Instant controlled pressure drop processing modulates phenolic composition and fatty acid profile in Amaranthus hypochondriacus grain

Abstract

Instant Controlled Pressure Drop (DIC) is a high-pressure/short-time thermo-mechanical pretreatment that can expand grain structure and improve phytochemical accessibility. This study assessed how DIC conditions (0.10-0.40 MPa/10-90 s) modulate phenolic composition and lipid fingerprints in two Amaranthus hypochondriacus grain varieties (“Criollo” and “Laura”) using a 13-run central composite design. Ethanolic extracts were profiled by HPLC, while GC-MS characterized the extracted oils. HPLC identified gallic acid, catechin, p-hydroxybenzoic acid, ellagic acid, trans-4-hydroxycinnamic acid, quercetin, and rutin, plus one additional peak quantified as rutin equivalents. DIC strongly affected total phenolics, with Criollo ranging from 346.14 μg/g (0.14 MPa/22 s) to 3035.2 μg/g (0.25 MPa/50 s) and Laura from 281.51 μg/g (0.36 MPa/78 s) to 2074.15 μg/g (0.25 MPa/50 s). Catechin and Rutin were the predominant compounds across treatments. GC-MS showed oils dominated by palmitic, oleic, and linoleic acids and squalene, with balanced saturated and unsaturated fractions. In both varieties, pressure-time interactions indicated that mild-to-moderate treatments were optimal, maximizing phenolic recovery without markedly altering the fatty-acid signature.

Graphical abstract

Highlights

• •DIC processing of two amaranth varieties boosted phenolic content, peaking at 0.25 MPa/50 s.
• •Major phenolics were quantified by HPLC, with catechin and rutin as predominant compounds.
• •GC–MS showed stable lipid fingerprints dominated by palmitic, oleic, and linoleic acids, with consistent squalene detection.
• •Pressure–time interactions identified mild-to-moderate conditions as optimal without major fatty-acid changes.

1. Introduction

Amaranth (Amaranthus spp.), a staple crop from pre-Columbian civilizations, has regained attention as a valuable pseudocereal for the development of nutrient-dense, gluten-free foods and ingredients. Amaranthus hypochondriacus, A. cruentus, and A. caudatus are among the best-known grain-producing species; they have attracted increased attention for their protein and mineral content and their wide range of bioactive compounds (Kumar et al., 2024). In this context, amaranth stands out for its phytochemical profile, which includes phenolic compounds and flavonoids, as well as a lipid fraction comprising unsaturated fatty acids and essential minor lipophilic constituents such as squalene and tocopherols. These compounds have been linked to antioxidant activity, which supports the growing interest in amaranth as a functional ingredient. However, it is important to note that the concentration and distribution of these phytochemicals are not constant; they may change depending on the species, genotype, and processing conditions. This shows how important it is to evaluate compositional changes under new technologies (Dinu et al., 2016; Kumar et al., 2024; Manyelo et al., 2020; Toimbayeva et al., 2025).

In cereal and pseudocereal matrices, phenolic compounds are generally found as soluble/free molecules and as insoluble “bound” forms that are esterified or otherwise linked to cell-wall polymers and other macromolecular structures. In many grain systems, the bound fraction can make up a large part of the total phenolics, and how much it contributes depends a lot on how much the matrix is broken during processing. In this regard, the distinction is especially pertinent for amaranth, as phenolics of nutritional significance (including hydroxycinnamic acid derivatives) may remain partially incorporated into structural fractions, leading conventional solvent extraction to underestimate the total pool unless processing or hydrolysis facilitates their release. This is consistent with findings indicating that cell wall disruption liberates bound polyphenols, thereby elevating quantifiable phenolic content (Paucar-Menacho et al., 2022; Schendel, 2018; Wang et al., 2014). Thermo-mechanical technologies, such as Instant Controlled Pressure Drop (DIC), create mechanical forces that break down cell walls and structures, creating a porous matrix that makes it easier for solvents to get in and compounds to leach out during extraction (Eikani et al., 2019; Mounir et al., 2014; Rochová et al., 2008).

Chromatographic analysis in amaranth matrices have reported compounds such as hydroxycinnamic acids, like p-coumaric, ferulic, caffeic, and chlorogenic acids; hydroxybenzoic acids including gallic, vanillic, and p-hydroxybenzoic acids; and flavonoid aglycones and derivatives, often linked to quercetin- and kaempferol-related compounds. Overall, targeted profiling in amaranth seeds supports the idea that the qualitative phenolic spectrum is broad, even though the quantitative distribution of individual compounds can be markedly influenced by the sample characteristics (species/variety) and by the extraction and analytical conditions applied (Dinu et al., 2016; Manyelo et al., 2020).

Consequently, processing techniques should be regarded as a viable experimental approach to modify phenolic accessibility and, in certain instances, to affect the phenolic profile, employing treatments that may change microstructure, pH, enzymatic exposure, and/or thermal kinetics, and at the same time, have these processing conditions meticulously regulated to mitigate undesirable degradation.

In addition to phenolic compounds, the lipid fraction of amaranth grain represents a relevant compositional feature that distinguishes this pseudocereal from many conventional grains. In general, amaranth seed oil is characterized by a predominance of unsaturated fatty acids, where C18:2 and C18:1 are commonly reported as the major components, followed by C16:0 and C18:0. This distribution is consistent with targeted characterizations of amaranth seeds, which, have similarly reported an overall dominance of unsaturated fatty acids, reinforcing the relevance of the grain lipid fraction when assessing compositional quality and the potential impact of processing (El Gendy et al., 2017; He and Corke, 2003; A. Hlinková et al., 2013a; Zhang et al., 2019).

