Sustainable Flour Innovation: Enhancing Product Safety and Market Position Through Low-Asparagine Wheat ^†

Abstract

Acrylamide, classified as a human carcinogen, forms mainly through the Maillard reaction between free asparagine and reducing sugars during baking. Wheat-based products are a major dietary source, and sulphur deficiency in soils can drastically increase asparagine levels in grain. This study evaluated a sustainable strategy to reduce acrylamide formation by cultivating wheat under sulphur fertilization across four sites in Northern Greece. Grain was milled and processed into bread, biscuits, and breadsticks, which were analysed for physicochemical and sensory attributes. Results showed 31–70% reductions in asparagine, while maintaining product quality and demonstrating strong market potential for safer bakery products.

1. Introduction

The discovery of acrylamide in heat-processed foods in 2002 raised serious concerns for public health and food safety regulation [1]. Acrylamide, classified as a Group 2A probable human carcinogen by the International Agency for Research on Cancer (IARC), has been associated with carcinogenic, neurotoxic, and reproductive effects. In Europe, bread and other wheat-based bakery products represent a major source of dietary acrylamide exposure.

Acrylamide forms primarily through the Maillard reaction between free asparagine and reducing sugars during baking, roasting, and frying. Consequently, the concentration of free asparagine in wheat grain is the main determinant of acrylamide-forming potential in flour. Agronomic factors, particularly sulphur availability in soil, strongly influence free asparagine accumulation, with severe sulphur deficiency increasing its concentration up to 30-fold.

Free asparagine has been identified as the key precursor in multiple studies. Halford et al. (2012) highlighted the multifactorial nature of asparagine accumulation, influenced by nutrient availability, stress, and genetic variation among wheat varieties [2]. Recent studies confirm that sulphur fertilization not only reduces free asparagine accumulation but may also improve flour quality. For instance, research on hard winter wheat reported significant effects of genotype × nitrogen × sulphur interactions on asparagine levels and dough rheology, with sulphur fertilization increasing solvent retention capacity (SRC) by 217–308% and farinograph stability from 9.2 to 14.6 min [3]. These findings reinforce the dual benefit of sulphur application in mitigating acrylamide risk while maintaining or even enhancing processing quality. Importantly, strong correlations (R² ≈ 0.99) have been found between free asparagine concentration and acrylamide levels in processed products [4].

While asparagine plays a role in nitrogen storage and transport within plants, sulphur deficiency induces disproportionate accumulation in wheat grain, thereby drastically increasing acrylamide formation in processed flour. Studies suggest that applying sulphur fertilization (~20 kg/ha) can effectively mitigate this risk [3]. Additionally, genotype × environment (G × E) interactions further influence free asparagine levels. Tafuri et al. (2023) reported variation from 0.55 to 2.84 mmol/kg dry weight across 54 wheat varieties in Italy, underlining the importance of selecting varieties with consistently low asparagine content [5].

The purpose of this article is to evaluate an integrated strategy for sustainable flour innovation. Specifically, we investigate:

(a)

the effect of sulphur fertilization on free asparagine accumulation in wheat;

(b)

the physicochemical and baking performance of low-asparagine flour;

(c)

the sensory acceptance of bakery products;

(d)

the techno-economic feasibility of positioning such products in the market.

The remainder of this paper is organized as follows: Section 2 describes the materials and methods, including experimental fields, fertilization regimes, milling procedures, analytical methods, and sensory evaluation. Section 3 presents the results on free asparagine reduction, flour quality, and consumer acceptance, supported by tables and figures. Section 4 provides a discussion, linking our findings to previous studies and highlighting their implications for food safety, agronomic practices, and market positioning.

2. Materials and Methods

Field trials were conducted by CHRISTIDIS S.A., a flour mill located in Ptolemaida, Greece, in four experimental wheat plots located in the region of Eordaia, Western Macedonia, selected according to agronomic criteria and the company’s long-standing experience in wheat cultivation. The location of the fields, their pH value, and their size are shown in the table below (

Table 1. pH variation in the selected fields.

Parcel	pH	Area (Acres)
Olympiada	4.7	22
Kryovrysi	5.7	20
Galateia	7.1	40
Anatoliko	8.2	20

).

All parcels were sown with the Anforetta wheat variety, and each field was evenly divided into two sections. Section A received conventional NPK fertilization without sulphur, while Section B received the same treatment supplemented with 20 kg of sulphur per acre, applied with a fertilizer spreader.

