Synergistic Effects of Azospirillum brasilense and Nitrogen Doses on Wheat Flour Characteristics and Yields of Reducing Sugars Obtained by Subcritical Water Hydrolysis from Bran

Abstract

The synergistic interactions between nitrogen doses and microbial inoculation in crops indicate the potential for integrated nutrient management strategies in plant cultivation. Therefore, this study investigated the interactive effects of nitrogen doses and Azospirillum brasilense inoculation on wheat flour characteristics in terms of the falling number and color parameters and yields of reducing sugars obtained by subcritical water hydrolysis (SWH) from wheat bran. The strip-plot experimental design, bifactorial with three replications, was applied. Factor A was three wheat cultivars: ORS Agile (AGI), ORS Feroz (FER), and TSZ Dominadore (DOM). Factor D was five nitrogen doses in the topdressing: 0, 20, 40, 60, and 80 kg ha⁻¹. The lowest value of falling number of 332 s was achieved with flour from FER cultivar using a nitrogen dose of 80 kg ha⁻¹ with A. brasilense inoculation. The SWH produced yields of reducing sugars (Y_RS) from wheat bran of up to 6.74 ± 0.18 g (100 g of wheat bran)⁻¹ for the cultivar DOM when using a nitrogen dose of 60 kg ha⁻¹ associated with A. brasilense inoculation. In this cultivation condition, the falling number was 408 s and the color parameters were L* of 92.49, a* of −0.26, and b* of 11.91. In the other conditions, the Y_RS ranged from 2.93 ± 0.63 to 6.52 ± 0.04 g (100 g of wheat bran)⁻¹. Both flour and bran are nutritional products with high application potential, and this study indicated SWH as a promising technique to dissociate the lignocellulosic complex of wheat bran without using hazardous solvents.

1. Introduction

On a global scale, there is a pressing demand to enhance crop yield and preserve soil fertility while mitigating environmental repercussions. The available nitrogen sources for crop production primarily encompass four key steps: (i) nitrogen mineralization from indigenous soil organic matter; (ii) nitrogen derived from biological fixation facilitated by legume-associated rhizobial bacteria; (iii) synthetic nitrogen fertilizers; and (iv) nitrogen-rich manures, composts, biosolids, agroindustrial by-products, and assorted organic wastes. Farmers can prioritize the utilization of these diverse nitrogen sources through strategic management practices [1].

Wheat (Triticum aestivum) stands as a cornerstone among field crops, demanding substantial nitrogen inputs to ensure both abundant yields and superior quality. However, some nitrogen sources are costly investments for producers and are susceptible to environmental losses that contribute to pollution and ecological decline. While optimal nitrogen levels are essential for cultivating top-tier wheat crops, the efficiency of nitrogen utilization remains modest, particularly at elevated fertilization levels. Unutilized nitrogen can exacerbate environmental issues through runoff, leaching, and volatilization, amplifying negative impacts on ecosystems [2]. Therefore, it is important to devise strategies aimed at augmenting plant nitrogen uptake from the soil, aiming to enhance both plant yield and the quality of flour and bran while mitigating adverse environmental impacts [3].

Biological nitrogen fixation offers a promising strategy for reducing nitrogen fertilizer use and mitigating natural resource pollution in wheat cultivation. Diazotrophic bacteria, such as Azospirillum spp., play a pivotal role in this process, promoting nutrient and water absorption and resistance to various stresses. Inoculation with A. brasilense has shown significant benefits in wheat, such as increased grain yield and nutrient content [4].

Wheat flour takes center stage in a wide array of food items, from bread and cakes to biscuits, doughnuts, crackers, cookies, pasta, and noodles [5]. The viscoelastic characteristics of wheat dough render it well suited for a variety of bakery items, with gluten proteins playing a pivotal role in defining the quality of wheat-based baked goods [6,7]. Wheat flour comprises high amounts of starch (up to 75%), water (approximately 14%), and proteins (up to 12%). Also, there are significant amounts of amylose and amylopectin in starch of up to 28% and 75%, respectively [8].

