Preliminary study of physicochemical, thermal, rheological, and interfacial properties of quinoa oil

Background: The growing popularity of nutrient-rich foods, among which is quinoa, is due to the increasing demand for healthier choices. Oils and hydrolyzed proteins from these foods may help prevent various health issues. The objective of this work was to perform extraction from the endosperm of the grain from high-protein quinoa flour by physical means via a differential abrasive milling process and extracting the oil using an automatic auger extractor at 160°C, as well as characterizing extracted oil. Methods: Quinoa oil extraction and physicochemical characterization were carried out. Chemical and physical quality indexes of quinoa oil were established, and both characterizations were conducted based on international and Columbian standards. Thermal properties were evaluated by differential scanning calorimetry, and rheological and interfacial properties of the oil were evaluated using hybrid rheometers and Drop Tensiometers, respectively, to determine its potential for obtaining functional foods. Results: The result was 10.5 g of oil/ 100 g of endosperm, with a moisture content of 0.12%, insoluble impurities of 0.017%, peroxide index of 18.5 meq O 2/kg of oil, saponification index of 189.6 mg potassium hydroxide/g of oil, refractive index of 1.401, and a density of 0.9179 g/cm 3 at 20°C. Regarding contaminating metals, it presented 7 mg of iron/kg of oil, a value higher than previously established limits of 5 mg of iron/kg of oil. The oil contained 24.9% oleic acid, 55.3% linoleic acid, and 4% linolenic acid, demonstrating antioxidant capacity. Quinoa oil showed thermal properties similar to other commercial oils. Conclusions: The interfacial and rheological properties were suitable for the stabilization of emulsions, gels, and foams, which are important in various industrial applications and could facilitate the development of new products. The extracted quinoa oil presented similar characteristics to other commercial oils, which could make it a potential product for commercialization and application in different industries.


Introduction
Quinoa is a dicotyledonous plant of the Amaranthaceae family, herbaceous, annual, or biennial.Its morphology, coloration and phenology depend on the agroecological zones where it is grown.It has a high plasticity to adapt to different edaphic conditions, drought, frost and salinity. 13][4] During the 1980s, quinoa was cultivated both commercially and noncommercially in seven countries.However, at present, the cultivation of quinoa has expanded significantly, with over 95 countries now reported to be involved in its production. 5Such a situation has been gaining importance in Colombia, where until a few years ago, research was limited by the lack of diversity in the genetic material present and cultivated within the country. 6ere are around 100 quinoa cultivars, whose grains are prepared in various ways for direct consumption and transformed into multiple derivatives. 7In most cultivars, the endosperm is rich in fats and proteins, components that are stored in the lipid and protein bodies. 8While, the perisperm contains uniform cells filled with starch granules. 9These granules are present as individual entities or as spherical aggregates. 10In order to achieve the separation of the anatomical parts of the grain, differential grinding processes have been implemented. 11,12This type of process is completely dry, without dissolution or filtration stages, which does not produce liquid or polluting effluents. 13By subjecting the grain to abrasive grinding, the content of protein, fat and ash increases in the separated endosperm compared to the whole grain, this process of separation of external grain tissues has been studied by other authors. 14,15e oil content in the quinoa grain ranges from 1.8% to 9.5%, with a calculated global mean of 5.8%. 16In the composition of lipids, unsaturated fatty acids dominate, highlighting its high content of linoleic acid (50.2-56.1%)and oleic acid (22.0-24.5%),and moderate of linolenic acid. 17The presence of linoleic and oleic fatty acids normally makes oils susceptible to oxidative rancidity.In the case of quinoa oil, its tocopherol content provides antioxidant protection against oxidative rancidity. 18The extraction of quinoa oil by means of supercritical carbon dioxide has shown good extraction performance while preserving the quality of the oil, 19 whereas the extraction of quinoa oil by the wet method (solid-liquid) and with ultrasound pretreatment are also effective. 20,21Authors such as Mufari et al., 22 have reported oxidative stability and characterization of quinoa oil extracted from wholemeal flours and from the endosperm (germ), the results showed a greater extraction from the endosperm.Because the information on the extraction and characterization of quinoa oil is limited, the objective of this work is to carry out the extraction by physical means and characterize the oil extracted from the endosperm of the grain.

Quinoa grain
The quinoa grain (Chenopodium quinoa Willd.), cultivar Nariño, supplied by SEGALCO SAS, was used.The germ was obtained by the abrasive milling process (GranEL, model C-100, Bogotá, Colombia), that had an abrasion chamber in order to obtain flours and polished grain from 1,000 kg of quinoa grains as described in Roa-Acosta et al. 13

Quinoa oil extraction and physicochemical characterization
A commercial type of automatic oil extractor machine (DULONG, Canton, China) was used, which has an alternating current motor of 450 W, handling a voltage of 110 volts, equipped with a worm screw and a thermal resistance, to perform a cold or hot extraction.It has a processing capacity of 2 kg/h.In total, 400 g of flour was recirculated from the germ to obtain a higher extraction.The resistance of the equipment was adjusted to 120°C. 21,22To clean the oil, it was subjected to centrifugation at 5,000 rpm for 5 minutes.Subsequently, it was decanted in 120 mL amber bottles for 24 hours at a lower temperature of 15°C, to separate the fine particles that were not eliminated in the centrifugation.

