The Use of Common Bean and Mesquite Pods Flours as Partial Substitute of Semolina, Impact of Their Proteins and Polysaccharides in the Physical, Chemical, and Microstructural Characteristics of Spaghetti Pasta

Abstract

Spaghetti pasta is a popular food; different ingredients than wheat have been explored to increase their nutritional value, the use of mesquite flour with pea protein remains unexplored. This study aimed to evaluate the impact of substituting semolina with mesquite pod flour and pea protein isolate on the techno-functional properties of spaghetti. Spaghetti was prepared using semolina hydrated to 35–40%, (15 cm strands), dried at 50 or 60 °C until 7–8% moisture. Semolina was substituted (0–30%), with pea protein isolate (PPI) (0–20%) and mesquite flour (0–25%). Guar and xanthan gum were added (0–1%). Proximate analysis, trypsin inhibitors, culinary properties, water absorption, texture profile, color, soluble protein, protein, starch digestibility, Raman and confocal microscopy were performed. The legume incorporation increased the protein content and digestibility of the pasta. Although the culinary properties were affected by legume substitution, levels of 75–85% substitution yielded acceptable results. Spaghetti containing PPI and mesquite flour, dried at 60 °C, showed similar secondary protein structure compared to the control. However, mesquite flour notably altered the color of the pasta. The combination of PPI, mesquite flour, and hydrocolloids improved protein availability while reducing available starch and enhancing the nutritional quality of the spaghetti.

1. Introduction

Spaghetti is a globally popular foodstuff whose production process has undergone significant evolution and adaptation over time. However, some European countries such as Italy have maintained the tradition that dry pasta is made exclusively from durum wheat semolina, allowing only the inclusion of 3% common wheat flour. In contrast, several countries, such as Mexico, define pasta as a product obtained by drying figures made of semolina and/or wheat flour with drinking water and allow optional ingredients such as eggs, additives, or flours other than wheat [1,2].

The use of alternative flours to improve the nutritional quality of the pasta is numerous. Legumes and cereals other than wheat are used, including beans; however, the use of mesquite has not been reported.

Mesquite, as it is commonly known in Mexico, belongs to the genus Prosopis spp., it is a tree native to Asia, Africa, and America that grows in arid and semi-arid areas [3]. Its pods are 7 to 20 cm long and 8 to 15 mm wide, have a yellowish-brown color, indehiscent, falcate, straight, or linear, and have a fleshy mesocarp, endocarp, and seeds [4]. It is used as wood, forage, rubber, and livestock feed. Pods are used as food for human consumption in natural form, flour for the preparation of several foods and even to produce drinks such as “mosquetole”, which is made from corn, water, and mesquite pods [5].

Some studies have been carried out using the mesquite pods’ flour of different species to give added value to various foods; it has been shown that it is a good source of fiber, essential amino acids, minerals, and phytochemical compounds with beneficial effects on health [6]. Sciammaro et al. [7]; use mesquite flour to obtain bread in blends of rice and corn flour. They observed that an increase in mesquite flour increases the protein content, diminishes adhesiveness, and creates a softer structure. Korus et al. [8]; reported that the use of mesquite flour in the bread obtention produces a diminished hard structure and an increase in the fiber content.

The mesquite flour has the presence of galactomannans, a gum obtained from mesquite composed of a series of water-soluble polysaccharides, it is like gum arabic, it is linked by branched bonds of β-D-galactose residues, linked by 1→3 bonds and/or with 1→6 branches with L-arabinose, L-rhamnose, β-D-glucuronate, and 4-O methyl-β-D-glucuronate [9]. It has been shown that galactans from mesquite and pea protein isolate can form a stable complex [10], thus, the use of pea flour could be useful to improve the stability of the structure obtained with mesquite flour in pasta.

The pea (Pisum sativum L.) is a legume with great potential for use in the food industry in the form of flour or isolate, due to its high protein, starch, and fiber content [11]. Nutritionally, the high content of essential amino acids such as lysine, leucine, and arginine and the low methionine content characteristic of legumes stand out [12]. Its proteins can be classified mainly into globulins (65–80%) and albumins (10–20%), and to a lesser extent prolamins and glutelins (3–5%). Globulins are the main storage proteins, composed mainly of legumin (320–410 kDa), vicilin (150 kDa), and to a lesser extent convicilin (180–210 KdA) [11].

