Probiotic Lactic Acid Bacteria Fermentation Modulates the Bioactive Properties of Sprouted and Unsprouted Amaranth Seed

TL;DR

This study shows that fermenting sprouted and unsprouted amaranth seeds with probiotic bacteria enhances their nutritional and antioxidant properties.

Contribution

The novel aspect is the combined effect of sprouting and probiotic fermentation on the bioactive properties of amaranth seeds.

Findings

Red amaranth samples showed higher levels of biologically active compounds than black amaranth.

Fermentation with L. rhamnosus MIUG BL38 increased protein background intensity, indicating protein hydrolysis.

Sprouting and probiotic fermentation improved the functional and biochemical properties of amaranth seeds.

Abstract

This study aims to investigate the functional and biochemical characteristics of sprouted and unsprouted red and black amaranth flours by fermentation with four probiotic strains (Lactiplantibacillus plantarum MIUG BL21, Lactiplantibacillus pentosus MIUG BL24, Lacticaseibacillus rhamnosus MIUG BL38, and Lactiplantibacillus paraplantarum MIUG BL74). Aqueous extracts from freeze-dried fermented products derived from sprouted and raw seed of two Amaranthus species (Amaranthus cruentus—red amaranth and Amaranthus hypochondriacus—black amaranth) were characterised for their acidification and phytochemical profiles by titrimetric, spectrophotometric and chromatographic methods, and their antioxidant activities by ABTS and DPPH assays. Water-soluble proteins were evaluated by SDS-PAGE analysis. Nine phenolic acids (gallic acid, protocathechic acid, syringic acid, ellagic acid, ferulic acid, cinnamic acid, caffeic acid, p-coumaric acid, and chlorogenic acid) and twelve flavonoids (epicatechin gallate, hesperitin, quercetin, apigenin, luteolin, naringenin, quercetin 3-glucoside, isorhamnetin, peonidin 3-O rutinoside, epicatechin, keracyanin, and rutin trihydrate) were identified in the extracts of amaranth samples. The titratable acidity ranged from 0.59 to 5.50 mL of 0.1 N NaOH. Total flavonoid content (TFC) varied from 1.09 to 4.67 mg CE/g DW; whereas, total phenolic content (TPC) fluctuated from 1.99 to 5.76 mg GAE/g DW. The spectrum of ABTS and DPPH values was from 17.49 to 56.82% and 0.60 to 35.50%, respectively. More biologically active compounds were found in red amaranth-based samples, both sprouted and unsprouted, compared to black amaranth-based samples. There was a moderate correlation between the TPC and the antioxidant activity. The fermentation of red amaranth with L. rhamnosus MIUG BL38 led to a global increase in the protein background intensity, consistent with protein hydrolysis. Overall, sprouting and probiotics fermentation improved the fermentative performance of the amaranth seeds, enabling their effective use as a nutritive food with potential health-promoting properties.

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1. Introduction

Amaranth (Amaranthus spp.) is an age-old pseudocereal cultivated by the Aztec, Inca, and Mayan civilisations and is now recognised for its distinctive nutritional and functional qualities [1]. Unlike traditional cereals, amaranth is gluten-free, making it a valuable alternative for individuals with celiac disease or gluten sensitivity [2]. Its seeds are rich in high-quality protein (15–18%), containing a high level of lysine, the limiting amino acid in most cereals, as well as dietary fibre, unsaturated fatty acids, and essential minerals such as iron, calcium, and magnesium [3,4,5].

Amaranth is a nutrient-rich, easily cultivated, and underutilised pseudocereal that can enhance climate change mitigation and food and nutritional security in Africa [6]. Instead, in Europe, amaranth seed production has rapidly increased, where it is considered a niche of pseudocereals market development. Germany is the primary consumer market for the seeds. Still, its popularity is growing in other countries such as the United Kingdom, the Netherlands, Belgium, Sweden, France, and Romania due to its valuable nutritional and functional benefits [7,8].

The complex biochemical profile of amaranth also includes bioactive compounds such as polyphenols, flavonoids, phytosterols, squalene, and antioxidant peptides, which help reduce oxidative stress and prevent cardiovascular, metabolic, and inflammatory diseases [1,2]. Because of these properties, amaranth is regarded as a new generation functional food with both nutritional and therapeutic potential [2,5]. Recently, the food industry has increasingly utilised the potential of this pseudocereal in gluten-free products and fortified nutritional formulas, including bread, biscuits, pasta, fermented drinks, sports products, and dietary formulas for patients with metabolic disorders [4]. At the same time, the high adaptability of amaranth plants to various climatic conditions and their agronomic benefits make it a strategic crop for global food security [3,4].

The importance of protein hydrolysates has grown owing to their bioactive properties and health benefits. They are an essential source of bioactive peptides which exhibit antibacterial, antihypertensive [9], anticoagulant, hypocholesterolemic, anti-diabetic [10], immunomodulatory, anti-inflammatory [11], anticancer, and antioxidant properties [5]. In summary, proteins and peptides derived from amaranth have shown potential as ingredients in functional foods and nutraceuticals, offering health benefits in the prevention of metabolic diseases.

The germination process is one of the most effective natural biotransformation methods for enhancing the nutritional and functional value of pseudocereals. During germination, the activation of endogenous enzymes leads to the breakdown of antinutritional compounds (phytic acid, tannins) and the production of bioactive metabolites, such as phenolic compounds, flavonoids, and antioxidant peptides [12,13,14].

Many studies have shown that germinating amaranth seeds significantly increases antioxidant activity, amino acid bioavailability, and the digestibility of protein and starch [12,13,15]. Additionally, bacterial fermentation, especially when performed with probiotic strains of lactic acid bacteria, is a biotechnological process that can further improve the functional properties of amaranth flour. During fermentation, microorganisms convert the plant substrate into bioactive compounds, decrease the levels of antinutrients, and release peptides with antioxidant, anti-inflammatory, and hypoglycaemic effects [14,16]. Recent research indicates that fermenting germinated amaranth with Lactiplantibacillus plantarum results in higher antioxidant activity and a more favourable phenolic profile compared to unfermented products [15,17].