Density
A 25 mL Gay-Lussac pycnometer was used for all density measurements.The volume of this pycnometer was calibrated between 4 and 45°C using deionized water.It was filled with oil to the limit avoiding the presence of bubbles when covering it with the stopper.Finally, it was weighed to the nearest 0.1 mg.The determination was carried out in triplicate. 23

Moisture content and volatiles
Oil (3 AE 0.001 g) was weighed and heated at 103°C for 1 h.Subsequently, the sample was cooled in a desiccator to room temperature and weighed at 1 hour intervals until a constant weight was achieved.The determination was carried out in triplicate. 24

Insoluble impurities
The oil was weighed (20 AE 0.01 g) and 150 mL of petroleum ether was added.Then, it was allowed to stand at 25°C for 30 minutes.The oil was filtered on a 589/3 grade paper, previously dried and weighed, with the help of a funnel and a vacuum pump.Finally, the filter paper was evaporated in a convection oven at 103°C for 1 hour and then weighed to the nearest 0.001 g.The determination was carried out in triplicate. 24

Color properties
The color of the samples was determined with a spectrophotometer (CM5 colorimeter, Konica Minolta, Tokyo, Japan).In this analysis, the color and appearance of transparent, opaque, or translucent samples were evaluated.The color attributes of the oil were measured using a spectrophotometer (CM5, Konica Minolta Inc., Tokyo, Japan) in reflectance mode.Standard black and clear quartz plates were used to calibrate the spectrophotometer.Samples were loaded into a 10 mm cuvette using standard D65 illumination as the light source and the standard observer setting of 10°(10°dihedral angle) was chosen.The results were expressed as CIELAB L*, a* and b* values by triplicate.The wavelength range was 360 to 740 nm.Calibration of the equipment was performed with a black plate provided by the manufacturer and zero and blank calibration was performed.In total, 10 mL of sample was taken in the quartz cell and the parameters L*, a* and b* were determined.Color was performed in triplicate.In color space, L* indicates lightness, a* and b* are the color directions: +a* is the red axis, -a* is the green axis, +b* is the yellow axis and -b* is the blue axis.Saturation was calculated with the equation described by Gutiérrez et al. 25 Saturation was calculated by using Equation 1.The color of sample was used as reference for the determination of the total color difference of each sample. 25All measurements were performed in triplicate.

Physical and chemical quality indexes 2.3.1 Refractive index
It was carried out according to the instructions of the Abbe Mark II refractometer equipment, on a scale of 1.3 to 1.7.
After each measurement, the prism surface was cleaned with a soft cloth and a cotton swab moistened with hexane.[28]

Peroxide index
This index was determined according to the procedure described in AOAC standard 965.33.We mixed 5.0 g of oil in 25 mL of an acetic acid/chloroform solution (3:2), stirring until complete dissolution.Then, 0.5 mL of a saturated solution of potassium iodide was added and allowed to stand in the dark for 60 minutes.Subsequently, 75 mL of hot water was added and 0.5 mL of 1% starch solution was added, forming a dark color.This solution was titrated with 0.01 N sodium thiosulfate until the color disappeared.The determination was carried out in triplicate.

Free fatty acid index
This determination was carried out according to AOAC 940.28.In an Erlenmeyer, 7.05 AE 0.01 g of oil was weighed, 50 ml of ethanol (95% purity), 2 ml of phenolphthalein and 15 drops of 0.1M sodium hydroxide were added.This solution was titrated with NaOH 0.25 M, stirring constantly until a permanent faint pink appears and persists.The determination was carried out in triplicate.The acid number is expressed as a percentage of oleic acid (molecular weight 280.4472 g/mol).

Saponification index
The saponification index was determined according to AOAC 920.160.A total of 2.3 g AE 0.01 g of sample was weighed into an Erlenmeyer flask, 25 ml of 4% ethanolic potassium hydroxide was added and refluxed at 80°C for one hour.It was dismantled and 25 ml of ethanol (95% purity) and three drops of phenolphthalein 1% were added.This solution was titrated with 0.5N HCl until a yellow coloration was observed, using an automatic titrator (848 Titrino plus, Metrohm, Switzerland).The determination was carried out in triplicate.

Iodine index
This determination was carried out according to AOAC 920.159.In an Erlenmeyer flask lined with aluminum foil, 0.08 g of oil was weighed.Then, 8 mL of a solution of hexane and acetic acid was added at a ratio of 1:1, and after shaking, 10 mL of Wijs solution was added.The Erlenmeyer was capped and placed at room temperature in the dark for 30 minutes.After this time, 8 mL of KI 15% were added and little by little 40 mL of previously boiled and cold water was added.
This solution was titrated with 0.1 N sodium thiosulfate until the yellow color disappeared.To this solution, 1 mL of 1% starch solution was added and titration was continued until the blue color disappeared.The determination was carried out in triplicate.

Metallic trace elements
The determination of traces of copper, iron and nickel in quinoa oil was carried out by the atomic absorption method.Previously, 20 μL of oil was diluted in petroleum ether in a 1:3 ratio and evaporated in a graphite furnace.The UV-Vis spectrometer (Genesys 10S, Thermo Scientific, USA) was calibrated with standard solutions of organic compounds of the metals tested.The wavelengths used for the determination of copper, iron and nickel were 327.7, 302.1 and 232 nm, respectively.For the determination of iron, a graphite tube coated with niobium on the inside was used.Addition of the synthetic antioxidant butylated hydroxytoluene (BHT) was performed at a concentration of 0.02%, described by Mousavi et al. 30 Samples of 3 g with and without addition of the antioxidant were taken and placed in the reaction cells in the Rancimat 743 apparatus (Metrohm, Herisau, Switzerland) at a temperature of 110°C and an air flow of 20 L/h.The induction time was obtained for quinoa oil with and without the addition of BHT.

