Introduction

Flaxseed (Linum usitatissimum L.), also known as linseed, is considered a functional food ingredient due to its numerous reported health benefits [1,2,3,4]. Several components of flaxseed including lignans, omega-3 fatty acids, and complex polysaccharides, support a wide range of biological activities such as antioxidant, antiviral, antibacterial, insecticidal, fungicidal, estrogenic and antiestrogenic effects, to mention a few [2,3,4,5,6,7,8]. Mucilage has been the subject of special attention due to its high water-holding capacity, foamability, amphiphilic properties, and temperature-dependent emulsification properties [9,10,11,12,13]. The uses of mucilage in food, agriculture, medicine, and other industries are well-documented. Mucilage is used for example in the treatment of constipation or diabetes. It is also an effective forming agent in gluten-free bread, and stabilizer in products such as stirred yogurt [1, 7, 14,15,16,17,18,19,20,21,22,23]. Several studies have aimed to optimize extraction of mucilage from flaxseed [19, 24,25,26,27,28,29,30,31,32,33].

Variation in flaxseed mucilage is largely dependent on genotype [27, 29, 31]. Genotypes with low mucilage content may be preferred for some types of animals, to avoid digestive problems [34]. It is also important to note that mucilage is negatively correlated with oil content [35]. Other traits are also relevant to seed mucilage, including: thousand-seed weight [36], seed coat morphology [34], seed color and seed size [21, 29], structural composition of mucilage secretory cells [9, 37, 38], and morphology of the mucilage envelope [37, 38].

Bearing the aforementioned in mind, the aim of this study was to investigate the quality of the mucilage of different flax genotypes by determining morphometric, biochemical, antioxidant and antimicrobial properties and then to hypothesize which properties are related to each other.

Materials and methods

Biological material

Flaxseeds (Linum usitatissimum L.) were provided by Agritec Plant Research, Ltd., Šumperk, Czech Republic. A total of 9 genotypes were used in the research, 4 of which originate from the Czech Republic (Agram, Agriol, Astella, Raciol), 3 from the Netherlands (Flanders, Libra, Lola), 1 from France (Natural), and 1 from Canada (CDC Bethune). All oilseed flax type genotypes except Flanders, Libra (technical use), and Natural (dual use) are intended for food use.

Morphometric analysis

For morphometric analysis, at least 500 dry, undamaged seeds were randomly selected for each genotype. Images of the dorsal side of each seed were acquired by a fully automated microscope ZEISS Stereo Discovery V20 with camera MRc5 and processed by software ImageJ 1.53q using a macro [39, 40]. Objects (seeds) in the image were segmented (Fig. 1) and subsequently measured in the following parameters—area (mm2), perimeter (mm), major (primary) and minor (secondary) axis of best fitting ellipse (mm), maximum and minimum distances (Feret) between any two points (mm), aspect ratio (\(\frac{Major}{Minor}\)), circularity\((4\pi \times \frac{Area}{{Perimeter}^{2}} )\), and roundness\((4\times \frac{Area}{{\pi \times Major}^{2}} )\).

Fig. 1
figure 1

Image processing workflow and measured morphometric parameters on dorsal side of flaxseed. A area (mm.2); B major and minor axis of best fitting ellipse (mm); C perimeter and maximum and minimum distances (Feret) between any two points (mm); scale bar (1 mm)

Full size image

Determination of the mucilage properties

Amount of mucilage released

The swelling index, based on European Pharmacopoeia (9th edition) methodology [41], and the TBO-agarose protocol [42] were used to determine the amount of released seed mucilage. Briefly, to determinate the swelling index, 1 g of flaxseed was weighed into a measuring cylinder, and 25 mL of distilled water was added. The initial volume was subtracted, and the seeds were allowed to absorb the liquid over the next 3 h. Then, the volume occupied by the seeds along with the adhering mucilage was read and the difference between the final and initial volume was calculated.

For the TBO-Agarose protocol (mucilage releasing), two aqueous solutions (agarose solution with tetraborate and toluidine blue with Tween®20, Sigma-Aldrich, Saint-Louis, Missouri, USA) were used. They were prepared separately and mixed only after the agarose solution was cooled. The agarose solution was composed of 0.6% (w/v) agarose in 50 mL ultrapure water and the other of 0.4% (w/v) toluidine blue; 0.1% anhydrated sodium tetraborate and 0.001% Tween®20 (Sigma-Aldrich) in 500 µL ultrapure water. After mixing the solutions, the resulting solution was poured into a Petri dish and air-dried for one hour. Subsequently, 4 seeds of each flaxseed genotype were enclosed in Petri dish to incubate at laboratory temperature overnight, and the diameter of the released mucilage was measured.

