Analytical Characterisation of Oat-Enriched Binary Composites of Wheat Flour and Their Processing Behaviour in Bread Making

Abstract

Oats (Avena sativa L.) are a rich source of β-d-glucans, dietary fibre, proteins, and lipids. However, the behaviour of these components in wheat–oat composite systems during baking, particularly with regard to matrix-dependent analytical responses, remains unclear. This study evaluated the compositional changes, technological performance, and sensory quality of wheat bread enriched with various forms of oat. Composite flours containing 5–15% wholegrain oat flour, commercial oat bran, milled commercial oat flakes, or milled sprouted oat grain (sprouted under laboratory conditions for three days at 25 °C) were prepared using the Slovakian oat cultivar ‘Peter’. The raw materials, flour blends, and baked breads were analysed for β-d-glucans (BG), total dietary fibre (TDF), starch, proteins, and lipids using standardised enzymatic, gravimetric, and polarimetric methods. Bread quality was assessed through loaf volume measurements and a sensory evaluation using a 5-point hedonic scale by seven trained panellists. Multivariate statistical analysis was applied to integrate compositional, technological, and sensory data. Compared to wheat flour (0.24% BG and 3.45% TDF), the incorporation of oats significantly increased the contents of BG, TDF, proteins, and lipids, with oat bran showing the strongest enrichment effect (owing to 15.69% TDF in the raw material). Baking induced oat-form-dependent changes in the measured BG and TDF content. The level of BG diminished in wholegrain oat blends but increased or remained stable in bran-rich systems. This reflects differences in matrix structure and analytical extractability, rather than true compositional gains. Meanwhile, starch content consistently declined across all composite breads. Fibre-rich formulations exhibited reduced loaf volume and altered both bread geometry and morphology, particularly at 15% substitution. Breads containing 5% oat flour or moderate levels of oat bran (5 or 10%) were considered the most acceptable in terms of nutritional enhancement and quality attributes. Germinated oat breads showed the greatest technological impairment and the lowest sensory scores. Overall, moderate oat enrichment strikes a balance between nutritional improvement and technological performance without significantly compromising sensory quality. These findings emphasise the significance of matrix effects when interpreting standard total dietary fibre and β-d-glucans analyses and offer an integrated analytical and technological framework for the rational design of fibre-enriched cereal products.

1. Introduction

The oat (Avena sativa L.) is increasingly recognised as a cereal with significant nutritional value due to its high protein content, its high content of unsaturated fatty acids, and its high dietary fibre content, particularly β-d-glucans (BG) [1,2]. In addition to these primary constituents, oats contain secondary metabolites that are relevant from a physiological perspective, such as the antioxidant-active compounds known as avenanthramides [3,4]. Continuous research has identified bioactive molecules and extraction strategies relevant to the development of functional foods [5,6,7].

A major nutritional advantage of oats lies in their protein composition. Unlike wheat and related cereals, oats contain storage proteins of predominantly globulin type, with a favourable essential amino acid profile and higher levels of lysine [8,9]. The lipid fraction comprises both neutral and polar lipids that exhibit antioxidant and emulsifying properties [10,11,12,13]. However, the high activity of endogenous lipolytic enzymes requires stabilisation through heat treatment to prevent hydrolysis and oxidation during storage [14,15].

Oats are also an important source of total dietary fibre (TDF), which includes insoluble fibre (IDF) and soluble fibre fractions (SDF). The composition and content of these fractions vary depending on the cultivar and processing method used, which in turn influence both the physiological and functional properties [16,17]. Dietary fibre contributes to the water binding of flour, the rheology of aqueous dispersion or dough, and the sensory properties of the final product; thus, it is widely used in the development of functional cereal products [18,19]. Among the fibre components, BG is particularly of importance. Their concentration varies from 1.8 to 7.0% across different oat cultivars [20,21], and their physiological effects depend on factors such as molecular weight, conformation, and viscosity in water [22,23,24,25].

The target polysaccharides BG have been linked to a reduced glycaemic response, lower cholesterol levels, modulation of the immune system, anti-inflammatory effects, and antioxidant activity [26,27], corresponding to the internal conformation. They also exhibit prebiotic properties and influence gut microbial metabolism [28,29].

Technological processing has a significant impact on the structure, solubility, and extractability of oat fibre components. Germination, fermentation, thermal treatment, and extrusion can alter the molecular weight of BG, their depolymerisation kinetics, and their interactions with the matrix [30,31,32,33]. During fermentation and baking, endogenous cereal enzymes can partially degrade polysaccharides, including BG, thereby reducing their molecular weight and influencing their physiological and functional properties [34,35,36]. The extent of these modifications depends strongly on the food matrix and the intensity of the raw material processing.

In wheat-based systems, incorporating oat fractions markedly affected dough water absorption, rheology, and bread quality. Oat flour, flakes, and bran increase hydration requirements and often reduce loaf volume at higher substitution levels [37,38]. However, these ingredients also enhance the nutritional value of bread by increasing just fibre and BG content [39,40,41]. While many studies have examined the effects of individual oat forms and fractions, few have only compared the impact of oat flour, bran, flakes, and germinated grain in a standardised study. Even fewer have combined nutritional profiling with the evaluation of processing effects and analytical behaviour in composite flour systems.

This represents a significant analytical gap and research opportunity. Standard enzymatic and AOAC methods for determining BG and total fibre have been primarily validated for relatively simple cereal matrices. The efficiency, robustness, and metrological accuracy of their extraction in composite wheat–oat systems with different processing histories (e.g., germination, fermentation, and baking) are not well described. Therefore, it is unclear the extent of the following: (i) reflecting the true composition in measured values; and (ii) influence of the matrix heterogeneity—alterations in macromolecular architecture propagate to changes in solubility, enzymatic activity, etc. Furthermore, the extent to which individual oat fractions differ in their analytical response and measurability after baking has not been systematically examined. Clarifying these matrix-dependent differences is essential for both cereal chemistry and the rational development of functional foods. Without such data, it is challenging to accurately assess the nutritional impact of various oat ingredients due to methodological and matrix effects that confound comparisons across studies. We hypothesise that the morphological and structural characteristics of oat components (flour, flakes, bran, and germinated grain) have a differential effect on the analytical accessibility of TDF and BG. This leads to apparent differences after thermal processing that reflect matrix-dependent analytical recovery rather than true compositional changes. Oat germination is associated with significant enzymatic and structural changes that may affect the accessibility of TDF and BG for analysis. The impact of these changes within oat composite systems after processing remains insufficiently explored.

The oat ingredients were selected to represent different material states, rather than to maximise compositional differences. Wholegrain flour, bran, flakes, and germinated grain differ primarily in terms of structural integrity and enzymatic modification, factors that affect hydration, enzyme accessibility, and polysaccharide extractability. A substitution level of 5–15% was defined as a technologically relevant range, allowing the assessment of structural and matrix effects without the introduction of significant processing artefacts or quality loss.

This study aims to comprehensively characterise the nutritional composition of wheat flour, commonly used oat forms, and their wheat–oat composite flours; it quantifies changes in major components, particularly BG, before and after baking; and evaluates differences in analytical response across matrices using identical standardised procedures. The goal is to describe the matrix effects; this study therefore provides the necessary data for the rational optimisation of functional bakery products, deepening our understanding of the limitations and capabilities of routine analytical methods for determining fibre and BG in technologically heterogeneous cereal systems.

2. Materials and Methods

2.1. Oat Cultivar and Derived Fractions

The selected oat ingredients—flour, flakes, bran, and germinated grain—were chosen to represent various levels of structural integrity and processing intensity relevant to cereal and bakery applications.