Therefore, combining germination and fermentation offers a promising synergistic approach to creating functional foods with high biological value. In the current climate of growing interest in sustainable, gluten-free products with proven nutritional benefits, amaranth presents a significant opportunity for developing innovative ingredients aimed at maintaining health and preventing diseases related to oxidative stress [3,17].

This study aimed to evaluate the effect of probiotic lactic acid bacteria fermentation, using four LAB strains, on the functional and biochemical properties of red and black amaranth flours, both germinated and ungerminated. The research is expected to identify the optimal combination of lactic acid bacteria strain and amaranth type, in order to obtain a fermented food ingredient with functional potential, intended to promote a healthy diet.

2. Materials and Methods

2.1. Plant Seeds and Amaranth Flour

The seeds of black amaranth (BA) (Amaranthus hypochondriacus) were collected from the rural commune of Ditinn, Dalaba prefecture (Guinea), in 2024 [18]. The seeds were divided into 500 g batches, vacuum-packed, and kept at room temperature in the dark [19].

The red amaranth (RA) seeds ‘Garnet Red’ (Amaranthus cruentus) were purchased from a commercial distributor S.C. XTREME ANG MARKETING S.R.L., Bucharest, Romania.

Red amaranth seeds (RA), Amaranthus caudatus L. var. Red Garnet, purchased from a local commercial distributor (Bucharest, Romania), was selected due to its high content of phenolic compounds, promoters of the antioxidant activity [20]. Meanwhile, black amaranth seeds (BA) (Amaranthus hypochondriacus), an invasive weed, collected from the rural commune of Ditinn, Dalaba prefecture (Guinea), were chosen based on their dietary minerals, vitamins, proteins, and bioactive compounds with potential health benefits [18,21].

Seed germination was performed using the EasyGreen EGL 55 automated germinator (Biovie Co., Langlade, France), following the method described by Ghinea et al. (2021), with modifications [22]. In brief, to prevent microbial contamination, seeds were exposed to UV radiation in a safety cabinet (SafeFAST Elite 209 S, Cornaredo, Italy) for 15 min, followed by washing with 2% sodium hypochlorite solution for 10 min and rinsing with distilled water for 20 min. Amaranth seeds (BA and RA) were allowed to germinate for 70 h at 23 ± 1 °C in semi-dark conditions. A high relative humidity (~90–95%) was ensured by spraying the seeds with water for 15 min every 4 h. Then, the seeds were dried at 40 °C for 24 h in an incubator (Stericell Typ 111, Munich, Germany).

Sprouting efficiency (%) was determined as the ratio between the number of germinated seeds and the total number of incubated seeds [23], calculated by the following equation:

$$[eqn]$$

For red amaranth seeds, the sprouting efficiency was 82.83 ± 0.45%, while for black amaranth, it was 72.53 ± 0.72%.

To obtain the flour from unsprouted amaranth seeds (BA and RA), the same disinfecting, washing, and drying steps as described above were carried out. Using an electric grinder (Heiner HCG150SS, Bucharest, Romania), the dried seeds and sprouts were ground, and the resulting flours were stored at 4 °C [18,19].

2.2. Reagents and Chemicals

The following reagents were purchased from Sigma-Aldrich (Hamburg, Germany): DPPH (2,2-Diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), aluminium chloride (AlCl_3_), sodium carbonate (Na_2_CO_3_), sodium nitrite (NaNO_2_), potassium chloride (KCl), sodium acetate (CH_3_COONa), calcium carbonate (CaCO_3_), Folin–Ciocalteau and Bradford reagents. Merck Millipore (Darmstadt, Germany) provided culture media, including De Man, Rogosa, and Sharpe (MRS) broth and agar. Ethanol and methanol, which were HPLC-grade solvents, were acquired from Scharlau (Barcelona, Spain).

2.3. Lactic Acid Bacteria Reactivation and Fermentation

Four probiotic strains, Lactiplantibacillus plantarum MIUG BL21, Lactiplantibacillus pentosus MIUG BL24, Lacticaseibacillus rhamnosus MIUG BL38, and Lactiplantibacillus paraplantarum MIUG BL74, from the Microorganisms Collection (abbreviated MIUG) at the Integrated Centre for Research, Expertise and Technological Transfer in Food Industry (BioAliment-TehnIA), “Dunărea de Jos” University of Galaţi, Galaţi, Romania, were examined (Table 1) [21]. Two of these strains, Lp. plantarum MIUG BL21 (LMG P-33745) and Lp. paraplantarum MIUG BL74 (LMG P-33746) were deposited at the Belgian Coordinated Collections of Microorganisms (BCCM), Gent, Belgium.

Several factors, including technological, functional, and safety-related properties, were considered when the probiotic strains were chosen. All strains were selected based on their previously demonstrated resistance to simulated gastric juice (low pH and bile salt tolerance) and adhesion to intestinal epithelial cells. Additionally, the selected strains showed no antibiotic resistance, no hemolytic activity, and antibacterial activity against common foodborne pathogens [21].

A volume of 2 mL of stock culture was transferred into 9 mL of MRS broth (Merck, Darmstadt, Germany) and cultivated for 48 h at 37 °C, using an incubator (Binder BF4000, Tuttlingen, Germany) to reactivate the stock cultures, which had been preserved in 40% (w/w) glycerol solution at a temperature of −80 °C.

A sterile loop was used to streak 1.0 µL of the reactivated culture onto MRS agar medium (Merck, Darmstadt, Germany) supplemented with 30 g/L CaCO_3_ to obtain single colonies [9].

The LAB inoculum was obtained by transferring a single colony onto 50 mL of MRS broth and incubating it stationary, for 48 h at 37 °C. A spectrophotometer (Biochrom, Libra 22, Holliston, MA, USA) was then used to measure and adjust the optical density at 600 nm (OD 600) to a value of about 2.0 (using fresh MRS broth), which indicates a cell concentration of 1 × 10^8^ CFU/mL solution [24].

