Lipid Process Markers of Durum Wheat Debranning Fractions

At present, whole grains are usually obtained by adding bran and middlings to refined flours, and this recombination leads to certain variations in the ratio of endosperm, bran and germ, resulting in flours with very different compositional characteristics and rheological properties. Therefore, this study focuses on the identification of specific lipid markers in different debranning fractions of Italian and Canadian durum wheat blends. The by-products obtained from five different debranning levels (3, 6, 9, 12 and 15%) had a high content of monounsaturated fatty acids and a higher concentration of tocopherols and sterols than the corresponding debranned grains. The Italian and Canadian durum wheat samples did not show significant differences in the content of these bioactive lipid compounds. In particular, palmitic acid, oleic acid, tocopherol isomers and total sterols could be useful biomarkers for evaluating the grain-to-tissue ratio in recombined flours.


Introduction
Durum wheat (Triticum turgidum L. subsp.durum) is widely cultivated worldwide, and it is an essential food source that plays an important role in the human diet, providing carbohydrates, proteins and energy.For this reason, it is milled to produce many staple foods.The traditional milling process of wheat grains produces refined flour by separating endosperm of the kernels from the bran and germ fractions.The separation of aleurone cells from the outer bran layers is very difficult; for this reason, the term bran usually includes also the aleurone layer, which is removed with the bran during traditional milling.In the 1990s, the debranning process was introduced upstream of the traditional milling process and its beneficial effects are now well known.Debranning involves the gradual removal of the outer skin from the surface of the inward kernels through friction or abrasion.This means that the aleurone layer could remain attached to the endosperm and the outermost layers, which could contain contaminants and microorganism, are removed [1,2].The debranning process is becoming increasingly popular in the milling and bakery industries, and several studies report its positive effects on flour quality compared to conventional milling, including an improved final flour colour [1,3], an increase of more than 8% in ash, a decrease of about 20% in α-amylase activity, and a decrease of 71% of the deoxynivalenol levels [1,4].Posner and Hoseney [5] also reported an increase in milling loads due to a reduction in different necessary stages in a milling flow sheet.Debranning also produces new valuable by-products and novel cereal-based foods with the use of debranned cereal kernels.Sun and co-authors [6] observed a positive effect of wheat debranning on the Foods 2023, 12, 3036 2 of 12 quality of bread after in vitro digestion, with a degree of debranning between 3% and 7% offering the highest nutritional value.
Flours currently on the market are obtained via a milling process that aims to extract/separate endosperm from wheat caryopsis to obtain refined flour or semolina.The other parts of the caryopsis (rich in fiber, vitamins and minerals) are considered by-products, including mainly bran and fine bran.In Italy, products obtained from durum wheat that are intended for human consumption are regulated by the Italian Presidential Decree No. 187/2001, which classifies flour according to the degree of refining, as expressed by the ash content.Currently, in most cases, wholemeal type flours are obtained by recombining appropriate amounts of bran and middlings with refined flours.However, this recombination results in products with very different compositional characteristics and technological properties, which also affects the organoleptic profiles of whole-grain end products.This is the consequence of the lack of a clear and precise definition of whole-grain flour in the Italian Presidential Decree No. 187/2001.Moreover, the European Food Safety Authority, in a whole-grain-related health claim opinion [7], provides the definition of the American Association of Cereal Chemists which states that "whole grain consists of the intact, ground, cracked or flaked caryopsis, whose principal anatomical components-the starchy endosperm, germ and bran-are present in the same relative proportions as they exist in the intact caryopsis".In processed whole-wheat products, this cannot be traceably and unambiguously transferred due to a lack of markers and biomarkers of different kernel fractions, such as germ, the aleurone layer and bran.Consequently, recombination per grain and variety leads to certain variations in the ratio of endosperm, bran and germ between flour and product lots, which have very different compositional and rheological characteristics.
For these reasons, in this study, the lipid components of the fractions obtained from different percentages of debranning of durum wheat kernels were characterized.In particular, qualitative and quantitative analyses of fatty acids, tocochromanols and phytosterols were performed in debranned grains and in debranning by-products to identify specific lipid markers.

Samples
Italian (I) and Canadian (C) blends of durum wheat (Triticum turgidum L. subsp.durum), which were obtained from a local distributor, were subjected to five different levels (3, 6, 9, 12 and 15%) of a debranning process, using a lab-scale debranner (TakaYama, Taichung, Taiwan), thereby recovering debranned durum wheat kernels (DG) and debranning by-products (DB).A summary of the samples is presented in Table 1.