Minor lipophilic constituents further underscore the importance of evaluating amaranth lipids beyond total lipid content and basic fatty acid distribution. Among these compounds, squalene is of particular interest (Levytsky et al., 2024; Preobrazhenskaya et al., 2016). Squalene, a bioactive compound, is found in significant quantities in amaranth oil and exhibits various therapeutic properties, including antioxidant, anti-inflammatory, hepatoprotective, and cardioprotective activities (Czaplicki et al., 2012; Levytsky et al., 2024).

Since unsaturated fatty acids and small lipids are susceptible to oxidation and heat, short-term, carefully controlled processing methods are especially appealing when the goal is to improve extractability or bio accessibility while keeping lipids intact.

One of these strategies is Instant Controlled Pressure Drop (DIC), which is a high-pressure, short-time thermomechanical process. In DIC, the product is briefly exposed to high-pressure saturated steam before being suddenly dropped to a vacuum (Fig. 1). This rapid drop in pressure causes the moisture inside to evaporate on its own, cooling it immediately. It also causes the material to expand and form microstructural alveolation. The microstructural effects are often the most important for achieving higher extraction yields and accessing compounds more easily (J. L. Pech-Almeida et al., 2021a; Rane et al., 2022; Rodriguez-Castillo et al., 2025; Berka-Zougali et al., 2014; Tienda-Vazquez et al., 2023).

Fig. 1.

DIC processing cycle: 1) introduction of food material to the reactor; 2) initial vacuum within the reactor; 3) injection of dry saturated steam until achieving the desired pressure and retaining it during the selected time frame; 4) instant controlled pressure-drop towards a vacuum, and 5) releasing to atmospheric pressure. Taken from Juan Leopoldo Pech-Almeida et al., 2021a.

DIC may enhance mass transfer by inducing expansion, increasing porosity, and disrupting compact structures, thereby facilitating solvent penetration into the matrix and promoting the release of phenolic compounds that would otherwise remain physically entrapped or weakly associated within the cell-wall network ((Mounir et al., 2014; Pech-Almeida et al., 2021a). Experimental evidence in legumes corroborates this mechanism: DIC treatment of green lentils resulted in significant increases in polyphenols/flavonoids and antioxidant capacity, aligning with improved extractability following thermomechanical expansion (Tienda-Vazquez et al., 2023).

A previous study indicated that DIC processing enhanced phytochemical-related metrics (e.g., phenolic/flavonoid-linked antioxidant capacity) in amaranth, demonstrating the viability of DIC as a method to augment the functional potential of amaranth ingredients (Rodriguez-Castillo et al., 2025).

In the case of amaranth grain flour, where unsaturated fatty acids and squalene are important parts of the composition, the main question is whether DIC can make target compounds easier to extract while keeping a good fatty-acid profile.

Accordingly, a focused study using A. hypochondriacus grain flour that incorporates (i) HPLC phenolic profiling and (ii) GC–MS fatty-acid profiling under defined DIC parameters can provide evidence regarding whether DIC facilitates quantifiable alterations in individual phenolic constituents while causing shifts in, or the preservation of, the fatty-acid. In this way, the work goes beyond global antioxidant indicators by connecting DIC-driven thermomechanical and microstructural effects to compositional fingerprints that are directly useful for making functional foods, positioning them nutritionally, and optimizing processes.

2. Materials and methods

2.1. Chemicals and reagents

Chemical reagents and standards were obtained from Sigma–Aldrich® (St. Louis, MO, USA).

2.2. Amaranth grains collection and preparation

Two subvarieties of A.hypochondriacus grains, “Laura” and “Criollo,” were obtained from the 2023 harvest in Puebla, Mexico. Before treatment, to avoid possible field contaminants, both samples were quickly washed with tap water for 1 min and dried at 50 °C for 8 h until they reached 10% humidity.

2.3. Amaranth treated by the instant controlled pressure drop process

The Instant Controlled Pressure Drop technique was used to pop 100 g of dried amaranth grains with the LAB DIC 0.1 equipment (ABCAR-DIC Process, La Rochelle, France). Selected operative parameters were saturated steam pressure (P) and processing time (t), ranging from 0.10 to 0.4 MPa and 10 to 90 s, respectively. For DIC treatment, the grains were first placed in the DIC reactor, then saturated steam was injected until the target pressure was reached, and the pressure was maintained for the selected time. Subsequently, samples were subjected to an instant controlled pressure drop (ΔP/Δt > 0.5 MPa.s⁻1) towards vacuum (0.003 MPa). Finally, the pressure was released to atmospheric pressure, and the samples were recovered. Once finished, the treated seeds were collected and dried in a hot air oven at 50 °C for 1 h to eliminate water and steam residues, and the dried samples were stored in sealed bags for further analysis.

2.4. DIC experimental design

To assess the impact of DIC treatments on both amaranth grains varieties, a central composite design with 4 factorial points (2ⁿ = 2^²=(−1/-1;-1/+1; +1/-1, and +1/+1), 4 start trials (2*n) = 2x2=(-α/0; +α/0; 0/-α and 0/+α); and 5 central points (0,0) was employed, resulting in a total of 13 experimental points. The analysis used both a response surface plot and a Pareto chart to assess the influence of variables on the responses, with a vertical line indicating statistically significant effects at the 95% confidence level. Steam pressure and processing time were considered as independent variables. Table 1 shows the experimental design for both amaranth grain varieties.

Table 1.

Experimental design for DIC treatment of A. hypochondriacus grains.