The fermentation media were previously autoclaved at 121 °C for 15 min, in an autoclave (Panasonic, MLS-3751L, Watertown, MA, USA). A 2% (v/v) LAB inoculum was added to the fermentation medium, which contained 5% (w/w) black and red amaranth seed flour, both sprouted and unsprouted in distilled water. Then, the mixture was incubated for 48 h at 37 °C and 100 rpm in an orbital shaker (Lab Companion SI-300, GMI, Minneapolis, MN, USA) [25]. Control samples were obtained using both red and black amaranth flour, including sprouted and unsprouted variants. Following fermentation, the fermented products were freeze-dried with a freeze-drier (Christ Alpha 1–4 LD plus, Osterode am Harz, Germany), at −42 °C and 0.10 mbar. Then, the moisture content was analysed with a moisture analyser (KERN, DAB 100-3, Balingen, Germany).

2.4. pH and Total Titratable Acidity (TTA) Measurement

The pH and the total titratable acidity of the fermented samples were measured with a pH metre (FiveEasy Plus FP20, Mettler Toledo, Greifensee, Switzerland) and an automatic titrator (TitroLine Easy, Schott Instruments, Mainz, Germany). To summarise, for the total titratable acidity, a mixture of 10 g of fermented products and 90 mL of distilled water was titrated to an endpoint of 8.50 [26]. The TTA was assessed in millilitres of 0.1 N NaOH [21].

2.5. Antioxidant Activity (AA)

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were used to evaluate the antioxidant activity of the freeze-dried samples [27,28].

Water extracts 1:10 (w/v) of the freeze-dried fermented products were prepared. After vortexing for 5 s, the mixture was incubated overnight at room temperature with shaking at 200 rpm. It was then sonicated for 30 min at 40 °C in an ultrasonic water bath (DU-32; ARGOLAB, Capri, Italy). The supernatant was separated by centrifuging at 7000 rpm for 10 min at 4 °C (Hettich Universal 320R, Tuttlingen, Germany). These aqueous extracts were globally characterised regarding antioxidant activity, total flavonoid content, total polyphenolic content and water-soluble proteins.

In short, 3900 μL of 0.004% (w/v) DPPH (in methanol HPLC) combined with 100 μL of the extract was incubated for 90 min in the dark, and then the absorbance was measured at 515 nm.

To perform the ABTS assay, 20 μL of the extract was mixed with 1980 μL of ABTS solution (which had a 0.7 absorbance at OD734), homogenised, and after 15 min in the dark, the mix’s absorbance at 734 nm was measured.

According to Equation (2) [28], the antioxidant activity was calculated as the radical scavenging activity (RSA, %):

$$[eqn]$$

where

A_control_—the absorbance of the control sample.

A_sample_—the absorbance of the analysed sample.

2.6. Total Flavonoid Content (TFC)

The aluminium chloride colorimetric assay was employed to determine the total flavonoid content. In summary, a mixture of 250 µL of the extracts, 1250 µL of distilled water and 75 µL of a 5% (w/v) sodium nitrite solution was homogenised and kept at room temperature for 5 min. In addition, 150 µL of a 10% (w/v) aluminium chloride solution was added to the samples, and after 6 min of incubation, 500 µL of 1.0 M sodium hydroxide and distilled water were added, up to a total volume of 3000 µL [29]. Using a spectrophotometer (Libra S22 UV-VIS, Biochrom, Cambridge, UK), the mixes’ absorbance was measured at 510 nm.

Using a calibration curve (y = 1.856x − 0.0201, R^2^ = 0.9951), TFC was calculated as mg catechin equivalents per gram of dry weight (CE)/g DW.

2.7. Total Polyphenolic Content (TPC)

The total polyphenolic content in the samples was evaluated with the modified Folin–Ciocalteu method. Briefly, 500 µL of Folin–Ciocalteu reagent was mixed with 200 µL of the extracts that had been diluted in 7900 µL of distilled water. After 5 min, 500 µL of a 20% (w/v) sodium carbonate solution was added, and the samples were left to stand in the dark for 60 min. Then the absorbance at a wavelength of 765 nm was evaluated [29].

Using a gallic acid standard curve (y = 1.3612x − 0.0205, R^2^ = 0.9838), TPC was considered as mg gallic acid equivalents per gram dry weight (GAE)/g DW.

2.8. Soluble Protein Content (SPC)

The soluble protein content of samples was assessed by the Bradford method. Briefly, 900 μL of Bradford reagent was mixed with quantities of 30 μL of the extracts. After 30 min of dark incubation at room temperature, the absorbance at 595 nm was measured. Using a bovine serum albumin calibration curve (y = 0.4382x + 0.0127, R^2^ = 0.9933), the protein content was measured and expressed as mg/g DW [30].

2.9. Probiotic Cell Counting

The viability of lactic acid bacteria in fermented sprouted and unsprouted amaranth products was established using the classical method by counting the colonies on Man, Rogosa and Sharpe (MRS) agar medium, supplemented with calcium carbonate [18]. Briefly, one gram of freeze-dried fermented products was aseptically homogenised with 9 mL of sterile 0.9% saline solution. Decimal dilutions (10^−1^–10^−7^) were made, and 1 mL of each dilution was distributed and homogenised with MRS agar containing 3% (w/v) CaCO_3_. The mixtures were cultivated at 37 °C for 48–72 h in aerophilic conditions and distinct colonies were enumerated in plates containing between 30 and 300 colonies and then expressed as CFU/gram of sample.

2.10. Reverse-Phase High-Performance Liquid Chromatography (HPLC) Analysis of Phenolic Compounds

The identification of phenolic compounds followed the method outlined by Mërtiri et al. [27], using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) that includes a degasser, quaternary pump, autosampler, column, and multi-wavelength detector. Separation was carried out on a BDS Hypersil C18 column (150 mm × 4.6 mm, 5 μm) with the following settings: 30 °C temperature, 10 μL injection volume, 1 mL/min flow rate. The mobile phases consisted of 100% methanol (solvent A) and 10% formic acid (solvent B). The gradient programme was as follows: 0–20 min, 9% A–91% B; 20–30 min, 35% A–65% B; 30–40 min, 50% A–50% B; 40–45 min, 9% A–91% B. Results are calculated as mean values ± STDEV for duplicate runs, in ng/μL, based on peak area and calibration curves with standard references.