Mucilage extraction

Based on previously described methods for flaxseed mucilage extraction [24,25,26,27], we attempted to develop a protocol that would minimize use of chemicals and other harsh treatments that might degrade the mucilage, while yielding sufficient quantity of mucilaginous matter suitable for further downstream analyses. The optimization centered on the ratio (ratios of seeds to water (1:2; 1:5; 1:10 and 1:20 w/v were compared), and a comparison of the use of either (i) pressing the seeds in gauze or (ii) centrifuging at 4 °C for 15 min at 2710 g. Each variant was tested in triplicates for three different genotypes. After establishing the optimal mucilage extraction methodology, this procedure was applied to all flax genotypes in three replicates to determine the volume (mL) of produced mucilage.

Measurement of sugar content in extracted flax mucilage

The extracted mucilage sugar content % (°Brix) was measured by Optronic Digital Handheld Refractometer (Kruss DR201-95) following the manufacturer’s instructions. Each sample was measured in five replicates under constant laboratory temperature.

Lyophilization of extracted mucilage

The mucilage obtained by the optimized procedure was poured into sterile plastic Petri dishes and lyophilized by the Alpha 2–4 LSCplus (CHRIST™) lyophilizer with the following parameters: freezing at − 30 °C for 20 min; warm-up at − 30 °C for 20 min; main drying at − 30 °C for 10 h, vacuum 0.1 mbar and final drying at 35 °C for 18 h, a vacuum of 0.01 mbar. Resulting lyophilizates were weighed by analytical balance.

The fiber content in lyophilized mucilage

Total, soluble and insoluble dietary fiber was determined by the enzymatic method in accordance with the AOAC 991.43 method from 0.1 g of lyophilized mucilage in triplicates. The nitrogen content in the soluble and insoluble fiber was determined using the Kjeldahl method. Subsequently, the blank and total nitrogen value were averaged and used in calculation.

The mineral content of the seeds and mucilage

Flaxseeds of 9 genotypes before and after mucilage extraction were used for mineral detection. Seeds were dried at 130 ± 0.2 °C to establish a constant mass (AOAC 925.10) and 0.5 g of each sample was eluted in 7 mL of HNO3 (65%) in a Teflon container [43]. Digestion of the samples was conducted in a CEM 6 microwave oven (Mars, CEM Corporation, Matthews, United States), at 210 °C, start-up time 15 min, hold time 15 min, pressure 800 psi, and power 900–1050 W. After digestion, demineralized water (Hydrolab System, Wiślina, Poland) was added. Concentrations of the minerals Ca, Fe, K, Mg, Mn, and Zn were determined using atomic emission spectrometry (Agilent MP-AES 4210) (Agilent Technologies, Melbourne, Australia) [44]. At least two calibration curves were prepared for each determination. The percentage of the released mineral into the mucilage was calculated according to a formula with the established threshold 15%.

$$\% Released=\frac{Seed before mucilage extranction-Seed after mucilage extraction}{Seed before mucilage extranction}\times 100$$

Total phenolic acid content and polyphenols profile

Total phenolic acid content and polyphenol profiles were determined from seeds and lyophilized mucilage samples before and after hydrolysis, obtained from 9 flax genotypes. Based on literature data, acid hydrolysis was used to release phenolic acids bound with mucilage polysaccharides. For this reason, seeds and mucilage (~ 0.4 g) were diluted in 8.9 mL of 80% (v/v) methanol solution, and 0.1 mL of 1 M HCl and shaking with a laboratory shaker S50 (CAT, Germany) for 30 min. Subsequently, the samples were centrifuged at 8000×g for 15 min. The supernatants were decanted, filtered (0.22 μm) and stored in a freezer at − 22 °C until use. Mucilage before hydrolysis underwent a similar preparation procedure, except for the use of 9 mL of 80% methanol (v/v) instead of 8.9 mL of methanol and 0.1 mL of 1 M HCl.

The total polyphenols content was measured using the Folin–Ciocalteu method [45]. It was worked in triplicates when 4 μL of the sample was diluted with 16 μL of water and 100 μL of 0.2 N FC reagent. In addition, 80 μL of sodium carbonate (75 g/L) was added after 3 min and incubation was carried out for 1.5 h. The absorbance was measured at 765 nm and a standard curve was made at concentrations of 1; 0.5; 0.25; 0.125; and 0.0625 mg/mL. The results were expressed as equivalents of ferulic acid (FAE) in mg per g of dry mass of samples.

