Wholegrain triticale sourdough: Effects of triticale:Wheat flour ratio and hydration level on bread quality

Abstract Triticale (×Triticosecale) is a hybrid between wheat (Triticum spp.) and rye (Secale cereale), producing higher grain yields than wheat in challenging environments. Triticale grain is also highly nutritious. Thus, the potential of triticale grain for an expanded range of food applications should be explored. Sourdough bread has unique functional and nutritional properties, but understanding the effects of partial substitution of triticale for wheat flour and varying levels of dough moisture content on sourdough quality requires further research. The aim of this study was to evaluate the wholegrain flour of contrasting triticale cultivars in comparison to that of a common wheat cultivar in commercial sourdough breadmaking. Two triticale cultivars (Goanna and Hawkeye, selected from a panel of Australian genotypes) and one wheat cultivar (Scout) were grown in a field trial in northern NSW, Australia. Differences in quantitative texture and color parameters of the dough and sourdough bread resulting from (1) substitution of commercial wholemeal wheat flour with different proportions of wholegrain triticale or wholegrain wheat (Scout) flour (0%, 60%, 70%, 80%, and 90%) and (2) varying the amount of water included in the dough preparation (70%, 80%, 90%, and 100 g water/100 g flour) were determined. Replacement of wholemeal wheat flour with 60% wholegrain Goanna flour (protein content 12.76%; c.f. 11.50% for Hawkeye, 12.40% for Scout), and addition of 100 g water/100 g flour in the dough preparation gave the highest quality sourdough bread based on specific volume, texture, and color parameters and had similar properties to the control made from wheat alone.