A set of 100 oat cultivars (Avena sativa L.), screened within the APVV-17-0113 project at the Vígľaš-Pstruša Breeding Station (Detva, Slovakia), was evaluated for TDF, BG, starch, protein, and lipid contents. The Slovak, yellow-hulled cultivar ‘Peter’ was selected, and dehulled grains produced at the same site (National Agricultural and Food Centre—Research Institute of Plant Production, Piešťany, Slovakia) were used to prepare wholegrain oat flour and germinated (sprouted) grain. Wholegrain flour was obtained by grain milling to a uniform particle size (ZM 100 ultra-centrifugal mill, Retsch GmbH., Haan, Germany). Germinated oat grains were prepared by soaking in distilled water for 12 h, incubating at 25 °C in darkness for 3 days with daily rinsing, and then finalised by drying and milling using the same device.

Commercial fine oat flakes (Mlyn Štúrovo, Štúrovo, Slovakia) and oat bran (Country Life, Prague, Czech Republic) were milled and sieved through a 0.5 mm screen using a ZM 100 ultra-centrifugal mill and a KS 1000 vibratory sieve shaker (Retsch GmbH., Haan, Germany). All oat materials were stored in sealed containers at 18 °C in darkness till further tests. Refined white wheat flour (WF), produced by commercial company Mlyn Pohronský (Ruskov, Slovakia), served as the control. Listed oat forms are coded as OFLO for the oat wholegrain flour (manufactured from a natural, non-treated grain), OB for the oat bran, OFLA for the oat flakes flour, and OSG for the germinated oat wholegrain flour, respectively. Composite wheat–oat flours were prepared by substituting wheat flour with 5, 10, or 15% (w/w) of the four supra-listed oat forms (wholegrain oat flour, bran, milled flakes, or germinated oat wholegrain flour) selected to ensure technological feasibility and comparability across all composite systems. All flour binary blends were homogenised horizontally on the BS–P06 device (Mezos, Hradec Králové, Czech Republic), pre-set to 100 RPM and 30 min. In cases of discussion of the results related to the breads (baked forms of the control WF and the blends WF + OFLO, … WF + OSG, letter ‘b’ was appointed (e.g., WF + 5OFLAb). After baking and sensory evaluation, the loaves were cut into 1 × 1 cm cubes manually and dried at 21 °C for 3 days, turning them twice a day to prevent uneven drying and mould formation. After drying, they were ground in a mill under the same conditions as other oat ingredients and stored similarly until the date of analysis. All plant samples (control flour, oat ingredients, flours, and loaves) were analysed using the same procedures and under the same conditions.

2.2. Considered Samples Analytes

Moisture was determined in duplicate using a semi-automatic moisture analyser (MA 45, Sartorius, Göttingen, Germany). The sample was dried at 110 °C until a constant weight was achieved, following the gravimetric AOAC Official Method 925.10 [42].

Protein content was measured in duplicate using a TruMac CNS analyser equipped by the Cornerstone software version 3.1.0 (LECO Corporation, St. Joseph, MI, USA), based on the Dumas method. Approximately 200 mg of homogenised samples were combusted, total nitrogen was quantified, and converted to protein using ‘N-to-protein’ factors 5.70 for wheat and 5.83 for oat in accordance with AOAC Official Method 992.23 [43].

The target polysaccharides BG were quantified enzymatically using the Mixed-linkage β-glucan Assay Kit (K-BGLU, Megazyme, Wicklow, Ireland) following the AOAC Official Method 995.16 [44], based on the procedure by McCleary and Codd [45]. Reagents included lichenase, β-glucosidase, and GOPOD (glucose oxidase, peroxidase, 4-aminoantipyrine). For each analysis, 100 mg of homogenised samples were suspended in 50% ethanol and 20 mM sodium phosphate buffer (pH 6.5), heated in a boiling water bath, and then incubated with lichenase at 50 °C. After dilution in 50 mM sodium acetate buffer (pH 4.0) and centrifugation (Universal 320R, Hettich Holding GmbH. & Co. KG, Kirchlengern, Germany), aliquots were incubated with β-glucosidase. Released d-glucose was quantified spectrophotometrically at 510 nm (SPEKOL 11, Carl Zeiss, Jena, Germany). The content of BG was expressed as a percentage of dry matter using a d-glucose standard.

The analyte TDF was determined using the enzymatic–gravimetric method according to AOAC Official Method 991.43 [46] using the Megazyme Total Dietary Fibre Assay Kit (K-TDFR, Megazyme, Wicklow, Ireland). Samples (1.0 g, in duplicate) were digested sequentially with thermostable α-amylase (100 °C), protease (60 °C), and amyloglucosidase in MES/TRIS buffer. Ethanol (96%, 60 °C) was added to precipitate the fibre. The residue was filtered through Celite-coated crucibles (Celite; Centralchem, Bratislava, Slovakia), washed with 78% and 96% ethanol and acetone, and dried at 103 °C in a laboratory oven (Memmert GmbH + Co. KG, Schwabach, Germany). The final residue was weighed and corrected for protein content, which was determined by the Dumas combustion method in accordance with AOAC Official Method 992.23 [43] using a TruMac CNS analyzer (LECO Corporation, St. Joseph, MI, USA).

Lipids were extracted using a hexane-based protocol adapted from Letellier and Budzinski [47] and Mandal et al. [48]. Briefly, 5.00 ± 0.01 g of sample was extracted in duplicate with n-hexane (Labo SK, Bratislava, Slovakia) under vigorous shaking, followed by centrifugation. The extracts were filtered through anhydrous sodium sulphate and evaporated to dryness using a rotary evaporator (Laborota 4001, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Lipid content was expressed as a percentage of dry matter.

Total starch was determined polarimetrically using the Ewers method in accordance with ISO 10520:1997 [49]. The milled sample (5.00 ± 0.01 g) was hydrolysed with diluted HCl, clarified with Carrez solutions I and II, filled to 100 mL, and filtered. Optical rotation was measured at 20 °C in a 200 mm tube (P 3001RS polarimeter, A. Krüss Optronic, Hamburg, Germany). Starch content was calculated using conversion factors of 5.47 for wheat and 5.51 for oats [49].

2.3. Preparation of Breads in a Semi-Industrial Regime

Bread dough variants were prepared from 250.00 g WF or binary blend, 12.50 g retail compressed baker’s yeast (Saccharomyces cerevisiae; commercial), 3.75 g kitchen salt, 2.50 g sugar, and 2.50 g commercial pork lard (ingredients as 5.0, 1.5, 1.0, and 1.0 wt.% of the WF basis, respectively). Water addition was adjusted according to the farinograph absorption (Farinograph-E, Brabender, Duisburg, Germany). Doughs were mixed in an RM 800 A-B mixer (Gastrolux, Ryomgaard, Denmark), fermented for 30 min at 32 °C and 80% RH in a BT 12 thermostat (1-CUBE, Havlíčkův Brod, Czech Republic), and divided into five pieces of the same weight. Moulding was carried out manually into small, rounded breads (buns) and proofed for 25 min at 32 °C and RH 70–80% in the above-mentioned device.

Buns were baked in a steam-injected deck oven (DOMINO, Bratislava, Slovakia) at 230 °C for 15 min and cooled down freely to room temperature for 2 h. Buns’ weight was recorded, and volume was determined by the rapeseed displacement method to express the specific bread volume (internal repeatability ± 15 cm³∙100 g⁻¹); the sensory evaluation followed. The sensory evaluation was conducted in accordance with the general recommendations for sensory testing environments, under controlled conditions ISO 8589:2007 [50], although the choice of hedonic scale and panel size followed study-specific design considerations. A trained sensory panel (N = 7), composed of assessors with long-term experience in bread quality evaluation, evaluated bread profiles using a five-point hedonic scale (1 pt.—the worst, 5 pt.—the best score) for 12 descriptors in total. Panellists were regularly trained persons and ‘calibrated’ using a reference bread sample before each of four evaluations. The simplified scale was chosen to facilitate practical interpretation in a technological context, which was considered sufficient for a trained panel. Samples were coded, evaluated under standardised laboratory conditions, and presented in a fixed order to ensure comparability. Assessed attributes included the bread’s overall appearance and shape, crust attributes (colour, thickness, and hardness), and the crumb ones (aroma, taste, elasticity, porosity, colour, firmness, and stickiness of mouthful), plus the overall impression. The partial results were summed into the 12th parameter sensory score (profile), i.e., to a point sum of all 11 attributes in a range of 15 to 55 pts. Considering the maximum of 55 pts. as the optimal quality, a relative comparison of the alternative products against the wheat control by the sensorial acceptability index was also carried out.