Thermal properties
The melting and crystallization point was determined was studied by using differential scanning calorimetry (DSC) analysis,.1-Butanol for presenting a low melting point of -89.8°C and a boiling point of 117.7°C used as a reference for the analysis.The sample size was 10 μL, with a temperature setting from -80 to 60°C, with a cooling and heating rate of 20°C/min and 10°C/min, respectively, with isotherms at the same for 1 min, repeated twice. 15

Rheological properties
The rheological properties of quinoa oil were measured using a rheometer (Discovery HR-3 rheometer, TA Instruments, USA) equipped with a parallel plate geometry (40 mm diameter).The quinoa oil was subjected to shear rate from 0.1 s -1 to 100 s -1 at 15°C, 25°C and 35°C.A strain sweep from 0.01% to 100% was performed to determine the region of linear viscoelasticity at a constant frequency of 0.1 Hz.Then, a frequency sweep was performed at a constant strain (0.1%) and in the range of 0.062 rad s -1 to 62 rad s -1 .The quinoa oil was subjected to a heating and cooling cycle from 15°C to 65°C at 6.2 rad s -1 .We measured viscous and elastic modulus for different frequency and temperature sweeps.We applied a power model to analyze both flow and viscoelastic tests. 3111 Interfacial properties and dilatational rheology 2.11.1 Adsorption kinetics 3][34] The sample was placed in a 10 mL quartz cuvette, and a 3 mm diameter stainless steel capillary tip was submerged inside to form an air bubble.All measurements were conducted at room temperature.The interfacial tension data measured as a function of time were analyzed using nonlinear numerical fitting expressed by the following equation 31,35 : where, y 0 represents the equilibrium interfacial tension and the exponential terms (decay rate) correspond to the known adsorption kinetics (or adsorption constants) for proteins and other macromolecules.Therefore, the penetration mechanism (K d ) of the interface is described by the first exponential decay, while the reordering mechanism (K p ) of the molecules in the interface is described by the second exponential decay.The individual decay rates (penetration and ordering) were obtained using the relationships K d = 1/t 1 y K p = 1/t 2 .

Dilatational rheology and viscoelastic properties
The dilatational rheology of the air/oil interface was analyzed by sinusoidally oscillating the volume of an air bubble in the Rising method setup.The method consisted of a periodic, automated, and sinusoidal compression and expansion of the interface, performed by decreasing and increasing the volume of the drop with deformation amplitudes of ΔA/A = 5 and 10% (the percentage change in area is in the linear viscoelastic range), with measurements taken at an angular frequency of 100 Hz.The sinusoidal oscillation for the measurement of the dilatational surface was performed with five 5 oscillation cycles followed by one 1 cycle of no oscillation.The average standard precision of the surface pressure was approximately 0.1 mNm À1 .This perturbation in the interface allows the adsorbed molecules to reduce the tension depending on the adsorption or desorption of the solution, giving rise to relaxation processes and the pseudo-thermodynamic equilibrium of the system.The surface rheological parameters E, E d , E v , and ϕ were measured as a function of the adsorption time (θ).
The interfacial dilatational modulus (E) derived from the change in interfacial tension (dilatational tension) corresponds to the resistance to surface perturbations α (Equation 3), resulting from the small change in surface area (dilatational tension) A (Equation 4) 32,36,37 : Where σ 0 and A 0 are the amplitudes of stress and strain, respectively, q is time, f is the phase angle between stress and strain, w is the angular frequency, and |E|, is the absolute modulus, and the ratio (σ 0 /A 0 ) is a measure of the total material unit of resistance to dilation deformation (elastic + viscous). 32The interfacial dilation modulus (E) is a complex quantity and composed of real and imaginary parts (Equation 5).The real part (E d ) is called the storage modulus (G 0 ) and represents the elastic energy stored in the interface (i.e., the elasticity of dilation), E d = |E|(cos).The imaginary part (E v ) is called the loss modulus (G 00 ) and represents the dissipation of energy in the relaxation process (i.e., the viscosity of dilation), E v = |E|(sin), and f is the phase angle between stress and strain, that is, a measure of the relative elasticity of the film.Therefore, for a perfectly elastic material, stress and strain are in phase, f = 0 and the imaginary term (G 00 ) is zero.
In the case of a perfectly viscous material, f = 90°, and the real part (G 0 ) is zero.The dynamic viscoelastic behavior can be more intuitively analyzed with the loss tangent (tan δ), which is equal to ) is dominant, and the interface mainly exhibits viscous properties.Conversely, when tan d < 1, the interface mainly exhibits elastic properties, E d (G 0 ), that is, if the film is purely elastic, the loss angle tangent is zero. 32,35,38 Results and Discussion

Extraction yield and physicochemical characterization
For the extraction of quinoa oil by the pressing method, it was necessary to obtain the germ of the grain by abrasive grinding as described by Roa-Acosta et al. 13 Then, it was necessary to condition the germ by heating.This is necessary to facilitate extraction due to the low granulometry of the germ flour.In addition, the effect of temperature exerts positively on the extraction yield, Adrianzén et al., 39 points out that the heat treatment of the seed facilitates the extraction process by mechanical means, because it coagulates the proteins of the cell walls, makes them permeable to the passage of the oil and also decreases the viscosity of the oil.The temperature of the resistance was 120°C, obtaining an oil with a temperature of 60°C.

Moisture and volatile compounds
Table 1 112 shows the results of physical and chemical analysis of quinoa oil.It was observed that quinoa oil has 0.121% moisture and volatile matter, this low water content gives stability to the oxidation of the oil during storage, since the production of free fatty acids is slowed down. 22The hydrolysis process in oils is directly proportional to the moisture content.According to Ogori, 40 water reacts with glycerides, producing the separation of fatty acids, monoglycerides and diglycerides, this being the reverse reaction to fat formation.On the other hand, the moisture value can be influenced according to the extraction method.Scorzza et al., 41 found a difference when extracting almond oil by pressing and solvent extraction, with moisture and volatile matter contents of 0.17 and 5.36%, respectively.This can be attributed to the traces of solvent, making the pressing method more suitable for the food industry.
On the other hand, quinoa oil is made up of a high proportion of unsaturated fatty acids, which upon degradation generate several compounds responsible for affecting the organoleptic properties of the oil, influencing its quality.][44] These compounds, mainly aldehydes and ketones, vary due to the type of seed used for vegetable oil extraction. 45

Soluble impurities
The content of insoluble impurities was 0.017%; low values favor oil quality and preserve shelf life, as insoluble substances deteriorate the oil. 41This value is considered low compared to 0.05% in vegetable oils, which indicates that quinoa oil is mostly free of mechanical, nitrogenous, mineral impurities or soil and seed particles that can generate a bad taste, odor, or bad appearance to it.On the other hand, it also indicates that the purification method carried out by filtering and decanting was adequate to obtain a quality oil.When comparing with a quality oil such as virgin olive oil, Serrano, 46 found a value for this parameter between 0.047-0.099%,being in the permitted range, but higher than that found in this study.This behavior can be attributed to variables such as the type of seed used, and the processes carried out before extraction of the vegetable oil.