Lipid Extraction
Oil was extracted using a Soxhlet apparatus (Behr Labor-Technik, Fischer Scientific Italia, Milano, Italy), as reported by Marzocchi et al. [8] and in accordance with the AOAC Official Method [9] (AOAC International Website).About 10 g of ground wheat fractions were placed in a cellulose thimble, and oil was extracted using a refluxing n-hexane.After using a rotary evaporator to remove the solvent, the oil was taken up with n-hexane/isopropanol (4:1 v/v) solution and stored at −18 • C until use.The oil content of the wheat fractions was expressed as percentage (%) on the total fresh weight of each sample.Each extraction was performed two times (n = 2).

Fatty Acid Analysis
The fatty acid composition from the oil of the wheat fractions was determined as fatty acid methyl esters (FAMEs) via capillary gas chromatography (CGC) analysis after alkaline treatment under the conditions reported by Marzocchi et al. [10].FAMEs were analyzed using a GC 2010 Plus gas chromatograph equipped with a flame ionization detector (FID) and an AOC-20s auto sampler (Shimadzu Corporation, Kyoto, Japan) and using a fused silica capillary column BPX70 (10 m × 0.1 mm i.d., 0.2 µm f.t.) from SGE Analytical Science (Ringwood, VIC, Australia).FAMEs were identified by comparing the peak retention time with GLC-463 and FAME 189-19 standard mixtures, and they were quantified by comparing the peak area of each compound with that of the internal standard (C13:0, 2 mg/mL).The FAME composition was expressed as weight percentage of total FAME (mg/100 mg of FAME), and two replicates for each lipid extract (n = 4) were analyzed.

Tocochromanol Analysis
Tocochromanols were determined using an HPLC (Agilent 1200 series, Palo Alto, CA, USA) equipped with a fluorimetric detector (FLD) as previously described by Ben Lajnef et al. [11].A total of 50 mg of oil was dissolved in 0.5 mL of n-hexane, and then the extracts were filtered using a 0.45 µm nylon filter.The chromatographic separation was carried out using a HILIC Poroshell 120 column (100 mm × 3 mm and 2.7 µm particle size; Agilent Technologies, Palo Alto, CA, USA) under isocratic conditions with n-hexane/ethyl acetate/acetic acid (97.3:1.8:0.9 v/v/v) as the mobile phase and with a flow rate of 0.8 mL/min.
The identification of tocochromanols was conducted by setting the excitation wavelength at 290 nm and the emission wavelength at 325 nm.Tocopherols were identified via co-elution with the standards, whereas tocotrienols were identified by comparing their retention times with that of tocotrienols in a barley extract obtained via hot saponification.α-Tocopherol standard solutions (from 1 to 100 µg/mL) were used for calibration curve construction for quantification.Two replicates for each lipid extract (n = 4) were performed.

Sterol Analysis
The procedure for sterol analysis was the same as reported by Marzocchi et al. [8].After the addition of 0.5 mL of 5α-cholestan-3β-ol (c = 2 mg/mL) as the internal standard, Foods 2023, 12, 3036 4 of 12 250 mg of oil were saponified with 10 mL of 2 N methanolic potassium hydroxide at room temperature for 20 h [12].Subsequently, the organic fraction was washed with 10 mL of diethyl ether and 10 mL of water.The unsaponifiable fraction was further extracted twice with 10 mL of diethyl ether, 10 mL of 0.5 N aqueous KOH and 10 mL of distilled water, respectively.The organic solvent was removed under vacuum, and the unsaponifiable fraction was silylated [13] and used for sterol analysis via GC/MS (GCMS-QP2010 Plus, Shimadzu, Tokyo, Japan) under the same chromatographic conditions as reported by Cardenia et al. [14].The identification of sterols was carried out by comparing mass spectra and retention time with those of the corresponding chemical standards and by comparing them to the GC/MS data reported in the literature [15].The concentration of each individual sterol was obtained via the use of the internal standard and expressed as mg/100 g of fat.The analysis was conducted in 2 replicates for each lipid extract (n = 4).