Phenolic acids, including caffeic, chlorogenic, p-coumaric, gallic, and ferulic acids were determined by LC–MS/MS [46]. Chromatographic separation was achieved on a Luna 3 µm C18 analytical column (150 mm × 2.0 mm I.D., Phenomenex Inc., Torrance, CA, USA) using 5 mM ammonium acetate in water (A) and methanol (B) as the mobile phase in gradient elution mode: the flow rate was 0.2 mL/min and the injection volume was 5.0 μL. The gradient program was: 50% B from 0 min to 2.5 min, increased to 100% B in 3 min and held for 0.5 min; the flow rate was 0.20 mL/min. MS–MS detection was performed in negative ionization mode. The analyte was detected using the following settings for the ion source and mass spectrometer: aperture gas 10 psi, nebulizer gas 40 psi, auxiliary gas 40 psi, temperature 400 °C, ion nebulizer voltage − 4500 V, and precipitation gas set to medium. Quantitative analysis of the compounds was performed in multiple reaction monitoring (MRM) mode. For analytes was chosen one transitions of the deprotonated molecular ion and their respective ion product. These transitions (m/z) with associated declustering potentials (DP) and collision energies (CE) and CXP are shown in Table 1. The content all compounds were calculated by a standard calibration curve.

Table 1 Optimum MS/MS working parameters for compounds
Full size table

For the LC–MS/MS method, there were determined validation parameters, i.e.: limit of detection (LOD), limit of quantification (LOQ) and the linearity range. The limit of detection (LOD) for all analytes, defined as the concentration that yielded S/N (signal/noise) ratio greater than or equal to 5, and the limit of quantification (LOQ), defined as the concentration that yielded S/N ratio greater than or equal to 10. Linearity was determined for a series of standard solutions of the analyzed all compounds content in the concentration range from 0.000025 μg/mL to 1 μg/mL. The equation of the calibration curves as well as the limits of detection (LOD) and the limits of quantification (LOQ) are shown in Table 2.

Table 2 Linearity range, limits of detection and quantification of selected phenolic acids
Full size table

Antioxidant analyses of extracted flaxseed mucilage

The radical scavenging activity of flax mucilage was based on the method of Sanchés-Moreno, using 2,2-diphenyl-1-picrylhydrazyl (DPPH) [47]. Extracted mucilage (0.4 mL) was mixed with 3.6 mL of the DPPH solution (0.025% DPPH diluted in methanol w/v). The absorbance of the reaction mixture was determined using a Jenway (6405 UV/Vis, England) spectrophotometer at 515 nm and calculated according to the following formula:

$$\% inhibition=\frac{\left({A}_{C}-{A}_{AT}\right)}{{A}_{C}}\times 100$$

AC—control absorbance of DPPH solution (time = 0 min; constant 0.7)

AAT—absorbance of solution after reaction with antioxidant (time=10 min)

Subsequently, the antioxidant activity was assayed against ABTS (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) reagent according to the method described by Re et al. [48] on samples of seeds and lyophilized mucilages before and after hydrolysis from the previous chapter. The radical cation ABTS was diluted to an absorbance value of 0.7 at a wavelength of 734 nm. An aliquot of 190 µL of this solution was mixed with 10 µL of sample in a 96-well microtiter plate). This was followed by a 30 min incubation at room temperature, in the dark, and with shaking at 1000 rpm. A decrease in absorbance at 734 nm was observed and the results are presented as percent inhibition of ABTS using the previous equation. TEAC test results were expressed as mmol Trolox/g dry sample.

Antimicrobial activity of extracted flaxseed mucilage

Extracted flaxseed mucilage was used against three Gram positive (G +) bacteria (Enterococcus faecalis CCM 4224, Staphylococcus aureus subsp. Aureus CCM 2461, Bacillus subtilis CCM CCM 1999), three Gram negative (G−) bacteria (Yersinia enterocolitica CCM 7204, Salmonella enterica subsp. Enterica CCM 4420, Pseudomonas aeruginosa CCM 3955), four yeasts (Candida albicans CCM 8261, Candida glabrata CCM 8270, Candida krusei CCM 8271, and Candida tropicalis CCM 8264) (Czech collection of microorganisms; Brno, Czech Republic) and evaluated using the agar standard disk diffusion method. Briefly, G + together with G− bacteria were cultivated in Mueller Hinton broth (Oxoid, Basingstoke, UK) for 24 h at 37 °C, and yeast was cultivated in Sabouraud’s dextrose broth (Oxoid, Basingstoke, UK) for 24 h at 25 °C. The density was adjusted to 0.5 McFarland (1.5 × 108 colonies forming units per ml). A volume of 100 μL of the prepared microorganisms was inoculated on Mueller Hinton agar (bacteria) and Sabouraud’s dextrose agar (yeast) and the extracted flaxseed mucilage (50 μL) was loaded onto disks. Samples were incubated for 24 h at 37 °C (bacteria) and 25 °C (yeast). The radii of the inhibition zones formed by the extract were measured in sextuplicate. A positive control with antibiotics (cefoxitin, 30 µg/disc, gentamicin 30 µg/disc, fluconazole 30 µg/disc; Oxoid, Basingstoke, UK) was used as well.

