Enzymatically Hydrolysed Common Buckwheat (Fagopyrum esculentum M.) as a Fermentable Source of Oligosaccharides and Sugars

Abstract

Common buckwheat (Fagopyrum esculentum M.) is highly rich in starches and polysaccharides. Nowadays, the use of common buckwheat in brewing processes and functional product development is gaining popularity as it is gluten-free and therefore suitable for those suffering from coeliac disease. Moreover, enzyme-assisted extraction for common buckwheat releases these oligosaccharides as well as bioactive substances, which can be further used for developing new products. This research aims to compare different enzymatic hydrolysis methods and their effect on roasted common buckwheat flour. The degradation of buckwheat flour using these hydrolytic enzymes was captured using SEM. Oligosaccharide and sugar molecular mass distributions were identified using HPLC-SEC. The extract with the highest fermentable monomeric sugar content was further fermented with ancient lactic acid bacteria starters: Tibetan kefir grains and birch sap. Ferment extracts were analyzed for antimicrobial activity against ten different pathogenic bacteria. The results indicated that the incorporation of enzymes into the extraction process lead to the release of a wide variety of DP3-DP4. Furthermore, the successful fermentation of these extracts with ancient starters revealed promising antimicrobial activity against nine different pathogenic bacteria, including E. coli and L. monocytogenes. In general, common buckwheat is a suitable ingredient for developing beverages and products with high value and has high potential in pharmaceutical applications.

1. Introduction

Fagopyrum Esculentum M., better known as common buckwheat, is a great source of proteins and fibers. However, secondary metabolites such as rutin or quercetin are also abundant in buckwheat grains, which have anti-tumor properties, e.g., lung adenocarcenoma [1]. According to Ikari et al. [2] quercetin extracted from buckwheat enhances autophagy against protein aggregation by suppressing mTORC1 activity. An experiment conducted by Wang et al., altering the diet of rats, showed that buckwheat consumption led to a higher concentration of short fatty acids (SCFA) and increased levels of Lactobacillus, Blautia, and Akkermansia strains in the gut. The same study also showed that supplementation of Tartary buckwheat improves colonic barrier function by initiating ZO-1 protein expression [3]. Moreover, buckwheat is considered to be involved in lipid metabolism and prevents obesity, fatty liver, and hypedimia [4]. Lately, buckwheat has been gaining popularity in beer production due to its gluten-free origin, making it suitable for those suffering from coeliac disease. However, brewing is only allowed if enzymes are incorporated into the process [5,6,7].

Oligosaccharides are an interesting area due to their prebiotic potential and fermentability [8,9]. Lactic acid bacteria (LAB) fermentation is known for increasing food biopreservation [10]. Organic acids, bacteriocins, biogenic amines, and other metabolites have antimicrobial properties. These properties are essential for the prevention of foodborne infections and for increasing overall human health [11,12]

Enzyme-assisted extraction is one of the most suitable ways to extract oligosaccharides from plant materials [13,14]. It has mild extraction conditions, requiring relatively lower temperatures of 40–70 °C and no harsh solvents. Enzyme-assisted extraction also increases the stability of bioactive compounds during the extraction process [15]. However, enzymes need to be carefully tailored to the chosen plant material for extraction. Usually, for grain materials, amylolytic and cellulolytic enzymes are selected. These enzymes cleave both starch and non-starch polysaccharides. They may cleave specifically, with the same bonds consistently cleaved between different grains, or randomly, where the cleaved bonds are inconsistent between different grains, requiring more scientific research to be implemented [16].

Plant fermentation is a valuable source of volatile compounds [17]. The interest in probiotics and fermented products has risen in the past decade due to the discovery of a link with obesity, diabetes, cognitive function, and more. Although fermentation has been known for ages, scientists are now suggesting more specific metabolic mechanisms to explain its effects. Spontaneous fermentation usually encompasses a symbiotic relationship between bacteria and yeast colonies [18]. However, depending on the substrate, microbiological alteration may differ [19].

Tibetan kefir grains (TFG) and birch sap (BS) contain LAB and yeast colonies, which induce spontaneous fermentation when an appropriate substrate and suitable conditions are prepared [20,21]. In general, LAB fermentation is known for bio-preservative compound formation [22]. For example, acetic, lactic, and propionic acids form the media unfavorable for many pathogenic and spoilage bacteria [23,24].