Statistical Analysis
The Statistica 8 software (2006, StatSoft, Tulsa, OK, USA) was used to calculate oneway ANOVA (analysis of variance) coupled with Tukey's honest significant difference (HSD) test.p-Values lower than 0.05 were considered statistically significant.All results were expressed as the averages of two replicates for each extract (n = 4 for each sample), and the analytical data were used for statistical comparisons.

Lipid Content
By removing individual bran layers from the kernels, the debranning process produces different levels of by-products with a significantly higher fat content than the remaining debranned kernels, because of the different chemical composition of inner and outer layers of caryopsis.Indeed, the results in Figure 1 show how debranning at 6, 9, 12 and 15% produced by-products with the highest fat content at a significant level (p < 0.05), from 6.3 (I-DB6) to 7.4% (C-DB15), compared to the debranning by-products at 3% that showed values equal to 5.4 and 5.3% for Italian and Canadian samples, respectively.All corresponding counterparts, and thus the debranned grains (DG) obtained at the five levels of debranning, presented a significantly (p < 0.05) lower lipid percentage compared to the stripped-off outer layers.These values ranged from 0.5 (I-DG15) to 1.6% (C-DG15), and they did not show significant differences between them.The Italian and Canadian wheat grains presented the same trend and lipid content in the different debranning fractions.
The difference in lipid content confirmed the different quantities of aleurone and germ present in the debranning by-product samples.Unlike the debranned grains, the DB3, DB6, DB9, DB12 and DB15 were probably characterized with the presence of germ that is known to contain more oil than the other bran layers [16].
Figure 1 shows the lipid content (%) in different products obtained from the debranning process of Italian (I) and Canadian (C) durum wheat.
As shown in Figure 2, in all the samples analyzed, PUFAs were present with a percentage ranging from 60 to 64%, and they were characterized by higher levels of linolenic acid (C18:3n3) in the debranning by-products compared to the corresponding debranned grains; less defined and clear was the difference in the content of linoleic acid (C18:2n6) in the various fractions of decortication.MUFAs (17-21%), represented mainly as oleic acid (C18c9), showed significantly higher (p < 0.05) levels in the debranning by-product samples.Finally, the highest levels of SFAs (17-20%), consisting principally of palmitic (C16:0) and stearic acid (C18:0), were found in the debranned grain (DG) samples for all the different debranning percentages.As for MUFAs, the SFA results showed the highest level of this class of FA.Italian and Canadian wheat did not show significant variation in the FA content and distribution in the different debranning samples analyzed.different debranning percentages.As for MUFAs, the SFA results showed the highest level of this class of FA.Italian and Canadian wheat did not show significant variation in the FA content and distribution in the different debranning samples analyzed.

Tocochromanol Determination
In all Italian and Canadian durum wheat decortication fractions, five tocochromanols were identified and quantified: three tocopherols (α-tocopherol, β-tocopherol, and γ-tocopherol) and two tocotrienols (α-tocotrienol and β-tocotrienol) (Figure S1).As shown in Table 3, the various debranning products reported significantly different tocochromanol contents.The debranning fractions showed β-tocotrienol (49-119 and 36-137 mg/100 g of fat in the Italian and Canadian wheat, respectively) and α-tocopherol (38-87 and 43-68 mg/100 g of fat in the Italian and Canadian wheat, respectively) as the principal ones, followed by α-tocotrienol, β-tocopherol and γ-tocopherol (Table 3).In particular, tocopherol isomers presented a higher content in the by-product samples at all different levels of debranning (DB3, DB6, DB9, DB12 and DB15); in contrast, αand β-tocotrienols were more concentrated in all the debranned grain (DG) samples.Considering the total tocochromanol content, the Italian and Canadian wheat showed the highest concentrations at a significant level in different fractions after decortication; in fact, the Italian wheat showed the highest (p < 0.05) concentration in the DG3 and DG6 samples (246.5 and 245.6, respectively), while the Canadian wheat exhibited the highest concentration in the DG12 sample (265.6 mg/100 g of fat).
Comparing the Italian and Canadian grains, the samples registered the same trend but with more significant (p < 0.05) difference in content between debranned grains and debranning by-products in the Canadian samples.Furthermore, the greatest differences (p < 0.05) between the debranned grain and debranning by-product samples were recorded for the level of debranning at 15%.
Additionally, the Italian samples generally presented a higher content of tocochromanols compared to the Canadian samples at the 3 and 6% of debranning levels.In contrast, higher levels of tocochromanols were registered for the Canadian fractions compared to the Italian ones at the 9, 12 and 15% of debranning levels.
At all debranning levels, the total tocochromanol content was higher (p < 0.05) in the DG fractions compared to the DB ones, and this result is mainly due to the high contribution of αand β-tocotrienols that are principally present in the endosperm of kernels.Indeed, according to the literature, the fractions with germ (decortication by-products) are usually richer in tocopherols while the fractions with more endosperm (debranned grains) are richer in tocotrienol [18][19][20].β-Tocotrienol has already been reported in the literature as the main vitamer in dehulled wheats and is known to reduce serum LDL cholesterol through its antioxidant action [19].