Statistical analysis

The data were processed by ANOVA model and Tukey’s range test in Rstudio 2023.06.0 Build 421 with use of package agricolae: Statistical Procedures for Agricultural Research [49]. The analysis of principal components calculated by factoextra: Extract and Visualize the Results of Multivariate Data Analyses [50] and Pearson’s correlations were visualised by package heatmaply: Interactive Cluster Heat Maps Using ‘plotly’ and ‘ggplot2’ [51]. The data from the antimicrobial activity were separately averaged for Gram positive bacteria, Gram negative bacteria and yeast. The values of ABTS and total phenolic content and hydroxycinnamic acid contents in mucilage were multiplied by coefficient derived from lyophilized mucilage weight, and averaged from data collected before and after hydrolysis.

Results and discussion

Morphometric analysis of flaxseed

Morphometric analysis of the samples, which were subsequently used for further analyses, identified significant differences between genotypes in each parameter measured (Table 3, Figs. 2, 3). Representative images are presented in Fig. 4. The largest area as well as the largest perimeter were observed in the genotype Lola (8.95 mm2 and 12.44) and the smallest area and perimeter were observed in Agriol (8.22 mm2 and 11.99). This genotype had the smallest values in all parameters except Aspect ratio (2.00). The most circular, and most round genotype was Agram. Genotypes Flanders and Lola were also described by [52, 53] with very minimal differences compared to our results.

Table 3 Morphometric parameters of dorsal side of flaxseeds
Full size table
Fig. 2
figure 2

Distribution of genotypes based on Maximum and Minimum distances (Feret) between any two points (mm)

Full size image
Fig. 3
figure 3

Distribution of genotypes based on Circularity and Roundness

Full size image
Fig. 4
figure 4

Dorsal side of seeds. Legend: A Agram; B Agriol; C Astella; D CDC Bethune; E Flanders; F Libra; G Lola; H Natural; I Raciol; scale bar (1 mm)

Full size image

Determination of the mucilage properties

The highest swelling index was observed in the genotype Astella (2.10 mL), which was statistically significantly different from the other genotypes. The genotypes Agram and Raciol reached the swelling index 1.10 mm, and CDC Bethune was 1.00 mm. On the other side, the lowest value was measured for Agriol (0.53 mL). Down to 1 mL were also Flanders (0.67 mL), Libra (0.73 mL), Lola (0.83 mL), and Natural (0.87 mL). The most released mucilage was measured for the genotype Agriol (1.38 cm) and Lola (1.38 cm) and the least CDC Bethune (0.90 cm), Raciol (0.83 cm), and Astella (0.98 cm) (Table 2).

The optimized procedure of the mucilage extraction was as follows: Ten grams of washed flaxseed per each genotype were transferred to sterile conical centrifuge tubes (50 mL). Subsequently, 40 mL of distilled water (1:5 ratio; seeds:water) was added and tubes were placed in a water bath at 40 °C for 5 h (without stirring). After incubation, they were centrifuged for 15 min at 4 °C and 2710 g, and the volume of produced mucilage was measured. The biggest volume reached genotype Raciol (22 mL) and the lowest Astella (10 mL) (Table 2).

It turned out that the volume of water was too small to allow for a sufficient swelling of the seed coats and formation of mucilage in the 1:2 seeds: distilled water ratio. On the other hand, the 1:10 and 1:20 (w/v) ratios resulted in excessively high volumes of distilled water, in which the mucilaginous mass was diluted. The 1:5 ratio was determined as optimal (unpublished data). Moreover, mixing the flaxseed seeds with water in 1:20 (w/v) ratio subsequently required steps to concentrate the resulting mucilaginous solution [24], it took longer for the seeds to produce mucilage (more than 13 h) [32, 33], or an application of high temperatures (65 °C, 80 °C, 100 °C) was necessary during the incubation of seeds in water [24, 26, 33]. Mucilage extracted at 100 °C was darker in colour and contained more oil, protein and ash [24]. Another tested variable in the mucilage extraction procedure was the separation of mucilaginous matter from the seed. Studies of this kind report either the use of filtration of mucilaginous matter through glass wool [24, 25], sieve [19, 27, 30] or a mesh screen (40-mesh, 200-mesh, plastic mesh 1 mm × 1 mm) [26, 28, 31]. In our procedure there was an increase in the amount of mucilaginous matter obtained by centrifugation after the incubation of seeds in water in comparison to pressing the seeds in gauze (unpublished data).