Systematic crop monitoring was carried out throughout the season to ensure plant health and manage potential diseases that could affect yield and grain quality. Harvesting took place in early July 2021, with the exact dates varying according to site-specific crop maturity. Using modern self-propelled combine harvesters, yields ranged from 200 to 400 kg per acre. The harvested grain was collected in large, big bags and did not require drying, since moisture levels remained below 13.5%. To ensure safe storage, the grain was cleaned of foreign matter, broken kernels, and contaminants. Representative samples were collected with precision for subsequent analyses, which included physical, chemical, and rheological tests.

The grain samples were milled into flour and used for the preparation of three categories of bakery products: bread, biscuits, and breadsticks. Standard formulations and baking procedures were applied to ensure comparability across treatments. Free asparagine concentration was quantified by high-performance liquid chromatography (HPLC) after extraction with aqueous ethanol. Additional physicochemical analyses included measurements of moisture, ash content, protein levels, and gluten strength, all performed according to AACC methods. Sensory evaluation was conducted by a trained panel of 20 assessors, who evaluated appearance, aroma, taste, texture, and overall acceptability using quantitative descriptive analysis on a nine-point hedonic scale.

Finally, a techno-economic analysis and SWOT assessment were undertaken to assess the feasibility of introducing low-asparagine flour into the marketplace. This included an evaluation of market potential, investment requirements, and consumer trends, providing an integrated perspective on both the agronomic and commercial viability of the innovation.

3. Results

The field experiments clearly demonstrated the effect of sulphur fertilization on the accumulation of free asparagine in wheat grain. Across all four sites, sulphur application reduced asparagine content by between 31% and 70%, with the strongest reductions observed in the acidic soils of Olympiada and Kryovrysi (see

Table 2. Asparagine content of the flour samples.

	Free Asparagine in Flour Samples (mg/kg)
Location	Without Sulphur Fertilization	Sulphur Fertilization
Olympiada	303 ± 13	110 ± 5 (63.6% lower)
Kryovrysi	482 ± 23	244 ± 28 (49.4% lower)
Galateia	336 ± 15	102 ± 5 (69.6% lower)
Anatoliko	237 ± 10	162 ± 7 (31.6% lower)

). This pattern confirms that soil chemistry strongly influences the response of wheat to sulphur nutrition and highlighted the importance of tailoring fertilization strategies to site-specific conditions.

The analysis revealed that flour derived from wheat cultivated without sulphur fertilization contained between 237 and 482 mg/kg of free asparagine. In contrast, flour from sulphur-fertilized wheat contained markedly lower levels, ranging from 102 to 244 mg/kg. This corresponds to a reduction of 31.6% to 69.6% in asparagine concentration. A paired t-test confirmed that the decrease associated with sulphur fertilization was highly significant (p < 0.01). The effect was consistent across all sites, with non-overlapping 95% confidence intervals, thereby underscoring the robustness and reliability of the findings.

Physicochemical analyses of the resulting flours showed that moisture and ash levels were well within industry standards, while protein concentration and gluten strength remained unaffected by sulphur treatment. These results indicated that lowering asparagine through fertilization did not compromise the functional qualities of the flour. On the contrary, flours derived from sulphur-supplemented plots consistently showed good bread-making performance.

The sensory evaluation further supported these findings. Bread, biscuits, and breadsticks produced from low-asparagine flour were well accepted by the trained panel, with ratings for appearance, aroma, taste, and texture either equal to or slightly higher than those of products made with conventional flour (see

Table 3. Sensory evaluation scores of bakery products (9-point scale).

Product	Appearance	Aroma	Texture	Overall Acceptance
Bread (control)	7.5	7.2	7.4	7.3
Bread (low asparagine)	7.8	7.4	7.6	7.7
Biscuits (control)	7.1	7.0	7.3	7.2
Biscuits (low asparagine)	7.4	7.3	7.5	7.5
Breadsticks (control)	7.0	7.1	7.2	7.1
Breadsticks (low asparagine)	7.5	7.3	7.6	7.6

). In particular, panellists noted improvements in crust colour and crispness, suggesting that the intervention may enhance certain quality attributes.

In addition to the agronomic and sensory findings, the feasibility study provided a strong indication of the economic benefits for CHRISTIDIS S.A., which is active both in wheat trading and flour production. The analysis demonstrated that the new low-asparagine flour can be marketed at €1.36 per kilogram, while the cost of acquiring wheat was €0.62/kg and the cost of flour production amounted to €0.10/kg. This pricing structure confirms the profitability and long-term sustainability of the product, enabling the company to generate significant added value by leveraging its dual role in both raw material sourcing and finished product markets. In

Table 4. Economic analysis of low-asparagine flour production over three years.