such as lysine, compared to grains of other cereals (Silva et al., 2020;Zhu, 2018).The valuable nutritional composition of triticale grain has made this hybrid cereal a replacement option for wheat (Galoburda et al., 2020;Goryanina, 2020).
The most common bread recipe consists of cereal flour, water, salt, and a leavening agent (Plessas, 2021;Yildrime & Arici, 2019), while the most common cereal flour used in breadmaking is wheat, which is rich in starch and has a higher protein content when compared to rice, maize, and rye (Järvan et al., 2017;Wang et al., 1999;Zörb et al., 2018).Moreover, the protein in wheat flour is mostly gluten, which is lacking in other cereals, and has the unique property of providing a viscoelastic dough critical for leavened bread.Substituting wheat flour with triticale flour is known to have an impact on the sensory and textural qualities of bread, including overall appearance, texture, volume, color, taste, and aroma (Fraś, Gołębiewska, Gołębiewski, Mańkowski, & Szecówka, 2016;Fraś, Gołębiewski, Gołębiewska, Mańkowski, Boros, et al., 2016).Replacement of a high proportion of wheat with triticale flour generally has a negative impact on sensory qualities, with bakery products becoming darker, stickier, and harder (Fraberger et al., 2020).Due to these potential effects, it is important to evaluate the optimum degree of wheat replacement with triticale to maintain bread quality and, therefore, consumer acceptance.
Sourdough fermentation, which began in ancient times and is still widely used in the baking industry (Chavan & Chavan, 2011), is based on the fermentation of dough by a combination of lactic acid bacteria (LAB) and yeast.Couch (2016) reported that the selection of different species of yeast and bacteria for sourdough breadmaking depends on the region of the world in which the bread is produced and the types of flour it contains.For example, Lactobacillus sanfranciscensis dominates the sourdough microflora of sweet leavened baked products and wheat breads produced in Italy (Alfonzo et al., 2016), and the same species has been found in traditional Greek wheat sourdough (Papadimitriou et al., 2019).Lactobacillus pentosus and L. plantarum dominate the sourdough breads produced in southern Italy that use durum wheat flour (Petrova & Petrov, 2020;Ventimiglia et al., 2015), while L. brevis is predominant in Turkish and Portuguese sourdough bread (Siepmann et al., 2018).Lactococcus lactis in sourdough bread fermentation has been reported to improve the sensory attributes of the bread.Zlateva et al. (2017) showed that applying a culture starter with Lactococcus lactis to sourdough wheat flour bread increased the biological value of the bread, extended shelf life, and improved sensory quality.Similar results were reported by Wu et al. (2022), who also used Lactococcus lactis as a culture starter in sourdough wheat bread and demonstrated a positive effect on the concentrations of aldehydes, ketones, and furans and improved sensory attributes.
Sourdough fermentation produces a bread of superior quality to standard leavened bread in terms of texture, flavor, nutritional attributes, and delaying microbiological spoilage.These improvements are due to specific proteins, including enzymes, as well as metabolites, including organic acids, exopolysaccharides, and even antimicrobial compounds, which are provided by the microbes in the dough (Demirkesen-Bicak et al., 2021;Gänzle & Zheng, 2019;Gobbetti et al., 2019).The metabolites can also contribute to the relatively low glycemic index of sourdough bread via a lowering of starch digestibility (Arora et al., 2021;D'Alessandro & De Pergola, 2014;De Vuyst et al., 2016).Overall, using sourdough fermentation leads to a higher specific volume (i.e., loaf volume), a softer and more elastic structure, and a longer shelf life than for standard leavened bread (Ma et al., 2021).Indeed, the use of sourdough as a leavening agent for food fermentation is considered the gold standard (Gobbetti et al., 2019).
Bread quality is partly a reflection of the softness/firmness of the crumb, which is influenced by the complexes between starch and protein.When wheat gluten is compared to the rye storage proteins (secalins and prolamins), the latter are found to be unable to create a dough structure involving starch-gluten complexes because of the lack of low-molecular-weight glutenin subunits; however, intermolecular disulfide bonds can still form among the rye storage proteins (Wang et al., 2017).Also, the presence of water-extractable arabinoxylans in rye flour plays an important structural role via the binding of water and forming a viscous dough.The low pH of rye sourdough increases the extractability and swelling properties of the arabinoxylans (Wang et al., 2016).
One of the main functions of a sourdough culture in rye breadmaking is to inactivate α-amylase (Mihhalevski et al., 2012).Under normal leavening conditions, rye has greater activity of α-amylase than wheat; thus, inactivating this enzyme is particularly important in rye.The inactivation of α-amylase via sourdough fermentation ensures that staling is slower than in normally leavened wheat breads (Deleu et al., 2020).
The aim of this research was to determine the effects on the texture and color properties of triticale sourdough bread made using (1) different ratios of wholegrain triticale flour to commercial wholemeal wheat flour and (2) different levels of water content in the dough (i.e., dough yield, defined as 100 parts of flour plus the amount of water (in parts) used for hydration).Our hypothesis was that sourdough bread of comparable quality but with higher nutritional value can be made by partial substitution of wheat with triticale flour.Wholegrain flour from two Australian triticale cultivars and one wheat cultivar grown in a field trial (to exclude

Practical application
Triticale is a hybrid cereal developed by breeders to combine the grain quality of wheat with the disease resistance and tolerance to harsh environments of rye.Triticale grain is highly nutritious, being rich in micronutrients and essential amino acids.For sourdough breadmaking, it is important to understand the effects on bread quality of varying the ratio between triticale and wheat flour, as well as the addition of different levels of water to the dough.environmental effects) was compared for sourdough quality against a background of commercial wholemeal wheat flour.Partial substitution of wholegrain wheat flour for wholemeal wheat flour was included in the experiments to determine any effects of the type of wheat flour in the dough on sourdough bread quality.

| Grain and wholegrain flour samples
Grains of the triticale cultivars Goanna (G) and Hawkeye (H), as well as the bread wheat cultivar Scout (S), were harvested from a field trial run in 2019 at the I. A. Watson Grain Research Centre, a facility of the University of Sydney in Narrabri, NSW, Australia (Lat. -30.274249, Long.149.809321).Triticale and wheat cultivars were grown on a gray vertosol in a randomized complete block design with two replicates.Each treatment plot was 1.8 × 3.8 m.Granulock Z® fertilizer (Incitec Pivot, Australia) was applied at 60 kg/ha at sowing, and trials were sprayed with pesticide and herbicide as required.The grains were harvested with a Haldrup C65 plot combine (Haldrup, Germany).Subsamples of harvested grain were cleaned with a Kimseed Vacuum Separator (Kimseed, Australia) to remove trash and broken grains.Grain was milled using a Newport Scientific Hammer Mill 6000 incorporating a 0.8-mm screen (Newport Scientific, Australia) and sieved using a mesh size of 350 μm (N°55, Shangyu, China) to produce a wholegrain flour for the two triticale cultivars (GF and HF) and one wheat cultivar (SF).To clarify, the wholegrain flour was made by milling the grain directly into flour, whereas the commercial wholemeal flour would have been milled into white (endosperm) flour, germ, and bran, with the white flour and bran fractions recombined (and the germ excluded).Milled samples were used immediately for breadmaking.Commercial wholemeal wheat flour (White Wings brand) (WF) was obtained from an IGA supermarket in Sydney, Australia.