Mucilage with the highest sugar content was produced by the seeds of Agriol (2.70%) followed by Flanders (2.40%), and Raciol (2.33%) (Table 4). The sugar content can be affected by the extraction procedure (pH, temperature) [24, 32, 33], but it is also a genotype-dependent parameter [12, 27, 29, 31].

Table 4 Determination of the mucilage properties for flaxseed genotypes
Full size table

The highest weight of lyophilized mucilage was measured for genotypes Flanders (0.39 g), Agriol (0.38 g) and Lola (0.34 g). The genotype with the lowest weight was Astella (0.18 g) (Table 4, Fig. 5).

Fig. 5
figure 5

A Liquid and lyophilized mucilage, B TBO-Agarose test

Full size image

Our observations correspond to the results of studies with varying mucilage yield in different flax varieties [27, 29, 31, 35]. The genotype-induced differences were related not only to the amount of mucilage collected, but also to its biochemical parameters (protein and sugar content) as well as other mucilage properties such as viscosity [11, 27, 29, 31]. Some studies confirmed the effects of environmental factors such as climate, the length of growing season and crop age, on mucilage yield and its properties [11, 27, 29].

The fibre content in lyophilized mucilage

The results suggested that mucilage is largely composed of soluble fibre (from 40.49% observed in the genotype Natural to 63.00% Lola) and contains a small amount of insoluble fibre (from 1.63%—Natural to 8.29%—Astella—nonsignificant). Overall, five genotypes (Agriol, Astella, CDC Bethune, Libra, and Lola) have total fibre ranging from 60 to 70%, three (Agram, Flanders, and Raciol) from 50 to 60%, and the remaining one (Natural) less than 50% (Table 5). The average content of nitrogenous substances bound in soluble fibre of flaxseed mucilage was determined to be 0.12% and insoluble fibre 0.01%.

Table 5 Contents of the soluble, insoluble and total dietary fibre in mucilage
Full size table

Flaxseed contains approximately 36.7–46.8% total fibre, of which approximately 30% is insoluble fibre and 10% is soluble fibre [54]. The mucilage from Ziziphus mauritania LAMK. contains an average of 89.76% insoluble fibre to which 0.27% nitrogenous compounds are bound [55]. The total fibre of freeze-dried chia seed mucilage was determined to be 75.3%, which is similar to the total fibre content of flaxseed mucilage. On the other hand, the content of nitrogenous substances bound to mucilage was significantly higher (9.7%) [56]. In addition, the mucilage from white spotted chia seeds was found to contain more total fibre (78.86%) than that from black spotted chia seeds (71.31%) [57].

The mineral content of the seeds and mucilage

Among the macrominerals, potassium reached the highest concentration in all varieties of flaxseeds before extraction (average concentration 716.34 mg/100 g). The concentrations of other macrominerals were as following Mg (286.82 mg/100 g), Ca (246.15 mg/100 g), and Na (24.25 mg/100 g). The average concentration for the microminerals were Cu—0.83 mg/100 g, Zn—3.74 mg/100 g, Mn—1.91 mg/100 g, Fe—4.77 mg/100 g. The percentage of the released K into the mucilage was in average 40.52% (marked in bold font in table). Among genotypes with the highest value of the individual mineral was Natural (Mg and Zn), Lola (Ca and K), CDC Bethune (Na), Flanders (Cu), Libra (Mn) and Raciol (Fe). Among genotypes with the lowest value was Agriol (Mg, Ca, Zn and Fe), Lola (Na), Agram (K), CDC Bethune (Cu), and Flanders (Mn) (Table 6).

Table 6 Mineral content (mg/100 g) and percentage of the released mineral into the mucilage (marked in bold font) (established threshold 15%)
Full size table

A low potassium/high sodium diet has been associated with hypertension, strokes, kidney stones, osteoporosis, digestive tract cancers, and others health disorders. This trend is due to the addition of industrially produced salt to foods and the suppression of potassium-rich foods (vegetables and fruits) [58]. Kaur et al. [59] detected a higher concentration of Mg than K from the macrominerals of flaxseeds, while the Na value was similarly low. Similar differences were also observed in the microminerals when the lowest Fe concentration was found. The other order of the elements by magnitude of concentration (Zn > Mn > Cu) was the same as we observed. The mineral concentrations observed by us were in greater agreement with those measured by Daun et al. [60] who determined K over a relatively wide range (550–1060 mg/100 g). On the other hand, the Na contents observed by us were slightly lower than those measured by Daun (320 and 410 mg/100 g). In addition, we observed similar Ca and Na concentrations as Morris [61], but his measured Mg values were slightly higher (350–431 mg/100 g). Pajak et al. [62]measured very different mineral values of flaxseeds. While the average value of Fe concentration measured by us was almost 5 times higher, all other values were lower. They determined the concentration of K to be 2501 ± 131 mg/100 g, confirming on the one hand that this mineral is the most abundant within flaxseeds, but their value is more than three times higher than our measured value. Although they determined mineral values from golden flaxseed, we did not observe statistical differences between our brown and golden varieties.