Wheat bran is the main by-product resulting from wheat processing in the flour production process. More than 150 million tons of wheat bran are produced every year. Even though most of it is discarded into the environment and is not essentially used, the use of wheat bran is due to the amounts of carbohydrates (up to 56%), starch (up to 24.9%), and protein (up to 18.4%). Furthermore, significant levels of fiber and phenolic compounds are found, which are highly important in indicating the nutritional quality of grains [9]. Nonetheless, wheat grain has restricted solubility in water, mainly due to a significant amount of hydrophobic amino acids. This scenario requires the modification of wheat grain to enhance its functional and textural properties and increase the quality of the final product [10].

Wheat bran constitutes a highly rigid matrix of cellulose (40–50%), hemicellulose (25–35%), and lignin (15–20%) [11]. Due to the solid and resistant structure of lignocellulosic biomass, the accessibility to individual components is highly limited. Therefore, considering the distinction in the composition of different biomasses of the same lignocellulosic material, the use of a certain pre-treatment tool for all biomaterials is uncertain. One of the accepted universal methods comprises the action of hydrolysis on the hemicellulose fragment or direct dissolution of lignin, significantly enhancing contact with the lignocellulosic material and promoting a high conversion of the compounds [12]. Among the different pre-treatment strategies for breaking down the outer layer, the exploration of hydrothermal technologies is an attractive alternative, since wet lignocellulosic biomass, together with high extraction temperatures, causes the degradation of hemicellulose and displacement of the lignin, encouraging higher accessibility to cellulose structures [13].

Hydrothermal technologies are physical and chemical pretreatment methods, such as steam explosion, autohydrolysis, wet oxidation, liquid hot water, and subcritical water hydrolysis (SWH). Subcritical water demonstrates efficacy in mass transfer processes like dissociating compounds from organic materials [14]. SWH emerges as an efficient method due to its ability to break down hemicellulose biopolymers into simple sugars like xylose and arabinose, as well as into smaller molecules suitable for downstream fermentation processes [15,16]. Some studies indicate promising results in the use of SHW for the conversion of lignocellulose into compounds of industrial interest. The use of SHW in a temperature range of 180–260 °C, water/solid mass ratio of 15–30 g water (g biomass)⁻¹, and reaction time of 15 min indicated up to 27.1 ± 6.9 g reducing sugars (100 g biomass)⁻¹ in pecan shells [12]. Furthermore, under experimental conditions of temperature (180–260 °C), water/solid mass ratio (9–18 g of water (g soybean straw)⁻¹ and 7.5–15 g of water (g soybean hull)⁻¹), and a reaction time of 15 min, up to 9.56 ± 0.53 g (100 g biomass)⁻¹ were obtained for soybean straw and 10.15 ± 0.50 g (100 g biomass)⁻¹ for soybean hulls, as well as an efficiency of up to 23.65 ± 1.32 g (100 g carbohydrates)⁻¹ [17]. Corn biomasses under temperature conditions of 220 °C, a water/solid mass ratio of 15 g water (g biomass)⁻¹, and a reaction time of 5 min indicated up to 6.9 ± 1.4 g reducing sugars (100 g biomass)⁻¹ for corn cob and 6.5 ± 4.6 g reducing sugars (100 g biomass)⁻¹ for corn straw [18]. This process holds promise as a cost-effective approach for industrial applications. Therefore, this work reports the characteristics of flour and yields of reducing sugars (Y_RS) from the bran of three wheat cultivars subjected to five amounts of nitrogen fertilization in topdressing in the Central Region of Rio Grande do Sul with and without inoculation of A. brasilense.

2. Materials and Methods

2.1. Experimental Design, Cultivars, Harvest, and Analysis

The experiment was conducted at the Agronomic Station belonging to the State University of Rio Grande do Sul, located in Cachoeira do Sul, Brazil (29°53′ S and 53°00′ W, altitude of 125 m). The local soil is dystrophic red loam and the region’s climate is subtropical, with an average annual temperature of 19.3 °C and an average annual rainfall of 1665 mm, with no defined dry season. For inoculation, Azospirillum brasilense strain Ab-V5 was employed with a concentration of 2 × 10⁸ colony forming units per milliliter (CFU mL⁻¹), with a foliar application at a dose of 0.3 L/ha. The inoculation procedure was conducted in association with the first nitrogen topdressing.