Density
The density for quinoa oil was 0.9179 g/cm 3 , which is determined by the size of the fatty acids present.The number of unsaturation's they contain, the higher the value for these, the higher the density of the oil. 44Another variable to take into account for the determination is temperature, because at higher temperatures there is a dilation of the matrix leading to a decrease in density. 39Quinoa oil is minimally below the density of soybean and sunflower oils.As mentioned by Alam et al., 47 the density of an oil is the product of the density of its components.The percentage present in the mixture of each one, for which variations can occur between them.Paucar et al., 48 highlights the importance of less dense oils for cold consumption, since it is more digestible and has a lower melting point.

Color properties
Color constitutes a fundamental quality in sensory analysis, which can be affected by the variety and the degree of maturation of the grain, the production area, the process of obtaining and conservation. 20,49The value obtained for the L* coordinate was 82 considered as a clear oil, which can reflect light. 50The intense color can be affected by the coordinates a* and b*, which specify a brownish-yellow color, since b* has a high value, with yellow being the most predominant.On the other hand, the change of the a* coordinate makes it take on a slightly reddish hue.The color of quinoa oil can be influenced by the presence of compounds such as carotenoids, riboflavin and betacyanins, among others.Tang et al., 51 states that the pigmentation of quinoa corresponds to betacyanins, being compounds that add functional value.These can be subdivided into betacyanins and betaxanthins.The first being responsible for the red-violet color and the second for the yellowish-orange color.García-Parra et al., 5 found values for betacyanins between 0.278 and 0.883 mg/100 g and for betaxanthins between 1.1 and 13.8 mg/100 g in white quinoa grain.The coloration of the quinoa oil under study may be due to these compounds, with yellow being the most notorious when measured.3][54] It is pertinent to state that color is not a parameter that defines the quality of the oil, however, it turns out to be important because it exerts a strong influence on the preferences of different consumers.

Refractive index
Another parameter used to control the identity and quality of oils is the refractive index.It is directly related to the degree of unsaturation, confirming the content of long-chain fatty acids and is useful for observing the progress of reactions such as lipid hydrogenation. 55In the quinoa oil, a refractive index of 1.4701 was observed, in accordance with the degree of unsaturation detailed in the fatty acid profile, which was 87.93%.According to Bruin, 56 when performing analyzes at 40°C, they found values close to those obtained in the present study, this difference can be attributed to the effect of temperature at the time of determination, since, as mentioned by Latif and Anwar, 57 the increase in temperature decreases the refractive index because heat destabilizes the configuration of the double bonds present in fatty acids.Similarly, Wechsler et al., 58 reported a value of 1.4736 for sunflower oil at 20°C.

Peroxide index
Regarding the peroxide index, a value of 18.5 meq O 2 /Kg oil was observed.This index reflects the degree of oxidation of the oil, which is affected by the extraction conditions, since the oxidation process is initiated by the action of atmospheric oxygen and favored by temperature and exposure to light.The oil of quinoa extracted at a temperature of 120°C, which could directly affect the primary phase of peroxide formation.Gisbert et al., 59 when comparing two extraction systems, found a higher peroxide index in the method that involves a temperature above 60°C and exposure to light.In contrast to what was reported by López-Biedma et al., 60 the peroxide index was 14.9 meq O 2 /Kg oil, being lower than that found in the present study.It can be stated that this index in quinoa oil tends to be high due to its high degree of unsaturation, which makes it more susceptible to oxidative degradation and the formation of other compounds such as aldehydes and ketones, which are responsible for unpleasant odors and flavors characteristic of oxidized fats. 61For this reason, proper handling of the flour before extraction is recommended, since, as mentioned by Ng, et al., 62 the production of free fatty acids in quinoa flour is highly conditioned by temperature and storage time.Among other factors that influence, may be the breaking of the cells and the greater surface of contact of the flour with the oxygen during the milling process and the time elapsed between the extraction and the analysis. 39The determinations were made after 24 hours while the samples completed the decantation process.

Free fatty acids
Regarding the index of free fatty acids, a value of 0.65% expressed in oleic acid was observed, which is within the maximum limit for natural crude oils (1%).Miranda et al., 63 reported values between 1 and 4.1% free acidity, which are much higher compared to what was found in this study.Similarly, this depends on the type of seed, variety, state of maturation, environmental conditions where it is produced, and storage conditions of the ground seed.García-Parra et al., 5 indicated that a high level of free acidity provides a signal regarding the state of the seed at the time of oil extraction.
It should be noted that the value obtained is close to some values for different food-type oils, such as olive (0.76%) and sacha inchi (1.1%), reported by Acosta et al., 64 and Gutiérrez et al., 65 respectively.
This index of free fatty acids indicates low availability of water, which corroborates the previously determined moisture percentage, since it shows the low availability of water in the oil matrix that can generate hydrolysis in the triglycerides that constitute the oil, leading to the generation of free fatty acids.According to the result obtained, it can be said that the acidity index is not greatly affected by temperature compared to the peroxide index.However, a high index of free fatty acids at the time of extraction favors the oxidative deterioration of the oil.Kara et al., 66 evaluated quality parameters of quinoa oil under storage under accelerated oxidation conditions at 60°C, where they found that there were no significant differences in terms of free acidity for 12 days.On the other hand, Xie et al., 67 mentions that the smoke point of an oil is related to the acidity index, since lower values of this last parameter lead to raising the smoke point of the oil. 68