Kaur et al. [31] determined the contents of copper, zinc, cobalt, lead, chromium and cadmium in flaxseed mucilage and found that of these minerals, zinc (ranging from 1.685 ± 1.32 to 5.343 ± 0.38 mg/100 g) and copper (ranging from 1.887 ± 0.40 to 14.808 ± 1.88 mg/100 g) were the most concentrated. The other minerals, except lead, did not exceed 1 mg/100 g. On the other hand, Troshchynska et al. [63], among the minerals Mg, Ca, Na and K studied, found that K is the most abundant in flaxseed mucilage (ranging from 1.96 to 5.86%), which agrees with our measurements. In addition, a relatively high potassium content (112 ± 1.4 mg/100 g) was observed in the mucilage of okra seeds. Okra mucilage also contains high concentrations of magnesium and calcium [64]. The mucilage from watercress seeds contains the highest level of Ca (0.17% of the total weight) but is immediately followed by K (0.062% of the total weight). In addition, Na and Mg were also detected at lower levels [65].

The phenolic content of seeds and mucilage and antioxidant activity

After calculating the amount of phenolic acids in the mucilage obtained from 0.4 g of seeds by means of the coefficient (Table 7), it was found that the mucilage after hydrolysis had a higher amount of phenolic acids than before hydrolysis. This suggests that hydrolysis with 1 M HCl could have a positive effect on the release of total phenolic acids in the mucilage. While there were no statistically significant differences among genotypes in seeds, the mucilages before and after hydrolysis were divided into several groups based on the amount of total phenolic acids.

Table 7 The phenolic acid content and antioxidant properties of seeds, and mucilages after and before hydrolysis
Full size table

Subsequently, we determined the amounts of phenolic acids (p-coumaric, caffeic, ferulic, chlorogenic, and gallic acids), in the mucilage and seeds after hydrolysis and in the mucilage before hydrolysis. The observed results show that there are differences in the composition of phenolic acids between the seeds and the mucilage obtained from these seeds, and that the amount of phenolic acids obtained is dependent on the method of sample treatment.

The amount of coumaric acid in all genotypes is slightly higher in mucilage before hydrolysis than after hydrolysis. Interestingly, while the highest amount of p-coumaric acid in seeds is found in the genotypes Lola, Astella, and Flanders (9.68, 9.22 and 9.14 μg/g weight, respectively), the highest amount of this acid in mucilage (both before and after hydrolysis) is found in the genotypes Raciol (2. 42 μg/g w/w—before hydrolysis and 1.83 μg/g w/w—after hydrolysis) and Agriol (1.07 μg/g w/w—before hydrolysis and 0.88 μg/g w/w—after hydrolysis). This indicates that the concentration of phenolic acids in seeds is not released in direct proportion to the mucilage.

While the ferulic acid content of the seeds was negligible (up to 3.14 μg/g by weight), a relatively high amount was present in the mucilage. Except for one genotype (Astella in mucilage after hydrolysis), all genotypes of mucilage contain a higher amount of ferulic acid than p-coumaric acid. Except for one genotype (Lola), the ferulic acid content is higher all genotypes of mucilage before hydrolysis.

The caffeic and chlorogenic acid content was negligible in both mucilage and seeds of the observed flax genotypes. Acid hydrolysis had a minimal effect on the release of these acids.

Of all the observed cinnamic acid hydroxyderivatives, gallic acid had the highest concentration in the seeds of all genotypes (from 44.04 to 65.57 μg/g of mass). In contrast, the mucilage before hydrolysis contained less of this acid than ferulic acid (0.87 versus 1.25 μg/g of mass on average) and only slightly more in the mucilage after hydrolysis (0.86 versus 0.76 μg/g of mass).

The antioxidant activity of flaxseed mucilage was determined by the DPPH method and expressed as a percentage of inhibition ranged from 23% (Raciol) to 70% (Lola). The strongest antioxidant activity was detected in the genotypes Lola (70%), Natural (66%) and Agram (65%), the medium strong in the Flanders (60%), CDC Bethune (58%), Astella (53%) and Libra (50%), and the weakest in Agriol (37%) and Raciol (23%).