2.11. SDS-PAGE Analysis

The SDS-PAGE assay was performed according to Laemmli [31]. For protein electrophoresis, all the samples were diluted with ultrapure water (1:2), mixed with Laemmli buffer containing beta-mercaptoethanol, and treated for 5 min at 95 °C [32]. The electrophoresis was carried out at 100 V for 120 min. Further, the protein gels were fixed for 90 min, stained with Coomassie Brilliant blue G-250, and destained in 10% acetic acid for 40 min.

2.12. Statistical Analysis

Minitab V19.1 (Minitab LLL, State College, PA, USA) was used to evaluate the data. The one-way ANOVA method was used to analyse the results, which were presented as mean ± standard deviation of three replicates. At a 95% confidence level, the data were examined for homogeneity of variances (Bartlett’s test) and normal distribution (Ryan-Joiner test), and then either the Tukey test (p < 0.05) or the Games–Howell test (p < 0.05). The data was assessed using linear multiple regression and Pearson correlation.

3. Results and Discussion

3.1. pH and Total Titratable Acidity (TTA) of the Amaranth Fermented Products

The RA-fermented products exhibited pH values ranging from 3.16 ± 0.04 to 4.35 ± 0.69, with no significant differences (p ˂ 0.05) among them. However, the lowest pH values were observed in fermented products (FPs) with sprouted RA seeds, as shown in Figure 1. While the BA-fermented products displayed pH values ranging from 3.07 ± 0.10 to 4.89 ± 0.04, with significant differences (p ˂ 0.05) between them. Visibly, the probiotics fermentation of sprouted RA flour produced the lowest pH value when compared with the fermentation substrates. Castro-Alba et al. [19] shown that using Lactobacillus plantarum 299v^®^ for fermenting unsprouted amaranth flour resulted in a pH drop above 4.0 after 48 h of fermentation.

Alternatively, sprouted RA fermented products with Lp. pentosus MIUG BL24, Lp. plantarum MIUG BL21, and Lp. paraplantarum MIUG BL74 showed the highest TTA values (5.28 ± 0.22 to 5.50 ± 0.11 mL of 0.1 N NaOH), with no significant differences (p ˂ 0.05) among them (Figure 1).

Similarly, sprouted BA fermented products with Lp. pentosus MIUG BL24 and Lp. plantarum MIUG BL21 showed the highest TTA values (4.03 ± 0.08 to 4.18 ± 0.01 mL of 0.1 N NaOH), with no significant differences (p ˂ 0.05) among them (Figure 2).

Further, the FPs derived from unsprouted seeds listed lower values for the total titratable acidity.

The results are in accordance with Owheruo et al. [33], who reported that the sesame seed flour during sprouting exhibits a decrease in pH with a simultaneous increase in TTA during sprouting, due to naturally occurring microorganisms that convert carbohydrates into an acidic medium through a metabolic mechanism. In a comparable study, employing sprouted and fermented Phalaris canariensis seeds, when compared to unsprouted seeds, resulted in a greater TTA value when fermented beverages were prepared [34].

Similarly, unsprouted BA dough fermented with Lp. pentosus MIUG BL24 demonstrated the highest TTA value of 12.33 ± 0.35 mL of 0.1 N NaOH, according to a recent study by Souare et al. [18].

Venturi et al. [35] reported that in the amaranth-based sourdough fermentation, the pH decreased from 6.20 to 4.35–4.60, while TTA reached higher values (6.45–6.90 mL NaOH 0.1 N), highlighting the differences between fermentations with monoculture of lactic acid bacteria and those with co-culture of lactic acid bacteria and yeast.

Overall, the results confirm that germination increases the availability of fermentable carbohydrates, which in turn increases acidity by stimulating the metabolism of lactic acid bacteria. The main role of LAB is to ferment carbohydrates into organic acids, mainly lactic acid, which increases the acidity and reduces the pH of the end product. Since pH and TTA are related to the antibacterial and antifungal properties that are derived from the organic acid content of fermented products, they are key indicators in food preservation, processing, and protection [36].

3.2. Antioxidant Activity (AA) of the Amaranth Fermented Products

The DPPH and ABTS radical-scavenging methods were used to assess the substantial changes in the fermented amaranth samples’ antioxidant activity. The results showed that the DPPH values ranged from 0.60 ± 0.60% to 26.28 ± 0.18% and the ABTS radical scavenging values for red amaranth FPs ranged from 17.49 ± 1.83% to 56.82 ± 5.21% (Figure 3). While the DPPH values ranged from 11.17 ± 2.13% to 35.67 ± 2.05%, the ABTS values for the fermented black amaranth products ranged from 33.03 ± 6.70% to 55.09 ± 9.97% (Figure 4).

The antioxidant potential of black amaranth FPs was greater than that of red amaranth FPs. Furthermore, compared to unsprouted seeds, seed germination leads to an increased antioxidant potential for both amaranth species [20].

The results are in accordance with Souare et al., who reported that black amaranth doughs fermented with the Lp. pentosus MIUG BL24 strain and the Lc. rhamnosus MIUG BL38 strain exhibited the highest inhibition of the ABTS radical (58.00 ± 1.81% and 58.92 ± 6.05%), nearly 2.2 times higher than the control [18].

Additionally, red amaranth leaves had the highest total antioxidant capacity (TAC) (ABTS+) concentration (34.72 TEAC µg/g DW) compared to green amaranth leaves (25.27 TEAC µg/g DW), according to a recent study [37].

RA fermented products exhibited lower antioxidant activity values when compared to germinated and non-germinated controls. The germinated control had the maximum antioxidant potential such as 56.82 ± 5.21% (ABTS) and 33.08 ± 0.84% (DPPH), respectively (p ˂ 0.05). Fermented products coded RG 24, RG 38, R38, and R24 demonstrated comparable RSA values (ABTS assay), with no statistically significant differences among them (p ˂ 0.05).