Pedrosa et al. [13] compared the technological properties of pea flours with their protein isolates. The results showed an increase in the swelling capacity, emulsifying capacity, emulsifying stability, and foam formation in the protein isolates. This suggests that pea protein isolate has multiple applications in the food industry, including pasta formulation thanks to its swelling capacity. Furthermore, the extraction process decreased the content of anti-nutritional factors.

The main objective of the present work was to evaluate the effect of the partial substitution of semolina with mesquite pods flour and pea protein isolate on the digestibility of protein and starch, microstructure, and techno-functional characteristics of spaghetti pasta.

2. Materials and Methods

Materials

Pea protein isolate was bought from a local grocery store (Sanjo, Durango, Mexico). Semolina was bought from a local supplier (Da Rivara, CDMX, Mexico), Pea Protein Isolate was bought from Healthy Superfoods (CDMX, México), Xanthan and guar gum (food grade) were supplied by Diquitra (Tlalnepantla, México).

Mesquite flour was obtained following the procedure described as follows. The mesquite pods were sanitized in a sodium hypochlorite solution (1%), with constant rinsing to eliminate any contamination. Subsequently, pods were dried at room temperature (32 °C, 36 h) to achieve a humidity of 5.5%, ground (2 mm) and vacuum-packed until its use.

Spaghetti pasta preparation

The spaghetti was made as described by Di Stefano et al. [14], with modifications: semolina was hydrated to a humidity between 35 and 40% and mixed in a mixer (Kitchen Aid model K45, San Pedro Garza García, NL, Mexico) for 10 min at room temperature. Subsequently, using a pasta machine (Kitchen Aid, San Pedro Garza García, NL, Mexico) to make fresh pasta, strands with a length of 15 cm were made.

The dough was dried at 50 or 60 °C in a dryer with relative humidity (HR) of 60–65%, depending on the experimental design, until equilibrium humidity between 7 and 8% (w/w) was achieved (10 h for 60 °C, 12 h for 50 °C), and an air flow rate of 2 m/s. The experimental conditions for the use of the ingredients (hydrocolloids, mesquite flour, semolina, pea protein isolate) are shown in

Table 1. Experimental conditions for creating spaghetti pasta.

Sample Name	Semolina (% w/w)	Pea Protein Isolate (% w/w)	Mesquite Flour (% w/w)	Gum Rate *	Drying Temperature (°C)
Control50	100	0	0	0	50
Control60	100	0	0	0	60
A	70	15	15	0.5	60
B	70	10	20	0.5	50
C	80	10	10	0.5	50
D	70	20	10	1	60
E	70	5	25	1	60
F	85	7.5	7.5	1	50

* Guar gum and Xhantan gum (1:1), mass conditions independent of the main ingredients.

.

The stabilization process of the pasta after drying was conducted at 30 °C, for 240 min at HR 60% and a constant air flow of 2.4 m/s.

Proximate analysis

Proximate analysis was conducted on the spaghetti pasta samples to determine its moisture, ash, crude protein, and lipids content through the application of established analytical techniques [15]. Dietary fiber was determinate following the method of Goñi et al. [16] and the total carbohydrates were obtained by difference.

Trypsin inhibitors

Trypsin inhibitor activity was measured following the procedure in [17]. Briefly, α-N-benzoyl-dl-arginine-p-nitroanilide hydrochloride (BAPNA) (Sigma, St Louis, MO, USA) was used as the substrate for trypsin. A total of 500 mg of ground experimental sample was extracted with 25 mL of 0.01 N sodium hydroxide for 3 h at room temperature in agitation. Extracts were centrifuged (17,500× g) at 4 °C for 20 min, supernatants were filtered through Whatman No. 1 filter paper. The extracts were diluted to 30% in distilled water so that 1.0 mL could inhibit 50% of trypsin activity in the conditions presented herein.