Saponification index
The saponification index is defined by the types of fatty acids that constitute the oil, the longer the chain, the higher the molecular weight, this means that there is a relatively smaller number of carboxylic groups.Which means that the value of the index decreases and vice versa. 69The value found for the quinoa oil under study was 189.65 mg KOH/g oil, which, through the lipid profile, confirms the presence mainly of long-chain unsaturated fatty acids.On the other hand, Pardo et al., 70 mentions that a high saponification index indicates the presence of a high percentage of free fatty acids.This being consistent with the results obtained, since a low presence of these was demonstrated, which allows the saponification index of quinoa oil not to exceed the established limits, considering that it has not been subjected to a process of addition of antioxidant agents that can counteract the degradation of the oil.The result was compared with sunflower and soybean oil, since these are similar in terms of fatty acid composition and for which ranges of 188-194 and 189-195 mg KOH/g oil, respectively, are established.The value of 189.65 mg KOH/g oil is within the ranges established for oils of vegetable origin intended for human consumption.Furthermore, 71 reported similar values for quinoa oil, 191 and 192 mg KOH/g oil, respectively.

Iodine index
The iodine value is an indicator of the degree of unsaturation in the oil.Values between 26 to 48 cg/100 g indicate oil saturation with a tendency to solidify, while values between 94 to 135 g I/100 g show a high level of unsaturation. 72This index is used to determine the purity and identity of fats/oils; for example, the iodine values for unsaturated fatty acids, such as oleic, linoleic and linolenic acids, are 90, 181 and 274 g I/100g, respectively. 73The aforementioned statement aligns with the results found in the present study, which indicate a content of 127.36 g of iodine per 100g of quinoa oil.This observation also corresponds to the fatty acid profile of quinoa oil, characterized by a notably high percentage of linoleic acid.Authors such as Caipo et al., 44 found values between 140 and 142 g I/100 g for quinoa oil, this variation may be related to the temperature and time of heat treatment during extraction, since Adrianzén et al., 39 mentions that, by increasing these two variables, properties such as the iodine index tend to decrease due to the oxidation of the double bonds.In industry, this property is useful because it determines whether it is a drying, semi-drying, or non-drying type oil, which determines whether or not it forms films when in contact with air.The Codex Alimentarius 210-1999, 74 does not establish specific values for quinoa oil, however, there are limits for soybean oil from 124 to 139 g I/100 g and for sunflower from 118 to 141 g I/100 g, which were taken as a reference because they present similarity in the composition of mainly unsaturated fatty acids as mentioned above.According to the above, quinoa oil meets the parameters of these regulations for edible oils.

Determination of trace elements
Regarding the metallic trace elements that were determined, low values were found for nickel and copper, <0.01 mg Ni/kg and < 0.0135 mg Cu/kg, respectively, which are below the maximum limit allowed by CODEX ALIMENTARIUS 210-199, which establishes values for nickel and copper not exceeding 0.4 mg/kg.Regarding iron, the presence of 7 mg Fe/kg was determined, which is a value that exceeds the limit established by the standards, being 5 mg Fe/kg.These parameters contained in the standard are not specific to quinoa oil.However, they are taken as a reference because they are the standards that currently indicate the health requirements that must be met by oils and fats intended for human consumption.According to Gomez-Pando et al., 75 quinoa stands out compared to cereals for its content of minerals such as copper, potassium, calcium and iron, the latter being equivalent to twice that of wheat and six times that of corn.For this reason, oils with a high content of this trace element can be obtained.Likewise, these contents depend on the type of seed, the variety, the soil where it is grown, and the chemical elements that are provided as part of its nutrition. 76,77From the nutritional point of view, copper and iron are important micronutrients in human nutrition in adequate doses, fulfilling a wide range of biological functions, 78 among which the transport and storage of oxygen in the blood, as well as the maintenance of the nervous and immune system with the help of copper.