2.4. Statistical Analysis

Data were analysed using IBM SPSS Statistics v. 22. Descriptive analysis of variance (three-factor ANOVA) was applied to reveal the differences in single quality parameters statistically, and Tukey’s HSD test was used for such post hoc comparisons (p = 95%). The considered factors were the Oat form (OFLO, OB, OFLA, OSG) and Oat addition as the WF replacement (0, 5, 10, or 15%) plus the Physical stage of flour (unbaked ~ natural, baked ~ bread). For an illustration of overall data dissimilarity among the main sample groups WF, WF + OFLO, … WF + OSG, WFb, WF + OFLOb, …, and WF + OSGb, the factor Oat addition was suppressed in the second ‘groups’ ANOVA calculation. In tables, all items of the ANOVA significance longer than 3 letters were shortened to the first and the last letter (e.g., samples cluster ‘ab’ or ‘abc’ was maintained, but ‘abcd’ was shortened to ‘a-c’; for the ‘groups’ two-factor ANOVA, similarly, ‘ABCD’ was shortened to ‘A-D’).

Relationships among variables (the quality parameters) were described by a correlation matrix (p = 95%); the ones between the variables and cases (the samples tested) were explored using Principal Component Analysis, in which both plots of variable scores and sample loadings were constructed. In the PCA plots, a system of concentric circles was newly applied to allow a rough estimation of the statistical importance of the variables or the statistical similarity (inter-closeness) of the samples tested.

3. Results

3.1. Composition of Raw Materials

The wheat flour control contained 0.24% β-d-glucans (BG), 3.45% total dietary fibre (TDF), 1.45% lipids, 78.28% starch, and 14.74% protein (dry matter basis; Figure 1,

Table 1. Effect of addition of four forms of oat and heat treatment on basic chemical composition of wheat flour (WF), for form of oat and wheat–oat binary blends (ANOVA, p = 95%).

Sample Form			Flour, Flour Mix, Bread	Oat Add. (%)	Lipids (%)				Starch (%)				Proteins (%)
Flour	Flour type	Control	WF	0	1.45	a–f	1.45	AB	78.28	n	78.28	D	14.70	d–i	14.70	A
		Oat form	OFLO	100	3.22	i	5.07	C	61.32	b	60.00	A	12.28	a	15.67	AB
			OB	100	5.81	j			51.17	a			17.69	n
			OFLA	100	5.55	j			64.79	cd			14.04	b
			OSG	100	5.72	j			62.74	bc			18.68	o
	Binary blend		WF-OFLO	5	1.72	c–h	1.89	AB	77.27	mn	75.39	D	14.82	g–j	14.87	A
				10	1.93	e–h			74.92	jkl			14.87	g–k
				15	2.02	fgh			74.00	ijk			14.93	i–l
			WF-OB	5	1.68	c–h	1.96	B	75.29	j–m	74.36	BCD	14.73	e–i	14.84	A
				10	1.96	fgh			74.45	jkl			14.87	h–k
				15	2.25	h			73.35	ij			14.92	i–l
			WF-OFLA	5	1.68	c–h	1.94	B	77.49	mn	76.36	D	14.59	c–g	14.52	A
				10	1.93	e–h			76.16	k–n			14.53	c–f
				15	2.21	h			75.43	j–m			14.43	cd
			WF-OSG	5	1.69	c–h	1.87	AB	76.37	lmn	75.59	D	14.81	f–j	14.94	A
				10	1.85	e–h			76.14	k–n			14.94	i–l
				15	2.08	gh			74.25	jkl			15.08	j–m
Bread	Flour		WFb	0	1.56	b–g	1.56	AB	71.71	hi	71.71	ABCD	14.74	e–i	14.74	A
	Binary blend		WF-OFLOb	5	0.95	a	1.02	A	69.46	fgh	69.97	AB	14.94	i–l	15.09	AB
				10	1.14	abc			69.40	fgh			14.97	i–l
				15	0.97	ab			66.97	de			15.36	m
			WF-OBb	5	1.74	d–h	1.81	AB	70.69	fgh	68.61	A	14.72	e–i	14.99	A
				10	1.82	e–h			69.29	fg			15.14	klm
				15	1.86	e–h			69.92	fgh			15.10	klm
			WF-OFLAb	5	1.36	a–e	1.43	AB	70.50	fgh	70.52	ABC	14.60	c–h	14.48	A
				10	1.36	a–e			70.99	gh			14.51	cde
				15	1.58	c–g			70.08	fgh			14.34	c
			WF-OSGb	5	1.22	a–d	1.34	AB	69.90	fgh	69.15	A	14.77	ei	14.97	A
				10	1.35	a–e			68.92	efg			14.97	i–l
				15	1.45	a–f			68.63	ef			15.18	lm

Note: a–o: in columns, different letters identify a statistically significant difference among samples (N = 30, p = 95%). A–D: different letters identify a statistically significant difference among the WF and averages of 5 binary flour blends plus the resulting breads (N = 10, p = 95%).

). In contrast, all oat-derived raw materials exhibited substantially higher BG, TDF, and lipid contents, accompanied by markedly lower starch concentrations (Figure 1, Table 1).

Among the oat fractions, oat bran had the highest BG content (8.19%), followed by wholegrain oat flour (4.97%), oat flakes (3.97%), and germinated oat flour. Contents TDF ranged from 10.11% in germinated oat flour to 15.69% in oat bran, representing a three- to fivefold increase compared to wheat flour. Lipid content ranged from 3.22% to 5.81%, again exceeding the control value. Conversely, starch concentrations were logically the lowest in oat bran (51.17%), ranging between approximately 62% and 65% in the remaining oat forms. Protein content varied from 12.28% in wholegrain oat flour to 18.68% in germinated oat flour, indicating substantial compositional modification during germination (Figure 1, Table 1).

These pronounced differences establish compositionally distinct analytical matrices, particularly in terms of fibre enrichment, starch dilution, and lipid co-occurrence, which underpin the behaviour of composite flours and breads examined below.

3.2. Total Dietary Fibre (TDF) in Composite Flours and Breads

The TDF content of all composite flours increased significantly compared with wheat flour (Figure 2). The highest value was observed in the formulation containing 15% oat bran (TDF 6.56%) in agreement of the lowest starch content in this form of oat. Positively, baking did not alter the TDF content of the wheat control flour (3.45%), suggesting that thermal treatment alone does not affect the refined wheat matrix.

In contrast, several composite systems exhibited higher TDF proportions after baking than in the corresponding flour blends. This effect was most pronounced in the formulation containing 15% wholegrain oat flour, where TDF increased from 4.31% to 5.76%. Oat bran produced substantial TDF enhancement from 10% substitution onwards, both before and after baking. Oat flakes and germinated oat flour resulted in smaller increases at low inclusion levels. However, formulations containing 10 or 15% oat bran showed clear and statistically significant TDF enrichment, with baking accentuating the differences relative to the control formulation (Figure 2).

Flour TDF was strongly correlated with bread TDF across all formulations (r = 0.85, p = 99.9%; § 3.6 Statistical Exploration of Data), indicating a close quantitative relationship between fibre levels in flour blends and the corresponding baked products.

3.3. β-d-Glucans in Composite Flours and Breads

The addition of oat fractions at levels of 5–15% significantly increased the measured BG content of composite flours compared to wheat flour (Figure 3), with values ranging from 0.45% to 1.21% prior to baking. The highest enrichment was consistently observed in the 15% oat bran formulation, similarly to the case of TDF.