Sterols
Sterols are known for their several health-promoting effects like lowering of blood cholesterol and anticancer properties.Because of the high amount typically consumed in the human diet, cereals are a good source of sterols [22].Significant differences in sterol content between the different debranning fractions of the Italian and Canadian durum wheat samples were noted (Table 4).According to the literature [17,21,23], eight phytosterols that are typically found in wheats were identified in all samples: three saturated sterols (campestanol, sitostanol and avenastanol), four ∆ 5 -sterols (campesterol, β-sitosterol, avenasterol and stigmasterol) and one ∆ 7 -sterol (∆ 7 -avenasterol) (Figure S2).
The grain fractions obtained after different levels of debranning showed β-sitosterol as the principal one (671-878 and 689-1002 mg/100 g of sterols in the Italian and Canadian wheat, respectively), followed by campesterol, campestanol, sitostanol, ∆ 5 -avenasterol, stigmasterol, ∆ 7 -avenasterol and avenastanol.In general, all debranning by-product samples (DB3, DB6, DB9, DB12 and DB15) presented significantly (p < 0.05) higher sterol content compared to the corresponding debranning grain samples (DG3, DG6, DG9, DG12 and DG15), both for the Italian and Canadian grains.This result is in line with the literature, and it is due to the presence of bran and germ in the debranning by-products, which are parts of the caryopsis that is rich in sterols [8,22,24].
When looking at the total sterol content, the DB6 and DB12 fractions of both Italian and Canadian wheat showed the highest concentrations at a significant level (p < 0.05): 2175 and 2126.6 mg/100 g of sterols for the Italian wheat, respectively, and 2538 and 2568 mg/100 g of sterols for the Canadian wheat, respectively.In addition, in the Canadian wheat, the DB15 fraction also showed a high concentration of sterols equal to 2633.4 mg/100 g of sterols, which is the highest value registered compared to all other samples.Debranning resulted in a generalized loss of phytosterol, and the highest variation was observed for the Italian grain processed at a 15% of debranning level.In particular, the Italian and Canadian samples showed the same difference between the DG and DB fractions at the levels of 3, 6, 9 and 12% of debranning: the DB3, DB6, DB9 and DB12 presented about 25, 17, 13 and 15% higher sterol content than the corresponding DG3, DG6, DG9 and DG12 samples.On the other hand, the Italian DB15 sample showed a 33% higher content than the DG15 sample, while the Canadian DB15 sample had a total sterol content that was only about 15% higher than the DG sample.

Figure 2 .
Figure 2. Content of fatty acid classes (mg/100 mg of FAME) in different fractions (DG: debranned grain; DB: debranning by-product) of Italian (a) and Canadian (b) durum wheat at different levels of debranning (0, 3, 6, 9, 12, and 15%).Abbreviation: SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.Data are expressed as means ± standard deviation (n = 4).Different lowercase letters mean significant differences (p < 0.05) in the SFA, MUFA and PUFA contents among the debranning fractions within the same durum wheat variety (Italian and Canadian); different capital letters mean significant differences (p < 0.05) in the SFA, MUFA and PUFA contents between the same Italian and Canadian durum wheat debranning fraction.

Table 1 .
Samples obtained from debranning process of Italian and Canadian durum wheat kernels.

Table 2 .
Fatty acid composition and content (mg/100 mg of FAME) of different samples of Italian and Canadian durum wheat at different levels of debranning.

Table 3 .
Tocochromanol composition and content (mg/100 mg of fat) in different samples of Italian and Canadian durum wheat at different levels of debranning.

Table 4 .
Sterol composition and content (mg/100 g of fat) in different samples of Italian and Canadian durum wheat after the debranning process.