Conversely, the germinated and ungerminated BA controls exhibited the lowest antioxidant potential, as measured by the DPPH assay. The germinated FPs based on black amaranth seed flour fermented with Lc. rhamnosus MIUG BL38 (coded BG 38) strain showed the highest antioxidant activity at a value of 55.09 ± 9.97%, by ABTS assay. Additionally, germinated FPs fermented with Lc. rhamnosus MIUG BL38, Lp. pentosus MIUG BL24, and Lp. paraplantarum MIUG BL74 (coded BG 38, BG 24, and BG 74) strains registered similar values of antioxidant potential by DPPH method (35.67 ± 2.05%, 35.50 ± 0.82%, and 35.04 ± 1.30%, DPPH), with no significant statistical differences among them (p ˂ 0.05).

The data align with those of Chauhan et al., who showed that optimised sprouted amaranth flour had more antioxidant compounds than raw flour [12]. Additionally, Vento et al. [20] noticed that germination increased antioxidant activity in quinoa and amaranth seeds (13.07% and 9.06%, respectively), as shown by the DPPH approach. Compared to non-germinated seeds, germination and fermentation may improve the antioxidant potential of amaranth seeds, enhancing their health benefits [20].

In a recent study, raw, cooked and fermented seeds and germinated seeds of Chenopodium quinoa Willd. var. Tunkahuan and Amaranthus caudatus L. var. Alegrìa were compared regarding the total antioxidant capacity. After 48 h without sterilisation, fermentation of germinated seeds inoculated with Lactobacillus plantarum demonstrated the maximum antioxidant capacity (55.3 ± 2.03%) compared to the control (9.06 ± 1.25%). In contrast, antioxidant activity dropped significantly up to 8.45 ± 1.1% when germinated seeds were first sterilised and then fermented with L. plantarum for 48 h at 30 °C, compared to the control (9.06 ± 1.25%). Overall, the results showed that both species’ antioxidant activity decreased after sterilisation when compared to unsterilized samples [20].

Rich in bioactive substances, amaranth may strengthen the body’s defences against free radicals, preserving and enhancing the body’s functions [4]. It has been demonstrated that phenolic antioxidants reduce oxidative stress and inflammation, which may help prevent chronic illnesses like diabetes, cancer, and heart disease [3].

Given that DPPH activity supposed electron transfer, while ABTS activity included hydrogen atom transfer, there may be a variation in antioxidant processes [9]. Additionally, various bacterial strains may have different antioxidant processes [38].

Overall, fermentation and sprouting are promising approaches for enhancing or generating antioxidant compounds and improving their bioaccessibility [39].

3.3. Global Characterisation of Amaranth Fermented Products

3.3.1. Soluble Protein Content (SPC) of Amaranth Samples

Soluble protein contents ranging from 26.15 ± 0.47 to 41.97 ± 8.81 mg/g DW were obtained from RA fermented products, with no significant differences upon them (p ˂ 0.05) (Table 2). However, ungerminated BA flour fermented with Lp. plantarum MIUG BL21 and Lp. pentosus MIUG BL24 (coded B 21 and B 24), showed a higher content of water-soluble proteins, reaching 50.82 ± 1.03 and 48.47 ± 4.44 mg/g DW, respectively.

The results displayed in Table 2 and Table 3 showed that, when compared to the unfermented control, the soluble protein content improves by fermenting the flour based on ungerminated black and red amaranth seeds. Rather, by fermenting the sprouted red and black amaranth seeds, their soluble protein content is comparable to that of the control samples and does not differ significantly (p < 0.05).

Similarly, the doughs fermented with Lp. pentosus MIUG BL24 strain and Lc. rhamnosus MIUG BL38 strain showed comparable effects on protein hydrolysis during the fermentation of BA flour in a previous research. Although it was lower than the control (1.23 ± 0.03 mg/g DW), the dough fermented with the Lc. rhamnosus MIUG BL38 strain had the highest protein content (1.18 ± 0.06 mg/g DW) [18].

The results obtained are consistent with Cruz-Casas et al. [9], who concluded that releasing protein hydrolysates through fermentation is a strain-dependent method. The data, especially in the case of red amaranth, agree with Maldonado-Alvarado et al., who found that germination increased protein content in three types of quinoa (white, red, and black) [40].

Lactic acid fermentation is a relatively underexplored technique for obtaining protein hydrolysates from amaranth. Owing to their unique proteolytic system, which consists of intracellular peptidases, transport mechanisms, and cell-envelope proteinases, lactic acid bacteria are crucial for fermentation-based production of protein hydrolysates and bioactive peptides. They contain GRAS-classified genera and easily adapt to plant substrates. Even within the same species, there is a great variation in LAB proteolytic activity due to strain dependence [9].

The literature highlighted that amaranth proteins have lower quantities of prolamins (about 0–13%) than albumins (11–51%), globulins (16–51%), and glutelins (7–36%). In particular, compared to typical grains like wheat, barley, and maize, amaranth has a greater amount of albumins and globulins and lower levels of prolamins and glutelins [10].

3.3.2. Total Polyphenol Content (TPC) of Amaranth Samples

There were notable differences in total phenolic content (TPC) between RA fermented products (Table 2). The FPs coded R 24, R 38, RG 24, and RG 74 had higher TPC values ranging from 3.85 ± 0.31 to 4.25 ± 0.11 mg GAE/g DW, with no significant differences between them (p ˂ 0.05). The control sample (RC) had the lowest TPC (1.99 ± 0.02 mg GAE/g DW).

Furthermore, TPC varied considerably amongst the BA variants (Table 3). Ungerminated BA fermented with Lp. plantarum MIUG BL21 (coded B 21) showed the highest TPC (5.76 ± 0.12 mg GAE/g DW), followed by B 24 (4.37 ± 0.27 mg GAE/g DW), which were statistically different (p ˂ 0.05). The control samples (BC and BGC) had the lowest values, 2.10 ± 0.22 and 2.54 ± 0.13 mg GAE/g DW, respectively. The phenolic compounds concentration of BA samples was generally positively impacted by fermentation and germination.