Treatment	1	2	3	4	5	6	7	8	9	10	11	12	13
Pressure (MPa)	0.25	0.40	0.25	0.25	0.36	0.36	0.25	0.14	0.14	0.25	0.10	0.25	0.25
Time (s)	50	50	90	50	78	22	50	22	78	50	50	10	50

2.5. Ethanolic extracts preparation

DIC-treated amaranth grain samples and control grains (raw material) were ground in a coffee grinder (Krups GX4100, Solingen, Germany) until a fine powder was obtained (particle size, 0.074 mm). Then 2 g of each sample was weighed in a 50 mL conical flask and mixed with 20 mL of ethanol. Subsequently, at room temperature (25 °C), samples were left covered from light for 24h of maceration. After that, they were mixed for 2 h under orbital agitation at 130 rpm (Junior orbit shaker 3520, Chennai, India). After agitation, samples were centrifuged at 10000 g for 10 min in a Heraeus Multifuge X1R centrifuge (Thermo Scientific, Massachusetts, United States). Finally, the supernatant was recovered and stored at −20 °C for future analysis. All samples were prepared in triplicate.

2.6. Phenolic profile through HPLC

Furthermore, the phenolic compounds profile was determined by high-performance liquid chromatography (HPLC) using an Agilent 1200 HPLC system (Agilent Technologies 1200 series, Palo Alto, CA, USA), equipped with a quaternary pump, autosampler, and a diode-array detector. Separation of phenolic compounds was performed using an Agilent Eclipse XDB-C18 column (150 mm, 5 μm, 4.6 mm) at 25 °C.

Extracts were previously filtered with a Captiva Syringe Filter PTFE membrane (25 mm diameter, 0.45 μm pore size); the mobile phases were also filtered using a 0.45 μm nylon membrane filter. The method described by House et al. (2020) was followed with slight modifications, employing two mobile phases: phase A consisted of water with 0.1% formic acid, and phase B consisted of methanol. The HPLC elution gradient used was as follows: 95% solution B and 5% solution A for 0 to 5 min, 70% solution B and 30% solution A from 5 to 10 min, 65% solution B and 35% solution A from 10 to 20 min, 50% each of solution B and A were used from 20 to 40 min and 90% of solution A was used till the completion of 60 min, at a flow rate of 1.0 mL/min. Two injections per sample were performed, each with an injection volume of 20 μL. The total run time was 60 min. Detection was carried out at 280 nm. Compounds were quantified using calibration curves constructed for each standard, with concentration ranges from 0.025 to 0.05 mg/mL.

2.7. Accelerated solvent extraction ASE

Amaranth oil extraction was accomplished with a Dionex ASE 350 Accelerated Solvent Extractor (Dionex, Sunnyvale, CA). The pressure was held at 10.34 MPa and the temperature at 110 °C. Each sample (1.5g) was ground with 1.5 g of diatomaceous earth and placed in a 10 ml extraction cell loaded with a cellulose filter. Each sample was extracted three times with 15 mL of hexane: isopropanol (3:2) with a static time of 2 min. Then, the solvents were evaporated in a Thermo Scientific Savant SpeedVac SPD140DDA concentrator (Thermo Fisher Scientific). The obtained fat was stored in Eppendorf tubes in the dark at −20 °C until analysis by gas chromatography.

2.8. Fatty acids GC-MS analysis

Fifty milligrams of oil were dissolved in 900 μL of high-performance liquid chromatography (HPLC)-grade chloroform. Esterification was carried out by adding 100 μL of 5 N sodium methoxide and stirring for 5 min. Fatty acids (FAs) analysis was performed using an Agilent 7890A capillary GC system (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 5975C inter MSD equipped with a split/splitless injector and a selective mass detector operating in electron impact mode at 70 eV. The FAs were separated using a capillary column (HP-88, Agilent, 100 m × 0.250 mm × 0.20 μm) with the following temperature program: initial temperature, 50 °C; increase to 85 °C at 2.5 °C/min; increase to 170 °C at 10 °C/min; constant 170 °C for 20 min; final increase to 250 °C at 10 °C/min; and a constant 250 °C for 25 min. Using a 2:1 split, 1 μL of the methylated sample was injected, with the injector temperature set to 250 °C. Helium was used as the carrier gas at a flow rate of 4 mL/min. Identification was conducted using the NIST 05 library. The area under each FA peak, relative to the total area of all FA peaks, was used to quantify the FAs identified. The results obtained are reported as a percentage of the total FA.

2.9. Statistical analysis

All data were processed using Statistica Software 2022 (TIBCO Software Inc., Palo Alto, California, CA, USA), adhering to a pre-established significance threshold of p < 0.05. Descriptive statistical methods were applied. Response surface methodology and Pareto charts were used to evaluate the effects of Instant Controlled Pressure Drop on the phenolic and FAs profiles of amaranth grains. Saturated steam pressure MPa) and thermal processing time (s) were studied as independent variables; meanwhile, the response variables were the different phenolic compounds and FAs.

An ANOVA was used to assess differences between the treatment and control groups. A post hoc Dunnett test was conducted to analyze the treatment differences relative to the control. For non-normal data, the Kruskal-Wallis nonparametric test was used.