The experimental design was in strips with subdivided plots with 3 replications in a bifactorial scheme. Factor A consisted of three wheat cultivars (ORS Agile (AGI), ORS Feroz (FER), and TSZ Dominadore (DOM)), and factor D consisted of five amounts of nitrogen fertilization in topdressing (0, 20, 40, 60, and 80 kg ha⁻¹). Visual information about the design, sowing, pesticide application, and harvest is presented in Figure 1.

The experiment was implemented after harvesting soybean (Glycine max L. Merrill). The sowing density was 130 kg seeds ha⁻¹ for all cultivars in a direct planting sowing system using a fine grain seeder with 17 rows and a sowing depth of 3 cm. The plots consisted of 17 wheat crop rows (Figure 1B), spaced 17 cm (3.15 m wide) and 3.33 m long, totaling an area of 10.5 m² per plot. On the same day after sowing, base fertilization was performed using NPK fertilizer in a 5-20-20 formulation at 300 kg ha⁻¹. Topdressing fertilization was performed at the tillering stage in a single application, using urea with 45% nitrogen as a source, manually applied to the developing crop and with the appropriate humidity factor for application. Urea and NPK applications were carried out based on soil analysis for an expected productivity of 3000 kg ha⁻¹ [4]. To maintain the health of the culture, its development was monitored (Figure 1C). The control of weeds, pests and diseases was carried out in accordance with crop recommendations using 2,4-D amine and Difeconazole. In the maturation phase, close to harvest, the height of the plants and the length of the main stem were evaluated (Figure 1D).

2.2. Flour Analysis

In the middle of each plot, 1 m² was harvested and threshed (Figure 1E) to obtain the wheat flour. The wheat flour’s moisture, Falling number, and color (L*, a*, and b*) were determined using the AACC 44-15A, 56-81B, and 14–22 procedures, respectively [4].

2.3. Subcritical Water Hydrolysis of Wheat Bran

The subcritical water hydrolysis (SWH) process was performed in a semi-continuous process using a technological apparatus described in previous studies [12,16]. For each hydrolysis test, approximately 20 g of wheat bran was loaded into a stainless steel reactor of 50 mL. The pressurization was performed with distilled water and, thereafter, it was heated. Both pressure and temperature were controlled to reach 30 MPa and 220 °C, respectively. This process condition was defined based on previous studies [12,16,17]. After reaching the desired pressure and temperature, a static time of 20 min was applied. In the sequence, the dynamic SWH was carried out for 45 min with a water flow rate of 15 mL min⁻¹. The hydrolyzed solutions were collected in glass amber flasks at intervals of 0.5 min, 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 40 min, and 45 min. The assays were conducted in triplicates and the mean values and standard deviation were considered for data presentation.

2.4. Total Reducing Sugars

The determination of the total reducing sugars was performed using the dinitrosalicylic acid (DNS) method [12,16]. Absorbance was measured with a spectrophotometer (UV-1900, Shimadzu, Kyoto, Japan) at a wavelength of 540 nm [18]. According to previous studies, for each hydrolyzed solution test, the yield of reducing sugars (Y_RS, g (100 g bran)⁻¹) was determined by Equation (1) [12].

$${\mathrm{Y}}_{\mathrm{R}\mathrm{S}}=\frac{{\mathrm{m}}_{\mathrm{R}\mathrm{S}}}{{\mathrm{m}}_{\mathrm{S}\mathrm{A}}}\times 100$$

(1)

where m_RS is the accumulated mass (g) of total reducing sugars in the accumulated hydrolyzed solution; m_SA is the initial mass (g) of wheat bran charged into the reactor vessel.

2.5. Statistical Analysis

The collected data of accumulated Y_RS at 45 min for each wheat cultivar were subjected to statistical analysis using the software Minitab^® 18.0. The data were evaluated in terms of homogeneity and normality. Thereafter, the Analysis of Variance (ANOVA) was applied at a 95% confidence level and, when significant, the means were compared through Tukey’s test.

3. Results

3.1. Wheat Flour

The results of moisture, falling number, and color parameters of wheat flour from the AGI, DOM, and FER cultivars with and without A. brasilense inoculation are shown in

Table 1. Moisture, falling number, and color parameters of wheat flour from the AGI, DOM and FER cultivars with and without inoculation of A. brasilense.