In seeds, the highest antioxidant activity against ABTS was in Astella (4.43 mmol Trolox/g of dry sample), which is statistically significantly higher compared to the other genotypes. In contrast, the antioxidant activity is different in the mucilage. It is on average half as high in the mucilage before acid hydrolysis as in the mucilage after hydrolysis (0.08 to 0.04 mmol Trolox/g of dry sample). While in the mucilage before hydrolysis, the genotype Agriol (0.12 mmol Trolox/g of dry sample) stands out for its antioxidant activity, after hydrolysis, they are the genotypes Lola and Flanders (0.06 and 0.05 mmol Trolox/g of dry sample) that have the highest antioxidant activity.

Flaxseed accumulates in its seed coat a large amount of potentially bioactive compounds such as lignans, flavonol, and flavonoid acids (p-coumaric, caffeic, and ferulic acid). The amount of ferulic acid glucoside, caffeic acid glucoside, and para-coumaric acid glucoside were determined to be 2.58 ± 0.04, 3.63 ± 0.14, and 5.04 ± 0.23 mg/g dry weight, respectively, in flaxseed husks [66]. Plant mucilage concentrates phenolic compounds, presumably to protect plant seeds from pathogens since the seeds of some plants are separated from the external environment by mucilage [67]. The total phenolic content of the extracted mucilage is dependent on the extraction conditions, increasing with increasing temperature. Hence the increasing maximum antioxidant activity at the highest extraction temperature. However, as the temperature increases, protein contamination of the mucilage also occurs. At a culture temperature of 86.10 °C, the mucilage from Trigonella elliptica seeds contained a total phenolic content of 2.20 ± 0.11 mg GAE/g with free radicals scavenging activity value of 63.89 ± 2.93% [68]. Lallemantia royleana seed mucilage (with extraction temperature 50 °C) has lower total phenolic acid content (82.56 ± 1.6 μg GAE/mg) and its antioxidant activity against DPPH is IC50 528.54 ± 0.35 μg/mL [69].

Bouaziz et al. [70] similarly determined the antioxidant activity (ABTS method) of soluble flaxseed gum in the Comtess variety and confirmed a strong activity with a value of 75.6%. HadiNezhad et al. [71] determined the antioxidant activity of flaxseed mucilage (flaxseed from commercial source—Bulk Barn, Ottawa and Canada) of 53.7% by the DPPH method, which is comparable with our results. Safdar et al. [72] determined that the main components of flaxseed gums are glucosamine, glucuronic acid, galacturonic acid, arabinose, xylose, rhamnose, glucose, mannose, fructose and galactose. These authors also confirmed the antioxidant activity of gums. Emadzadeh et al. [73] determined a similar activity of mucilage in yellow and brown flaxseed (seeds from Sepakan Pakan Bazar company, Iran) by the DPPH method. The antioxidant activity ranged from 12.65% to 43.65%.

Antimicrobial activity of extracted flaxseed mucilage

The highest antimicrobial activity of flaxseed mucilage against Gram-positive bacteria E. faecalis (6.82 mm) was found in Agriol. The mucilage of Lola exhibited the strongest antimicrobial activity against S. aureus (5.98 mm) and CDC Bethune was most effective against B. subtilis (6.36 mm), however these last two results were not significantly different from the other genotypes tested. The most effective mucilage against Gram-negative bacteria Y. enterocolitica was from genotype Raciol (6.11 mm), against S. enterica from the CDC Bethune (5.52 mm) and against P. aeruginosa from Flanders (6.25 mm—not significant). The strongest activity against C. albicans was found in the mucilage from CDC Bethune (5.69 mm). From the other yeast, C. krusei was the most sensitive to mucilage from Lola (5.45 mm), C. glabrata to the mucilage from Astella (7.95 mm), and C. tropicalis nonsignificant to mucilage extracted from Lola (5.98 mm) (Table 8).

Table 8 Antimicrobial activity of flaxseed mucilage extract against Gram-positive bacteria
Full size table

The study of antimicrobial activity of flaxseed against foodborne pathogens has received much attention in various studies [8, 74, 75]. Abbas et al. [76] pointed out the antimicrobial ability of flaxseed oil extracts and thus confirmed the inhibitory effects against Streptococcus mutans and they have drawn attention to the differences in the impact on individual microorganisms. Similarly, Al-Mathkhury et al. [77] also discussed and investigated the various activities of flaxseed oil extracts on bacteria, yeast, fungi and biofilm. The flaxseed oil extract did not exhibit any activity against E. coli or E. faecalis, however, it exhibited antimicrobial activities against selected oral bacteria [78]. Very clear differences were identified between the effects of different extracts in the study [74]. In addition, Al-Mathkhury et al. [77] conducted a study to investigate the antibacterial and antibiofilm activity of the flaxseed oil extract on some locally isolated bacterial pathogens. In different study the in vitro effect of flaxseed oil on the bacterial isolates shows that there is no inhibitory effect against E. coli or E. faecalis, perhaps due to the resistance genes, which requires a further analysis. In the same study the flaxseed extract shows an antibacterial activity against the following selective oral pathogens [78]. The results of the study by Gaafar et al. [74] show that the flaxseed extracts were inactive against C. albicans and Aspergillus niger. Our study indicates that flaxseed mucilage was effective against tested pathogens in all cultivars.