3. Results and discussion

3.1. Criollo variety: phenolic and fatty acid profile

The HPLC method was used to separate, identify, and quantify the phenolic acids and flavonoids in the ethanolic extracts of A. hypochondriacus grain. Table 2 shows the results of the HPLC analysis. Among all the amaranth extracts, DIC-treated and control, eight peaks were observed, of which seven of them were well identified with the standards as Gallic Acid, Catechin, P-dihydroxybenzoic Acid, Ellagic Acid, Trans-4-Hydroxycinnamic Acid, Quercetin, and Rutin. The eighth peak, found at minute 44.7, was expressed as equivalent to the closest one, being Rutin. It was possible to highlight that DIC 7 (0.25 MPa/50s) showed the highest phenolic compound concentration (3035.2 μg/g). Furthermore, DIC 11 and 13 showed the greatest variety of compounds, accounting for seven of the eight peaks identified. Moreover, Catechin with a retention time of 5.2 min was the most abundant compound in all samples. Chromatograms from all samples, treated and control can be seen in Fig. 1S from the supplementary material. A 2024 study analyzed phenolic content across 9 amaranth species cultivated in 2 years. Their results revealed that concentrations among the different varieties were different, and in the case of A. hypochondriacus, it presented 7 hydroxybenzoic acids, 5 hydroxycinnamic acids, and 5 flavonoids; the concentrations varied between the seeds harvested in 2018 and those from 2019 (Bang et al., 2024). This is in accordance with the fact that the quantitative distribution of individual compounds can be markedly influenced by the sample characteristics (species/variety/environmental conditions).

Table 2.

Identification and quantification of phenolic compounds in A. hypochondriacus, “Criollo” variety extract by high-performance liquid chromatography (HPLC).

	RT	DIC 1	DIC 2	DIC 3	DIC 4	DIC 5	DIC 6	DIC 7	DIC 8	DIC 9	DIC 10	DIC 11	DIC 12	DIC 13	Control
Pressure (MPa)		0.25	0.40	0.25	0.25	0.36	0.36	0.25	0.14	0.14	0.25	0.10	0.25	0.25	NA
Time (s)		50	50	90	50	78	22	50	22	78	50	50	10	50	NA
Gallic Acid (μg/g)	1.7	42.3 ± 1.2a	36.6 ± 1.9a	38.4 ± 2.6a	45.1 ± 1.1a	42.9 ± 2.9a	41.5 ± 2.1a	46.1 ± 1.9a	8.57 ± 0.7	40.4 ± 1.6a	47.5 ± 1.9a	67.5 ± 4.2a	38.6 ± 3.1a	81 ± 6a	25.1 ± 1.3
Catechin (μg/g)	5.2	1508 ± 89.8a	2139 ± 58.9a	1394 ± 18.8a	2357 ± 299 a	1868 ± 172 a	1784 ± 16 a	2704 ± 38.6a	337 ± 42	1416 ± 80.3a	847 ± 10	708 ± 31	1080 ± 70	1623 ± 143a	679 ± 29
P-Hidroxybenzoic Acid (μg/g)	5.5	-	-	-	-	-	-	-	-	-	-	19.6 ± 0.3	8.56 ± 1.3	8.65 ± 2.6	-
Elagic Acid (μg/g)	6.7	-	-	-	-	-	-	-	-	56.3 ± 7.7	55.9 ± 4.9	37.2 ± 5.1	67.3 ± 5.3	55.3 ± 5.3	-
Trans-4-Hidroxycinamic Acid (μg/g)	7.4	8.62 ± 0.1a	5.65 ± 0.6a	5.90 ± 0.4a	17.5 ± 2a	5.68 ± 0.1a	7.26 ± 0.7a	8.13 ± 0.2a	-	13.19 ± 1.3a	6.65 ± 0.1a	4.11 ± 0.1a	13.02 ± 0.8a	7.34 ± 0.6a	-
Quercetin (μg/g)	9.8	25.4 ± 0	-	-	30.3 ± 0	34.2 ± 1.6	48.5 ± 3.7a	86.1 ± 1.1a	-	-	120 ± 10a	55.1 ± 7.6a	-	55 ± 4a	32.0 ± 0.8
Rutin (μg/g)	27	174 ± 9.8a	368 ± 24a	210 ± 19a	244 ± 28a	385 ± 42a	246 ± 24a	190 ± 20a	-	116 ± 13	234 ± 18a	130 ± 13	-	511 ± 52a	95 ± 1.5
Peak 8 (μg/g)	44.7												65.2 ± 2.4
Total Peaks		5	4	4	5	5	5	5	2	5	6	7	6	7	4
Total concentration (μg/g)		1776.33	2549.96	1649.42	2359.29	2336.38	2128.18	3035.2	346.14	1643.16	1312.64	1041.56	1170.11	2347.97	828.38

†DIC: Instant Controlled Pressure Drop; RT: retention time (min).

^aMeans statistically higher than the control (Dunnett, p < 0.05). The results are the average of triplicates.

Regarding the DIC sample extracts' results, it can be pointed out that under the selected study's parameters of steam pressure (P) and treatment time (t), there was an important variation in the polyphenol concentration of each extract, varying from 346.14 μg/g (DIC 8) to 3035.2 μg/g (DIC 7). The lowest performance was shown by DIC 8 (0.14 MPa/22s), demonstrating that low pressure coupled with a short time frame could affect the release of the phenolic compounds; in contrast, DIC 7 (0.25 MPa/50s) shows us that, at least in this specific amaranth variety, moderate pressure and time parameters are the best option to enhance the extraction and quantification of phenolic compounds. Fig. 2 displays the Pareto charts with their respective response surface graph of the 4 principal compounds found, being gallic acid, catechin, quercetin, and rutin. In these graphs, it can be appreciated how the conditions of pressure and time influenced the concentration detected in the amaranth grains; as mentioned before, the combination of moderate to high time frame and pressure yield the best results (Equations (1)–(4)). Similar results were found in a previous study in which these same grains were assessed, resulting in improved antioxidant activity and total phenolic/flavonoid content after treatment with DIC at moderate to high pressure and a moderate time frame (0.36 MPa/22s) (Rodriguez-Castillo et al., 2025). Also demonstrated by Martínez-Meza et al. (2021), combinations of high pressure and a wide range of time frames enhance the extraction of phenolic compounds from grape pomace. Similar findings were reported by Şahin (2024), who found that pressures of 0.5 MPa combined with 120s improved the concentration of bioactive components in oleaster flour.