In the wheat control, baking caused only a minor, non-significant reduction in BG content (from 0.24% to 0.19%), indicating limited thermal effects on BG recoverability in the refined wheat matrix. In composite systems, the response to baking was strongly fraction-dependent. Wholegrain oat flour blends showed consistent decreases in measured BG after baking across all inclusion levels. In contrast, wheat–oat bran blends exhibited a nonlinear response, with reduced BG at 5% substitution and increased measured BG at 10% and 15% substitution. Oat flake blends showed stable or slightly increased BG values after baking, whereas germinated oat blends exhibited minor decreases at higher substitution levels and a slight increase at 5% substitution (Figure 3).

Flour BG content was strongly correlated with bread BG and negatively correlated with specific bread volume (r = 0.89 and r = −0.75, respectively; p = 99.9%; § 3.6). Our results highlight the close relationship between bread’s technological performance and its BG enrichment.

3.4. Macronutrient Composition of Composite Flours and Breads—Starch, Protein, and Lipids

The incorporation of oat-derived fractions substantially altered the macronutrient composition of composite flours and breads compared to the wheat control (Table 1). Across all formulations, the starch content decreased due to dilution by non-starch components. Baking reduced the starch content of the wheat control from 78.28% to 71.71% (Table 1). In wholegrain oat flour blends, the starch content generally diminished with increasing substitution, although a slight numerical increase was observed at a 15% substitution compared to 10%. Oat bran formulations exhibited the lowest starch content consistently, while germinated oat blends showed pronounced reductions in starch content from ≥10% substitution onwards, which persisted after baking (Table 1). Flour starch content was negatively correlated with flour TDF and BG (r = −0.71 to −0.77, p = 99.9%) and positively correlated with specific bread volume (r = 0.60, p = 99%). This suggests that starch dilution is a factor in determining technological performance.

The protein content increased in most composite flours, particularly in those containing wholegrain oat flour, oat bran, and germinated oat flour. The highest value was observed in the formulation containing 15% wholegrain oat flour (14.90%; Table 1). Baking induced formulation-specific changes without a consistent directional trend. Significant protein enrichment relative to the control occurred in oat bran and germinated oat blends with ≥ 10% substitution; however, oat flake blends consistently exhibited lower protein content (Table 1). Flour protein content was negatively correlated with bread shape (h/d; r = −0.60, p = 99%) and sensory acceptability (r = −0.44, p = 95%).

Lipid content increased in all composite flours compared to the wheat control, with the greatest enrichment observed in oat bran and germinated oat flour blends (Table 1). Baking slightly increased the lipid content of the control flour, whereas lipid concentrations decreased after baking in the wholegrain and germinated oat blends, though they remained above control values. Oat bran breads retained the highest lipid content after baking at 15% substitution (Table 1). Flour lipid content showed a strong positive correlation with flour BG (r = 0.78, p = 99.9%) and a negative correlation with specific bread volume (r = −0.42, p = 95%), suggesting an interplay between lipid-rich fractions and fibre-driven technological properties. All supra mentioned correlations are summarised into the correlation matrix (§ 3.6).

3.5. Baking Trial Results and Sensory Evaluation

The overall appearance of the control WF bread together with all 12 wheat–oat variants was documented by a digital camera. The physical parameters of the bread variants are summarised in

Table 2. Wheat bread physical parameters as affected by the addition of four forms of oats at three replacement levels.

Flour, Flour Mix	Oat Add. (%)	Bun Height (cm)				Bun Diameter (cm)				Bread Weight * (g)				Bread Volume * (cm3)
WFb	0	5.7		e; C		8.7		abc; AB		355.1		a; AB		1170		e; B
WF + OFLOb	5	4.3	bcd	4.1	A	9.1	bc	9.1	B	356.8	a	358.4	B	1130	de	1028	AB
	10	4.1	a–d			9.3	cd			358.2	a			1035	bc
	15	3.9	abc			9.0	abc			360.3	a			920	a
WF + OBb	5	5.0	de	4.8	B	8.7	abc	8.6	A	361.6	a	362.8	B	965	ab	1000	A
	10	4.6	cd			8.4	a			360.0	a			990	ab
	15	4.9	de			8.7	abc			366.8	a			1045	bcd
WF + OFLAb	5	4.9	de	4.8	B	8.7	abc	8.7	A	355.0	a	356.2	B	1055	bcd	1045	AB
	10	4.8	d			8.6	ab			358.7	a			1030	bc
	15	4.6	cd			8.7	abc			355.0	a			1050	bcd
WF + OSGb	5	4.2	a–d	3.7	A	9.7	de	10.0	C	339.6	a	340.5	A	1130	de	1097	B
	10	3.6	ab			10.3	e			342.2	a			1110	cde
	15	3.4	a			10.2	e			339.6	a			1050	bcd

Note: WF—wheat flour; Oat forms: OFLO—wholegrain oat flour; OB—oat bran; OFLA—(milled) oat flakes; OSG (milled) sprouted oat grain; “b” means “baked”—e.g., WF + 5OSGb means bread baked from this binary flour blend. *: For both parameters, 5 pieces of small bread (buns) measured together; a–e: in columns, averages of 13 bread samples signed by the same letter are not statistically different (p = 95%). A–C: In columns, averages of 5 groups of samples signed by the same letter are not statistically different (p = 95%).

. The wheat control sample had the greatest loaf height and bread volume, reflecting the desired optimal technological performance. Composite breads generally showed reduced bun height and volume, with the most pronounced reductions observed in the oat bran and germinated oat formulations, particularly at ≥10% substitution. It can be deduced by reasoning that the reduction in the wheat dough elasticity and, conversely, its extensibility, caused by all forms of oats tested, group-averaged height-to-diameter ratios confirmed progressive loaf flattening in these complex biomaterial systems (Figure 4 and Figure 5).

The sensory evaluation results are partially presented in

Table 3. One and two-factor ANOVA of the selected results of sensory analysis—effect of fortification of wheat bread by four types of milled oat grain at three distinctive levels (p = 95%).

Bread Variant	Oat Add. (%)	Bread Shape and Appearance (pts.)				Crust Colour (pts.)				Crust and Crumb Taste (pts.)				Crumb Stiffness (pts.)
WFb	0	5.00 d; C				4.86 c; B				4.14 a; AB				4.43 a; A
WF + OFLOb	5	4.00	bcd	3.86	B	4.86	c	4.67	B	4.43	a	4.08	AB	4.57	a	4.14	A
	10	3.86	bcd			4.71	bc			4.10	a			4.00	a
	15	3.71	bc			4.43	abc			3.71	a			3.86	a
WF + OBb	5	4.86	cd	4.76	C	4.86	c	4.81	B	4.71	a	4.81	B	4.29	a	4.29	A
	10	4.71	cd			4.57	abc			5.00	a			4.14	a
	15	4.71	cd			5.00	c			4.71	a			4.43	a
WF + OFLAb	5	4.29	cd	4.33	BC	4.57	abc	4.62	B	4.71	a	4.71	B	3.86	a	3.95	A
	10	4.57	cd			4.71	bc			4.71	a			3.86	a
	15	4.14	bcd			4.57	abc			4.71	a			4.14	a
WF + OSGb	5	3.00	ab	2.38	A	3.57	a	3.67	A	3.86	a	3.76	A	4.14	a	4.00	A
	10	2.13	a			3.71	ab			3.71	a			4.00	a
	15	2.00	a			3.71	ab			3.71	a			3.86	a

Note: Bread variant: WFb—wheat control; WF + OFLOb—wheat–oat flour one; WF + OBb—wheat–oat bran one; WF + OFLA—wheat–oat flakes one; WF + OSG—wheat–oat sprouted-grain one. a–d: between 13 bread samples, averages in columns, signed by the same letter, are not statistically different (p = 95%). A–C: between 5 bread groups, averages in columns, signed by the same letter, are not statistically different (p = 95%).

and by radar plots. The wheat control achieved the highest scores across all attributes. Wheat-wholegrain oat flour breads retained relatively favourable flavour and aroma profiles, whereas oat bran and germinated oat counterparts showed significant declines in terms of shape, crumb texture, and overall acceptability (Table 3, Figure 5, Figure 6 and Figure 7). Among composite breads, 5% substitution consistently yielded the highest acceptability within each oat fraction (Table 3, Figure 7). Distribution and variability analyses further demonstrated increasing dispersion and reduced sensory performance with higher substitution levels, particularly for bran and germinated oat formulations (Figure 5 and Figure 6). In detail, differences among the sensory attributes of the tested breads offer the radar plots (Figure 8).