Cultivar	Nitrogen Dose (kg ha−1)	Inoculation of A. brasilense	Moisture (%)	Falling Number (s)	L*	a*	b*
AGI	0	Yes	14.1	440	90.64	0.52	10.97
		No	14.2	399	91.26	0.32	11.63
	20	Yes	14.1	392	91.50	0.24	11.42
		No	14.2	389	91.55	0.27	11.30
	40	Yes	14.2	400	91.54	0.33	11.24
		No	14.2	498	90.71	0.50	11.34
	60	Yes	14.1	392	90.88	0.41	11.67
		No	14.2	408	90.82	0.36	12.27
	80	Yes	14.2	417	90.50	0.51	11.73
		No	14.1	413	90.44	0.51	12.03
DOM	0	Yes	14.1	398	92.75	−0.36	11.58
		No	14.2	413	92.55	−0.41	11.86
	20	Yes	14.2	380	92.63	−0.38	11.86
		No	14.2	412	92.85	−0.46	11.84
	40	Yes	14.1	402	92.82	−0.37	11.76
		No	14.1	401	92.64	−0.36	12.08
	60	Yes	14.2	408	92.49	−0.26	11.91
		No	14.2	382	92.16	−0.39	12.94
	80	Yes	14.1	418	92.04	−0.20	12.20
		No	14.2	422	92.04	−0.24	12.30
FER	0	Yes	14.1	412	92.34	0.13	10.08
		No	14.1	412	92.02	0.21	10.00
	20	Yes	14.2	420	92.09	0.23	10.15
		No	14.2	429	92.28	0.21	9.82
	40	Yes	14.1	332	92.16	0.12	10.50
		No	14.2	418	91.87	0.28	10.11
	60	Yes	14.1	412	91.92	0.16	10.94
		No	14.2	411	91.66	0.25	10.31
	80	Yes	14.2	399	92.45	0.10	10.46
		No	14.2	396	91.83	0.24	10.61

. All parameter results evaluated were similar to the results reported by the research group in a previous study [4], and are within the standard parameters for wheat flour.

3.2. Subcritical Water Hydrolysis of Wheat Bran

The kinetics of the Y_RS obtained under different doses of nitrogen (0–80 kg ha⁻¹) with and without bacteria inoculations are shown in Figure 2, and the statistical comparisons are presented in

Table 2. Comparisons of means of Y_RS for hydrolyzed solutions at 45 min of three cultivars of wheat bran inoculated or not with Azospirillum brasilense and submitted to different doses of nitrogen (0–80 kg ha⁻¹).

Nitrogen Dose (kg ha−1)	Inoculation of A. brasilense	YRS (g (100 g bran)−1) *
AGI
80	No	6.52 A
40	Yes	6.11 A
80	Yes	6.08 A
20	Yes	5.61 AB
0	Yes	5.37 AB
60	Yes	4.60 ABC
40	No	4.21 ABC
0	No	3.86 BC
60	No	3.20 BC
20	No	2.93 C
DOM
60	Yes	6.74 A
20	No	6.67 A
20	Yes	6.61 A
0	Yes	6.41 A
80	Yes	6.26 AB
40	No	6.06 AB
60	No	5.57 ABC
40	Yes	4.07 BC
80	No	4.04 BC
0	No	3.64 C
FER
40	Yes	6.01 A
80	Yes	5.67 AB
40	No	5.53 ABC
20	Yes	5.35 ABC
0	Yes	4.56 BCD
60	No	4.42 BCD
60	Yes	4.33 BCD
0	No	4.04 CD
80	No	3.53 D
20	No	3.46 D

* Different uppercase letters within a cultivar indicate significant differences at a 95% confidence level through Tukey’s test; AGI: ORS Agile; DOM: TSZ Dominadore; FER: ORS Feroz (FER).

.

Considering the AGI cultivar, seven treatments presented the highest values of Y_RS, of which six were inoculated with A. brasilense. Similarly, for FER cultivar, four treatments presented the highest values of Y_RS, of which three were inoculated with A. brasilense (Table 2). Otherwise, for the DOM cultivar, no pattern was seen because seven samples did not statistically differ from each other (equal uppercase letter, A), which comprises either a low dose of nitrogen with bacteria inoculation or a high dose of nitrogen without bacteria inoculation.