Five portions of extracts (0, 0.6, 1.0, 1.4, and 1.8 mL) were pipetted into test tubes and the final volume was adjusted to 2 mL with distilled water. Trypsin solution (2 mL, 20 mg/L in 0.001 M HCl) was added and the tubes were placed in the water bath at 37 °C, followed by the addition of 5 mL of N-a-benzoyl-dl-arginine-p-nitroanilide (BAPNA) solution (0.4 mg/mL in Tris-buffer 0.05 M, pH 8.2) previously warmed to 37 °C. After exactly 10 min the reaction was stopped by adding 1 mL of 30% acetic acid to each test tube.

The absorbance was read at 410 nm and the reagent blank was prepared by adding 1 mL of 30% acetic acid to a test tube containing trypsin and water (2 mL of each) before the BAPNA solution was added. One trypsin unit was arbitrarily defined as an increase of 0.01 absorbance unit at 410 nm per 10 mL of the reaction mixture under the conditions used herein.

Culinary properties

Optimal cooking time (OCT)

A weight of 5 g of spaghetti was cooked in 60 mL of boiling water and every 30 s the spaghetti was squeezed between two Petri dishes to determine the OCT, which corresponded to the disappearance of the white core (non-gelatinized starch) [18].

Swelling index (SI)

Five pieces of cooked spaghetti (5 cm in length) were weighed, dried for 24 h to constant weight in an air oven at 65 °C. The SI was established following the following equation [19].

SI = (Cooked pasta (g) − Cooked pasta after drying (g))/Cooked pasta after drying (g)

Cooking weight loss

Cooking weight loss was performed according to the methodology in [20]. Briefly, the organic material lost during cooking was analyzed. For this purpose, the cooking water was dried to constant weight in an air oven at 65 °C. The remainder (solid loss) was weighed and expressed as a percentage following the next equation:

% weight loss due to cooking = (Solids lost (g))/(raw sample (g)) × 100

Techno-functional properties

Texture profile

The TA-XT2 texture analyzer (Stable Micro System, Godalmine, UK) was used. Briefly, four spaghetti samples were placed adjacent to each other in the center of the compression plate of the pasta firmness/stickiness equipment. The test and post-test speeds were 1 mm/s. The resulting force–time curve was used to extract several texture parameters, both primary (hardness, cohesiveness, and adhesiveness) and secondary (brittleness, chewiness, gumminess, and resilience). The TPA test was performed at least three times [21].

Water absorption

The percentage of water absorption (WA) indicates the maximum amount of water that the dough can absorb. WA is an important functional property of flour that improves the softness and digestibility of the product [22].

To determine WA, five pieces of cooked spaghetti (each 5 cm long) were weighed, and the weight was recorded. The WA (%) was determinate using the next equation:

% Water Absorption = (Cooked pasta (g) − raw pasta (g))/(raw pasta (g)) × 100

Color

The color of the dried and cooked pasta was measured using the Konica Minolta colorimeter (Konica, Ramsey, NJ, USA), as described in [23], performing the determination of the values of L*, a* and b*, where L* defines lightness, a* defines red-green and b* defines blue-yellow and using the next equation:

ΔΕ = √(〖(ΔL)² + (〖Δa²) + 〖(Δb²))

Soluble protein

Soluble protein measurement was performed according to [24], with minor modifications. Briefly, 0.5 g of finely ground sample was placed in 10 mL of phosphate buffer (0.05 M, pH 7) containing 0.1 M NaCl and 8 M urea or 8 M urea and 0.01 M dithiothreitol (DTT). The suspensions were shaken for 30 to 60 min at 25 °C. Subsequently, they were centrifuged at 10,000× g for 20 min at 20 °C. The supernatant was recovered, and the soluble protein content was quantified by dispensing 2 µL on the Take 3TM plate for microvolumes. The pre-programmed protocol in the Gen5 protein software was used for quantification.

Raman Microscopy

Raman microscopy was performed as described by [25]. Briefly, an Xplora Plus Raman microscopy (Horiba, Lyon, France) equipment was used, and a Syncerity scientific camera (Horiba Scientific, Lyon, France) was used as detector. The cooked pasta was placed on a microscope slide and observed under a 10× objective, illuminated with a 532 nm laser, and the spectra were scanned from 400 to 3900 cm⁻¹. The analyzed spectra were averaged over three recorded spectra.