Properties bioactive
The fatty acid composition of quinoa oil obtained by gas chromatography is presented in Table 2. Next, Table 3 shows the type of fat and its content per 100 g of oil.The fatty acids present in quinoa oil are mainly unsaturated, where 59.69% were identified as polyunsaturated, with linoleic acid being the most present, while 28.24% represented monounsaturated, standing out among these, oleic acid.Saturated fatty acids represented 12.07% of the total composition.The results obtained are similar to those reported by Tang et al., 79 for white quinoa oil, which were 12.82%, 26.82% and 60.50% for saturated, monounsaturated and polyunsaturated fatty acids, respectively.On the other hand, Tang et al., 80 found close contents in the three solvent extraction methods, for linoleic and oleic fatty acids, being 54.80% and 24.82%, for each one, instead there was a difference to the result for linolenic acid (Alpha) between 0.49-1.82%,being lower in terms of what was obtained in the present study, which was 4%.However, Caipo et al., 44 reported a value between 4.66-5.74%for α-linolenic acid, therefore, this value is more in agreement with the data obtained from the oil from flour of the endosperm.These results may vary slightly depending on the climate, soil and variety of the vegetable.
According to the content of fatty acids, it can be said that quinoa oil is more susceptible to oxidation, since it has a considerable percentage of fatty acids with more than one double bond in its structure (polyunsaturated).However, Paucar et al., 48 reports that oils with a higher presence of monounsaturated fatty acids, that is, with a single double bond in the chain, are less prone to oxidation, have greater stability and shelf life.In the case of oleic acid, it provides characteristics of resistance to chemical decomposition caused by high temperatures, 39 this is corroborated by Ceylan et al., 81 when subjecting extra virgin olive oil, sunflower, corn and peanuts, cooked, where the most resistant oil was extra virgin olive oil, as it maintains a high concentration of oleic acid; the most susceptible was the sunflower because it had a higher content of polyunsaturated fatty acids.
From a nutritional point of view, polyunsaturated fatty acids are important, for example linoleic acid, which fulfills the function of controlling and reducing cholesterol from accumulated saturated fats, as well as being fundamental in the formation in the formation of nerve and eye tissue.α-linolenic acid allows the development of human intelligence from the fetal stage, since more than half of the fat in the brain is omega 3. 48 According to the National Institutes Of Health, an adult should consume 1.6 g per day; therefore, quinoa oil would supply a quarter of your requirement.However, they have disadvantages due to their instability under the effect of temperature, atmospheric oxygen and direct incidence of light, which give rise to primary oxidation initiating compounds (peroxides) 81 that alter the quality of the oil and lead to adverse effects on human health.Therefore, quinoa oil should avoid procedures that modify the nature of the oil or analyze its behavior in mixtures with other oils.
Lipid peroxidation, in addition to deteriorating the taste and smell of the oil, also leads to emphysema, mutagenesis, heart disease and cancer. 79To counteract the effects of oxidative deterioration, various studies have been carried out to evaluate the antioxidant capacity, thus determining the bioactive compounds that can trap free radicals in food.When comparing the antioxidant capacity of the control, which was quinoa oil without additives, against another containing a concentration of BHT, it was found that the control had an induction time of 20.3 h.On the other hand, by adding BHT to quinoa oil, it was observed that the sample exceeded 21 h in its oxidative stability.However, the use of this synthetic antioxidant would not be necessary to improve this characteristic since the difference in the induction time is not considerable.
Components such as phenols, carotenoids, sterols, amino acids, and oligosaccharides can improve the functional properties of vegetable oils. 80Quinoa being a considerable source of vitamin E, which in addition to helping prevent lipid oxidation, is fat-soluble, which makes its presence in the oily matrix easier.Caipo 44 found a concentration of 1,300 ppm total tocopherols, with α-tocopherol being the most prevalent with 993 ppm.On the other hand, Abderrahim et al., 82 found that betalains contribute to antioxidant activity.This makes quinoa oil acquire a good barrier against oxidative degradation due to rancidity.The oil showed greater oxidative stability compared to sacha inchi oil (5.1 h), soybean (17.4 h) and sunflower oil (13.1 h) added with hydroxytyrosol derivatives, this being a polyphenol with antioxidant capacities. 83The results reported by Rubio (2006) for quinoa oil were 6.4 h and 8 h, which depend on the variety, these being lower than those found in the present study.Low molecular weight lipids with surfactant activity, such as phospholipids, glycerides, fatty acids, synthetic surfactant molecules, among others, are amphiphilic molecules that can adsorb weakly or strongly at the interface. 84This behavior depends on their chemical structure, that is, their lipophilic and hydrophilic balance (HLB).5][86] Structural changes undergone by proteins may be due to the difference in molecular weight between them and lipids (minimal structural changes), and similarly, to their cooperative nature of adsorption.Therefore, protein adsorption and desorption are significantly slower than lipids at both interfaces. 87,88Consequently, the presence of proteins may generate competitive adsorption at the interface.It has been reported that proteins interact with surfactant molecules in the solution differently at the surface/interface, through a hydrophobic and/or electrostatic interaction that can produce conformational changes of protein molecules in solution and at the surface/interface. 87me authors have described that oils obtained by mechanical extraction (virgin oils) contain different components, including secondary metabolites, proteins, among others. 891][92] Similarly, the presence of proteins in the oil contributes to the reduction of interfacial tension. 93,94The adsorption dynamics of virgin oil are shown in Figure 1 at the air-oil interface.The curves obtained show a reduction in interfacial tension up to an equilibrium value of 27.1 and 27.3 mNÁm -1 for 10 % and 5% deformations, respectively.This decrease was small compared to equilibrium values of highly surfactant molecules such as lipids and proteins reported by other authors. 87,88e experimental data of the adsorption mechanisms were fitted using Equation 2. The relaxation times t 1 and t 2 were 85.13839 s and 477.18531 s, respectively, with a coefficient of determination (R 2 ) of 0.9996 for a deformation amplitude of 5%.Meanwhile, the relaxation times t 1 and t 2 for a deformation amplitude of 10% were 0.98487 s and 112.1569 s, respectively, with a coefficient of determination (R 2 ) of 0.9994.These parameters demonstrate the adsorption mechanisms during the decrease of dynamic interfacial tension.It has been observed that increasing the deformation amplitude from 5% to 10% (Figure 2A) increases the adsorption kinetics.This could be attributed to the fact that the molecules exhibit greater molecular movement and therefore diffuse (Figure 2B) more rapidly due to a greater fluid perturbation, causing the molecules to penetrate the interface more quickly, promoting the formation of a more stable interfacial film.Consequently, in both established deformations, t 1 was significantly smaller than t 2 , suggesting that the adsorption of the molecules was not limited by the velocity of dynamic interfacial tension compared to the reorganization of the molecules.
This means that oil molecules exhibited slow diffusion and adsorption at 5% deformations, which was not observed at 10% deformations, due to the increased molecular movement and enhanced diffusion and adsorption at the interface.

Dilatational rheology
The analysis of the adsorption behavior of quinoa oil at the oil/water interface was performed using dilatational oscillations.Deformation sweeps experiments, analyzed in a classical manner, provide useful information only about the linear viscoelastic regime (LVR), where the viscoelastic moduli are independent of the applied deformation or stress.This response is necessary to correctly estimate E d (G 0 ) and E v (G 00 ) (Figure 3A and B).Consequently, it was observed that  the oscillations obtained from the interfacial tension at 5% deformations showed a smaller sinusoidal response compared to 10% deformations.Similarly, it was observed that the behavior in the tangent of delta (tan δ < 1) is less than 1, indicating a more elastic behavior (ϕ = 12), which is similar for both deformations.This behavior is ideal for the formation of emulsions with oil mixtures containing other components such as fibers (cellulose), proteins, or polysaccharides, which promote changes in structural properties within the mixtures, increasing the viscoelastic properties of this dispersed system.It is understood that when incorporating oily phases, it is necessary to stabilize them with surfactants and/or cosurfactants, depending on the industrial application.5][96][97][98][99] This may be due to the different intermolecular interactions between the components that modify the structure of the dispersed system, and in turn, the linear behavior (LVR) of the dispersed system (emulsion) modifying the sinusoidal behavior of the blend.The sinusoidal curves of the dilatational rheological behavior at the different amplitudes of deformation studied, it was observed that the molecular movement of the oil molecules, at these amplitudes, respond kinetically to a thermodynamic pseudo-equilibrium from 50 s, indicating the adsorption of the molecules in a constant way that helps to form the interfacial film.It is more evident at a deformation amplitude of 10% (Figure 3C and D).This behavior agrees with the trend shown in the dilatational modulus and the real component (E d ), which evidences a higher mechanical strength at higher strain amplitude.
In Figure 3A and B, a clear observation emerges: the modulus of expansion appears to be independent of the strain amplitude, and there is minimal change in the measured phase angle.These findings suggest that the adsorbed layer exhibits an inherent elastic behavior.Notably, the dilatational modulus shows an increase with rising strain amplitude.
As is commonly understood, this modulus serves as a measure of resistance against the creation of surface tension gradients and the rate at which these gradients can dissipate.
Specifically, for low molecular weight surfactants, the time required for surfactant molecules to diffuse into the perturbed region or for surface molecules to reorganize and establish equilibrium decreases as the external frequency increases.Consequently, the modulus of expansion increases in response to the amplified strain amplitude.
On another note, the phase angle represents the ratio between the loss modulus and the storage modulus.Due to the shorter time of the diffusion-exchange relaxation process as the extrinsic frequency rises, the contribution of the loss modulus is reduced.This leads to a decrease in the phase angle value.Consequently, the viscous component, E v , decreases with increasing strain amplitude (Figure 3B).Notably, despite these changes, the phase angle itself remains constant, indicating a higher fluidity in the interfacial film formed by the quinoa oil molecules.
To summarize, the results indicate that the adsorbed layer possesses elastic behavior, and the behavior of the dilatational modulus and phase angle is influenced by the surfactant's diffusion-exchange relaxation process with increasing external frequency.These insights shed light on the intricate properties of interfacial films, specifically those formed by quinoa oil molecules.

Curves flow and frequency sweep
Figure 4 shows the flow curves (Figure 4A, C and E) at different temperatures.It was observed that the viscosity decreases as the stress increases as a function of temperature.The flow and consistency behavior of the oil at the temperatures studied is similar, presenting a pseudoplastic behavior (n < 1) and indicating that the structure of the oil is modified as the temperature increases, presenting a shear-thinning flow.
Likewise, it was observed that the consistency index (K) decreases as the temperature increases, but the fluid index (n) of the oil increases as the shear increases.This behavior agrees with that observed in the dilatational rheological behavior of quinoa oil at 25°C.As for the frequency sweep study (Figure 4B, D and F), it was observed that the behavior of the loss modulus (G 00 ) is similar for all the temperatures studied.However, the delta for all samples presented a value greater than 1 (tang > 1), the tendency of this result shows that quinoa oil has a viscous behavior.Similar behavior was observed in the dilatational rheology.

Thermal properties
The warming and cooling temperature behavior of heat flow in the fusion and crystallization process has been reported for two cycles.Two exothermic peaks were identified for crystallization (Table 4), where the temperature values for the phase transition of the sample are between -14 and -32°C (first peak) and between -32 and -46°C (second peak).The crystallization is conditioned by the composition of fatty acids since a higher presence of polyunsaturated fatty acids (linoleic and linolenic) results in a decrease in the freezing point of the oils. 102ystallization occurs due to the initiation of microcrystal formation (peak 1) and the freezing temperature of the sample (peak 2). 103Another reason for the presence of different peaks is the cooling rate because higher cooling rates primarily exhibit exothermic behaviors, unlike lower rates where the heat flow behavior of the oil has more time to reach its thermal equilibrium, promoting its solidification with a peak of lower intensity compared to peaks at higher rates. 104milarly, triglycerides have the ability to adopt various crystalline forms, including α, β', and β, in increasing order of stability. 105This polymorphic behavior generates specific properties for each form, allowing them to be differentiated from one another.Therefore, in the crystallization process, despite having the same chemical composition, the variation in their crystalline structural configuration can also influence the presence of different exothermic peaks.Polymorphic transformations depend on different variables but mainly on the cooling rate and final temperature. 106 the melting temperature, two peaks were obtained, and the starting and ending temperatures for both peaks are between -47 and -30°C (peak 1) and between -31 and -8°C (peak 2).According to Cofrades et al., 107 the first endothermic manifestation is determined by the content of tri-unsaturated fatty acids (linolenic), as the melting point decreases with increased unsaturation of the fatty acid, indicating that a higher number of double bonds implies higher energy, and therefore, no energy is required for melting. 108On the other hand, Romero et al., 109 indicates that the first peak can be assigned to the fusion of one of the metastable polymorphic forms (unstable form) of a fat, and it is followed by a rearrangement of the molecules to form a new crystalline form that melts at higher temperatures.This can be correlated with the second peak (heating curve).Regarding the sum of mono-unsaturated (oleic) and di-unsaturated (linoleic) triglycerides, their composition in quinoa oil is significant since they are the most involved in the phase transformation that causes the second peak temperature, a behavior related to the appearance of a small peak due to the increase in the rate at which this process occurs. 110e enthalpies calculated from the area under the curve determined on a baseline for crystallization and melting were 74,399 and 88,436 J/g, respectively, which are the sum of all transitions in the two profiles.These values are similar to those reported by Huang et al., 89 for olive oil, with a melting peak of 75 J/g, despite the DSC curves having different behaviors and the polyunsaturated/monounsaturated fat ratio being lower.Durango-Giraldo et al., 111 mention that these values are related to the chemical composition of the oil.These behaviors are related to those observed in flow curves where the consistency index and the flow index depend on the molecular ordering of the lipid molecules present in quinoa oil.