GAμg/g = −33.4 + 266.5P - 301.3P² + 2t - 0.01t² - 2.5 Pt + 0. (Eq. 1)

Catechin μg/g = −2421 + 15018.3P - 12892.9P² + 61t - 0.3t² - 80.7 Pt + 0. (Eq. 2)

Quercetin μg/g = −136.8 + 806.7P - 1478.6P² + 4.05t - 0.03t² - 1.1 Pt + 0. (Eq. 3)

Rutin μg/g = −363.3 + 1158.7P - 580.3P² + 12.46t - 0.1t² + 1.8 Pt + 0. (Eq. 4)

Fig. 2.

Pareto Charts with their respective Response Surface graphs, Criollo Variety;

Gallic Acid (a, b), Catechin (c, d), Quercetin (d, e), Rutin (g, h).

To assess the impact of DIC treatment on amaranth, lipid fraction composition was analyzed by gas chromatography-mass spectrometry (GC-MS) as an exploratory method to identify the predominant fatty acid (FA) families in each treatment. The analysis confirmed the presence of fourteen major FAs and an important triterpenoid in Criollo amaranth grain extracts across all samples. Table 3 confirms the components and their classifications based on their saturation degree. While most identified FAs were consistently present across all samples, some exceptions were observed. Notably, palmitic acid, stearic acid, linolenic acid, arachidic acid, heneicosanoic acid, and docosanoic acid were absent in specific treatments, suggesting potential alterations in FA composition due to DIC treatment. This is highly consistent with the findings of Teresa-Martínez et al. (2025), who reported that DIC treatment in cardamom seeds also led to the absence of stearic acid, linolenic acid, and arachidic acid under certain conditions.

Table 3.

Lipid Fraction composition Criollo variety.

Compound	Name	CAS	Classification	Grain composition percentage %
(C13:0)	Tridecanoic acid	000638-53-9	SFA	0.01 ± 0
(C15:0)	Pentadecanoic acid	005129-60-2	SFA	0.2 ± 0
(C16:0)	Hexadecanoic acid/Palmitic acid	000112-39-0	SFA	11 ± 0
(C17:0)	Heptadecanoic acid/Margaric acid	001731-92-6	SFA	1 ± 0
(C18:0)	Octadecanoic acid/Stearic acid	000112-61-8	SFA	10 ± 0
(C18:1 cis-Δ9)	Oleic Acid	000112-80-1	MUFA	27 ± 1.1
(C18:2 Δ8,11)	8,11-Octadecadienoic acid	056599-58-7	PUFA	0.01 ± 0
(C18:2 cis,cis-Δ9,12)	Linoleic acid	000112-63-0	PUFA	21 ± 1.2
(C20:0)	Eicosanoic acid/Arachidic acid	001120-28-1	SFA	1 ± 0
(C20:1 Δ11)	11-Eicosenoic acid	003946-08-5	MUFA	1 ± 0
(C21:0)	Heneicosanoic acid	006064-90-0	SFA	0.01 ± 0
(C20:2 Δ11,13)	11,13-Eicosadienoic acid	056599-57-6	PUFA	2 ± 0
(C22:0)	Docosanoic acid/Behenic acid	000929-77-1	SFA	0.1 ± 0
(C18:2 cis, cis-Δ9,12)	9,12-Octadecadienoic acid	002462-85-3	PUFA	0.01 ± 0
Squalene		007683-64-9	Terpenoid	26 ± 1.2

Percentages are the average of triplicates.

† SFA= Saturated Fatty Acid, MUFA = Monounsaturated Fatty Acid, PUFA= Polyunsaturated Fatty Acid.

Analysis of amaranth grain extracts exposed to different DIC treatments revealed significant variation in fatty acid profiles across samples. The predominant components identified in the samples (treated and control) include the important triterpenoid squalene, unsaturated FAs such as Oleic and Linoleic acids, and the saturated FA palmitic acid. These results align with findings from a study evaluating the fatty acid composition of amaranth grains grown over two consecutive years. In this study, the dominant components of the A. hypochondriacus grain oil were Palmitic acid (31.3%), Oleic acid (23.8%), and Linoleic acid (40.1%) in both years analyzed (Hlinková et al., 2013a). These results are also in accord with Szabóová et al. (2020), who reported Palmitic acid 20.4%, Oleic acid 26.25%, and Linoleic acid 46.06% as the dominant components of A. hypochondriacus seed oil; they also highlighted the importance of the high squalene content in amaranth seeds.

Remarkably, some saturated fatty acids, such as stearic and arachidic acid, are present in lower amounts; stearic acid was detected in all samples except on DIC6 (0.36 MPa/22s), and DIC11 (0.1 MPa/50s), and arachidic acid was detected in less than half of the samples. This could indicate that DIC treatments have a selective impact on the retention or extraction of these less abundant fatty acids.

