Structural and nutritional portrayal of rye‐supplemented bread using fourier transform infrared spectroscopy and scanning electron microscopy

Abstract In the present study, four different variants, namely Gp‐1, Gp‐2, Gp‐3, and Gp‐4, were characterized for their nutritional and fatty acid profile. Later on, the nutritionally superior variant was used for bread preparation. Purposely, composite flour was prepared with different ratios of wheat and rye (100:0; 90:10; 80:20). Furthermore, structural characterization of bread was done using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Results showed that the Gp‐2 was more nutritional among the four variants. Furthermore, the spectra of composite flour bread were scanned in the range of 4000–600 cm–1. All the bread samples presented almost similar spectra for major peaks corresponding to wavenumbers in the functional group. The SEM micrographs showed the presence of small and large starch particles with compact structures. Conclusively, rye flour supplementation has a significant impact on the nutritional and structural attributes of the bread.

a quarter of the entire rye crop is consumed as food, primarily in bread. In the "Rye Belt" countries, rye consumption ranges from 10 to 30 kg per person/year (Deleu et al., 2020).
The rye and wheat composite flours at different extraction rates are used for the preparation of rye bread. The dietary fiber content in rye bread is in the range of 6%-16% (Grabiński et al., 2021). Rye grain can offer diversity to the cereal diets available to humans while also increasing the levels of DF and other bioactive chemicals. As customer perception of rye and whole-grain foods' health benefits increases, the cereal food sector is being challenged to offer novel nutritious rye products with better and various sensory profiles (Miedaner and Geiger, 2015).
Rye is a good source of phenolics, dietary fiber, minerals, and vitamins and is commonly eaten as whole grain products. Rye bread has been found to offer physiological benefits, particularly in terms of glucose metabolism and satiation. Despite significant variations in chemical and technological properties, rye (Secale cereale, L.) is very close to wheat among cereal grains. The husk on both grains is generally removed by threshing. The pericarp and testa, which contain the germ and endosperm (aleurone and starchy endosperm), are the outermost layers of the bare kernel (Shewry and Bechtel, 2001).
As a result, rye is a major source of dietary fiber (DF) in many countries. Sour and dark bread, crisp bread, flakes for porridges, loaf bread with sifted rye flour, and breakfast cereals are the most common rye dishes (Ghiafeh Davoodi et al., 2021). Owing to the nutritional profile of the rye flour, the present study was planned to explore the rye flour supplementation on different attributes of bread.

| Procurement of raw material
Rye grains were purchased from Forage Section, Ayub Agriculture Research Institute, Faisalabad. Four different gene pools named as RJS-10001, RJS-10002, RJS-10003, and RJS-10004 were coded as GP-1, GP-2, GP-3, and GP-4. All the chemicals required for the analysis were laboratory grade. H 2 SO 4 and methanol were purchased from the Sigma-Aldrich Chemical Co., whereas petroleum ether and n-hexane were purchased from Merck. The seeds were cleaned physically to expel dirt, dust particles, seeds of different harvests, and outside matter.

| Milling
Hammer-type laboratory mill 120 perton was used for the milling of rye grains at Ayub Agriculture Research Institute Faisalabad, Pakistan. Sieves of 0.5-2.0 mm are used for the milling of rye grains.

| Proximate composition
All variants of rye flour samples analyzed for moisture, crude ash, crude protein, crude fiber, crude fat, and NFE were determined through AACC (2000) methods.

| Fatty acid profile
The fatty acid profile of rye was determined by GC-FID as described by Medeiros and Simoneit (2007). Briefly, methyl ester was prepared by using the 2 g oil sample in 5 ml methanolic H 2 SO 4 (40:10).
Afterward, these were heated at 80°C for 2 h. After cooling the samples at room temperature, samples were transferred into test tubes and 1 ml of petroleum ether was added thrice in each test tube. Two milliliters of distilled water was added to the test tube and mixed by using a vortex mixer. For the complete formation of layers, 5-10 min stay time was given to the samples. The upper layer was removed and run in GC-FID. For further analyses, flame ionization detector, Agilent DB-5 with methyl polysiloxane polymer phase column, and nitrogen gas (N) as a carrier gas at a flow rate of 1.3 ml/min were used.

| Blended flour and dough development
Among four variants, the nutritionally superior rye variant was selected for the preparation of composite flour and later on for bread preparation (Table 1).

| Preparation of bread
The ingredients, composite flour (rye+wheat), compressed fresh yeast, sugar, salt, improver, shortening, and water, were mixed with a mixer, and bread was prepared with a straight dough method at Ayub Agriculture Research Institute Faisalabad, Pakistan, using method no. 10-10.03 AACC (2000) (Figure 1).

| Bread analysis
The bread samples were analyzed for the physicochemical analysis, i.e., bread loaf volume, water activity, moisture, crude protein, ash, crude fat, crude fiber, and NFE content, through AACC (2000) methods.

| Structural characterization
The bread was structurally characterized using the Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).

| Fourier transform infrared spectroscopy
As stated in the Instruction Manual IR Prestige-21, functional components of each sample were identified using FTIR. The Shimadzu Fourier transform infrared spectrophotometer-FTIR 8400 S was used to determine functional units. The scanned sample traveled through infrared, where it was detected as a continuous wave by a computer-connected detector and the spectrum of the tested sample was reported. Typically, samples were scanned in the absorbance range of 600-4000 cm-1. As the basis of spectrum type, the findings of the investigation included the chemical structure, molecular binding form, and specific functional groups of the examined material (Sivam et al., 2013).

