Nutritional, antioxidant and metagenomic analysis of 'superfoods' produced with sprouted soybeans

Abstract

This research was aimed at developing different ‘superfoods’ from sprouted soybeans and determining their nutritional, antioxidant and microbial characteristics. The specific objectives were to optimize the sprouting conditions (soaking and germination) of soybeans for the production of soymilk using response surface methodology, determine the effect of these optimized sprouting conditions of soybeans on the nutritional and quality attributes of resulting superfoods, determine their antioxidant activities, identify and evaluate the potentials of optimum sprouting conditions in reducing soy allergens and to determine the microbial community of fermented soy-based superfoods using next generation sequencing approach. In all cases, soy-based foods from unsprouted soybeans served as control samples. For sprouted soymilk production, soaking (12-24 h) and germination times (48-96 h) were optimized with response surface methodology, at a constant temperature of 25°C using central composite rotatable design. The optimum sprouting conditions of soybeans obtained were 12 h soaking and 52 h germination using desirability concept. Soymilk made from optimized conditions had 17% increase in total proteins, 50% reduction in phytic acid, 1.7% increase in total phenolics and a colour change of 4.89 compared to the control produced from unsprouted soybeans. There was a significant reduction in trypsin inhibitor activity (0.03 mg/g TI), with increase in total amino acids and similar rheological properties in optimized soymilk to the control.

Soy-based foods, including tofu, naturally fermented soymilk and soymilk-kefir, were produced from soymilk obtained at optimized conditions of soybean sprouting and their nutritional and colour attributes were investigated. Total protein contents of sprouted products were higher than their controls and there were no significant differences in the ash contents (p ≤ 0.05). In vitro protein digestibility ranged between (85.70-99.82%) for sprouted and (81.33-99.50%) for unsprouted ones, with fermented products having the highest digestibility. Generally, no significant difference in amino acid contents (p ≤ 0.05) was recorded for all sprouted soy foods and their controls. Total flavonoids were higher in sprouted tofu coagulated with CaCl2 (60.41mg/g) and sprouted fermented soymilk (48 h) (8.93 mg/g) and there was a general reduction in total condensed tannins for all sprouted products.

Colour deviation of sprouted products from their controls were minimally perceivable, as the highest ∆E value was 5.30. Soy products obtained from sprouted soybeans at optimized conditions had nutritional advantage over unsprouted ones, with negligible colour deviations.

Antioxidant activities of soy-based foods produced at optimized conditions of soybean sprouting were investigated using different assays. Total phenolic content (TPC) was determined using Folin Ciocalteau method and samples (0.5-5 mg/ml), in appropriate buffers were tested for their abilities to scavenge free radicals. Sprouted soy-based food products had significantly higher (p ≤ 0.05) TPC (13.21-14.21 mgGAE/g) when compared to their controls (13.01-14.07 mgGAE/g). Sprouted soy-based foods scavenged free radicals and demonstrated higher diphenyl-1- picrylhydrazyl (DPPH), superoxide and hydroxyl radical scavenging activities. Over 50% increase in metal ion chelation was observed in all sprouted soy-based food products, however, ferric reducing antioxidant power (FRAP) in both sprouted and unsprouted products were not significantly different (p ≤ 0.05). Production and consumption of soy foods from soybeans sprouted at these optimized conditions as functional foods could be a promising means of preventing the excessive production of reactive oxygen species.

The allergenic proteins in optimized sprouted soy-based foods and their respective controls were identified and their levels were evaluated. Protein was extracted from each soy-based food and their concentrations were determined using Bradford assay. Aliquots of proteins previously dissolved in appropriate buffers were separated according to their molecular weight and ionic strengths using 2-dimensional electrophoresis. Protein spots were then excised from the gels and made to undergo complete tryptic digestion to produce peptides. Identities of peptides were determined using MALDI-TOF mass spectrometry and the volumes of protein spots in each gel was determined using PDQuest image analyzer. Protein concentration significantly increased in sprouted products (up to 149% in SSK). Eight differentially expressed protein spots were selected and identified by MALDI- TOF/TOF mass spectrometry as glycinin subunit G2, β- conglycinin (α, α' and β subunits), trypsin inhibitor, 34 kDa soy seed protein and sucrose- binding proteins.

Higher extent of breakdown of soy storage protein was obtained in sprouted soy products, as shown by higher protein concentration and spot volumes. Lower spot volumes were obtained in all fermented products. This study suggests higher potentials for soybean-storage-protein breakdown and reduced allergen contents in sprouted soy-based foods.

The microbial community of fermented soy-based foods from sprouted soy foods and their respective controls was determined using rep-PCR and high throughput amplicon sequencing (HTS). Samples of spontaneously fermented soymilk and soymilk-kefir were collected at 6 h interval from 0-48 h and subjected to analyses. From rep-PCR, identified LAB species include Weissela cibaria, Lactococcus lactis, Leuconostoc lactis, Leuconostoc messenteroides, and Lecleria adecarboxylata while yeast species are Saccharomyces cerevisae, Pichia fermentans and Torulaspora delbrueckii. In addition to the genera revealed in rep-PCR, HTS showed the presence of Bacillaceae and other bacteria involved in spontaneous and kefir fermentation of soymilk. It could be concluded from the findings of this research that sprouting of soybeans at the suggested optimized conditions has potential for extension of use of soybeans in the production of soy-based foods with enhanced nutritional and functional properties.