Recently, Souare et al. [18] reported that fermentation with selected probiotic bacteria reduced the TPC in the doughs when compared to the control. BA ungerminated dough fermented with Lc. rhamnosus MIUG BL38 had the highest TPC (2.33 ± 0.20 mg GAE/g DW), suggesting that both the strain and fermentation system have an impact on polyphenol content.

The results obtained are in agreement with those of Vento et al., who found that by fermenting sterilised, ungerminated amaranth seeds with Lactobacillus plantarum, a significantly higher total phenolic content (8.55 ± 0.49 mg QE/g DW) than the unfermented control (2.35 ± 0.33 mg QE/g DW) occurred. On the other hand, fermentation of germinated seeds with the same strain resulted in a lower TPC value (5.58 ± 0.54 mg QE/g DW), after 48 h. Overall, these findings indicate that lactic acid fermentation led to a higher content of phenolic compounds in ungerminated amaranth seeds compared with germinated ones [20].

The increased phenolic content observed in fermented ungerminated amaranth seed samples may be attributed to the release of phenolic compounds that are originally bound to the seed cell wall matrix [41]. The enzymatic activity of lactic acid bacteria, combined with the acidic conditions generated during fermentation, likely enhances the release of these bound phenolics into soluble free phenols [42]. Additionally, endogenous enzymes produced during sprouting promote the release of phenolic compounds [43]. Overall, fermentation and germination increased the total phenolic content and improved nutritional value of amaranth through activation of endogenous enzymes. By changing the bioactivity and digestibility of foods, fermentation modulates their nutritional and anti-nutritional properties. Particularly, polyphenols are mostly found in bound forms, coupled with proteins, lipids, or sugars, in unfermented cereals. These compounds can be released into free forms during bioprocessing stages, which increases their bioavailability and antioxidant potential. However, free phenolic content may also drop as a result of degradation or interactions with other elements of the food matrix, depending on the type of bioprocess and biocatalyst properties [20].

During germination, procyanidins and catechins are among the phenolic chemicals whose concentration decreases as a result of condensation and polymerisation, which are aided by enzymes such as polyphenol oxidases. On the other hand, hydrolytic enzymes linked to total phenolic content, such as cellulases, amylases, hemicellulases, glucanases, and polyphenol oxidases, become more active and support the polymerisation and modification of phenolic compounds. Furthermore, germination increases the availability of free phenolic chemicals by breaking cell wall polymers. Additionally, germination triggers secondary metabolic processes, including those linked to phenolic metabolism, such as the oxidative pentose phosphate route, glycolysis, acetate/malonate pathway, shikimate pathway, and phenylpropanoid pathway. This activation increases the content of phenolic compounds and their antioxidant activity in germinated seeds, as does the creation of enzymes such as phenylalanine ammonia-lyase [43].

Through fermentation, due to the breakdown of bonds with grain cell wall components, activities of enzymes such as β-glucosidase, decarboxylases, esterases, hydrolases, and reductases, as well as the metabolic activity of fermenting microorganisms, bound phenolic compounds are converted from their linked or conjugated forms to free ones [44]. The released free aglycones can increase antioxidative activity, and phenolic compounds are more bioaccessible in their free form. On the other hand, because free phenolic compounds can associate with other molecules in the food matrix, either hydrolysed by particular microbial strains, or broken down by microbial enzymes, a decrease in their amount may occur during fermentation [45]. Overall, the combined application of germination and fermentation leads to increased availability and transformation of phenolic acids and flavonoids, contributing to enhanced antioxidant potential.

3.3.3. Total Flavonoid Content (TFC) of the Amaranth Samples

Total flavonoid content (TFC) varied among the RA fermented products. The control sample (RC) showed the lowest TFC (1.09 ± 0.03 mg CE/g DW), while higher values were generally observed in coded samples R 38, R 74, RG 21, and RG 24, which were obtained from germinated and non-germinated RA, fermented with Lp. plantarum MIUG BL21, Lp. pentosus MIUG BL24, Lc. rhamnosus MIUG BL38, and Lp. paraplantarum MIUG BL74 strains, indicating that fermentation and sprouting enhanced flavonoid levels, with no significant differences among them (p ˂ 0.05).

TFC variations were more noticeable in BA samples. Coded samples B 21 and B 24 exhibited the highest flavonoid content (5.28 ± 0.44 and 4.67 ± 0.62 mg CE/g DW, respectively), whereas the control samples (BC and BGC) had the lowest values. In summary, the amount of flavonoids in BA samples was positively impacted by both fermentation and germination.

A recent study stated that the total flavonoid content of black amaranth dough, based on ungerminated seed flour, fermented with Lc. paracasei MIUG BL4 strain, Lc. paracasei MIUG BL13 strain, and Lp. pentosus MIUG BL24 strain was higher than that of the control, with no statistical difference among them (p < 0.01) [18].

Total flavonoid content was found to be similar in raw and cooked amaranth seeds (1.8 mg QE/g DW) but sprouting significantly increased it to 5.0 mg QE/g DW. Most fermentation variants further increased TFC, although decreases were seen after fermentation with S. cerevisiae alone and in combination with L. plantarum. The highest levels of TFC were obtained through spontaneous fermentation in both ungerminated seeds (6.1 mg QE/g DW) and germinated seeds (9.41 mg QE/g DW), corresponding to increases of nearly 2.6- and 1.9-fold when compared to the controls [20].

Studies show that germination significantly improves total phenolics, total flavonoids, and antioxidant activity in a variety of grains, with nutritional benefits. Soaking, ultrasound, and thermo-alkaline hydrolysis are recommended pre-germination treatments that modify the structure of grains to influence germination outcomes, promoting the accumulation of flavonoids and phenolic acids during germination and increasing antioxidant activity [43].

Increased levels of phenolic compounds and flavonoids as a result of microbial hydrolysis are primarily responsible for the improvement of antioxidant activity during fermentation [38] and germination [43].

