). In addition, other authors have reported that soy fermented beverages containing probiotics present good general acceptability by potential consumers (Shimakama et al. 2003). Among other parameters, the pH influences the stability, aroma, flavor, texture, and shelf-life of the fermented soymilk products.
Both bacteria and also their spent culture supernatants employed in the present work, showed inhibitory effect against E. coli, Salmonella and S. aureus. In general, the antimicrobial activity of Lactobacillus strains is well documented; for instance, Bao et al. (2012) demonstrated that the strain Lactobacillus plantarum IMAU70004 presented antagonistic activity against the foodborne pathogens Salmonella typhimurium S50333, Escherichia coli O157 882364, and Staphylococcus aureus AC1.2465, among others. Shokryazdan et al. (2014) found different degrees of antagonism against twelve pathogen strains, including S. aureus and E. coli exerted by nine Lactobacillus strains. It is known that the degree of inhibition against different types of pathogens depends on the probiotic strain (Ebhodaghe et al. 2012). Shokryazdan et al. (2014) also described the antimicrobial effect of Lactobacillus supernatants against pathogens and concluded that the inhibitory effect was due to their organic acid productions. Michetti et al. (1999) described that the whey-based culture supernatant from L. acidophilus La1 inhibited H. pylori growth in vitro. On the other hand, Bian et al. (2011) reported that the cultural supernatant of the strain L. reuteri DPC16 grown in MRS broth significantly inhibited the growth of selected food-borne pathogens, possibly due to acidic effect as the activity was pH-dependent. Considering the antimicrobial effect of the strains and their spent culture supernatant, the effect of the fermented beverages obtained was also evaluated and these products also exibited antimicrobial activity against the three pathogens analyzed. In line with the results obtained in the present work, Kumari and Vij (2015) reported antimicrobial activity against S. aureus and E. coli 0157:H7 ATCC 35150 of soymilk fermented with L. rhamnosus C6, and showed higher inhibition for S. aureus than for E. coli. In line with these results, soymilk fermented with L. helveticus V3 presented growth inhibition of S. aureus and S. typhi, while soymilk fermented with S. thermophilus MD2 evidenced antibacterial effect against E. coli (Hati et al. 2018). Ebhodaghe et al. (2012) evidenced inhibitory effects of soymilk fermented with Bifidobacterium longum against E. coli and S. aureus. The antimicrobial activity against pathogens exhibited by the fermented beverages could be attributed either to the presence of organic acids (e.g., lactic acid, acetic acid) produced by the strains (Ebhodaghe et al. 2012) and also to bioactive peptides produced during soymilk fermentation (Singh et al. 2015).
The antioxidant capacity of the soy-based formula and the fermented products obtained in this work was moderate and no increase of this activity was obtained after fermentation. In contrast, Marazza et al. (2012) informed that a more significant antioxidant activity increase occurred after 9 h of fermentation of soymilk with L. rhamnosus, reaching a 29.5% of DPPH inhibition after 24 h at the end of the fermentation process. Also, the percentage of inhibition of DPPH radical reported by Zhao and Shah (2014) were between 15 and 36% for soy protein extract fermented with L. acidophilus CSCC 24, L. paracasei CSCC 279, L. zeae ASCC 15820, and L. rhamnosus WQ2. However, Rani and Pradeep (2015) reported a higher DPPH radical scavenging activity for soymilk fermented with L. paracasei KUMBB005 during 48 h respect to the unfermented beverage. Other authors reported higher antioxidant activity of soymilk fermented with LAB and bifidobacteria compared with the unfermented soymilk as determined by different techniques such as inhibition of ascorbate autoxidation and scavenging of superoxide anion radicals and hydrogen peroxide (Wang et al. 2006). As Monajjemi et al. (2012) suggested, the DPPH radical scavenging activity seems to have a positive correlation with fermentation time. Also, the antioxidant activity might be associated with proteolysis of soymilk proteins due to microbial metabolic activity during fermentation (Singh et al. 2015). However, given that the fermentation time and proteolysis degree affect the organoleptic and bioactive characteristics of the product, a compromise relationship is generated between these parameters. Choudhary et al. (2019) reported that the antioxidant activity of inulin-supplemented soymilk fermented with L. paracasei CD4 increased during the storage under refrigeration conditions. Finally, on the other hand, the passage through the gastrointestinal tract of the fermented soymilks in another factor that could increase in the antioxidant activity due to larger hydrolysis of soy proteins and the release of bioactive peptides (Capriotti et al. 2015).