Fig. 3 proves the impact of pressure “P” and processing time “t” on the yield of each dominant lipid constituent, squalene, palmitic acid, oleic acid, and linoleic acid. As illustrated in Fig. 3ab, the quadratic effect of time suggests a medium to high time frame to achieve a higher percentage of palmitic acid (Equation (5)). Fig. 3cd displays the influence of time, its quadratic effect, and its interaction with pressure, with low pressures and long time frames being the best parameters for achieving higher linoleic acid percentages (Equation (6)). Fig. 3e and f demonstrate how no effect from the DIC parameters studied was significant to the behavior of Oleic acid and Squalene. DIC6 (0.36 MPa/22s) portrayed the highest percentage of Palmitic acid, whereas DIC9 (0.14 MPa/78s), DIC12 (0.25 MPa/10s), and DIC 10 (0.25 MPa/50s) exhibited the highest percentages for Linoleic acid, Oleic acid, and Squalene, respectively. This analysis highlights the role of different processing parameters in modulating lipid fraction composition; for instance, pressure has been shown to play a key role in the extraction efficiency of linoleic acid, oleic acid, and squalene. While the effect on linoleic acid is minimal, higher pressures commonly tend to enhance the extraction yields of oleic acid and squalene (Akgün, 2011; Al-Darmaki et al., 2012; Dorado Achicanoy et al., 2018; Hartati et al., 2018; Hrnčič et al., 2019; Norhidayah et al., 2012).

Palmitic acid = −2668.44 - 5746.39P + 4410.20P² - 8.28t - 0.011t² - 12.74 Pt + 0. (Eq. 5)

Linoleic acid = -165.34 + 2467.39P - 1862.13P² + 809t - 0.007t² - 36.86 Pt + 0. (Eq. 6)

Fig. 3.

Pareto Charts with their respective Response Surface graphs, Criollo Variety; Palmitic Acid (a, b), Linoleic Acid (c, d), Oleic Acid Pareto chart (e), Squalene Pareto chart (f).

Based on the composition percentages presented in Table 3, the comparison between saturated and unsaturated fatty acids shows that saturated fatty acids occupy only 22% of the total oil components, 29% are monounsaturated fatty acids (MUFAs), basically Oleic acid, 23% are polyunsaturated fatty acids (PUFAs), essentially Linoleic acid, and Squalene represents 26% prevail in the samples. The balanced presence of saturated and unsaturated fatty acids in A. hypochondriacus oil, along with its squalene content, makes it a versatile and valuable ingredient for various applications (Hlinková et al., 2013a; Lourembam et al., 2025).

As shown in Table 3, the percentages of the main amaranth oil composition, in accordance with Fig. 3 and as mentioned previously, are highest for Squalene, Linoleic acid, Oleic acid, and Palmitic acid (hexadecenoic acid).

3.2. Laura variety: phenolic and fatty acid profile

Table 4 shows the results of the HPLC analysis for the Laura variety. Amid all the amaranth extracts, DIC treated and control, ten peaks were observed, from which seven of them were well identified with the standards as Gallic Acid, Catechin, Vanillin, P- Coumaric Acid, Trans-4-Hidroxycinamic Acid, Quercetin, and Rutin, peak 2, found at minute 3.6 was expressed as equivalent to the closest one, being Gallic Acid, peak 8, found at minute 11.6 was expressed as equivalent to the closest one, being Quercetin, peak 10, found at minute 44.7 was expressed as equivalent to the closest one, being Rutin. DIC 10 (0.25 MPa/50s) showed the highest concentration of phenolic compounds (2074.5 μg/g); however, it didn't differ significantly from the untreated group, which showed a total concentration of 2050.24 μg/g. Furthermore, DIC 6 (0.36 MPa/22s) showed the widest range of compounds, accounting for nine of the ten peaks identified. As noted, in this variety, two additional peaks were detected compared to the Criollo variety, once again demonstrating that compound distribution and concentration depend on factors such as variety, growing conditions, etc. (Dinu et al., 2016; Manyelo et al., 2020). Moreover, Catechin, with a retention time of 5.2 min, and Rutin, with a retention time of 27 min, were the most abundant compounds in all samples, which is consistent with the Criollo variety. Chromatograms of all samples, treated and conttrol can be seen in Fig. 2S from the Supplementary material.

Table 4.

Identification and quantification of phenolic compounds in A. hypochondriacus, “Laura” variety extract by high-performance liquid chromatography (HPLC).