Particularly, for RA fermented products, TFC had a strong and moderate, respectively, positive correlation with SPC (r = 0.632, p < 0.05) and TPC (r = 0.435, p < 0.05), suggesting that total flavonoid content increased with soluble protein content, as an effect of bioprocessing modification of vegetal structure. TPC showed moderate positive correlations with DPPH (r = 0.376, p < 0.05), indicating that phenolics may contribute to the increment of the antioxidant activity (Figure 5).

A strong negative correlation was observed between antioxidant activity (ABTS) and water-soluble proteins (r = −0.386, p < 0.05), in the case of black amaranth FPs. Other strong correlations, such as those between SPC and TFC or TPC (r = 0.881, p < 0.05, r = 0.819, p < 0.05 were statistically significant (Figure 6).

According to regression analysis, soluble proteins significantly affected ABTS activity (p = 0.037), while total phenolic content was the main factor influencing DPPH activity (p = 0.015). These findings demonstrate various antioxidant response patterns (Table 4).

3.4. The Probiotic Cell Enumeration of the Amaranth Samples

The statistical analysis revealed significant differences (p < 0.05) between the mean values of probiotic viability (log CFU/g) from FPs based on red and black amaranth, germinated and ungerminated. With values ranging from 7.68 ± 0.11 to 8.15 ± 0.49 log CFU/g, the samples fermented with unsprouted RA coded R21, R24, and R74 had the highest cell concentrations; there were no significant differences between them (p < 0.05). When compared to the other samples, ungerminated BA seeds fermented with the Lp. paraplantarum MIUG BL74 strain had the highest cell concentration (8.84 ± 0.06 log CFU/g), which was statistically significant. The fermented products based on sprouted red and black amaranth seeds exhibited significantly lower cell concentrations, up to 3.11 ± 0.04 and 5.60 ± 0.24 log CFU/g, respectively (Table 5). Instead, among the sprouted FPs, those based on black and red amaranth (RG 38 and BG 38) exhibited the greatest probiotic viability.

Probiotic viability in FPs derived from ungerminated amaranth fell within ranges previously reported for amaranth- and pseudocereal-based fermentations, with cell counts of 2.60–8.54 log CFU/g in spontaneously fermented amaranth sourdough [46] and approximately 8 log CFU/g in amaranth-based purees [47]. According to these results, unsprouted amaranth flour might be a suitable substrate for the growth of lactic acid bacteria under optimal fermentation conditions [48].

Additionally, the biochemical changes that occur in amaranth seeds during germination impacted the resilience of probiotic lactic acid bacteria. Similar results regarding the effect of pretreatment (including sprouting) and substrate type on probiotic viability have been reported by Nkhata et al. [49] and Petrova and Petrov [50], who agreed that both the characteristics of the probiotic strain and the degree of processing of the pseudocereal influence the probiotic’s survivability following fermentation.

3.5. RP-HPLC Characterisation of Amaranth Samples

For subsequent analyses, fermented products from both sprouted and unsprouted red and black amaranth with the strain Lc. rhamnosus MIUG BL38 were chosen, along with the controls. The selection criteria included balanced acidification, enhanced ABTS and DPPH antioxidant activity, high total phenolic and flavonoid contents, and a probiotic threshold of at least 6 log CFU/g.

In the analysed RA fermented products, the nine phenolic acids that were identified were four hydroxybenzoic acids (gallic acid, protocathechic acid, syringic acid, ellagic acid), and five hydroxycinnamic acids (ferulic acid, cinnamic acid, caffeic acid, ρ-coumaric acid, chlorogenic acid), as described in Table 6 and Table 7. The fermentation and germination processes revealed twelve flavonoids, as follows, epicatechin gallate, hesperitin, quercetin, apigenin, luteolin, naringenin, quercetin 3-glucoside, isorhamnetin, peonidin 3-O rutinoside, epicatechin, keracyanin, and rutin trihydrate.

The amount of phenolic acids and flavonoids in the examined samples varied significantly. More biological active compounds were found in red amaranth-based samples, both germinated and ungerminated, than in black amaranth-based samples.

The current findings are supported by Sarker and Oba [51], who reported abundant phenolic acids such as salicylic acid, vanillic acid, protocatechuic acid, gallic acid, gentisic acid, β-resorcylic acid, p-hydroxybenzoic acid, syringic acid, ellagic acid, sinapic acids, chlorogenic acid, trans-cinnamic acid, m-coumaric acid, caffeic acid, p-coumaric acid, and ferulic acid, as well as flavonoids like rutin, hyperoside, isoquercetin, myricetin, quercetin, apigenin, kaempferol, and catechin in the red-coloured amaranth leaves, which were much higher compared to the green-coloured amaranth.

Both the germinated and ungerminated RA-controls displayed a variety of phenolic compounds, with the maximum quantity of epicatechin (4518.68 ± 15.86 ng/µL) and protocatechuic acid (2000.3 ± 2.38 ng/µL) in the germinated control (RGC). Gallic acid was the most prevalent, with the highest concentration of 833.08 ± 1.23 ng/µL in the germinated RA sample fermented with Lc. rhamnosus MIUG BL38 strain (coded sample RG38), followed by chlorogenic acid (110.67 ± 3.28 ng/µL). However, the FPs included small amounts of phenolic acids such as syringic, protocatechuic, and chlorogenic acids. Instead, only in controls, ferulic, cinnamic, and caffeic acids, quercetin, and apigenin were identified (Table 6).

Gallic acid levels in the FPs derived from germinated seed were significantly higher in BG38 (288.45 ± 2.18 ng/µL) than in control, coded BGC (47.92 ± 0.06 ng/µL). Fermented products based on ungerminated BA seed showed the same pattern, as seen in coded sample B38, which had a concentration of 97.16 ± 0.69 ng/µL compared to the control’s concentration of 38.57 ± 0.14 ng/µL. The main flavonoid in coded sample B38 was epigallocatechin (1349.76 ± 3.14 ng/µL), but epicatechin was mostly found in BG38 (619.86 ± 11.62 ng/µL), suggesting that the distribution of flavonoid compounds varies depending on the sample (Table 7). Overall, the HPLC data showed notable qualitative and quantitative variations in the phenolic acid and flavonoid content of the examined FPs, indicating that the phytochemical profile is significantly influenced by processing (fermentation and germination).