This research aims to compare the effects of different hydrolytic enzymes on common buckwheat oligosaccharides before and after in-vitro digestion. The graphical experimental design is presented in Figure 1. Hydrolyzed buckwheat extracts were further fermented with TKG and BS. The fermented extracts were then collected for evaluation of potent antimicrobial properties against ten different pathogenic bacteria.

2. Materials and Methods

2.1. Plant Material (Dependent Sub-Sample)

This study was performed using common organic buckwheat (F. esculentum M.) harvested in Lithuania in 2020 (Ekofrisa, Prienai District Municipality, Kaunas, Lithuania). The experiment was carried out at least twice. A total of 1 kg batches of the aforementioned buckwheat were prepared from stock by the supplier for the experiment. Prior to the extraction, plant materials were ground with an ultra-centrifugal mill, ZM 200 (Retsch, Haan, Germany), using a sieve with 0.5 mm holes.

2.2. Enzyme Products (Independent Sub-Sample)

GRAINZYME NL is a classical multienzyme product used as an effective substitute for lowering viscosity in grain products. The cocktail’s main enzymatic component is cellulase. The product is reported to have cellulase activity of 5000 U mL⁻¹. However, it is also known to contain various additional active substances including hemicellulase, endo-xylanase, exo-xylanase, beta-glucanase, mannanase, galactosidase, and pectinase (Suntaq, Guangzhou, China).

SQzyme GL is a glucoamylase monocomponent [EC3.2.1.3] derived from the fermentation of wild Aspergillus niger (Suntaq, Guangzhou, China). The product is reported to have a glucoamylase activity of 150,000 U mL⁻¹.

SQzyme BAL is a food-grade bacterial α-amylase [EC3.2.1.1] derived from the fermentation of wild Bacillus subtilis (Suntaq, Guangzhou, China). The product is reported to have a glucoamylase activity of 180,000 U mL⁻¹.

2.3. Enzyme-Assisted Extraction and Spontaneous Fermentation Using Tibetan Kefir Grains and Birch Sap

Enzyme-assisted extraction, was performed as described by Streimikyte et al. [25]. Two different batches were analyzed. Milled common buckwheat (>0.5 mm) was homogenized with distilled water at a ratio of 1:5. Following that, 0.15% of AL and 0.15% NSP were added to the first batch, and 0.45% AL + AG as well as 0.15% NSP were added to the second batch. After 2.5 h of enzymatic extraction at 68 °C, the liquid and solid fractions of the buckwheat extract were collected. The liquid and solid parts were separated using a 100-micron sieve and were then frozen and freeze-dried, respectively, until analysis.

Following the enzyme-assisted extraction, but prior to freezing, the liquid phase was taken for further fermentation. Specifically, 10% of Tibetan Kefir grains were incorporated into the liquid samples. In parallel, the oat liquid fraction was homogenized with fresh birch sap at a ratio of 1:1. The fermentation was carried out in an incubator at a fixed temperature of 28 °C for five days. Initial and end in the sample with birch varied from pH 6.5 ± 0.1 to 4.5 ± 0.1; with Tibetan kefir grains varied from 6.3 ± 0.2 to 3.9 ± 0.1. After five days, the samples were filtered and frozen at −20 °C until analysis.

2.4. Scanning Electron Microscopy (SEM) Analysis

The morphological structure of the tested plant was examined using the images obtained with the SEM FEI Quanta 200 FEG (FEI Company, Hillsboro, OR, USA). The samples were analyzed in low vacuum mode operating at 3.0 kV using an LDF detector. Common buckwheat spent grain samples from the control and the enzymatically hydrolyzed residue groups were spread on an aluminium table and measured at three different locations.