	RT	DIC 1	DIC 2	DIC 3	DIC 4	DIC 5	DIC 6	DIC 7	DIC 8	DIC 9	DIC 10	DIC 11	DIC 12	DIC 13	Control
Pressure (MPa)		0.25	0.40	0.25	0.25	0.36	0.36	0.25	0.14	0.14	0.25	0.10	0.25	0.25	NA
Time (s)		50	50	90	50	78	22	50	22	78	50	50	10	50	NA
Gallic Acid (μg/g)	1.7	16.6 ± 0.5	22.2 ± 0.8	12.4 ± 1.5	26.8 ± 2.1	22.2 ± 0.6	21.3 ± 0.5	21.2 ± 2	28.3 ± 1.5	32.7 ± 1.4	19.1 ± 1.7	41 ± 4.2a	35.7 ± 2.4	22.5 ± 1.9	31.3 ± 2
Peak 2	3.6	-	-	-	-	-	11.5 ± 0.5	-	-	-	-	-	-	-	-
Catechin (μg/g)	5.2	1457 ± 121	423.0 ± 52	217.8 ± 13.3	1743 ± 154	217.5 ± 24	663 ± 11	903.0 ± 32	175 ± 11	1169 ± 67	1709 ± 175	267.2 ± 32	1700 ± 3.6	1465 ± 14	1857 ± 95
Vainillin (μg/g)	5.9	-	-	3.32 ± 0.5	-	-	61.3 ± 3.8	-	-	-	-	-	-	-	-
P-Coumaric Acid (μg/g)	6.8	1.07 ± 0.06	0.23 ± 0.04	-	-	-	-	-	-	-	-	-	-	-	-
Trans-4-Hidroxycinamic Acid (μg/g)	7.4	1.15 ± 0.1	1.40 ± 0.2	5.66 ± 0.5a	1.52 ± 0.1	-	9.85 ± 0.9a	17.58 ± 0.3a	6.77 ± 0.7a	6.19 ± 0.5a	8.61 ± 0.5a	6.52 ± 0.7a	4.70 ± 0.5	5.02 ± 0.1	2.94 ± 0.3
Quercetin (μg/g)	9.8	-	-	-	-	-	35.6 ± 1.0	-	5.7 ± 0.9	64.9 ± 2.6	-	-	18.0 ± 1.9	-	51 ± 2.4
Peak 8	11.8	-	-	-	-	-	125 ± 8.1	-	-	-	-	-	-	-	-
Rutin (μg/g)	27	272 ± 28a	136 ± 0.8	167 ± 12.1	281 ± 3.5a	41 ± 0.7	486 ± 4.3a	221 ± 8.4a	301 ± 14.7a	330 ± 6.6a	337 ± 18.5a	185 ± 18.0a	229 ± 10.0a	283 ± 25a	107 ± 7.2
Peak 10 (μg/g)	44.7	-	-	-	-	-	369 ± 21	22.1 ± 2.8	234 ± 11	58.9 ± 5.7	-	61 ± 5.1	63.5 ± 4.6	12 ± 1.8	-
Total Peaks		5	5	5	4	3	9	5	6	6	4	5	6	5	5
Total concentration (μg/g)		1749.46	583.85	406.43	2053.85	281.51	1783.67	1185.49	751.47	1662.82	2074.15	561.65	2051.69	1789.98	2050.24

†DIC: Instant Controlled Pressure Drop; RT: retention time (min).

^aMeans statistically higher than the control (Dunnett, p < 0.05). The results are the average number of triplicates.

Regarding this DIC sample extracts' results, it can be pointed out that under the selected study's parameters of steam pressure (P) and treatment time (t), there was an important variation in the polyphenol concentration of each extract, varying from 281.51 μg/g (DIC 5) to 2074.15 μg/g (DIC 10). The lowest performance was shown by DIC 5 (0.36 MPa/78s), not only was this sample the one with the lowest total concentration, but it also displayed only three peaks from the ten detected, demonstrating that high pressure coupled with long time frame could accelerate bioactive compounds degradation, it should be noted that even though DIC 5 applied the same pressure parameter as DIC 6, which is the one who exhibited the highest number of peaks, the time frame in DIC 5 was more extended, which could have been one of the reasons why compounds lost is seen¸ comparable to this Chatur et al. (2022) found a decrease in phenols, flavonoids and even antioxidant activity after processing chickpeas with high pressure conditions for long time frame (5min). In contrast, DIC 10 (0.25 MPa/50s) demonstrates that, at least in this specific amaranth variety, moderate pressure and time parameters are the best option for enhancing extraction and preserving the quantification of phenolic compounds. Fig. 4 displays the Pareto charts with their corresponding response surface graph of the 3 principal compounds found, being gallic acid, catechin, and rutin, in these graphs it can be appreciated how the conditions of pressure and time influenced the concentration detected in the amaranth grains, as mentioned before combination of moderate time frame and pressure, yielded the best results (Equations (7)–(9)). This was comparable to the Criollo variety, which also showed enhanced concentrations in medium DIC conditions.

GAμg/g = 58.82-195.36P + 328.52P²-0.14t+0.0009t²-0.28 Pt+0. (Eq. 7)

Catechin μg/g = -2668.44-5746.39P+4410.20P²- 8.28t-0.011t²- 12.74 Pt+0. (Eq. 8)

Rutin μg/g = -165.34 + 2467.39P-1862.13P²+809t-0.007t²-36.86 Pt+0. (Eq. 9)

Fig. 4.

Pareto Charts with their respective Response Surface graphs, Laura Variety. Gallic Acid (a, b), Catechin (c, d), Rutin (e,f).

Passing to the lipid fraction of this amaranth variety, GC-MS analysis confirmed the presence of thirteen major FAs and an important triterpenoid in Criollo among all samples.

Table 5 confirms the components and their classifications based on their saturation degree. While most identified FAs were consistently present across all samples, some exceptions were observed. Notably, PUFAs, 8,11-Octadecadienoic acid, and palmitic acid, 11,13-Eicosadienoic acid, were only present in some of the DIC-treated samples, and not in the control. On the other hand, stearic acid, margaric acid, and behenic acid were absent in specific DIC treatments, suggesting potential alterations in FA composition.

Table 5.

Lipid Fraction composition of Laura variety.