Conclusions
Quinoa oil extracted from hyperprotein flour has great potential for commercialization.It has important functional characteristics due to its content of vitamin E, betalains, carotenoids, among others.In addition, this oil showed a content of mainly unsaturated fatty acids, highlighting oleic acid (24.9%), linoleic acid (55.29%) and linolenic acid (4%), which makes it suitable for cold human consumption.Likewise, according to its physicochemical, thermal, interfacial and rheological properties characteristics, it would be useful in the formulation of new innovative products in the cosmetic industry.Are sufficient details of methods and analysis provided to allow replication by others?
• All presented methods are developed in detail and can be repeated.If the authors used other methods that were not developed by them, information about them was provided in the cited literature.
If applicable, is the statistical analysis and its interpretation appropriate?
• The values of the physicochemical parameters of quinoa oil are given after statistical processing.Mean values and standard deviations are provided.Fatty acid profile of quinoa oil contains average data.They were probably made in only one repetition.The oil color parameters were statistically processed and include average values and standard deviations.Rheological tests were carried out meticulously and statistical analysis of the results was carried out.Thermal properties tests were performed in one repetition, probably due to the high time consumption.
Are all the source data underlying the results available to ensure full reproducibility?
• The authors have posted files with source data at the indicated e-mail address.The address is active and the reviewer has checked access.
Are the conclusions drawn adequately supported by the results?
• Individual tasks and the obtained results are extensively discussed in the Results and Discussion chapter.The results were compared with those of the cited authors.The conclusions are modest and constitute a short summary of the results obtained.
• Research in the field of the interfacial and rheological properties is original in relation to consumer oils.The information provided is of great value to the materials industry for the stabilization of emulsions, gels, and foams and other materials.

Notes to Authors:
• Due to the high content of unsaturated fatty acids in the oil, the proposed production technology does not seem to be the best solution due to the relatively high extrusion temperature (120 0 C) and the possibility of oil contact with air.A better solution would be to use another technology, e.g.supercritical CO2 extraction, which is unfortunately a more expensive solution.
• Moreover, the proposed pressing technology is suitable for raw materials with fatty acid content above 15%.However, there is quite a lot of oil left in the cake, often at the level of 5-8%.To improve efficiency, high pressing temperatures of up to 200 0 C are often used, which can significantly deteriorate the quality of the oil and residue.
• In the tested raw material, the acid ratio (omega 3):(omega 6) is 4.16:55.29 (see table 3), which corresponds to approximately 1/13.Meanwhile, for consumption purposes, the optimal ratio of (omega 3): (omega 6) for consumers is considered to be 1:1 to 1:5 (see Trends in Food Science & Technology 120 (2022) 140-153).Therefore, for consumption purposes, for health safety reasons, this oil should be blended with other oils to obtain the desired or similar omega 3/omega 6 composition.
• The conclusions stated that the oils additionally contain carotenoids, betalains and vitamin E, but these are only minor mentions.The same applies to the mention of the uses of oil in the cosmetics industry.This information should not be included in the summary because it was not the subject of broader research and was not discussed in the text of the publication.But they could possibly be included in the chapter on expected development trends in research on other biologically active compounds in quinoa oil.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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Figure 1 .
Figure 1.Estimated of L*, a*, b* coordinates by CIE-Lab of quinoa edible oil.

Figure 2 .
Figure 2. (A) Dynamic interfacial tension (γ) at the air-oil interface as a function of time for quinoa oil at room temperature.(B) The adsorption mechanisms are indicated at logarithmic time scale, as defined for individual proteins (diffusion, adsorption and penetration, and interface reorganization and equilibrium) as a function of deformation (DA/A, %).

References 1 .
Patel A, Desai S, Mane V, Enman J, et al.: Futuristic food fortification with a balanced ratio of dietary ω-3/ω-6 omega fatty acids for the prevention of lifestyle diseases.Trends in Food Science & Technology.2022; 120: 140-153 Publisher Full TextIs the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?PartlyCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Supercritical extraction technology, bioactive properties of natural extracts, process optimisation, analytical methods.

Table 1 .
Values of physicochemical parameters of quinoa oil.

Table 2 .
Fatty acid profile of quinoa oil.

Table 3 .
Percentage by fatty acid group in quinoa oil. 100,101

Table 4 .
Initial temperature, final temperature, and enthalpy of the different peaks of quinoa oil.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? and
chemical quality indexes, peroxide index, free fatty acis index, saponification index, iodine index, metallic trace elements, bioactive properties, thermal properties, rheological properties, interfacial properties and dilatational rheology.The authors cited 112 references.The literature is well selected thematically and is up-to-date.There are even a few entries from 2023.Is the study design appropriate and does the work have academic merit?•The study design is designed correctly.Due to the detailed descriptions of research methods and materials used, the paper has great academic value both in terms of research, teaching and practice.The presented research methods can be used by both students and employees of research laboratories.
Hamdy A ZahranFats and Oils Dept., Food Industries and Nutrition Research Institute, National Research Centre, Dokki, Egypt Dear Author, -My comments were mentioned on the attached PDF file found here -sufficient details of methods and analysis are required especially for oil content calculation for your matrix -the conclusions aren't adequately supported by the results, it needs some revision -some results for oxidative stability are concerning Regards Is