2.5. Static In Vitro Digestion

The static in vitro digestion (IVD) of enzymatically hydrolyzed hydrophilic extracts of F. esculentum was carried out in three different stages as described by Minekus et al. and Braford et al. [26] Modifications to the procedure, as described by Streimikyte et al. in a study conducted in 2020, were incorporated [27]. Briefly, 5 mL of the sample with 2 g of glass beads were mixed with simulated saliva fluids (SSF) and pre-incubates at 37 °C for a few minutes. Amylase was added, and the mixture was incubated for 2 min in a rotary mixer. After the samples were mixed with simulated gastric fluid (SGF) containing pepsin (2000 U mL⁻¹ of digest), the pH was adjusted to 3 using 5 mol L⁻¹ of HCl. The final digest volume was adjusted to 20 mL. The mixtures were placed into a temperature-controlled thermostat with a continuous rotator at 600 rad s⁻¹. After two hours of incubation, the final intestinal step was carried out by adding simulated intestinal fluid (SIF) supplemented with the following individual enzymes: trypsin (100 U mL⁻¹ of digest), chymotrypsin (25 U mL⁻¹ of digest), pancreatic amylase (200 U mL⁻¹ of digest), porcine pancreatic lipase (2000 U mL⁻¹ of digest), and bile salts (10 mM of digest). The pH was adjusted to 7 by adding 5 mol L⁻¹ of NaOH. The final volume of the sample was 40 mL. IVD was then performed for 180 min. The digestion process of the samples was stopped at gastric phase points by adjusting the pH to 7 and by cooling the samples; the digestion process for the intestinal phase samples was stopped only by cooling the samples to 0–4 °C in ice water. After cooling, the samples were centrifuged at 10,000 rpm at +4 °C and filtered. The soluble fraction of the digest was collected, frozen, lyophilized, and stored at +6 °C prior to analysis. The digestion procedure was performed twice. Lyophilised samples were further stored at −20 °C until high-pressure liquid chromatography size exclusion for molecular mass distribution analysis.

2.6. High-Pressure Liquid Chromatography Size Exclusion for Molecular Mass Distribution (HPLC-SEC)

HPLC-SEC was applied to the enzymatically hydrolyzed and lyophilised samples of the common buckwheat extracts before and after in vitro digestion. A Dionex Ultimate 3000 HPLC (International Equipment Trading Ltd., Mundelein, IL, USA) equipped with a column oven with integrated size-exclusion Ohpak SB-802 HQ (8 × 300 mm (8 μm)) (Shodex, Munich, Germany) and Ultrahydrogel 500 (7.8 × 300 mm (10 μm)) (Waters, Wilmslow, UK) columns were used. Sugars were detected with the RI detector Optilab T-rEX (Wyatt, Santa Barbara, CA, USA). As a standard, in order to identify the degree of polymerization (DP), the following monomeric units were used: DP1-D-(+)-glucose (≥99.5%; G8270; Sigma, St. Louis, MO, USA); DP2-D-(+)-mannose (≥95%; 92,683; Supelco, Bellefonte, PA, USA); DP3-maltotriose (>99%; GLU313; Elicityl S.A, Crolles, France) DP4-maltotetraose (>99%; GLU314; Elicityl S.A, Crolles, France). The following main parameters were applied: 0.05 M NaNO₃ eluent, a flow rate of 0.5 mL min⁻¹, the temperature of 40 °C, work pressure of 50–52 bar, RI detector, and an injection volume of 50 μL. A sample solution containing 2 to 20 mg g⁻¹ (1% DMSO-dimethylsulphoxide as internal standard) of the lyophilized matter was used for the test. The samples were stirred with a magnetic stirrer for 30 min. The required sample volume (~1 mL) was filtered through a 0.45 μM syringe filter into a chromatographic flask (the filtrate had to be precise). Results were obtained by analyzing the chromatograms. In this case, the retention times (tR) of each peak and internal standard (DMSO) were essential. These values and the chromatogram data were exported in the csv format and other required data (baseline and coordinates) were generated in a standard calculation file. The molecular weight distribution curve was plotted against sample concentration curves based on regularly checked and updated calibrations.