| Wholegrain flour protein content
The total protein content of the milled flours GF, HF, and SF, plus the control WF, was determined using a nitrogen/protein Vario MACRO Cube analyzer (Frankfort, Germany).Flour samples were analyzed following the AOAC method 968.03.Thirty milligrams of each sample were prepared in 12 × 6-mm pressed aluminum capsules.The samples were combusted with oxygen and nitrogen oxide and collected in a ballast tank.A helium gas carrier was used alongside the nitrogen oxide combustion gas, which was reduced to nitrogen.
Water and carbon dioxide were then removed by passing the sample through magnesium perchlorate and sodium hydroxide on a silicate carrier.A thermal conductivity detector was utilized to measure the nitrogen content, using helium as a reference.The measured nitrogen content was converted to total protein content by applying a conversion factor of 5.81 for wheat and 5.7 for triticale (Fujihara et al., 2008;Wrigley et al., 2016).Measurements were performed in triplicate and expressed as mean values.

| Flour substitution ratio and water content
Sourdough breads were made using 100% wholegrain triticale flour using each of the two triticale cultivars (GF and HF), 100% wholegrain wheat flour (SF), and 100% wholemeal wheat commercial flour as controls, each with a water content of 100 g/100 g flour.
Breadmaking was based on a process requiring kneading.The dough was prepared by hand-mixing the wholegrain triticale flour (180 g) or wholegrain wheat flour (180 g) with wholemeal wheat flour (20 g) and water (191 g) together for 2 min, then allowing the dough to incubate at room temperature for 45 min.The starter (4 g) and dough were combined slowly over 2 min.Salt (3 g) and malt (2 g) were added, and the dough was mixed slowly over 1 min and then rapidly until the dough was developed.The final dough (400 g) was allowed to bulk ferment for 2 h at room temperature and allowed to retard at 4°C for 12 h.Then the dough was proofed at room temperature for 3 h and baked in an oven (XECC-0523-E1R, Unox, Australia) at 150°C for 20 min.Three sourdough breads were prepared per treatment.

| Dough pH, moisture content, water activity, and specific volume
Dough (D) pH was measured in a dough extract at room temperature using a pH meter (HI 9025, Hanna Instruments, Woonsocket, RI, USA) (Jayaram et al., 2013).
Moisture content for GH, HF, SF, and WF was measured using a gravimetric method.Samples (3 g) were weighed on a paper muffin liner and placed in an oven at 105°C for 3 h.Samples were analyzed in triplicate.
where H 0 represent the initial mass and H f the final mass of the flour.
The water activity (A w ) of the sourdough loaves (L) was measured using a water activity meter (Model AquaLab Pre; Decagon Devices, Pullman, Washington, USA).The specific volume of the sourdough bread was measured using the rapeseed displacement method (AACC Method 10-05.01)after cooling the loaves to room temperature.Measurements were performed in triplicate.

| Dough and sourdough bread color
Color parameters were measured for the dough, as well as the sourdough bread crumb (C) and crust (CT) using a chromometer (Minolta Chroma Meter [Konika Minolta, USA]).Each sample was tested in triplicate for lightness (L*), redness/greenness (a*), and yellowness/ blueness (b*).The brownness index (BI) and chroma (∆E) were derived using the following equations:

| Sourdough bread texture
Texture parameters were quantified for the crumb of the sourdough loaves for each treatment using a texture analyzer (TMS-Touch; Food Technology Corporation, USA) fitted with a 500-N load cell.
Samples were cut into circular shapes (40 mm diameter × 20 mm height) using a stainless-steel round cutter.
For the crumb, the middle portion of each loaf was used for texture determination.Samples were analyzed using a 75-mm-diameter probe, with a compression ratio of 30%, a pre-test speed of 50 mm/ min, a test speed of 72 mm/min, a distance of 13 mm, and a trigger force of 0.2 N. Hardness (HARD), springiness (SPRING), chewiness (CHEW), resilience (RES), cohesiveness (COH), and gumminess (GUM) were calculated using the TMS-Touch software.The average values of eight samples were calculated.