Year	Wheat Purchase Price (€/kg)	Flour Selling Price (€/kg)	Project Participation Cost (€) *	Extra Milling Line Cost (€)	Sales Volume (t)	Net Additional Profit (€)
1st	0.62	1.36	53,660	25,000	250	106,340
2nd	0.62	1.36	26,830	30,000	300	165,170
3rd	0.62	1.36	0	35,000	350	224,000

* It should be noted that the ‘Participation to project’ column refers to the company’s own contribution to the funded program, which amounted to €80,490 over a period of 1.5 years, distributed as €53,660 in the first year and €26,830 in the second.

the results of the economic analysis of low-asparagine flour production are presented, showing the relationship between costs, sales volumes, and the resulting net additional profit over a three-year period. The SWOT assessment revealed significant strengths in terms of food safety, consumer health orientation, and compliance with emerging regulatory requirements. Opportunities were identified in contract farming schemes, differentiation in domestic markets, and export development. Challenges included the need to increase consumer awareness of acrylamide risks and to balance production costs when scaling up. Together, these results demonstrated both the scientific validity and the commercial relevance of the approach.

4. Discussion

The findings of this study provide strong evidence that targeted sulphur fertilization can substantially reduce the free asparagine content in wheat, thereby lowering the potential for acrylamide formation in baked goods. Across all experimental sites, flour from wheat without sulphur fertilization contained 237–482 mg/kg of asparagine, whereas sulphur-fertilized wheat flour contained only 102–244 mg/kg, representing a statistically significant reduction of 31.6–69.6% (p < 0.01). The consistency of these reductions, confirmed by non-overlapping 95% confidence intervals, highlights the robustness of the effect across diverse soil conditions. These findings are in line with earlier research demonstrating the central role of sulphur in asparagine metabolism [5,6].

The biochemical basis of acrylamide formation is well established. Acrylamide is produced mainly through the Maillard reaction, particularly via the Strecker-type degradation of free asparagine in the presence of reducing sugars during high-temperature processing [7,8]. Halford et al. (2012) emphasized that free asparagine is unusual among amino acids due to its propensity to accumulate under nutritional imbalances, particularly when nitrogen is abundant, but sulphur is limited [2]. Under such conditions, wheat plants utilize asparagine as a temporary nitrogen store, a physiological mechanism that inadvertently increases acrylamide risk. Additional stress factors, such as drought, salinity, or heavy metals, may further elevate asparagine concentrations, illustrating the multifactorial nature of this food safety issue.

At the same time, varietal differences and genotype × environment (G × E) interactions are equally critical. Curtis et al. (2018) reported substantial variation among commercial wheat varieties in both their baseline free asparagine levels and their responsiveness to sulphur fertilization [9]. Some varieties consistently maintained low concentrations across years and environments, while others displayed large increases under stress. These results suggest that the selection of stable, low-asparagine varieties, in combination with sulphur fertilization, could provide a dual strategy to minimize acrylamide formation. Breeding programs should therefore integrate asparagine stability as a trait of interest, complementing agronomic management.

Equally important, reducing asparagine through fertilization did not compromise flour quality or consumer acceptance. Physicochemical parameters such as protein content, gluten strength, and ash values remained within industry standards, while sensory evaluation confirmed that bread, biscuits, and breadsticks produced from low-asparagine flour were rated as highly acceptable, with some improvements in appearance and crispness. This confirms that safety-driven innovations can be introduced without trade-offs in product quality, addressing one of the main challenges in food safety management [2].

Beyond agronomic and technological aspects, the economic analysis carried out as part of the project demonstrates the feasibility and sustainability of this innovation. The feasibility study showed that low-asparagine flour could be marketed at €1.36/kg, with total costs (wheat acquisition and flour production) amounting to €0.72/kg, resulting in a profit margin of €0.64/kg. More detailed modelling across a three-year horizon indicated net additional profits of €106,340 in the first year, €165,170 in the second, and €224,000 in the third, once the initial project-related costs are no longer present. These results not only highlight profitability but also confirm the long-term sustainability of the business model. It should be noted that the “Participation to project” costs correspond to the company’s co-funding of €80,490, distributed as €53,660 in the first year and €26,830 in the second, reflecting its investment in innovation and future competitiveness.

The integration of agronomic practices, sensory evaluation, and techno-economic analysis underscores the need for holistic approaches in tackling food safety challenges. According to CHRISTIDIS S.A., the production of low-asparagine flour not only reduces public health risks but also creates strategic opportunities for differentiation in domestic and international markets. Contract farming schemes and transparent supply chains can strengthen consumer trust and position the company at the forefront of sustainable agri-food innovation. Furthermore, by aligning with European Commission recommendations on acrylamide reduction, the approach anticipates regulatory developments and transforms compliance into a source of competitive advantage.