Studies comparing the leaves of red genotypes of A. tricolour and the green genotypes of A. lividus show that the red genotypes are richer in flavonoids and phenolic acids. In particular, phenolic contents in red accessions ranged from 85 to 312 µg/g DW, which was higher than the range of 71 to 220 µg/g DW in green accessions. Gallic acid, vanillic acid, salicylic acid, protocatechin, caffeic acid, p-coumaric acid, ferulic acid, and trans-cinnamic acids constituted the majority of these compounds. Additionally, red accessions were rich in flavonoids such as rutin, quercetin, myricetin, and kaempferol [51,52].

According to a recent study by Suoare et al. [18], black amaranth fermented doughs had higher contents of flavonoids than phenolic acids, with epigallocatechin being the most prevalent, followed by epicatechin and catechin. The levels of epigallocatechin were 7789.88 ± 17.00 ng/μL in the control, 6942.47 ± 5.63 ng/μL in the dough fermented with Lc. rhamnosus MIUG BL38, and 4983.16 ± 7.29 ng/μL in the dough fermented with Lp. pentosus MIUG BL24. Only the control had minor phenolic acids, such as p-coumaric and 4-hydroxybenzoic acids.

Assessing other plant parts (leaves, stalks, seeds, and flowers), sprouts contained the fewest identifiable phenolic chemicals; only gallic acid, protocatechuic acid, and rutin were quantified [53]. Cinnamic acid derivatives (ferulic and chlorogenic acid) were less common and generally at lower concentrations in sprouts of A. caudatus.

Phenolic compounds, including flavonoids, hydroxycinnamic acids (ferulic acid, cinnamic acid, caffeic acid, p-coumaric acid, chlorogenic acid), and hydroxybenzoic acids (gallic acid, protocathechic acid, syringic acid), are commonly found in Amaranthus species [54].

Sprouting (germination) induced nutritionally favourable changes, making amaranth sprouted seeds nutritionally superior compared to non-sprouted seeds. Consequently, they represent promising ingredients for developing foods with enhanced nutrient and antioxidant properties.

3.6. SDS-PAGE of the FPs Extract Analysis

The SDS-PAGE evaluation of water-soluble proteins extracted from black and red amaranth FPs highlighted a few distinct protein bands between the germinated, ungerminated and fermented samples, respectively (Figure 7).

More precisely, in the ungerminated black amaranth control (BC), a pronounced background smear of molecular weight proteins smaller than 15 kDa were observed, which was reduced in the germinated control (BGC). In addition, two distinct protein bands of ~35 kDa and ~27 kDa were maintained across all the samples, including the red amaranth controls. The ~35 kDa band may correspond to the acidic subunits of 11S globulin [55,56], one of the major storage proteins found in amaranth seeds, while the ~27 kDa band represents a smaller globulin fraction, both of which were resistant to germination process and remained visible after germination. Moreover, the germination process led to a reduction in the intensity of proteins bands having a molecular weight between 10 and 15 kDa and a decrease in the background smear, suggesting a partial hydrolysis of storage proteins. Different electrophoretic profiles have been documented in the literature, highlighting variations in amaranth genetic diversity, extraction solvents and processing conditions. For instance, Bojórquez-Velázque et al. [57] showed that the hydrophilic protein fraction of the seeds contains a wide range of proteins, with major bands at ~33 kDa, 37 kDa, and 52 kDa, while the hydrophobic fractions have different bands which can be clustered in three regions (20–24 kDa, 32–35 kDa and 50–70 kDa), with clear species-dependent differences in band presence. Making a comparative proteomic analysis of amaranth seeds, Bojórquez-Velázque et al. [56] found that the hydrophilic fraction contained abundant small- and medium-molecular-weight proteins of 10–15 kDa and 25–40 kDa, respectively. The main proteins from the high-variation region were the 11S globulin, granule-bound starch synthase I and the Vicilin-like seed storage proteins, respectively [56].

Additionally, the fermentation process with L. rhamnosus MIUG BL38 probiotic strain further changed the protein’s background. In ungerminated black amaranth (B38, line 5), two clear bands of 15 kDa and 50 kDa were emphasised, along with a faint one of 75 kDa. In contrast, when the germinated black amaranth was used as substrate in the fermentation, the protein background became uniform, with no sharp protein bands visible, suggesting that an extensive hydrolysis occurred (line 6). Further, in red ungerminated amaranth, the fermentation with L. rhamnosus MIUG BL38 (R38—line 7) led to an overall increase in the protein background intensity, indicating the presence of a greater number of soluble proteins. This pattern suggests that the fermentation process generates a range of polypeptides and peptides through partial proteolysis, rather than complete protein degradation. Furthermore, the fermentation of germinated red amaranth produced a proteins profile similar to that of fermented germinated black amaranth, indicating an extensive hydrolysis of storage proteins in both varieties.

4. Conclusions

The current investigation shows that the functional and biochemical properties of red and black amaranth seeds are effectively improved by the combined use of sprouting and probiotic fermentation. Acidifying potential, antioxidant activity, phenolic and flavonoid profiles, water-soluble protein content and probiotic viability were all greatly influenced by sprouting and fermentation processing. The results are highly strain- and amaranth variety-dependent. Black amaranth-derived fermented products demonstrated stronger antioxidant activity but sprouted and unsprouted red amaranth generally showed higher levels of bioactive compounds. Lacticaseibacillus rhamnosus MIUG BL38 had the most balanced technological and functional performance among the strains studied, promoting favourable acidification, increased soluble protein release, improved antioxidant potential, and proper probiotic viability. Overall, the results indicate that red amaranth, Amaranthus cruentus sprouts fermented with Lc. rhamnosus MIUG BL38 represents a promising gluten-free functional food ingredient with numerous potential health benefits, suitable for incorporation into innovative probiotic and nutraceutical food formulations.