| Statistical analysis
Differences between all samples were evaluated using Analysis of Variance (ANOVA).Differences were calculated by Tukey's post hoc test with a significance level of p < .05.Pearson's coefficient correlations of two variables were performed with a significance level of p < .05.All statistical tests were conducted using SPSS-Advanced Statistics 20 software (SPSS Inc, Chicago, IL).

| Protein content in triticale and wheat grain flour
The highest protein content (%) among the two triticale wholegrain flours was that of GF (12.76 ± 0.01, db), with HF having a substantially lower value (11.50 ± 0.01, db), while the wheat samples had the following protein contents: SF (12.40 ± 0.01, db) and WF (11.50 ± 0.01, db).Consistent with the results for the two triticale cultivars, Dennett et al. (2013) reported that the protein content in triticale grains is due partly to genotype, not only environment (e.g., soil available nitrogen).It was shown that the protein content of 11 triticale genotypes from a specific year of a field trial was 10.5%-14.6%(db), while with nine genotypes in another year of the same trial, the protein content was 11.8%-15.2%(db).Frás, Gołębiewska, Gołębiewski, Mańkowski, and Szecówka (2016) reported a protein content of triticale flour between 9.8 and 13.9% (db).

| Moisture content, A w , pH, and specific volume
Higher levels of moisture content (%) were observed for WF (13.03 ± 0.01), followed by HF (12.35 ± 0.02), SH (12.13 ± 0.01), and GF (12.01 ± 0.02) (p < .05).The ranges of A w values for the sourdough loaves for which commercial wholemeal wheat flour was substituted with triticale or wheat wholegrain flour at different ratios were GL 0.90-0.95,HL 0.91-0.95,and SL 0.90-0.96.The values for the 100% wholegrain and 100% wholemeal flours were WL (0.92), GL (0.91), HL (0.91), and SL (0.92).When the water content was varied, the ranges of A w values were GL 0.90-0.97,HL 0.90-0.96,and SL 0.90-0.97.A w values increased slightly (p < .0001)with higher levels of replacement of wholemeal wheat flour, and, as expected, A w increased when water content increased.pH ranged from 5.70 to 5.72; that is, no significant differences in pH were observed for dough samples among cultivars and treatments.
The specific volumes of the loaves (in cm 3 /g) with different ratios of wholegrain triticale or wheat flour to commercial wholemeal wheat flour (Figure 1a) and varying the water content for SL, HL, and GL (Figure 1b) were determined.When the specific volumes of the samples with different flour ratios and varying water content were compared to the 100% wholemeal wheat flour control, results (1) showed that specific volume increased from T 1 to T 4 as follows: for GL by 4.0%-6.5%,HL by 4.0%-5.7%,and SL by 4.0%-6.2%.For W 1 to W 4 , the increases were as follows: GL by 4.7%-6.0%,HL by 4.2%-5.5%,and SL by 4.2%-6.0%.The specific volume for 100% wholemeal wheat flour and triticale wholegrain flour were as follows: WF (4.7%), GL (3.2%), SL (3.4%), and HL (2.9%).GL and SL had higher specific volumes than HL, which ranked the same as protein content.
Loaves with 60% wholegrain Goanna triticale flour and 40% wholemeal wheat flour (i.e., T 4 ) appeared to have a greater volume than loaves with Scout wheat or Hawkeye triticale also at T 4 (Figure 1a).
As expected, loaves made from 100% triticale wholegrain flour leavened very poorly, with specific volumes much lower than those of T 1 , which is why these data are not shown in  2002) also reported that the optimal substitution rate of triticale to wheat flour for bread is up to 50%.
Specific volume depends on flour protein quality, particularly levels of gliadin and glutenin in the gluten (Banu et al., 2011).These proteins are responsible for dough rheology, gas retention, and bread volume (Ortolan & Steel, 2017).Pattison et al. (2014) reported that among 17 modern triticale cultivars, the average white flour protein content was 16% lower in triticale compared to wheat, despite similar wholegrain protein contents, suggesting that triticale stores a lower proportion of protein in the endosperm.
Our results showed that using higher substitution levels of wholegrain triticale or wheat flour for wholemeal wheat flour, as well as higher levels of water in the dough, increased specific volume.
The addition of more water to a dough (up to a point) is expected to create a stronger network between proteins, allowing a major retention of gas when leavening with sourdough starter, which will increase the volume in the bread (Biel et al., 2020).GL showed a higher specific volume compared to HL, which may have been due to the higher protein content of GL. and W 1-4 ): GL (r = 0.991), HL (r = .998),SL (r = .997),GL (r = .989),HL (r = .989),and SL (r = .991).