In another hand, inulin is mainly added before the soymilk fermentation, so it is available to be hydrolyzed and employed as fermentable substrate by bacteria (Battistini et al. 2018; Bedani, Rossi, and Saad, 2013; Saarela et al. 2006). In the present work, to avoid inulin from being hydrolyzed during the fermentation and to analyze the effect that inulin polymerization degree has on the bacterial viability, this compound was added after the bacterial soy-based formula fermentation. The soy-based beverage with post-fermentation addition of inulin obtained in this work presented significantly higher viable counts than in the soy-based beverage without inulinafter storage, regardless of the polymerization degree of the inulin added. These results can be attributed to a physical protection the bacterial cells undergo in the inulin polymer structure, reducing their damage during storage. In line with these observations, Khorasany and Shahdadi (2021), reported that the post-fermentation adittion of medium-chain inulin (at 1 and 1.5%w/v) contributed to increase the probiotic viability in a yoghurt sample. Moreover, these authors described that although the pH values and acidity decreased during refrigerated storage, no differences were detected between samples with or without inulin additon. Other authors have also reported that inulin added before fermentation improved the viability of L. plantarum CECT 220 in fermented vegetal juices after 20 days of refrigerated storage (Oliveira et al. 2011; Valero-Cases and Frutos, 2017). Also, Paseephol and Sherkat (2009) observed that adding inulin at 4% w/v before fermentation of cow milk improved the viability of L. casei LC-01 during refrigerated storage. Donkor et al. (2007) showed that the addition of inulin in fermented soymilk exerted a protective effect on LAB viability and activity during fermentation. The same results were also described by Oliveira et al. (2011), who reported that the presence of inulin in fermented skim milk with probiotic cocktails of L. rhamnosus, L. bulgaricus, and B. lactis improved the counts of these strains during two weeks of storage at 4°C, in comparison with the product without inulin. One of the advantages of the post-fermentation addition of inulin can be related to keeping the chemical structure of this ingredient to profit from the technological and bioactive properties that it can confer to the final product.
The metabolic activity of the LAB cultures employed for soymilk fermentation is reduced during refrigerated storage, however some variation of the pH of the final products may take place. The difference between the pH values obtained after 30 days of storage of each fermented soy-based beverage obtained, could be related to the metabolic activity intrinsic of each strain. Other authors demonstrated that fermented soy-products acidity is commonly maintained or decreased during storage (Capriotti et al. 2015; dos Santos et al. 2019). Also, it was observed lower final pH values for the fermented soy-based beverages with inulin than those obtained for the soy-based beverages without inulin after 30 days of storage at 4°C. Saito et al. (2014) reported a post-acidification after 27 days of refrigerated storage of a soy-based beverage fermented with L. fermentum and L. plantarum. Despite this post-acidification, the general appearance of the fermented products with or without inulin was essentially the same during storage. Mishra and Mishra (2018) showed that no significant changes were detected in pH values of inulin-supplemented fermented soymilk even after 28 days of storage at 4°C. Other authors reported different variations in the pH values during storage of soymilk fermented with different bacterial cultures with the addition of inulin (Capriotti et al. 2015; dos Santos et al. 2019) suggesting that the bacterial behavior during storage in those cases is strain-dependent. Moreover, as suggested by other authors there is a negative correlation between the titable acidity and pH with the acceptability (Souza et al 1991). The fermented beverages with a pH under 4 resulted more stable against spoilage (Raja et al. 2009), giving rise to a typical refreshing product with a mild acidic taste (Simova et al. 2002).
Probiotic foods must demonstrate the ability to preserve the viability of the strains after passage through the gastrointestinal tract (Buriti et al. 2010). It was determined that the soy matrix contributed to improve the survival of both strains when compared with their survival as free cells (Iraporda et al. 2019). Other authors reported that the survival rate of L. acidophilus La-5, B. animalis Bb-12 (Bedani et al. 2013), and L. casei (Guo et al. 2009) after simulated gastrointestinal conditions was higher in a fermented soy beverage than in culture media. Moreover, in the present work it was observed that the post-fermentation addition of inulin (either of low or high DPn) contributed to increase the percentage of survival of L. plantarum after simulated gastrointestinal treatment. In another hand, the post-fermentation addition of inulin with high DPn produced a higher increase of the survival of L. paracasei in fermented soy-based beverage than when inulin of the lower DPn was added. Therefore, the mean molecular weight, and hence the DPn of the inulin, could be correlated with the degree of protection that this polysaccharide exerts on bacterial tolerance to gastrointestinal conditions, as suggested by Valero-Cases and Frutos (2017). This result leads to highlighting a differential action with regards of the inulin structure; thus, post-fermentation inulin incorporation could be advantageous in order to prevent it from being hydrolyzed by bacteria and to maintain the initial soluble fiber level.