| Scanning electron microscopy
The bread was sputter-coated using gold in a sputter coater after being mounted on a stub with double-sided sticky tape (JEC-3000FC). A scanning electron microscope (cube series company, Emcraft) was used to image the samples (800) at a 5-kV accelerating voltage. The images were recorded as previously described by (Wang et al., 2017).

| Statistical analysis
The resultant data were statistically analyzed by CRD using Minitab statistical package described by (Steel, 1997). The results were presented as mean values ± standard deviation (SD).

| Proximate analysis of rye
The results regarding the proximate analysis of rye are shown in Table 2. Results showed that the moisture content in the different F I G U R E 1 Bread prepared with supplemented flour rye flours ranged from 7.01% to 7.6%. The maximum moisture content (7.6%) was present in the GP-2, whereas the lowest (7.01%) was observed in the GP-1. The ash content ranged from 1.47% to 1.97% in the different rye flours. The maximum ash content (1.97%) was observed in Gp-3, whereas the lowest ash content (1.47%) was observed in the Gp-1.
The crude protein content in the various rye content ranged from 9.13% to 9.56%. Gp-3 showed the maximum protein content (9.56%); meanwhile, minimum protein content (9.13%) was noticed in the Gp-1. The crude fat content ranged from 1.73% to 1.86% in the different rye flours. The maximum crude fat content (1.86%) was noticed in the Gp-2, whereas the lowest (1.73%) was observed in the Gp-4. The crude fiber in the rye flour ranged from 2.5% to 2.8%. The found that the moisture, ash, protein, fat, and NFE were 9.3, 1.27, 8.2, 1.31, and 89.2 g/100g.

| Fatty acid profile of rye flour
The results concerning the fatty acid content of rye are shown in

| Bread analysis
The results regarding the physicochemical analysis of the composite bread are shown in Table 3. Results showed that the bread loaf volume of the bread was ranged from 231.3 to 236.31 cm 3 . The control bread (T0) showed the maximum bread loaf volume (236.31 cm 3 ), whereas the T2 (231.3 cm 3 ) presented the lowest bread loaf volume.
As the rye flour in the bread increases, the loaf volume decreases just because of the high fiber content in the rye flour. The water activity of the bread ranged from 0.932 to 0.945. The maximum water activity of the bread was noticed in the T2 (0.945), whereas T1 (0.932) showed the lowest water activity. Bread's water activity reveals the microbiological growth's lower limit in terms of water availability.
The moisture content of the bread ranged from 35.3% to 41.2%.
The maximum moisture content (41.2%) was present in the T2, while the lowest moisture content (35.3%) was noticed in the T0. The ash content in the bread ranged from 0.8% to 1.2%. The maximum ash content (1.2%) was present in the T2, whereas minimum ash content (0.8%) was observed in the T1. The crude protein in the bread ranged from 9.7% to 11.6%. The maximum crude protein content (11.6%) was present in the T2; meanwhile, the lowest (9.7%) was noticed in

| Fourier transform infrared analysis
The Fourier transform infrared (FTIR) spectroscopic analysis was conducted to support the proximate data for possible variation in the composition of major fractions such as moisture, proteins, fats, and saccharides of composite bread (Figure 2). The spectra of wheat flour and rye composite bread were scanned in the range of 4000-600 cm -1 , and almost 11 nominal peaks corresponding to the constituents were evaluated. All the samples of bread presented almost similar spectra for major peaks corresponding to wavenumbers in the functional group region (4000-1200 cm -1 ) and the unique pattern also called fingerprint region (1200-600 cm -1 ) (Cocchi et al., 2004;Guo et al., 2015;Sujka et al., 2017). The wavenumber ranging from 1200 to 800 cm -1 is assigned to the presence of polysaccharides in the bread flour. The intense peak at 1014 cm −1 is originated from the stretching of C-O and twisting vibrations of CH 2 in the -CH 2 OH units (Wang & Somasundaran, 2007). A shoulder peak of less intensity was also noticed at 1077 cm −1 in all the samples, attributed to the C-O-H bending vibrations of the glycosidic linkages. Similarly, another shoulder at 1151 cm −1 has been correlated with the C-H stretching of the starch (Mathlouthi & Koenig, 1987). Interestingly, these peaks between 1200-800 cm −1 shifted to a higher transmittance for the composite bread compared to the control wheat bread

| Scanning electron microscopy
Scanning electron microscopy studies of all the samples were done to characterize the bread structure. Scanning electron micrographs of prepared bread samples are shown in Figure 3. The micrographs of control bread showed the presence of small and large starch particles. The gluten-developed structure can be seen in the micrographs of the control bread sample. Likewise, the sample prepared with rye flour supplementation showing a more compact structure and low bread volume compared to the control. Furthermore, the addition of 20% flour supplementation showed a more compact structure.
The results of the study are in line with other researchers who proposed that the micrographs of control bread varied from treated bread samples. Similarly, in another study, it was reported that dough treated with enzymes gave different micrographs compared to control. Micrographs of the study showed that starch particles were embedded in gluten structure (Ahlborn et al., 2005). The presence of supplemented rye flour exhibited a significant effect on the micrographs of different bread compared to control.

| CON CLUS ION
In the four different variants of the rye, Gp-2 was found to be more nutritionally superior. Rye flour supplementation has a significant impact on the nutritional and structural attributes of the bread.