| Color parameters
Bread color is considered an important visual factor, influencing consumer preferences and purchasing decisions.Analysis showed the influence of different substitution rates of triticale and varying water content on L*, a*, b*, BI, and chroma values for dough, crust, and crumb color (Tables S1-S3).The brownness index decreases in crust and crumb when the water content of bread increases; water content plays an important role in the Maillard reaction and caramelization, which lead to changes in color during the baking process (Sahin et al., 2020).The addition of water leads to a decrease in the extent of the browning reaction because of the dilution of the browning compounds (Fatemi, 2003).Zain et al. (2023) reported that, in wholemeal bread, the brownness index in the crust decreased when higher levels of water (80%-105%) were added during breadmaking.L* values increased as water addition increased, indicating increased crust brightness.This suggests that the color parameters were less affected at higher water amounts.The author stated that the addition of 80% water improves color parameters in breadmaking.
Changes in color are also due to reactions occurring at high temperatures, such as protein denaturation, enzyme inactivation, evaporation of moisture, caramelization, and the Maillard reaction (Dennett & Trethowan, 2013;Mezaize et al., 2009;Navarro-Contreras et al., 2014).Darker breads associated with increasing levels of triticale flour can be due to pigments in the triticale flour and a lower content of water (Sheikholeslami et al., 2020).
be related to the less extensive gluten matrix and greater fiber content in Hawkeye triticale flour compared to wholemeal wheat flour (Burešová et al., 2017;Patil et al., 2016).It can also be associated with the ability of triticale to absorb less moisture than wheat (Naeem et al., 2002) or the lower ability of starch to react with water (Huang et al., 2010).
In general, our results showed that, in terms of sourdough bread texture, Goanna triticale was superior to Hawkeye and resembled Scout wheat.Changes in texture, especially in hardness, can be associated with moisture content, strength of the gluten matrix, fiber content, and starch in the blended flour (Cauvain, 2004;Dvořakova et al., 2012).The effects of adding different amounts of wholegrain triticale flour as a replacement for wholemeal wheat flour and varying the amount of water allowed us to create sourdough bread with similar properties to traditional wholemeal wheat sourdough bread.

| CON CLUS ION
A blend of 60% wholemeal triticale flour and 40% wholemeal wheat flour with 100 g water/100 g flour in the dough preparation created sourdough bread with the most favorable specific volume, texture, and color among the treatments tested.Replacement of wholemeal wheat flour with wholegrain triticale flour at a 60:40 substitution level is a good option in sourdough breadmaking as it allows a bread to be made with similar quality to a traditional wholemeal wheat sourdough bread (or to a sourdough bread made with wholegrain wheat flour and wholemeal wheat flour at 60:40 substitution level).
From a nutritional perspective, wholegrain triticale flour is richer in vitamins, minerals, phosphorus, and essential amino acids when compared to wholemeal wheat flour, and thus substitution should be encouraged.Among the two Australian triticale cultivars tested, Goanna was superior to Hawkeye.
Future studies will include nutritional and functional properties of the flour and sourdough bread samples, such as amino acid profiles, gluten quantification, and rheology, to better understand the effects of including wholegrain triticale flour in sourdough bread making.
Stankowski et al. (2017) reported the influence of fertilizer type and nitrogen nutrition level on the baking and technological properties of spring triticale grain cv.Nagano grown in the Szczecin Lowland in north-western Poland.Increasing amounts of nitrogen fertilizer increased the protein content up to a maximum of 12.7% in the triticale grains, a value consistent with those obtained here for GF.
Figure 1.Tohver (2005) conducted baking tests with substitutions of 10%-70% wholegrain flour from 12 triticale genotypes for wholegrain wheat flour.Those breads with up to 50% triticale flour showed similar quality, including specific volume, as those made from wheat flour only.Incorporation of triticale flour over 50% substitution negatively affected quality parameters.The author made the point that the protein content of flour is extremely important because almost all flour properties, including gluten content, water absorption, mixing requirement, and specific volume of breads, are highly correlated with protein content.Doxastakis et al. ( Pearson's correlation (p < .05)with two factors, specific volume (cm 3 /g) and A w , for samples with different substitution levels of wholegrain GL, HL, and SL flour and varying water content showed a positive correlation between specific volume (cm 3 /g) and A w .High r values were observed among all the samples and treatments (T 1-4