Regarding the kinetics of Y_RS (Figure 2), the SWH presented different behaviors for the different conditions of nitrogen dose (kg ha⁻¹) and inoculation of A. brasilense. For example, for conditions such as 80 kg ha⁻¹ of nitrogen with and without inoculation for AGI, 60 kg ha⁻¹ of nitrogen with inoculation for DOM, and 80 kg ha⁻¹ of nitrogen with inoculation for FER, the Y_RS remained increasing with high rates until the final hydrolysis time of 45 min. For instance, for the cultivars AGI, DOM, and FER, maximum absolute values of Y_RS of 6.52, 6.74, and 6.01 g (100 g wheat bran)⁻¹ were obtained, respectively, at 45 min of SWH. Otherwise, for conditions such as 20 kg ha⁻¹ of nitrogen without inoculation for AGI and FER, and 0 kg ha⁻¹ of nitrogen without inoculation for DOM, the Y_RS became approximately constant after 20 min of reaction. Therefore, based on the kinetic profile (Figure 2), most of the wheat bran samples from conditions with A. brasilense inoculation at higher doses of nitrogen presented higher Y_RS rates in the first 30 min of SWH.

4. Discussion

A variation in moisture, falling number, and color parameters (L*, a*, b*) of wheat flour was obtained as a function of different cultivation conditions. In the case of AGI, A. brasilense inoculation tends to slightly reduce moisture levels, with non-pattern effects across different nitrogen doses. Different responses in falling number and color parameters are observed, indicating interactions between inoculation and nitrogen levels in terms of quality attributes. The falling number represents the diastatic potential of wheat flour, through correlations between the viscosity of gelatinized starch and the activity of the alpha-amylase enzyme. Low falling number values, generally lower than 200 s, are not desired, as starch breakdown will be rapid and there will be an excess of sugar in the process, leading to uncontrolled fermentation and accentuated caramelization of the dough during baking (Maillard reaction).

Commonly, the use of wheat flour is based on its falling number. Flour with high diastatic activity can generate soft dough. Otherwise, flours with low diastatic activity generate hard dough, which directly interferes with the texture and uniformity of the final product. For values ranging from 200 to 250 s, the application is for sweet cookies and cakes. For 225 to 275 s, the application is for fermented biscuits and breads. For values larger than 350 s, the recommended use is for pasta. Therefore, in this study, the falling number ranged from 332 to 498 s, which is a characteristic that indicates the use of flour for pasta preparations. Overall, the falling number presented different values between the conditions tested, especially related to nitrogen levels. For AGI and FER cultivars, the inoculation of A. brasilense at a nitrogen dose of 40 kg ha⁻¹ resulted in values of falling number approximately 20% lower than the same condition without inoculation. This is most likely caused by a favorable combined condition of biological nitrogen fixation with such bacteria, causing an influence on the vegetal tissues and, consequently, grains characteristics. In such a case, it produced “softer” grains with higher enzymatic activity. However, for the DOM cultivar under this same experimental condition, the values of falling number were very similar. Therefore, it suggests that there are specific interactions between wheat genetics and fertilization, without a clear pattern.

The color parameters L*, a*, and b* indicate a wheat flour with luminosity near to white and tonalities slightly tending to yellow. The lower the extraction, the lighter the color of the flour, with wheat flour corresponding to approximately 80% extraction, and wholemeal flour corresponding to that with 100% extraction. In general, grains with a hard texture produce darker flours and soft grains produce lighter flours. For the fresh pasta segment, the preference is for flours with a lighter color because the consumer evaluates color as a synonym for quality.

For the SWH of wheat bran, according to the kinetic performance, the Y_RS for the reaction time increased up to 45 min of hydrolysis. The conditions applied in the study indicated an accumulated solvent-to-feed mass ratio of approximately 22.5 g water (g bran)⁻¹. The temperature used in the process (220 °C) was defined taking into account previous studies [12,16]. High temperatures reported a drop in Y_RS because the compounds can suffer severe thermal degradation, reducing the amount of sugars available in samples. Lower temperatures cannot reach the desired dissociation of longer chains of cellulose. Therefore, the temperature of 220 °C in the SWH procedure resulted in the rupture of water molecules into ions, significantly increasing the concentration of H₃O⁺, and enhancing the conversion of carbohydrate molecules into sugars of lower chains [19].