Statistical consideration on mucilages properties

Analysis of the principal components and Pearson´s correlations within morphometric parameters and mucilage properties revealed a close relationship between lyophilized mucilage weight and mucilage releasing (0.67), as well as Aspect ratio (0.67), and Mucilage volume (0.60). The remaining variables were found to be closely related to seed size parameters (Figs. 6 and 7).

Fig. 6
figure 6

Analysis of the principal components within morphometric parameters and mucilage properties

Full size image
Fig. 7
figure 7

Heatmap of Pearson´s correlations between morphometric parameters and mucilage properties. P value: < 0.001*** < 0.01** < 0.05

Full size image

Parameters such as lyophilized mucilage weight, mucilage releasing, aspect ratio, and Mucilage volume were selected to determinate the correlations between mucilage and various factors including: antioxidant activity (ABTS and DPPH); antimicrobial activity (Gram positive bacteria, Gram negative bacteria and yeast); sugar and fiber content (soluble, insoluble and total fibre); total phenolic content, and the content of p- coumaric, caffeic, ferulic, chlorogenic, gallic acid and potassium including percentage of its release into the mucilage (Figs. 8 and 9). The results revealed strong correlations between DPPH and hydroxycinnamic acids (p- coumaric acid—0.88, caffeic acid—0.79 and ferulic acid—0.79), as well as with sugar content—0.70. A high correlation was observed between ABTS and total phenolic content—0.68. The antimicrobial activity against Gram positive bacteria was likely influenced by fibre content (0.83), especially soluble fibre (0.83), and also by potassium content (0.72) which may also be associated with insoluble fibre (0.21) against yeast (0.27). The correlation between potassium and insoluble fibre was found to be 0.69. The amount of bound potassium in mucilage is likely influenced by total fibre content (0.83). The percentage of released potassium may have a connection to seed size parameters. Gallic acid showed correlations with other hydroxycinnamic acids, while chlorogenic acid appeared to be effective against Gram negative bacteria (0.70). The correlation differences observed between the ABTS and DPPH antioxidant assay could be attributed to the different basis of these two assays as well as a weak correlation between them each other and higher correlation of ABTS with TPC in general [79,80,81].

Fig. 8
figure 8

Analysis of the principal components within biochemical properties of the flax mucilage

Full size image
Fig. 9
figure 9

Heatmap of Pearson´s correlations among biochemical properties of the flax mucilage. P value: < 0.001*** < 0.01** < 0.05*

Full size image

Conclusion

The mucilaginous substances of flaxseeds are composed mainly of water-soluble fiber, the amount of which varies slightly among genotypes. Of the minerals, the mucilage is most rich in potassium, the release of which from the seeds is mainly determined by the amount of total fiber. It is the amount of fiber and its potassium content that have a positive effect against Gram-positive bacteria. The amount of chlorogenic acid in the mucilage has a significant effect against Gram-negative bacteria. The antimicrobial properties of mucilage against some bacteria are significantly different among genotypes (E. faecalis, Y. enterocolitica, and S. enterica) and, conversely, for others, the differences are only minimal (S. aureus, B. subtilis, and P. aeroginosa).

In addition to antibacterial properties, flaxseed mucilage also exhibits antioxidant properties against ABTS and DPPH. While the antioxidant properties of the mucilage against DPPH are mainly influenced by the hydroxycinnamic acid content (mainly p-Coumaric acid), other phenolic compounds are active against ABTS, as they are most strongly correlated with the total phenolic content. While there are minimal differences in total phenolic content between the seeds of the selected genotypes, these substances are already released into the mucilage in different amounts, mainly depending on the sugar content of the mucilage.

Of the mucilage quantity detection methods, the weight of lyophilized mucilage is the most accurate, as it demonstrates the actual weight of mucilage that can be extracted from the seeds. This weight is primarily dependent on the volume of mucilage that is released from the seeds into the water. However, in order to determine the volume or weight of the mucilage, a relatively large quantity of seeds is required. If it is necessary to determine from a small number of seeds which flax genotype will release the largest amount of mucilage, the most accurate methods are the TBO-Agarose protocol (mucilage releasing), and the aspect ratio between the major (primary) and minor (secondary) axis of the best fitting ellipse (mm).