2.7. Antibacterial Activity Assay

Antibacterial activity in-vitro was evaluated using agar diffusion against ten different bacteria and fungi: Staphylococcus aureus ATCC 25923; Staphylococcus epidermidis ATCC 12228; Enterococcus faecalis ATCC 29212; Escherichia coli ATCC 25922; Klebsiella pneumoniae ATCC 13883; Pseudomonas aeruginosa ATCC 27853; Proteus vulgaris ATCC 8427; Bacillus cereus ATCC 11778; Listeria monocytogenes ATCC 19115; Candida albicans ATCC 10231. The antimicrobial activity assay was performed in a solid Mueller-Hinton medium on a plate. A total of 10 mL of medium were infused with 2 mL of sample, which was diluted 12 times. The plates were incubated at 37 °C for 24 h. Bacterial growth was assessed following incubation and where no growth or little growth was seen, with no crowded, dense, colonies, antimicrobial activity was recorded.

3. Results and Discussion

3.1. Scanning Electron Microscopy (SEM)

The microstructure of common buckwheat spent grain was observed by taking SEM images, which can be seen in Figure 2. In the control sample (Figure 2a) the appearance of common buckwheat particles is that of irregular geometric shapes with a rough surface appearance. Moreover, microfibrils and amorphous zones can be seen on the surface [28]. This demonstrates the complex structure of common buckwheat flour, which traps peptides and secondary metabolites [29].

Enzyme-assisted extraction cleaves the polymeric chains of buckwheat flour and releases metabolites, which can be clearly observed in the SEM images (Figure 2b–d). The crystalline and round structures can be identified as substances of protein origin as well as crystalline sugar molecules. Enzyme-assisted extraction is also used for protein extraction from plants [30]. It has been reported that carbohydrases (Cellulase, Pectinase, Viscozyme L) increase the extraction yield by releasing the proteins attached to the polysaccharide matrix in the plant materials [31,32,33].

In general, chemical degradation and disruption of plant cell wall matrices using acid or base hydrolysis are both commonly practiced methods that lead to increased extractability and thus, increased bio-functional activity [34,35].

3.2. High-Pressure Liquid Chromatography Size Exclusion for Molecular Mass Distribution (HPLC-SEC)

Both Common buckwheat control and enzymatically hydrolyzed extracts were lyophilized, and identification of molecular mass distribution was evaluated and performed using HPLC-SEC (Figure 3). Buckwheat is a pseudocereal with a high starch content of starches. However, in origin, buckwheat typically has low levels of amylases, which must be implemented and added when used in beer production [6,7]. In this study, we compared two different enzymatic hydrolysis methods for common buckwheat flour. By incorporating amylase and a non-starch polysaccharide enzyme cocktail into the extraction, long-chain carbohydrates were cleaved from 7,000,000 kDa to ≤7000 kDa (Figure 2a,c). This process mainly created DP3 length oligosaccharides, which were found in lower concentrations in the control extract. However, when additional glucoamylase enzymes were incorporated into the buckwheat flour extraction process, the product contained mostly monomeric sugars (Figure 3e). Interesting molecular mass distribution was observed in all the extracts after IVD. The control extract demonstrated cleavage of soluble polysaccharides resulting in higher DP3-DP4 oligosaccharide content. Also, the formation of DP2 and DP3 after IVD was observed in samples to which amylase, glucoamylase, and non-starch polysaccharide enzyme cocktails were added. This means that additional isomerization and metabolic pathways were formed with the digestive fluids. The water-soluble fibres of buckwheat seeds mostly consisted of pectins, arabinogalactans, and xyloglucans, which in turn consist of monomeric sugars such as glucose, xylose, arabinose, and galactose [36,37]. This composition indicates possible variation in sugar composition ranging between DP1 and DP4 following enzymatic extraction.

3.3. Antimicrobial Properties against Gram—Positive and Gram—Negative Pathogenic Bacteria

Extracts with the highest monomeric sugar content were further fermented with Tibetan kefir grains and birch sap. Both are known for containing lactic acid bacteria and commence to naturally ferment in suitable conditions. Fermentation is the process of transforming complex substrates into simple compounds [38]. In our study, extracts were incubated for 5 days at a temperature of 28 °C. Following that, the filtered ferment extract was analyzed for potential antimicrobial properties against ten different pathogenic bacteria (

Table 1. Potential of antimicrobial activities of common buckwheat fermented with Tibetan kefir grains and birch sap.