An increase in Y_RS is seen for most assays until the total reaction time of 45 min. For all cultivars, especially AGI, the inoculation with A. brasilense is a promising strategy to intensify wheat production through increased nitrogen fixation [20], phytohormone synthesis [21], higher availability of nutrients [22], and higher drought tolerance [23], producing bran with better characteristics to hydrolysis. Nonetheless, studies indicate that inoculation with A. brasilense does not always increase productivity and, consequently, the amount of free sugars [24]. Direct inoculation responses are active in plants in diverse situations, such as drought, foliar diseases, competition with weeds, and deficiencies in solar radiation, water availability, and nutrients [25]. This scenario may be related to the maximum absolute expression of Y_RS for the DOM cultivar, which presented up to 6.74 g (100 g bran)⁻¹ with A. brasilense inoculation. When the abiotic conditions at the experiment site are adverse, the DOM and FER cultivars may have benefited significantly from A. brasilense inoculation. It promotes the direct production of the plants’ antioxidants, which can help protect the plant from oxidative stress by increasing the activity of antioxidant enzymes and stimulating the synthesis of growth hormones, increasing the plants’ stress tolerance capacity [25].

It is not possible to make a straightforward comparison of yields because no study was found in the scientific literature with a similar approach. Some studies used ultrafast hydrolysis in supercritical water to recover sugars from wheat bran. One example is the FASTSUGARS process, which uses supercritical water at 400 °C and 25 MPa to hydrolyze wheat bran into sugars at reaction times lower than 1 s. The conversion of cellulose and hemicellulose into sugars of lower chain ranged from approximately 61 to 65% [26]. The conversion of wheat bran into soluble saccharides such as glucose, xylose and arabinose at 400 °C and 25 MPa with reaction times between 0.2 and 1 s was also reported. The highest recovery of cellulose and hemicellulose as soluble sugars (73% w/w) was achieved at 0.19 s of reaction time. An increase in the reaction time decreased the yield of cellulose and hemicellulose. The main hydrolysis product was glycolaldehyde, yielding 20% (w/w) at 0.22 s of reaction time [27]. Although these studies present faster reactions, the temperatures are high. Also, the values of Y_RS were not provided in such studies. Only the conversion rates were presented, which limits a straightforward comparison of mass yields of recovered sugars. Therefore, the current work advances knowledge by demonstrating good values of Y_RS using SWH at 220 °C and 25 MPa from samples of bran after the wheat was submitted to different treatments in the field.

Increasing doses of nitrogen have direct effects on plant growth and biochemical composition, including levels of reducing sugars in wheat biomass [28]. Nitrogen is a critical nutrient for plant growth and development, playing a key role in protein synthesis, enzyme activity, and general metabolism. Fertilization with nitrogen can promote plant growth and improve photosynthesis, leading to increased production of carbohydrates, including reducing sugars such as glucose and fructose [4]. The results with inoculation of A. brasilense may have resulted in excessive doses for the plants, which could cause serious imbalances in the absorption of nutrients by the plants, potentially affecting their metabolism [29]. High levels of nitrogen enhance the wheat’s initial growth, based on the accumulation of carbohydrates in storage organs, such as grains. This scenario may result in lower levels of reducing sugars in the biomass, as a large portion of the nutrients are diverted to the leaves and structural components, rather than to productive reserves [30,31].

5. Conclusions

This study presents a strategy between nitrogen doses and microbial inoculation in producing wheat flour and bran for specific applications. The flour has characteristics to be used for pasta production, while the bran can be dissociated through SWH to produce reducing sugars. A Y_RS of 6.74 g (100 g wheat bran)⁻¹, obtained by SWH at 220 °C and 30 MPa, was related to the cultivar DOM under the conditions of 60 kg ha⁻¹ with A. brasilense inoculation, which is a favorable condition to produce the raw material for hydrolysis. The integration of microbial inoculation with varying nitrogen fertilization levels exerts a positive influence on yield indicators and metabolic components, signifying the potential for integrated nutrient management strategies in enhancing wheat production efficiency. These results highlight the importance of tailored nutrient management approaches and the significant role of microbial inoculation in optimizing crop performance and sustainability. Many integrated products can be reached, with flour and bran being able to produce many other derivatives, from candies to platform chemicals.