A multivariate analysis that integrated compositional, technological, and sensory variables revealed a primary axis characterised by fibre-related parameters (TDF and BG). Bread structure and sensory attributes, meanwhile, were captured along an orthogonal component (Figure 9 and table of communalities).

Based on 91 sensory scores, the summary sensory index called sensory score demonstrated a normal distribution of the 88% of input data (scores 40–55 pts.), as the 12 scores in range 25–40 pts. could be considered to be randomly occurring outliers. The most frequent category was the one with an average of 44 pts., representing 45% of the responses. Scores in intervals of 40–45 pts. and 50–55 pts., which represented 20% and 23% responses, were statistically equal in their percentages of all collected answers (20% and 23%, respectively; Figure 7).

3.6. Statistical Exploration of Data

In the correlation matrix (

Table 4. Correlation analysis between the wheat–oat flour composition, bread physical parameters, and wheat–oat bread composition.

Variable	Flour TDF		Flour BG		Flour Lipids		Flour Starch		Flour Proteins		Bread Shape h/d		Bread Specific Volume		Bread Sensory Acceptability
Flour TDF	1		0.72	***	0.68	***	−0.71	***	0.26	ns	0.13	ns	−0.44	*	0.18	ns
Flour BG	0.72	***	1		0.78	***	−0.77	***	0.08	ns	0.07	ns	−0.75	***	0.08	ns
Flour lipids	0.68	***	0.78	***	1		−0.75	***	0.15	ns	−0.27	ns	−0.42	*	−0.04	ns
Flour starch	−0.71	***	−0.77	***	−0.75	***	1		−0.46	*	0.31	ns	0.60	**	0.18	ns
Flour proteins	0.26	ns	0.08	ns	0.15	ns	−0.46	*	1		−0.60	**	0.02	ns	−0.44	*
Bread shape h/d	0.13	ns	0.07	ns	−0.27	ns	0.31	ns	−0.60	**	1		−0.21	ns	0.54	**
Bread specific volume	−0.44	*	−0.75	***	−0.42	*	0.60	**	0.02	ns	−0.21	ns	1		−0.12	ns
Bread sensory acceptability	0.18	ns	0.08	ns	−0.04	ns	0.18	ns	−0.44	*	0.54	**	−0.12	ns	1
Bread TDF	0.85	***	0.82	***	0.82	***	−0.81	***	0.27	ns	−0.13	ns	−0.57	**	0.05	ns
Bread BG	0.86	***	0.89	***	0.76	***	−0.68	***	−0.05	ns	0.21	ns	−0.67	***	0.19	ns
Bread lipids	0.63	***	0.22	ns	0.21	ns	−0.26	ns	−0.11	ns	0.48	*	−0.18	ns	0.28	ns
Bread starch	−0.70	***	−0.54	**	−0.63	***	0.69	***	−0.59	**	0.37	ns	0.23	ns	0.28	ns
Bread proteins	0.37	ns	0.29	ns	0.23	ns	−0.53	**	0.92	***	−0.45	*	−0.24	ns	−0.30	ns

Note: TDF–total dietary fibre, BG—β-d-glucans; ^ns, *, **, ***: pair correlation non-significant (p < 95%), or significant at p = 95%, 99% or 99.9%, respectively (N = 26).

), there were 48 non-significant pair relationships and 10, 10, and 28 significant at p = 95%, 99%, or 99%. The percentages from a total count of 96 correlations are as follows: 50, 10, 10, and 30%, respectively. In the upper part of Table 4, as presumed, those frequencies confirm a tight interdependence of the rates of the analytes tested in flour, which logically predetermines their proportion in yeasted bread (left bottom corner of the matrix). Three basic constituents’ threesome—TDF, BG, and lipids—showed a negative correlation with bread specific volume; only the starch as a source of fermentable sugars supported the dough (right bottom corner of Table 4). Although the count of verifiable correlations was lowered by a subjective character of the sensory analysis, both the PCA plot of the variable scores and sample loadings (Figure 9a, Figure 9b, respectively) allow a meaningful interpretation (PCA based on the correlation matrix).

The first and the second principal components (PCs) covered 48 and 24% of the input data variability, overcoming a recommended border of 70%; PC3 absorbed 9% of data variability, but its interpretation is not necessary in general (

Table 5. Communalities, a percentage of explained data scatter by the first three principal components (PC).

Variable	PC1	PC2	PC3	Sum PC1–3
Flour TDF 1	79%*	5%	11%	95%
Flour BG 2	78% *	5%	9%	92%
Flour lipids	70% *	0%	4%	74%
Flour starch	82% *	3%	1%	86%
Flour proteins	14%	67% *	8%	89%
Bread shape h/d 3	2%	73% *	3%	78%
Bread specific volume	41% *	8%	18%	67%
Bread sensory acceptability	0%	49% *	1%	50%
Bread TDF 1	87% *	0%	0%	88%
Bread BG 2	75% *	16%	2%	93%
Bread lipids	15%	27%	47% *	89%
Bread starch	60% *	10%	12%	82%
Bread proteins	26%	43% *	1%	70%
Average	48%	24%	9%	81%

^{1, 2} Total dietary fibre and β-d-glucan content, respectively; ³ bread shape (vaulting) as the height-to-diameter ratio. Note: * Pair correlation of the variable and principal component is significant at p = 95% (other pair correlations were statistically insignificant); underlined italics value format signifies a negative relationship within the proper pair.

). This third PC has a middle tight relation to the specific bread volume but a crucial role for the bread lipid content (18 and 47% of the feature scatter, respectively). Primarily, the PC1 is related to the polysaccharide constituent of the wheat–oat flour composites, confirming a positive effect of starch content on the specific bread volume (vice versa for TDF and BG). The second coordinate, PC2, is connected with protein and lipid content, which determines the loaf shape (the h/d ratio) and results of sensory testing (Figure 9a). Variability of the specific bread volume was unequally distributed between PC1 and PC5 (41, 8, 18, 15, and 10%, respectively); in the PC1 × PC2 plane, the parameter position is located close to the border of 75% of explained scatter. A mutual position of the bread shape h/d and the bread sensorial acceptability pair to the parameter specific bread volume is almost rectangular. According to the PCA method definition, an angle of 90° indicates zero correlation, i.e., this pair of variables could be considered as independent (cos 90° = 0). Second, a reciprocal closeness of the bread sensorial acceptability and the bread lipid content confirmed a generally known fact, that fat is connected to the pleasant taste as it is a carrier of taste (here, in fact, some typical oat and wholegrain taste could affect the sensory score itself).

In Figure 9b of the samples’ PCA scores, the higher the WF replacement ratio by any oat form, the lower the quality/statistical similarity of the binary flour blends, and from them resulted breads as presumed. It implies a clustering of samples in agreement with the fortification. Owing to the oat bran (OB) nature of “pure fibre”, the quality of the WF + 15OB bread was the most different per contra to the control (100% statistical dissimilarity). The inserted circles show approximate statistical similarity of the fortified flours/breads to the WF control from 75% up to 0% (from the centred WF pair). By a distribution of the statistical similarity, three most frequent categories were demonstrated between <60; 70), <50; 60), and <40; 50) percent; these categories collected six, three, and five samples, respectively (58% of all 13 samples pictured in duplicates as measured). Only 1 sample, a pair of WF + 5OFLA, differed from the WF control by less than 25%. In an overview, the milled oat flakes (OFLA) seemed to be the most promising form of oat for a recipe modification in praxis—the most enriched flour binary blend/bread WF + 15OFLA demonstrated the similarity to the WF control was roughly 50%. In comparison to wholegrain oat flour (OFLO), the technological steps of oat grain soaking followed by flaking itself represent an extra cost. At the same time, cereal grain sprouting is a traditional process applied in the brewery; in this direction, semi-industrial germination of oat grain instead of barley and wheat ones could be carried out at a professional, hygienic, and safe level.

4. Discussion

This study investigated the effects of incorporating distinct oat fractions, differing in both structure and composition, into wheat flour on the fibre content, macronutrient balance, technological performance, and sensory quality of composite breads. Through a combination of compositional analysis, baking trials, and multivariate statistical evaluation, this study provides an integrated view of how the type of oat fraction and the level of substitution modulate nutritional attributes and bread quality.

The results showed the fibre enrichment in wheat–oat systems depended not only on the number of oat-derived components present but also on matrix-dependent effects related to starch dilution, lipid co-occurrence, and changes in analytical recoverability induced by processing. These interactions underpin the fraction-specific responses observed during baking and explain the technological and sensory trade-offs associated with increased oat incorporation levels.

4.1. Composition of Raw Materials

The pronounced compositional differences observed between wheat flour and oat-derived raw materials are consistent with the well-established anatomical and biochemical heterogeneity of cereal grains, particularly concerning fibre localisation and the effects of starch dilution. Refined wheat flour is characterised by a predominance of endosperm tissue, explaining its high starch content and low concentrations of BG, total dietary fibre, and lipids [51,52].

In contrast, oats retain substantial proportions of bran, aleurone, and germ tissues, which are the primary reservoirs of mixed-linkage BG, non-starch polysaccharides, and lipids [53,54]. The markedly higher BG and TDF levels measured in oat bran and wholegrain oat flour reflect the preferential localisation of BG in subaleurone and cell wall structures in the outer kernel layers [55]. The intermediate BG and TDF levels observed in oat flakes and germinated oat flour are consistent with the partial removal or structural modification of these tissues during the sprouting and flaking processes.

The substantially lower starch concentrations in all oat fractions compared to wheat flour are primarily due to dilution by fibre, protein, and lipid components rather than absolute starch depletion. This starch dilution effect is a critical compositional feature as it alters the nutritional profile and functional behaviour of composite flours during dough formation and baking [56]. In germinated oat flour, the pronounced reduction in starch is further amplified by enzymatic mobilisation during sprouting, which activates α- and β-amylases and increases the proportion of non-starch components [57,58].

The elevated lipid content observed in all oat-derived materials, particularly oat bran and germinated oat flour, is another feature that distinguishes oats from wheat. Concentrated in the germ and aleurone layers, oat lipids are rich in polar lipids and unsaturated fatty acids. These lipids may interact with starch and fibre components, influencing analytical recoverability and technological performance [59,60]. The co-occurrence of lipids and polysaccharides is increasingly recognised as an important factor affecting enzyme accessibility and extractability in fibre and BG analytical methods [53,61,62].

The variability in protein content among the oat fractions, particularly the elevated protein content of germinated oat flour, reflects compositional remodelling that occurs during sprouting. In this process, starch degradation leads to the relative enrichment of nitrogenous compounds rather than de novo protein synthesis [9,63]. These changes result in a fundamentally different macronutrient balance compared with refined wheat flour.

Overall, oat-derived raw materials represent distinct compositional and analytical matrices characterised by fibre enrichment, starch replacement, and lipid co-occurrence. These features are not merely nutritional characteristics, but they are also expected to significantly impact enzymatic accessibility, analytical response, and processing behaviour in composite flours and breads. The compositional contrasts identified here therefore provide an essential framework for interpreting the effects observed on a per-fraction basis during baking and subsequent analytical determination of fibre-related components.

4.2. Total Dietary Fibre (TDF) in Composite Flours and Breads

In all composite flours, the increase in TDF content observed reflects the high fibre content of oat-derived fractions, particularly oat bran and wholegrain oat flour. These mill streams are characterised by their high levels of non-starch polysaccharides, including BG, arabinoxylans, and cellulose-rich insoluble fibre. Similar dose-dependent increases in TDF following the incorporation of oats into wheat-based matrices have been reported consistently in previous studies on composite flours and breads [18,22,64,65].

The absence of any measurable change in TDF content in the wheat control after baking confirms that thermal processing alone does not alter the fibre fraction of refined wheat flour as determined by gravimetry. This finding is consistent with earlier reports demonstrating the high thermal stability of wheat fibre components under conventional baking conditions when analysed using AOAC enzymatic–gravimetric methods [62,66].

In contrast, several composite systems exhibited higher TDF values after baking than in the corresponding flour blends. This phenomenon, most pronounced in formulations containing wholegrain oat flour and oat bran, is unlikely to reflect de novo fibre formation. Instead, it can be attributed to matrix- and method-dependent effects that affect fibre extractability and analytical recovery during the enzymatic–gravimetric determination process. Baking-induced structural disintegration of the starch–protein matrix, together with heat- and moisture-driven loosening of cell wall architecture, may increase the accessibility of fibre polymers to enzymatic digestion and subsequent gravimetric precipitation. This can lead to an apparent increase in measured TDF [67,68].

Wholegrain oat flour exhibited the most significant rise in TDF levels after baking, potentially reflecting the combined effects of partially disrupted endosperm cell walls and the enhanced solubilisation of fibre components during this process. Oat bran, characterised by a high proportion of insoluble fibre and intact cell wall structures, exhibited consistently elevated TDF levels at ≥10% substitution, both before and after baking. This indicates a more stable fibre contribution, which is largely independent of changes in extractability induced by processing. Similar behaviour has been reported for bran-enriched wheat products, where fibre enrichment is dominated by the absolute contribution of insoluble fractions rather than processing-induced transformations [69,70].

Oat flakes and germinated oat flour produced more modest TDF increases at lower substitution levels, which may be related to their distinct structural and compositional features. In flakes, prior hydrothermal treatment during flaking may enhance fibre accessibility, limiting changes during baking. In germinated oat flour, partial enzymatic degradation of cell wall polysaccharides during sprouting may reduce the proportion of high-molecular-weight insoluble fibre recovered by gravimetric methods, particularly at low inclusion levels [63,71,72].

The strong correlation between flour and bread TDF levels across all formulations (r = 0.85, p = 99.9%) confirms that fibre enrichment in baked products is primarily driven by the initial flour composition rather than processing variability. At the same time, the baking-associated amplification of TDF differences among formulations highlights the importance of considering analytical recoverability and matrix effects when interpreting fibre data in thermally processed cereal products. Therefore, increases in measured TDF after baking should be interpreted with caution, primarily as an analytical response to matrix modification rather than as an actual increase in dietary fibre available to consumers [73,74,75].

From a mechanistic perspective, the apparent increase in measured TDF after baking can be explained by several concurrent matrix transformations. Firstly, the thermal and moisture-induced disruption of cereal cell walls can expose fibre polymers that were previously embedded within intact tissues, particularly in whole grain and bran-rich matrices [67,68]. Secondly, starch gelatinisation during baking can alter the physical entrapment of fibre within the starch–protein network, either releasing fibre fractions or enabling them to be included in the gravimetric residue recovered during AOAC analysis [67,68,69,70]. Thirdly, protein denaturation and weakening of the gluten–starch matrix can enhance enzyme penetration during the enzymatic–gravimetric procedure further [69,70]. Taken together, these effects increase analytical accessibility rather than fibre mass, resulting in apparent, rather than compositional, increases in TDF.

4.3. β-d-Glucans in Composite Flours and Breads

The increase in β-d-glucans (BG) content observed following the incorporation of oat fractions is consistent with the well-documented enrichment of mixed-linkage BG in oat tissues, particularly in the aleurone and subaleurone layers. These layers are retained to varying degrees in wholegrain flour, flakes, and bran fractions [22,61,76]. The magnitude of BG enrichment observed in the present study, with composite flours reaching 1.21% BG at 15% oat bran substitution, is similar to values reported for wheat–oat composite systems within a similar substitution range [77,78].

The minimal and statistically non-significant reduction in BG content observed in the wheat control after baking indicates that thermal processing alone does not substantially affect BG recoverability in refined wheat matrices. This is consistent with previous findings that wheat-derived BG, present at low concentrations, exhibits limited susceptibility to thermal degradation under standard bread-making conditions [79,80]. In contrast, the fraction-dependent responses observed in composite systems demonstrate that BG behaviour during baking cannot be interpreted as a simple function of temperature exposure; rather, it reflects matrix-specific interactions and analytical accessibility.

The consistent decrease in measured BG content after baking in wholegrain oat flour blends suggests partial depolymerisation or reduced enzymatic recoverability of BG in these matrices. Heat-induced cleavage of β-(1 → 3)(1 → 4)-linkages, as well as interactions with gelatinising starch and denaturing proteins, have been shown to affect the solubility of BG and the efficiency of its enzymatic hydrolysis in mixed cereal systems [26,81]. In contrast, the nonlinear response observed in oat bran blends, with increased measured BG content at higher substitution levels after baking, strongly indicates enhanced extractability or analytical release of BG from bran-rich matrices following thermal processing. Previous studies have attributed similar apparent increases in BG content after baking to matrix loosening, disruption of cell wall integrity, and improved accessibility of BG to lichenase-based analytical methods rather than de novo BG formation [26,36,81].

The comparatively stable BG levels observed in oat flake formulations and the modest changes detected in germinated oat blends further highlight the importance of fraction-specific structural organisation. In oat flakes, partial pre-processing (steaming and rolling) may promote BG accessibility to some extent, thereby reducing the magnitude of further changes induced by baking [53,82]. In germinated oats, enzymatic activity during sprouting may lead to partial BG modification before baking, resulting in attenuated responses during subsequent thermal treatment [57,83].

The strong positive correlation between flour BG and bread BG confirms that BG is efficiently transferred from composite flours to baked products. This supports the nutritional relevance of BG enrichment strategies in bread making. Conversely, the pronounced negative correlation between flour BG content and specific bread volume highlights the well-known technological trade-off associated with BG enrichment. Increased BG levels increase dough viscosity, restrict gas cell expansion, and interfere with gluten network development, ultimately resulting in reduced loaf volume [84,85] as well as firmer, more chewable crumb.

Taken together, these findings show that changes in measured BG content during baking depend on a complex interplay between oat fraction type, substitution level, and matrix-dependent analytical recoverability. From a technological perspective, BG enrichment enhances the nutritional value of bread but imposes structural constraints on quality. From an analytical standpoint, however, post-baking BG values must be interpreted with caution as they may reflect altered extractability rather than true changes in BG mass. These insights are critical for the rational design of fibre-enriched bakery products and for correctly interpreting BG data in processed cereal matrices (e.g., during a sourdough production stage).

For BG, changes in measured content induced by baking can be explained by differences in the molecular organisation and matrix interactions of the fractions. In wholegrain matrices, partial depolymerisation of BG, interactions with gelatinising starch, and physical entrapment within the developing gluten network may reduce enzymatic recoverability [26,81,84,85]. Conversely, in bran-rich systems, the thermal loosening of cell wall architecture may increase the accessibility of BG to lichenase-based assays, resulting in an apparent increase in the measured amount of BG [26,36,81]. These opposing trends demonstrate that post-baking BG values primarily reflect matrix-dependent analytical behaviour rather than the true degradation or formation of BG.

4.4. Macronutrient Composition of Composite Flours and Breads

The reduction in starch content observed across all composite systems is primarily due to the dilution effect caused by incorporating oat-derived fractions that are enriched in non-starch components, such as dietary fibre, lipids, and proteins. This effect was most pronounced in oat bran formulations, where the high proportion of cell wall polysaccharides displaced a significant amount of starch in both the flours and the bread. Similar effects have been consistently reported in wheat-based composite systems following the incorporation of oat bran and are considered a driver of altered dough rheology and bread structure [86,87].

However, the pronounced starch reduction observed in germinated oat blends from ≥10% substitution cannot be attributed solely to dilution; rather, it reflects biochemical modification during germination, particularly the degradation of starch reserves by amylolytic enzymes. Sprouting activates endogenous α- and β-amylases, leading to partial starch hydrolysis and an increased proportion of soluble carbohydrates before baking [57,58,88]. The persistence of reduced starch levels after baking indicates that these compositional changes are retained through thermal processing, contributing to the weakened gas retention and reduced loaf volume observed in germinated oat breads.

The strong positive correlation between flour starch content and specific bread volume highlights the pivotal role of starch as the primary structural polysaccharide in wheat-based breads. Starch gelatinisation during baking provides mechanical stability to the crumb structure; moreover, it is a source of simple fermentable sugar, metabolised by yeasts to carbon dioxide. However, its progressive dilution by fibre-rich oat fractions compromises this function [69,89]. The inverse relationship between starch and both TDF and BG confirms that displacement of starch represents a key compositional trade-off that underlies the technological limitations of high-fibre formulations.

Protein enrichment in most composite flours reflects the higher natural protein content of oat fractions compared to refined wheat flour [90]. The strongest effects are observed with oat bran and germinated oat flour. In germinated oats, protein enrichment is additionally driven by starch mobilisation during sprouting, increasing nitrogenous compounds [91]. However, despite their higher protein content, negative correlations between flour protein content and both bread shape and sensory acceptability suggest that oat proteins do not functionally compensate for gluten dilution. Unlike wheat gluten, oat proteins support the elastic part of bread dough bionetwork required for sufficient (optimal) retention of fermentation gases; their incorporation tends to weaken rather than reinforce the dough structure [38,83].

The increase in lipid content across all composite systems is consistent with the high lipid content of oat grains, particularly in the germ and aleurone layers that are retained in the bran and wholegrain fractions. Depending on their concentration and interaction with starch and proteins, lipids are known to exert complex effects in bread making, acting both as crumb softeners and as potential inhibitors of gluten network development [92,93]. The strong positive correlation between lipid and BG content indicates that these components are located in the same area of oat tissues, particularly in bran-rich formulations. The negative correlation observed between flour lipid content and specific bread volume indicates that lipid enrichment may exacerbate structural weakening in fibre-rich matrices, possibly through competition for water and interference with gluten–starch interactions.

Taken together, these findings demonstrate that shifts in macronutrients induced by the incorporation of milled forms of oat grain are closely linked, with the partial replacement and dilution of starch, the enrichment of fibre, the co-occurrence of lipids, and the enrichment of non-gluten proteins acting synergistically to influence the structure and quality of final yeasted bread. From a technological perspective, starch reduction is the main cause of volume loss, while protein and lipid enrichment affect crumb structure and sensory attributes in a fraction-specific manner. These results highlight the importance of considering macronutrient balance rather than individual components when formulating new, fibre-enriched wheat–oat composite breads.

4.5. Baking Trial Results and Sensory Evaluation

In composite breads, the reductions observed in bun height and bread volume (particularly those containing oat bran and germinated oat fractions at ≥10% substitution levels) are consistent with the well-documented disruptive effects of high fibre content on gluten network development and gas retention capacity in wheat-based doughs [67,89,94]. The presence of insoluble fibre-rich fractions, such as oat bran, physically interferes with gluten continuity and competes for water. This limits dough development and extensibility, as well as later reduces oven spring [95,96]. This mechanism plausibly explains the pronounced loaf flattening and reduced height-to-diameter ratios observed in these formulations (Figure 4 and Figure 5).