Compound	Name	CAS	Classification	Grain composition percentage %
(C13:0)	Tridecanoic acid	000638-53-9	SFA	0.01 ± 0
(C15:0)	Pentadecanoic acid	005129-60-2	SFA	0.01 ± 0
(C16:0)	Hexadecanoic acid/Palmitic acid	000112-39-0	SFA	20 ± 1.0
(C17:0)	Heptadecanoic acid/Margaric acid	001731-92-6	SFA	1 ± 0
(C18:0)	Octadecanoic acid/Stearic acid	000112-61-8	SFA	7 ± 0.2
(C18:1 cis-Δ9)	Oleic Acid	000112-80-1	MUFA	24 ± 1
(C18:2 Δ8,11)	8,11-Octadecadienoic acid	056599-58-7	PUFA	0.01 ± 0
(C18:2 cis,cis-Δ9,12)	Linoleic acid	000112-63-0	PUFA	25 ± 1.2
(C20:0)	Eicosanoic acid/Arachidic acid	001120-28-1	SFA	0.1 ± 0
(C20:1 Δ11)	11-Eicosenoic acid	003946-08-5	MUFA	3 ± 0
(C21:0)	Heneicosanoic acid	006064-90-0	SFA	1 ± 0
(C20:2 Δ11,13)	11,13-Eicosadienoic acid	056599-57-6	PUFA	0.01 ± 0
(C22:0)	Docosanoic acid/Behenic acid	000929-77-1	SFA	0.1 ± 0
Squalene		007683-64-9	Terpenoid	19 ± 1

Percentages are the average of triplicates.

† SFA= Saturated Fatty Acid, MUFA = Monounsaturated Fatty Acid, PUFA= Polyunsaturated Fatty Acid.

Similar to the Criollo variety, the main components were Palmitic, Oleic, and Linoleic acids, along with the triterpenoid Squalene.

Fig. 5 proves the impact of pressure “P” and processing time “t” on the yield of each dominant lipid constituent. As illustrated in Fig. 5ab, the quadratic effect of time and pressure suggests that moderate conditions are required to achieve a higher percentage of linoleic acid (Equation (10)). Fig. 5cd shows the influence of pressure and its quadratic effect, with lower pressures yielding higher squalene percentages (Equation (11)). Fig. 5e and f demonstrate how no impact from the DIC parameters studied was significant to the behavior of Palmitic acid and Oleic acid. Analyzing these effects, it is noted that DIC3 (0.25 MPa/90s) portrayed the highest percentage of Palmitic acid, whereas DIC4 (0.25 MPa/50s), DIC13 (0.25 MPa/50s), and DIC9 (0.14 MPa/78s) exhibited the highest percentages for Linoleic acid, Oleic acid, and Squalene, respectively. With this, it is shown that at least with the Laura variety, low-medium pressures play a crucial role in the extraction efficiency of linoleic acid, oleic acid, and squalene (Akgün, 2011; Al-Darmaki et al., 2012; Dorado Achicanoy et al., 2018; Hartati et al., 2018; Hrnčič et al., 2019; Norhidayah et al., 2012).

Linoleic acid = - 2668.44 - 5746.39P + 4410.20P² - 8.28t - 0.011t² - 12.74 Pt + 0. (Eq. 10)

Squalene = - 165.34 + 2467.39P - 1862.13P² + 809t - 0.007t² - 36.86 Pt + 0. (Eq. 11)

Fig. 5.

Pareto Charts with their respective Response Surface graphs, Laura Variety. Linoleic Acid (a, b), Squalene (c, d), Palmitic Acid Pareto Chart (e), Oleic Acid Pareto Chart (f).

Based on the composition percentages presented in Table 5, the comparison between saturated and unsaturated fatty acids shows that saturated fatty acids occupy 30% of the total oil components, 26% are monounsaturated fatty acids (MUFAs), basically Oleic acid, 25% are polyunsaturated fatty acids (PUFAs), essentially Linoleic acid, and Squalene represents 19%. These results correspond to those of the Criollo variety. Once again, we observe a balanced presence of saturated and unsaturated fatty acids, along with its squalene content, making amaranth, in its different varieties, a possible functional ingredient for various applications.

Finally, as seen in Table 5, the percentages of the main Laura variety oil composition, in accordance with what was mentioned previously, the highest percentages are those from Squalene, Linoleic acid, Oleic acid, and Palmitic acid (hexadecenoic acid).

4. Conclusion

DIC processing significantly modulated the phenolic fingerprint of A. hypochondriacus grain extracts, with pressure–time influence and variety-specific responses. In Criollo, total quantified phenolics varied widely (346.14–3035.2 μg/g), and moderate conditions (0.25 MPa/50 s) produced the highest recovery, while low pressure/short time reduced phenolic release. In Laura, the same moderate condition (0.25 MPa/50 s) yielded the maximum phenolic concentration (2074.15 μg/g). In contrast, a more severe treatment (0.36 MPa/78 s) resulted in the lowest concentration and reduced peak diversity, suggesting degradation under harsher conditions. Across treatments, catechin remained the dominant compound, indicating that DIC primarily shifts availability and relative distribution rather than eliminating key constituents. GC-MS profiling showed that the lipid fraction remained nutritionally favorable, dominated by palmitic, oleic, linoleic acids, and squalene, with balanced saturated and unsaturated families. Altogether, these findings reinforce amaranth's value as a promising functional ingredient. Applying pretreatments such as DIC can further enhance this functional potential by increasing the extractability and measurable concentration of bioactive phenolics through structural modification of the grain matrix. DIC is an effective, tunable strategy for improving the nutraceutical quality of amaranth-based ingredients while maintaining desirable nutritional attributes.

Author contributions

Patricia Rodríguez-Castillo: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Giselle Teresa- Martínez: Methodology, Conceptualization. Maritza Alonzo-Macías: Visualization, Resources, Methodology, Conceptualization. Carmen Téllez-Pérez: Visualization, Resources, Methodology, Conceptualization. Anaberta Cardador-Martínez: Writing – review & editing, Visualization, Validation, Supervision, Resources, Methodology, Data curation, Conceptualization.

Funding sources

The APC was funded by Tecnológico de Monterrey Grants for Scientific Papers Publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. Maria Corradini

Appendix A. Supplementary data

The following is the Supplementary data to this article: