Introduction

Bridging the gap between demanded diverse functionality of food and at the same time maintaining the consumer acceptability of such is the main challenge that the food industry is facing nowadays. To combat this challenge, the exploration and exploitation of ancient nutrient-dense plant resources such as alfalfa or lucerne (Medicago sativa L.) is of great interest to the food industry. Apart from being rich in protein (34–42%), lipid (3–10%), and total dietary fibre (4–13%) [1, 2], alfalfa seed contains considerable amounts of minerals (Ca, Fe, Zn) and vitamins (E, B1, B2, C), essential polyunsaturated fatty acids, and diverse bioactive constituents (phenols, flavonoids and saponins) [3] demonstrating antioxidant, anticancer, antiatherosclerotic, antidiabetic and antiobesity activities [4].

Although approved for use in the human diet in the form of edible seeds, sprouts and protein concentrates, alfalfa still did not reach its full potential for food applications. Major limiting factors for alfalfa’s dietary exploitation are the existence of antinutrients (tannins, lectins, trypsin inhibitor and phytic acid) and beany flavour. As an ancient and well-known practice, germination is reintroduced in wider food product applications nowadays representing a green processing technique of emerging interest. During the germination of legumes, many biochemical changes occur affecting macromolecular structure, functionality, bioavailability of nutrients and bioactive constituents, digestibility, antinutrients content, as well as sensory perception of the resultant legume flours [1, 2, 5, 6]. All mentioned changes are considered positive and open new possibilities for the widespread application of alfalfa and other legumes in diverse food products which in turn can potentially contribute in attenuating adverse lifestyle disease outcomes within the population.

Similarly to germination, sourdough fermentation regained popularity as another biotechnological process offering numerous advantages towards bread quality enhancement, including pleasant flavour, extended shelf life, and increased protein quality and antioxidant activity [7]. Additionally, reduction in antinutrients content resulting in enhanced mineral bioavailability, as well as a decrease in glycaemic response upon bread consumption and aid in the management of irritable bowel syndrome are another benefits attained upon sourdough fermentation involving usage of lactic acid bacteria and yeasts [8].

Incorporation of various legume flours (lentil, chickpea, pea, soybean) in both germinated and non-germinated form, as wheat flour substitutes in bread-making, has been the subject of several research studies conducted to date [9]. In general, regardless of the legume flour type used, the resulting wheat-legume flour blends and breads have markedly enhanced protein, lipid, dietary fibre and mineral contents compared to wheat counterparts [9]. Conversely, technological functionality of doughs and obtention of satisfactory bread loaf volume, crumb structure and hardness is greatly dependent on the type and level of wheat flour substitution by legumes [9]. Furthermore, only a few studies highlighted the potential of combined germination and sourdough processes in bread production involving legumes such as broad beans [7], lentils [6], and chickpeas [5] reporting promising results.

In the studies of Djordjević et al. [1] and Sahni and Sharma [3], the influence of germination conditions and drying technique on alfalfa flour techno-functional properties at nutritional and structural levels were analysed. Hence, the application of germinated alfalfa seeds may be a new technological approach providing the development of an innovative functional and low-cost ingredient appropriate for bread-making. However, an insight regarding alfalfa seed flour’s role, both non-germinated and germinated, in formulating wheat flour-alfalfa blends intended for bread-making has not been fully addressed in the scientific literature. Therefore, this study explored the feasibility of alfalfa seed flour’s (non-germinated and germinated) usage in bread-making, by assessing the proximate composition, mineral profile, antioxidant and Mixolab rheological properties of wheat flour-alfalfa blends, together with a preliminary assessment of physical and textural characteristics of the resulting bread. Additionally, the effects of germination and sourdough fermentation treatments on alteration in the corresponding blends’ and breads’ parameters were also evaluated.

Materials and methods

Raw materials

Basic ingredients used for bread production were: white wheat flour (T 405, GoodMills Polska Sp. z o.o., Stradunia, Poland), fresh yeast, salt and sugar from the Polish market. As starter culture, Livendo™ starter LV1 was employed (LIS by Lesaffre, Cérences, France). Alfalfa seeds originated from domestic fields (Vojvodina, Serbia) and were obtained from the Institute of Field and Vegetable Crops (Novi Sad, Serbia). After damaged seeds removal, seeds were germinated according to the procedure described by Djordjević et al. (2023) and dried in convective hot air at 70 °C for 10 h (Iskraterm 2 K, Ljubljana, Slovenia). Non-germinated seeds were also dried under the same conditions. Germinated (GASC) and non-germinated (ASC) alfalfa flours with particle size < 315 µm were obtained in the MultiDrive B S000 laboratory mill (IKA, Staufen, Germany) by milling at 10 000 rpm for 2 min, and stored in sealed bags at 8 °C until use. Wheat flour-alfalfa blends (WFAB) were made by substituting 5 g and 10 g/100 g of wheat flour with ASC (WF_5A and WF_10A) or GASC (WF_5G and WF_10G).

Proximate composition

The proximate composition of flours and WFAB was evaluated according to AACC International [10] methods. The moisture content was determined by oven drying at 105 °C; ash content was determined upon incineration in a furnace at 550 °C for 5 h; total nitrogen content was estimated by the Kjeldahl method (behrotest®InKjel; behrdistilation unit S5, behr Labor-Technik, Düsseldorf, Germany) and protein content was calculated by its multiplying with a conversion factor of 6.25 for alfalfa flour and 5.70 for WFAB; lipid content was determined by the Soxhlet extraction. The starch was determined using Lintner's method [11]. Starch content in the solution was calculated according to Eq. (1):

$$C=\frac{\alpha \times 100}{l\times {\left[\alpha \right]}_{D}^{20}}$$
(1)

where C is starch content in solution (g/100 g), α is the recorded angle of rotation of polarized light, l is the tube length (dm), and \({\left[\alpha \right]}_{D}^{20}\) is the specific rotation of starch taken as + 202º.

Starch content in the examined flour samples was calculated as follows (Eq. 2):

$$S=\frac{C\times 100}{A}$$
(2)

where S is the starch content in flour sample (%), C is starch content in the solution (g/100 g), and A is the flour sample weight (g).

The total dietary fibre (TDF) was determined by a gravimetric-enzymatic method according to AOAC 985.29 [12] using a commercially available TDF Assay Kit (TDF100A; Sigma-Aldrich, Missouri, USA). All results were expressed on a dry weight basis.

Mineral profile

Macro (Ca, Na, Mg, K) and microelements (Fe and Zn) content in flours and WFAB was determined by the ISO 6869:2000 [13] method consisting of dry ashing, subsequent ash dissolution in HCl followed by dilution (50 mL) and measurement using atomic absorption spectrometer (SpectrAA 10, Varian Inc., Australia). Results were expressed on a fresh weight basis.

Total phenolic content and antioxidant activity

Extract preparation

Flours and WFAB were extracted with an aqueous solution of methanol at a concentration of 80% (v/v) with 1% HCl. The ratio between the weight of the sample and the amount of solvent was 1:10. The samples were extracted for 24 h and subjected to sonication twice (10 min) (Polsonic, Warsaw, Poland) immediately after combining the sample with the solvent. Separation of the solid phase from the extraction mixture was performed by centrifugation (3500 rpm, 10 min) (MPW-350, MPW Med. Instruments, Warsaw, Poland).

Total phenolic content

Total phenolic content (TPC) of flours and WFAB was determined according to the spectrophotometric method described by Gao et al. [14]. Gallic acid was used as a standard, and the results were expressed as mg gallic acid equivalents /100 g.

Antioxidant activity assays

Antioxidant activity of flours and WFAB was estimated throughout ABTS and FRAP assays following the procedure described by Re et al. [15] and Benzie and Strain [16], respectively. Results were expressed as μmol Trolox equivalents /100 g.

Mixolab rheological properties

The physical dough properties of wheat flour and WFAB containing 5 g and 10 g/100 g ASC or GASC were evaluated using the Mixolab device from Chopin Technologies (Paris, France). The rheological behaviour of the dough during mixing and temperature changes was measured using AACCI 54–60.01 method [17]. The Mixolab analysis recorded the dough resistance and temperature every second, and the resulting graph was used to calculate rheological parameters. The parameters such as water absorption, dough development time, dough stability and softening, peak viscosity, activity of amylolytic enzymes, and starch retrogradation were evaluated using the Chopin + protocol lasting 45 min. The dough weighed 75 g, and the torque resulting from the water absorption of the flour was 1.1 N m. The temperature, time, and torque were determined at specific points (C1-C5) in the process.

Standard and sourdough bread-making process

Wheat flour was substituted by 5 g and 10 g/100 g of ASC or GASC flours to obtain desired blends for bread-making. Other ingredients expressed on 100 g of the basic blends were: fresh yeast (3.12 g), salt (1.5 g), and water in an amount to reach a consistency of 300 ± 20 FU.

The standard bread samples (C, A5, A10, G5 and G10) were prepared by mixing the dough ingredients in Brabender farinograph (Brabender, Duisburg, Germany; bowl for 300 g of flour) for 3 min at 63 rpm after achieving the 300 FU consistency. Dough was subsequently transferred to the oiled mould and proofed for 60 min with kneading after 30 min. Bread samples were baked at 220 °C for 30 min with steaming in the first minute of baking (IBIS GT 600 oven, IBIS, Szubin, Poland). Subsequently, the breads were taken out of the moulds and cooled for approximately 2 h at 20 °C.

The sourdough bread samples (SC, SA5, SA10, SG5, SG10) preparation included fermentation of the equal parts of the wheat flour/prepared blends and water for 24 h at 30 °C with the addition of LV1 starter (0.5 g/100 g). Afterwards, sourdough was mixed with the rest of the flour blends and water in a Brabender farinograph, proofed, and baked following the procedure described for the preparation of standard bread samples.

Characteristics of bread containing alfalfa flour

Bread specific volume

After weight measurement, the volume of bread samples was estimated by the millet displacement method using the SA-WY volumeter (ZBPP, Bydgoszcz, Poland). Specific volume was calculated as loaf volume and bread weight ratio. Overbake was calculated as bread mass to flour mass percentage ratio, and bake loss was calculated as indicated in Eq. (3):

$$Bake loss (\%)=\frac{(a-b)\times 100}{a}$$
(3)

where a is the initial weight of dough before baking (g), and b is the weight of baked and cooled bread (g).

Crumb texture profile analysis

The properties of the bread crumb were determined using the TPA method performed on ZWICK ROEL Z010 apparatus with 100 N load cell (Zwick Roell, Ulm, Germany). A sample of crumb in the form of a cylinder with height of h = 13 mm and a diameter of Ø = 17 mm was subjected to double compression (40% of compression) by a flat plate (Ø = 35 mm) with a falling speed of 60 mm/min with an interval of 0.5 min between compression cycle. Based on the collected data, the following parameters were obtained: hardness [N]; springiness [−]; cohesiveness [−]; chewiness [N] and gumminess [N]. All samples were tested in 8 replicates.

Statistical analysis

The statistical analysis of the results was conducted in Statistica 14.0.0.15 software (TIBCO Software Inc., California, US). The results are presented as the mean values of three replicates unless otherwise stated, and subjected to a one-way analysis of variance (ANOVA). Duncan’s multiple range test was applied to assess the significant differences set at p ≤ 0.05 between the mean values and homogeneous groups. For Mixolab parameters two-way analysis of variance (ANOVA) was carried out at p = 0.05 with the type of substitute (ASC or GASC) and its amount (5 g and 10 g/100 g) as factors. In addition, principal component analysis (PCA) by centroid-linkage agglomerative method was used based on correlation matrix. To determine the distance between the samples employed the Euclidean measure.

Results and discussion

Proximate composition

The proximate composition of wheat, ASC and GASC flours and WFAB is summarized in Table 1. Alfalfa germination during 48 h resulted in a significant increase in moisture and protein content, and a decrease in starch with no statistically significant changes in ash and lipid content (GASC, Table 1). Higher moisture levels induced by germination represent a consequence of water uptake needed for metabolic functions and were previously reported for legumes [18]. However, a safe moisture level of 10 g/100 g [19] was attained for germinated alfalfa and corresponds to germinated lentil [18] and Bambara groundnut [19] flours (ASC and GASC, Table 1).

Table 1 Proximate composition, mineral profile and antioxidant activity of flours and blends containing non-germinated and germinated alfalfa seeds
Full size table

Furthermore, the observed enhancement in protein content is consistent with previous findings in germinated alfalfa [2] and Bambara groundnut [19] flour. However, there are also studies without detected changes in germinated alfalfa regarding protein content [1, 20]. The assumption is that increased protein content is associated with storage protein hydrolysis, water-soluble proteins increase and amino acid synthesis all taking place during germination [2, 18]. Additionally, the protein content obtained herein was found to be higher compared to previously studied alfalfa [1, 2, 20]. High protein content of alfalfa flour samples is reflected in blends where an increase is observed with the rise in the alfalfa inclusion level (WF_5A and WF_10A, WF_5G and WF_10G, Table 1).

The tendency of starch content reduction after germination due to the action of α- and β-amylases is consistent with previous findings in lentil and chickpea [5] while for alfalfa data were not reported. Although statistically not significant, the lipid content slightly increased upon germination in line with previous results for alfalfa [1, 20]. TDF content found in ASC and GASC flours (36.78 and 33.00%, respectively, Table 1) was significantly higher compared to previously reported for non-germinated and germinated alfalfa seeds [1, 2] as well as for chickpea and lentil [5]. A decrease in TDF content upon germination observed is consistent with previous reports on chickpea and lentil [5]. As regards to blends, a trend of increase in TDF content with increase in alfalfa flour levels was recorded (WF_5A and WF_10A, WF_5G and WF_10G, Table 1). Considering the above stated, alfalfa flour regardless of type, could be a valuable ingredient in terms of protein and TDF content enrichment in bakery products.

Mineral profile

Germination induced changes in the mineral profile of alfalfa seeds (Table 1). An increase in Ca and Zn content was detected upon alfalfa seeds germination during 48 h with simultaneous reduction of Mg, K, Na and Fe content (ASC and GASC, Table 1). The corresponding changes represent a consequence of minerals leaching during seed soaking, cell structure modification, antinutrients reduction as well as redistribution and remobilization of minerals acting as cofactors in enzyme catalysis of carbohydrates and proteins during germination [1]. Previously, a positive influence of germination on Ca content was reported for Bambara groundnut flour [19], while an increase in Zn content was detected in alfalfa [1]. Moreover, the obtained Ca content (580.65 mg/kg) is higher than noted for germinated Bambara groundnut flour [19]. Zn content obtained herein (35.89 mg/kg) corresponds to other germinated legumes such as bean, lentil, lupine, and chickpea [18]. Despite noticed reduction after germination, the Mg content (1694.65 mg/kg) was higher than in germinated Bambara groundnut flour [19], comparable to various legumes [18], but reduced when compared to previous results on alfalfa [1] probably due to differences in seeds variety and origin. Furthermore, a decline in Na and K content was noticed after alfalfa germination (GASC, Table 1) consistent with previous studies [1, 2], however, it was still higher compared to other legumes [18]. Nevertheless, Fe content (75.17 mg/kg) higher than previously reported for germinated alfalfa [1], and bean, lentil, lupine, and chickpea [18] was obtained herein, but it was lower than detected in Bambara groundnut flour [19].

A significantly higher content of macro and micro elements was noted for ASC and GASC when compared to wheat flour (Table 1). As regards to WFAB, the same trend was observed with the exception of K and Fe content in blends with 5 g/100 g ASC and GASC (WF_5A and WF_5G, Table 1) suggesting an insufficient substitution level for flour enrichment in corresponding minerals. Nevertheless, evident enhancement in the entire mineral profile compared to wheat flour was observed at 10 g/100 g substitution with ASC and GASC (WF_10A and WF_10G, Table 1) which will consequently reflect positively on the mineral profile of produced bakery products.

Total phenolic content and antioxidant activity

Germination induced a decrease in TPC as well as the reduction in antioxidant activity of alfalfa samples assessed by ABTS and FRAP and consequently noticed changes reflected on the corresponding parameters of the blends (Table 1). The TPC content of germinated and subsequently dried alfalfa was three times lower compared to the sample subjected to drying treatment only (GASC and ASC, Table 1), but still higher than reported for alfalfa upon 48 h germination [2]. A significant loss in TPC was also previously observed for alfalfa subjected to soaking and germination [2,3,4]. Conversely, prolonged germination (6 days) resulted in increased TPC content of alfalfa sprouts [20]. The corresponding discrepancies could be explained by changes occurring during germination where TPC reduction is associated with compounds conversion due to the activation of endogenous enzymes in order to defend seedlings from external stress [4], while TPC increase represents a defensive mechanism against favourable conditions for microbial growth [20]. As regards to TPC of blends, with increasing alfalfa flour levels, regardless of type, a rise in TPC was observed and it was more prominent for blends containing ASC flour (Table 1).

Reducing ability determined by FRAP (2127.70 µm TE/100 g) was higher than previously detected in germinated alfalfa [2], but complies with the trend related to the reduction in FRAP value upon germination disclosed by the same authors. Conversely, a significant decrease in ABTS upon alfalfa germination (from 3406.09 to 1225.91 µm TE/100 g) observed herein was contrary to the earlier reports regarding the germination effect on the same legume [2, 4]. Disclosed observations on reducing ability and antioxidant activity apply for blends as well, with significant differences in favour of ASC inclusion (WF_5A and WF_10A, WF_5G and WF_10G, Table 1). Diverse factors including treatment and treatment conditions (thermal and biological in this case), the reactivity of potentially formed radicals and their detection method, influence the relation between bioactive compounds content and antioxidant activity [3]. Hence, a straightforward dependence among increased bioactive compounds content and antioxidant activity may not be established as noticed here for 48 h geminated alfalfa.

Mixolab rheological properties

When studying the effect of alfalfa flour substitution at concentrations of 0, 5, and 10 g/100 g, it was observed that the water absorption capacity of the samples remained consistent, ranging from 59.3% to 62.6% (Table 2). This indicates that the presence of alfalfa did not significantly impact the flour's ability to absorb water and reach the desired dough texture.

Table 2 Mixolab rheological parameters of doughs depending on the alfalfa flour type and amount
Full size table

The effect of alfalfa flour inclusion on dough development time (time to reach point C1) was examined, revealing that the alfalfa flour type did not yield a significant impact (Fig. 1). However, the alfalfa flour amount demonstrated a notable effect on dough development time. Specifically, when the alfalfa flour content reached 10 g/100 g, the dough development time significantly increased to 5.51 min compared to both the control sample with 0 g/100 g alfalfa and sample containing 5 g/100 g alfalfa flour with dough development times of 1.57–1.56 min (Table 2). This suggests that higher concentrations of alfalfa flour in the flour blend resulted in a prolonged time required for dough development. The presence of fibre compounds in alfalfa (36.78 and 33.00% for ASC and GASC respectively, Table 1), such as hemicellulose and lignin, can create physical barriers that hinder the formation of the gluten network and starch gelatinization, both of which are crucial for proper dough development.

Fig. 1
figure 1

Mixolab rheological profile of wheat flour blends containing 5 and 10 g/100 g non-germinated (WF_5A and WF_10A) (a), and germinated alfalfa flour (WF_5G and WF_10G) (b)

Full size image

In terms of dough stability, the addition of alfalfa flour, whether ASC or GASC, did not lead to a significant effect. However, it was observed that the incorporation of GASC flour resulted in slightly lower dough stability (8.13 min) compared to ASC (9.40 min, Table 2, Fig. 1). This suggests that GASC flour may have exerted a slight influence on the overall stability of the dough compared to ASC flour. The process of germination involves enzymatic activity and metabolic processes that can lead to compositional changes in alfalfa. These alterations have the potential to affect the interaction between gluten proteins and other dough components, thereby contributing to a slight reduction in dough stability compared to dough with ASC flour [4].

The measurement of dough properties at point C2, which assesses protein weakening as a function of mechanical work and temperature, revealed no significant effect of the alfalfa flour inclusion (Fig. 1). The lack of effect may be attributed to the possibility that the impact of alfalfa on protein weakening is overshadowed by other factors or mechanisms involved in dough development at this stage. Point C2 primarily focuses on the balance between gluten protein degradation and the formation of a stable gluten network, processes that may not be substantially influenced by the addition of alfalfa flour. It is also plausible that the incorporation level of alfalfa flour used in the study did not exceed the threshold required to produce a discernible effect on protein weakening at this particular stage. The properties and characteristics of alfalfa, such as its fibre content or enzymatic activity, may not directly affect protein weakening during this stage of dough development. Other factors, including the composition of the flour blend and the conditions of mixing and processing, could play a more dominant role in determining the protein weakening properties observed at point C2 [21].

The C3 torque and C3 temperature measurements were not significantly affected by the alfalfa flour type and amount (Table 2, Fig. 1). The lack of significant effects may be attributed to the interactions between the added alfalfa flour and the dough matrix, which may not lead to substantial changes in the rheological properties at this stage. The torque and temperature at C3 are influenced by factors such as gluten protein interactions, starch gelatinization, and water absorption, which were not strongly influenced by the alfalfa flour inclusion. Starch characteristics, amylase activity, and competition for water between starch and fibre can influence the gelatinization parameters of dough at different hydration levels [22].

The C4 time measurement exhibited significant differences among the different alfalfa flour type and amounts included. The addition of 10 g/100 g alfalfa flour resulted in a significantly longer C4 time (33.98 min) compared to both the control (0 g/100 g alfalfa) and the 5 g/100 g addition (32.57–32.58 min, Table 2, Fig. 1), indicating that a higher alfalfa flour content resulted in prolonged C4 time and increased gel stability [23]. The observed variations in C4 time and torque can be attributed to the higher fibre content when 10 g/100 g alfalfa flour was included, which may have increased the dough viscosity and resistance to flow, resulting in a longer C4 time. Additionally, the enzymatic activity associated with alfalfa flour, particularly when germinated, can modify its composition, leading to potential changes in protein degradation, starch modification, and other biochemical reactions that can influence the rheological properties of the dough, thus affecting C4 time and torque [23].

The significant differences observed in the C5 torque measurement among the different types and amounts of included alfalfa flour indicate that the presence of alfalfa influenced the rheological and thermal properties of the dough, which in turn can impact the resulting bread quality and shelf-life [24]. The lower torque value at point C5 for the incorporation of 10 g/100 g alfalfa flour (1.83 Nm) compared to the control (3.14 Nm) and the 5 g/100 g (2.47 Nm, Table 2) suggests a lower degree of starch retrogradation. Overall, these results indicate that the addition of alfalfa flour, particularly at higher concentrations, can influence the rheological properties, thermal behaviour, and starch retrogradation in the dough, ultimately affecting bread quality and shelf-life [25].

Characteristics of bread containing alfalfa flour

Bread specific volume

The specific volume of bread samples was generally positively affected by ASC and GASC flour inclusion and ranged between 2.96 and 3.16 cm3/g for standard, and between 3.23 and 3.62 cm3/g for sourdough bread (Table 3). Although all standard bread samples containing alfalfa flour had higher specific volume compared to control wheat bread (Table 3), the effect of both alfalfa flour substitution level and flour type on specific volume was noticed. Increase in ASC content from 5 to 10 g/100 g led to an increase in the specific volume (samples A5 and A10, Table 3), conversely to previous results reported for chickpea flours at substitution level up to 15 g/100 g [26]. This result could be explained by the higher protein content in alfalfa seeds flour compared to the abovementioned legumes, and the adequate water amount required for their foaming activity resulting in the formation of stable gluten-legume protein network capable to entrap gases and consequently greater volume [27]. This assumption is potentially confirmed by Mixolab results since with ASC flour incorporation alteration of water absorption capacity and dough stability was minimal.

Table 3 Technological quality parameters of bread samples containing non-germinated and germinated alfalfa seeds
Full size table

Inclusion of 5 g/100 g GASC yielded bread with higher specific volume (sample G5 3.16 cm3/g, Table 3) in accordance with results reported on germinated soybean flour at same inclusion level [28, 29]. It is assumed that the greater impact of germinated flours on specific volume increase could be ascribed to its superior foaming and emulsifying activity due to the enhanced protein solubility upon germination [1, 27]. Furthermore, a rise in the content of fermentable sugars available for yeast, as a consequence of the amylases action on starch triggered during germination, results in the release of a larger amount of CO2, and thus greater bread volume [28]. Further increase in GASC content (10 g/100 g) induced a volume-depressing effect in line with results reported for germinated chickpea and yellow pea flours incorporated at same and higher quantities [26, 27]. This effect is probably a consequence of slight dough weakening induced by gluten dilution due to the presence of legume proteins in larger quantities, as well as the partial degradation of starch granules during germination affecting dough viscosity [26]. However, the increase in GASC content did not have a detrimental effect on specific volume since it was still higher compared to control wheat bread (samples, G10 and C, Table 3).

Regardless of the used alfalfa flour type and its quantity, sourdough bread samples exhibited greater specific volumes compared to corresponding bread samples obtained by the standard bread-making process (Table 3). These results comply with the ones previously reported in literature when raw or germinated lentil and chickpea flour [5, 6], as well as blend of raw chickpea, lentil and bean was used in sourdough bread production [30]. The increase in bread specific volume is mainly attributed to the physicochemical changes in the protein network occurring during sourdough fermentation enabling greater dough expansion [8]. However, a slight volume-depressing effect compared to control sourdough bread was observed in sourdough bread samples containing 10 g/100 g ASC and GASC flour (samples SA10 and SG10, Table 3), suggesting that this substitution level can be the highest applied without pronounced detrimental effect on specific volume. However, mentioned bread samples still had higher specific volumes than corresponding standard bread counterparts (Table 3).

The bake loss values for all bread samples ranged between 7.45% and 12.41% (Table 3). Although the lowest bake loss was observed in the control sourdough sample (7.45%), sourdough bread samples containing alfalfa flour exhibited greater bake loss compared to corresponding bread samples obtained by the standard bread-making process (Table 3). Overbake values were greater for standard bread samples containing ASC and GASC flour ranging between 46.5 and 53.26%, whilst the corresponding sourdough bread samples had lower overbake ranging between 42.99 and 50.67% (Table 3).

Crumb texture

The texture parameters of the standard and sourdough bread samples containing ASC and GASC flour are shown in Table 3. Conversely to the previous findings regarding hardness increase in bread enriched with non-germinated or germinated bean and yellow pea [27, 30], results obtained in the present study showed a decline in crumb hardness which was even more pronounced in sourdough bread samples (Table 3). It is assumed that the positive influence of alfalfa flour on crumb hardness can be ascribed to the dietary fibres presence (36.78 and 33.00% for ASC and GASC respectively, Table 1) and potential hydrogen bonding between fibre and starch enabling a delay in starch retrogradation. Furthermore, improved crumb softness was previously observed in breads from germinated soy, and explained by triggering the activity of hydrolytic enzymes during germination, especially the α-amylase activity which reduces amylopectin retrogradation and thus, the crumb firming rate [29]. Greater specific volumes of sourdough bread samples containing alfalfa flour were followed by lower crumb hardness (Table 3). Conversely, Rizzello et al. [30] reported an increase in crumb hardness in sourdough bread samples containing a blend of chickpea, lentil and bean. The observed difference can be a consequence of higher legume flour quantities used in mentioned studies (15–30 g/100 g) compared to the present study (5–10 g/100 g).

Alfalfa flour quantity exhibited a greater effect on crumb hardness than alfalfa flour type. Accordingly, a more pronounced decrease in hardness with increase in alfalfa flour content from 5 to 10 g/100 g was recorded in standard bread samples (samples A10 and G10, Table 3), whilst a reverse trend was observed in sourdough bread samples (samples SA10 and SG10, Table 3). This observation is supported by the Mixolab results where a lower torque value at point C5 were observed in blends containing 10 g/100 g alfalfa flour implying a reduced degree of starch retrogradation (Table 2). However, a greater crumb softening effect in sourdough bread samples containing 5 g/100 g alfalfa flour could be explained by the presence of proteins and fibres in an optimal amount to interact between themselves and with other dough constituents and provide better crumb texture and softness through the abovementioned mechanisms.

Changes in crumb cohesiveness (0.79–0.85) and springiness (0.95–0.97) upon inclusion of alfalfa flour were minimal, irrespective of alfalfa flour content and type, as well as bread type (Table 3). Presented cohesiveness and springiness values were higher compared to those obtained for sourdough bread containing non-germinated and germinated lentil flour fermented with different LAB strains [6]. Gumminess and chewiness decreased significantly in all alfalfa containing bread samples following the same trend as observed for hardness when different quantities of alfalfa flours were used, with generally lower values in sourdough bread samples (Table 3). Obtained results suggest the good capability of the bread crumb containing alfalfa flour to spring back and withstand deformation (high springiness and cohesiveness values), and be easily chewable at the same time (lower hardness, gumminess and chewiness values).