In addition to the mechanical dilution of gluten and the physical effect of fibre particles, the evaluation of changes in loaf volume and crumb structure should also consider biochemical and physicochemical interactions occurring during mixing, fermentation, and baking [93,97]. Although the lipid content of oats and composite doughs remains low at the applied substitution levels, oat lipids, particularly polar lipids from bran and germ, may influence interfacial properties within the dough, such as gas cell stability and the behaviour of the continuous phase during oven spring [97,98].

Biopolymer BG plays a significant role. Due to its high water-binding capacity and ability to increase the viscosity of the aqueous phase, it alters water distribution among starch, proteins, and fibre. This can delay starch gelatinisation and disrupt crumb setting, leading to reduced loaf volume. These effects are pronounced in fibre-rich and germinated oat formulations, where the combined influence of BG-mediated water retention, starch dilution, and enzymatic weakening of structure impairs gas retention and crumb stabilisation [99,100].

Germinated oat formulations exhibited comparable or even stronger negative effects on loaf geometry, which may reflect additional enzymatic weakening of the dough structure. Sprouting is known to enhance amylolytic and proteolytic activities, leading to excessive starch degradation and protein modification. This can impair dough stability and gas-holding capacity during fermentation and baking [57,71,88]. The combined impact of fibre enrichment and enzymatic activity likely underpins the pronounced technological deterioration observed in germinated oat breads.

Sensory evaluation outcomes closely mirrored these technological trends. The consistently high acceptability of breads containing 5% oat substitution across all fraction types suggests that low-level enrichment remains within the tolerance threshold of the wheat bread matrix, preserving desirable texture and flavour attributes (Table 3; Figure 6). Similar substitution limits have been reported previously for oat fibre enrichment; beyond these limits, consumer acceptability declines sharply due to textural hardening and reduced loaf volume [89,96,101,102].

Wholegrain oat flour formulations retained relatively favourable flavour and aroma profiles, even at higher substitution levels. This may be due to the presence of lipid-associated volatile compounds and Maillard reaction precursors, which contribute to a pleasant cereal flavour [103,104]. By contrast, oat bran breads showed clear sensory deterioration, especially in terms of crumb firmness and texture, which is consistent with the high insoluble fibre content and reduced porosity that were observed using instruments (Figure 5, Figure 6 and Figure 7). These results are consistent with previous studies that have linked the enrichment of bread with insoluble fibre to increased crumb hardness and reduced sensory acceptance [67,83].

Germinated oat breads received the lowest sensory scores for most attributes, including bread shape, crumb texture, and taste. Beyond structural defects, atypical or slightly acidic flavour notes reported by panellists may reflect biochemical changes during germination, such as the formation of organic acids and altered lipid oxidation pathways [105,106]. While such changes are nutritionally relevant, they may negatively affect consumer perception of conventional bread products.

Multivariate analysis, which integrates compositional, technological, and sensory variables, further supports these interpretations. The dominance of fibre-related parameters (TDF and BG) along the primary principal component highlights fibre enrichment as the main factor influencing technological performance and sensory differentiation (Figure 9; Table 5). Bread structure and sensory attribute loading along an orthogonal axis indicate that consumer perception is modulated by factors beyond fibre concentration alone, including starch dilution, lipid co-occurrence, and protein–fibre interactions [107,108,109].

Overall, these results demonstrate that the technological and sensory feasibility of oat-enriched wheat breads depends heavily on the type of oat fraction used and the level of substitution. While moderate inclusion (approximately 5%) is compatible with acceptable bread quality, higher levels, especially of commercial bran of traditional rough granulation spectrum or whole sprouted-grain flour, pose significant structural and sensory challenges. These challenges must be addressed through formulation or processing strategies if the higher fibre content is desired (and it surely is).

4.6. Integrated Analytical and Technological Framework—Practical Application

This framework links component, technological, and sensory data, serving as a guide for product development and the interpretation of analytical results. It provides food manufacturers with recommendations on suitable oat forms and replacement levels for various products. For standard wheat bread, a low to medium proportion of whole grain oat flour (approx. 5%) appears to be optimal, as this allows for fibre enrichment without significantly compromising volume or quality. Conversely, oat bran and sprouted oat flour are better suited to dense or specialty products, where a firmer crumb and smaller volume are acceptable or desirable.

For analytical laboratory evaluation, the framework highlights the importance of considering matrix effects when determining the levels of fibre and BG in baked goods. It recommends introducing uniform sample pretreatment procedures, such as controlled drying, standardisation of particle size, and clearly defined extraction conditions, to ensure comparable and reliable results. By combining technological and analytical aspects in this way, the framework provides a practical basis for recipe development and more accurate data interpretation.

5. Conclusions

This study shows that the nutritional profile, technological behaviour, and final quality of wheat–oat composite breads are not solely determined by the level of oat substitution but are primarily influenced by the structural and compositional characteristics of the milled forms of oat grain used. Incorporating oat ingredients resulted in a significant increase in β-d-glucan and total dietary fibre contents, accompanied by a consistent reduction in starch proportion. However, the extent of these changes and their technological implications varied significantly depending on the oat fraction tested. Oat bran provided the greatest fibre enrichment, while germinated oat flour exhibited the most significant shifts in macronutrient balance due to starch mobilisation during germination.

Interpretation of the results indicates that the behaviour of wheat–oat composite systems is governed by two weakly coupled mechanisms only. The first mechanism is associated with the content and physicochemical nature of dietary fibre, particularly β-d-glucans. These influence water binding and dough rheology, which consequently affect the specific volume and crumb structure of bread. The second mechanism relates to compositional changes within the starch–protein matrix, including starch dilution, the presence of lipids, and protein–fibre interactions. These changes are strongly reflected in the sensory and shape-related characteristics of the breads. These distinct mechanisms explain why comparable levels of fibre enrichment can result in markedly different technological and sensory outcomes, depending on the type of oat form used. This was confirmed and visualised by multivariate statistical analysis. As the wheat flour still represented a substantial component of binary flour blends and resulting breads, a majority of tested oat-enriched variants demonstrated a statistical similarity to the wheat control in a range of 30–55%.

From an analytical perspective, the study highlights the significant impact of matrix effects in technologically heterogeneous systems. Any increase in the measured content of dietary fibre and β-d-glucans after baking should primarily be interpreted as a consequence of changes in the extractability and analytical accessibility of the polysaccharide components, rather than as an actual increase in their mass. These findings emphasise the limitations of routine enzymatic and AOAC methods when applied to composite flour systems with different processing histories and highlight the need for analytical data to be interpreted cautiously in the context of matrix-dependent changes.

From a formulation standpoint, a moderate substitution level of approximately 5% was identified as the most suitable compromise between nutritional enrichment and preservation of technological and sensory quality across all tested oat fractions. Higher substitution levels result in pronounced technological limitations, particularly with regard to bran and germinated fractions. Overall, this study provides a framework grounded in analysis for the rational optimisation of fibre-enriched bakery products, contributing to a deeper understanding of the capabilities and limitations of standard analytical methods for determining dietary fibre and β-d-glucans in complex cereal matrices.

The limitations of the study include a relatively small, albeit trained, sensory panel and the use of a simplified hedonic scale. In addition, the number of replicates per treatment was limited to two; however, the variability between replicates was minimal and did not exceed the deviation permitted by the relevant analytical standards. Important areas for future research to optimise recipes include evaluating the glycaemic index and shelf life of oat products, given the proven positive effect of β-d-glucans on glycaemic control and the lower stability of oat products due to the higher susceptibility of fats in oats to oxidation.