L
* and b* dough values slightly increased from T 1 to T 4 , and a* and BI decreased from T 1 to T 4 .For the crust, L* decreased from T 1 to T 4 , and a*, b*, and BI increased from T 1 to T 4 for GCT, HCT, and SCT.For the crumb, L* and b* increased from T 1 to T 4 , while a* and BI decreased from T 1 to T 4 for GC, HC, and SC.Color parameters for the dough, crumb, and crust of the sourdough bread were also influenced by water content.For the dough, L* values increased from W 1 to W 4 ; a*, b*, and BI decreased from W 1 to W 4 for GD, HD, and SD.For the crust, L*, b*, and BI decreased from W 1 to W 4 , and a* increased from W 1 to W 4 for GCT, HCT, and F I G U R E 1 Specific volume of sourdough breads containing (a) different ratios of Goanna (triticale), Hawkeye (triticale), and Scout (wheat) wholegrain flour to commercial wholemeal wheat flour and (b) varying water content.GL, Goanna loaf; HL, Hawkeye loaf; SL, Scout loaf; T 1 , 90% wholegrain triticale flour and 10% wholemeal wheat flour; T 2 , 80% wholegrain triticale flour and 20% wholemeal wheat flour; T 3 , 70% wholegrain triticale flour and 30% wholemeal wheat flour; T 4 , 60% wholegrain triticale and 40% wholemeal wheat flour; W 1 , 70 g/100 g flour; W 2 , 80 g/100 g flour; W 3 , 90 g/100 g flour; W 4 , 100 g/100 g flour.For the experiment in panel b, the ratio of triticale or wholegrain wheat flour to commercial wholemeal wheat flour was held constant at 60%:40%.The dashed line represents 100% wholegrain wheat flour (WL).SCT.For the crumb, L* increased from W 1 to W 4 , and a*, b*, and BI decreased from W 1 to W 4 for GC, HC, and SC.

Figure 2
Figure2shows BI for the crumb and crust for the various cultivars and treatments.For the crust, BI values for the loaves containing wholegrain triticale or wheat flour were below the control sample, whereas for the crumb, the BI values were above the control sample.In traditional wholegrain triticale bread, increasing amounts of triticale in the flour blend have been shown to have a significant effect on the color of the crust(Sheikholeslami et al., 2020).L* values decreased when the proportion of triticale flour was increased, leading to a darker crust.a* values increased when wholegrain triticale content was increased, while b* values increased after increasing the triticale flour proportion.The crumb color was also darker in samples containing higher amounts of wholegrain triticale flour.Pycia et al. (2018) also reported that L* values for triticale bread fell as the proportion of triticale increased.The lower values of L* were thought to result from the substantial content of monosaccharides in the dough, which are a substrate for the Maillard reaction during baking at high temperatures.
Table1) showed the influence of different substitution levels of wholegrain triticale or wheat flour for wholemeal wheat flour, as well as varying water content, on texture parameters (HARD, COH, GUM, SPRING, CHEW, and RES).HARD, Texture parameters for sourdough bread crumb containing different ratios of Goanna (triticale), Hawkeye (triticale), or Scout (wheat) wholegrain flour to commercial wholemeal wheat flour and varying dough water content.