Fermented probiotic beverage based on quinoa : Functionality, hygiene, and health effects

Sammanfattning: White quinoa grains were used as a source of novel beneficial microorganisms and as a matrix for development of a fermented plant-based drink. Studies on the quinoa grain microbiota were performed by applying spontaneous fermentation to allow isolation of autochthonous Lactiplantibacillus strains with potential to be used as starter cultures. The quinoa-based drink was fermented with the commercial probiotic strain Lactiplantibacillus plantarum DSM9843 (=299v), or with autochthonous Lactiplantibacillus strains. The efficiency of the strains as starter cultures were determined by monitoring changes in the bacteriological community during fermentation. Additionally, interactions between polyphenols and the strains during fermentation were analysed in the beverage and finally, in an attempt to categorize one of the strains as future probiotics, the modified microbiota composition in healthy volunteers was determined after consumption.The experimental procedure was designed to characterize the grain microbiota on the surface as well as inside the grains through preparation of a liquid quinoa dough fermented spontaneously at 30 °C for 8 days. Samples were cultured and viable cells were isolated and genetically analysed applying Sanger sequencing. A consortium of potential pathogenic and beneficial bacteria co-existed and changed positively during quinoa dough fermentation, with the Lactobacillaceae family overtaking the niche. Isolates of autochthonous Lactiplantibacillus strains were further investigated and a phenotypical characterization of the enzymatic capacity of the strains on different carbohydrates and on degradation of tannins were performed. The results showed that the L. plantarum strains were able to ferment a large array of carbohydrates, including xylose and glycerol. Furthermore, tannase degradation was also observed for most of the tested strains.When using starter cultures to ferment the quinoa-based beverage, the commercial strain L. plantarum DSM9843 proved to be a strong inhibitor of undesirable microorganisms for up to 28 days of storage time. However, viable cells of Enterococcus spp., remained present, questioning the efficiency of the commercial strain as starter culture and the limitations of using pH values of 4 or below as parameter of safeness. As a second approach, the quinoa grains were toasted, mixed with sterile water, and inoculated with four of the previously isolated strains: L. plantarum 3, L. plantarum 5, L. plantarum 9, and L. plantarum 10, respectively. Mapping the bacterial community by next generation sequencing (NGS) showed that Firmicutes dominated after fermentation. Changes in the content of polyphenolic compounds were analysed using reversed phase high-performance liquid chromatography (RP-HPLC). No significant variation was observed in the total content of polyphenols or flavonoids between beverages fermented with the different strains, but significant variations were observed for different compounds such as gallic acid, vanillic acid, syringic acid, quercetin 3O-glucoside and rutin.Furthermore, one of the isolated strains, L. plantarum 3, was tested as a first step to categorize the bacterium as a future probiotic strain. Healthy volunteers consumed the fermented quinoa-based beverage for 14 days. Saliva and stool samples were collected and analysed qualitatively and quantitatively by quantitative-polymerase chain reaction (q-PCR), terminal restriction fragment length polymorphism (T-RLFP) and NGS. A modified microbiota composition was found and the increase of the amount of Lactobacillaceae species may indicate that the bacterium did survive the transit through the gastrointestinal tract