Sample Names	Incubation Period	Staphylococcus aureus ATCC 25923	Staphylococcus epidermidis ATCC 12228	Enterococcus faecalis ATCC 29212	Escherichia coli ATCC 25922	Klebsiella pneumoniae ATCC 13883	Pseudomonas aeruginosa ATCC 27853	Proteus vulgaris ATCC 8427	Bacillus cereus ATCC 11778	Listeria monocytogenes ATCC 19115	Candida albicans ATCC 10231
		1	2	3	4	5	6	7	8	9	10
		Inhibition Zone, mm
FB+BS	5 days	4.5 ± 0.3 a	3.7 ± 0.2 a	1.5 ± 0.1 a	0.3 ± 0.1 a	0.4 ± 0.1 a	0.4 ± 0.1 a	0.3 ± 0.2 a	2.5 ± 0.5 a	2.6 ± 0.5 a	0
	12 days	0	0	0	0	0	0	0	0	2.8 ± 0.3 a	0
FB+TKG	5 days	0	0	2.7 ± 0.3 b	0.5 ± 0.3 a	0.7 ± 0.2 b	0.8 ± 0.2 b	0.6 ± 0.2 b	1.1 ± 0.3 b	1.3 ± 0.2 b	0
	12 days	0	0	2.6 ± 0.3 b	0.3 ± 0.2 a	0.6 ± 0.2 b	0	0	0	0	0
TKG	5 days	0	2.5 ± 0.1 b	0	0	0	0	0	0	2.3 ± 0.1 a	0
	12 days	0	2.4 ± 0.1 b	0	0	0.8 ± 0.2 b	0	0	0	2.6 ± 0.4 a	0
BS	5 days	3.5 ± 0.2 b	2.7 ± 0.2 b	0	0	0	0	0	0.7 ± 0.3 c	0	0
	12 days	3.6 ± 0.2 b	2.8 ± 0.2 b	0	0	0	0	0	0.6 ± 0.2 c	0.5 ± 0.6 c	0

Note: FB + BS—Fermented buckwheat extract with birch sap; FB + TKG—Fermented buckwheat extract with Tibetan kefir grains; TKG—Tibetan kefir grains fermented in water; BS—fermented birch sap. Different letters within the same column indicate significant differences (one-way ANOVA and Tukey’s HSD test, p < 0.05).

). The fermentation process then continued for another 7 days and samples were analyzed again on day 12 for antimicrobial properties. Bacteriostatic and bacteriocidic properties were recorded in Table 1.

On day 5, antimicrobial properties were recorded in the extract containing fermented buckwheat with birch sap (FB + BS) against nine different pathogenic bacteria and in the extract containing fermented buckwheat with Tibetan kefir grains (FB + TKG) against eight different pathogenic bacteria. By day 12, the extract containing FB + BS retained antimicrobial activity against Listeria monocytogenes only whereas the extract containing FB + TKG retained antimicrobial activity against three of the 10 microorganisms: Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae. Extracts containing TKG alone as well as BS alone had lower antimicrobial properties with activity recorded on day 5 against two and three microorganisms, respectively, and on day 12 against three and four microorganisms, respectively. Results demonstrate that the diversity of lactic acid bacterias in TKG and BS releases a large panel of metabolites through the Embden-Meyerhoff, phosphoketolase, and Leloir pathways [39]. These metabolites include antimicrobial compounds against Gram-positive and Gram-negative bacteria. Scientific literature identifies that lactic acid bacteria, during fermentation, release bacteriocins and bioactive peptides and convert phenolic compounds into organic acids [40,41]. Moreover, metabolic pathways enhance antioxidant and antimicrobial activities in ferment extracts [39,40].

4. Conclusions

Common buckwheat contains a wide variety of polysaccharides, which, after in-vitro digestion, have been demonstrated to breakdown into oligosaccharides. The incorporation of enzymes during the fermentation process results in fermented products with a wide variety of antimicrobial properties against various pathogenic bacteria, including S. aureaus, E. Coli, and L. monocytogenes. Moreover, this study has shown, using SEM, that the fermentation process leads to increased degradation of the polymeric matrix of roasted buckwheat flour, leading to increased compound release, which impacts further spontaneous fermentation metabolic pathways and different antimicrobial activity. The results of this study demonstrate promising applications for roasted common buckwheat flour in creating novel food products and developing new pharmaceutical solutions with potential significance for patient diet and health.