PCA analysis

The variability of the blends obtained as a result of the addition of processed alfalfa seed flour in terms of the examined characteristics was explained by two main components as much as 96.7%, which consists of PC 1: 88.26% and PC 2: 8.44% (Fig. 2a). The alfalfa flour (ASC, GASC) and wheat flour used differ significantly from each other (Fig. 2a). Their addition to wheat flour in the non-germinated and germinated form and amount of 5 and 10 g/100 g differentiated the mixtures to a small extent. However, differences were noted between individual blends with different amounts and alfalfa flour type.

Fig. 2
figure 2

Principle component analysis (PCA) showing score (a) and loading plot (b) describing the relationship between the flours and blends and their different characteristics. WF-wheat flour, ASC-alfalfa seeds convective dried, GASC-germinated alfalfa seeds convective dried, WF_5A-blend containing 5 g/100 g ASC, WF_10A-blend containing 10 g/100 g ASC, WF_5G-blend containing 5 g/100 g GASC, WF_10G-blend containing 10 g/100 g GASC. TDF-total dietary fibre content, TPC- total phenolic content, FRAP- Ferric Reducing Antioxidant Power Assay, ABTS-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-based assay

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There was a very strong correlation between the content of Mg, Zn, K and the content of protein, lipids, fibre, total phenolic, and the antioxidant (ABTS) and reducing (FRAP) potential of blends with the addition of alfalfa (Fig. 2b). The content of Na correlated with the content of Mg, Fe, ash, lipids and fibre.

The analysis of principal components explained 64.90% (PC1 48.02%; PC2: 22.82%) of the assessed variability of the bread characteristics obtained by the direct and indirect method from the WFAB (Fig. 3a). Performed PCA showed that both control samples (C, SC) differ from each other (Fig. 3a). It was also found that the greater the proportions of ASC and GASC flour in the blends, the smaller the differences in the bread obtained by different methods.

Fig. 3
figure 3

Principle component analysis (PCA) showing score (a) and loading plot (b) describing the relationship between the breads and their different characteristics. C-control wheat bread, A5-wheat bread containing 5 g/100 g ASC, A10-wheat bread containing 10 g/100 g ASC, G5-wheat bread containing 5 g/100 g GASC, G10-wheat bread containing 10 g/100 g GASC, SC-Control wheat sourdough bread, SA5-wheat sourdough bread containing 5 g/100 g ASC, SA10-wheat sourdough bread containing 10 g/100 g ASC, SG5-wheat sourdough bread containing 5 g/100 g GASC, SG10-wheat sourdough bread containing 10 g/100 g GASC

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Bread characteristics such as overbake, bake loss or specific volume correlated with other characteristics to a small extent (Fig. 3b). Specific volume correlated with bread crumb characteristics determined by TPA. It positively correlated with cohesiveness and springiness and negatively with gumminess, chewiness, and hardness.

Conclusions

This study assessed the effect of non-germinated and germinated alfalfa flour usage in bread-making through the determination of compositional, antioxidant and rheological properties of wheat flour-alfalfa blends and the physical and textural characteristics of the resulting standard and sourdough bread. Alfalfa seeds germination induced an increase in protein, Ca and Zn content, and decline in starch, Mg, K, Na, Fe, TPC and antioxidant activity, while ash and lipid content remained unchanged. Enhancement regarding the corresponding valuable compositional features was achieved in all wheat flour-alfalfa blends. However, blend with non-germinated alfalfa flour at 10 g/100 g inclusion level was established as the most valuable regarding nutrients. Although alfalfa flour used in the study did not greatly modify dough rheological properties, prolonged dough development time and C4 time with the lower torque value at point C5 was observed when 10 g/100 g of alfalfa flour was incorporated. Overall, improvements in bread specific volume and crumb softness were obtained regardless of the alfalfa flour type used and were rather more dependent on its quantity and produced bread type. Sourdough bread samples were characterised by greater specific volumes and lower hardness compared to standard bread samples at the same alfalfa flour type and inclusion level. The reported findings evidenced improvements in nutrients composition of blends and specific volume and crumb softness of bread indicating that both non-germinated and germinated alfalfa flour are promising nutrient-rich ingredients with nutritional and health benefits that can be imparted into bakery products which is the subject of further research.