The spectral data were analyzed as follows: the spectrum of the control pasta was subtracted from each of the signals of the formulations and corrected by applying a double 7-point Savitzky–Golay smoothing. Subsequently, a baseline was plotted, and a normalization of the data was performed, all using OriginPro 2024 software (Northampthon, MA, USA).

Laser confocal microscopy

Laser confocal microscopy was performed as described by [26]. Briefly, a cooked spaghetti sample was cut thinly (approximately 1 mm). Subsequently, it was stained for 30 min with fluorescein (FITC) at 0.05% by weight of the sample (previously dissolved in water) and rhodamine at 0.05% by weight of the sample (previously dissolved in ethanol). The former stained the starch granules while rhodamine stained the proteins. Samples were rinsed in distilled water for 15 min, mounted, and covered on slides and coverslips.

An Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan) with excitation and emission at 488 and 561 nm was used, and the samples were observed under a 10× objective.

Digestibility properties

Protein digestibility

Protein digestibility was carried out following the method in [27]. Briefly, distilled water was added to the cooked pasta sample at a rate of 1 mL per 6.25 mg of protein, then the pH was adjusted to 8 and the sample was maintained at 37 °C. An enzyme mixture of trypsin, protease, and chemotrypsin (1.6, 1.3, and 3.1 mg/mL, respectively) was prepared. The sample suspension was mixed with the multienzyme solution at a ratio of 5:0.5, the pH change was monitored during a timeframe of 0 to 10 min, and protein digestibility (PD) was determined following the next equation:

PD = 210.46 − (18.10 × (X)).

where X is pH change and PD is protein digestibility.

Starch digestibility

Starch digestibility was obtained following the method described in [27]. Briefly, a 2.5 g sample of cooked spaghetti with size 3–5 mm was added to 30 mL of distilled water, and stirred under heating at 37 °C for 10 min. Subsequently, 1 mL of 10% pepsin solution in 0.05 mol/L HCl was added and shaken at 130 rpm at 37 °C for 30 min. A sample of 1 mL of the solution was removed to mix with 4 mL of methanol (time 0). Next, 0.1 mL of amyloglucosidase and 5 mL of 2.5% pancreatin were added to 0.1 M maleate buffer solution. An aliquot was withdrawn after 20, 60, and 120 min, to which 4 mL of methanol was added to stop any further reaction. The reduction in sugar content was analyzed using 3,5-dinitrosalicylic acid (DNS). A graph of glucose release versus time and area under the curve (ABC) was analyzed.

Statistical Analysis

Prior to the analysis, it was verified that the data obtained conformed to a normal distribution, so for the results of the nutritional, anti-nutritional, and soluble protein factors a t-student test was performed (with a significance level of p < 0.05). Response surface plots were constructed for techno-functional and culinary properties using the general linear model (GLM), with a significance level of p < 0.05, also a Dunnett’s multiple comparisons test was used with a significance level of p < 0.05. All statistical data were processed in the Statistica 12.5 software (StatSoft, Tulsa, OK, USA).

3. Results

The results of the proximate analysis of the main ingredients and spaghetti pasta samples are shown in

Table 2. Proximate analysis of the main ingredients used, spaghetti pasta samples, and controls.

Main Ingredients	Drying Temp (°C)	Protein (% w/w)	Lipids (% w/w)	Carbohydrates (% w/w)	Ash (% w/w)	Humidity (% w/w)
Semolina	NA	9.18 ± 0.50	1.34 ± 0.43	77.69 ± 2.23	0.73 ± 0.05	11.06 ± 0.60
Mesquite flour	NA	11.91 ± 0.00 *	2.55 ± 0.06	74.37 ± 0.89	4.24 ± 0.00 *	6.93 ± 0.57 *
Pea protein isolate (PPI)	NA	73.59 ± 2.32 *	0.55 ± 0.18	14.91 ± 4.25 *	3.72 ± 0.05 *	7.23 ± 0.46 *
Spaghetti samples
Control50	50	9.30 ± 0.75	0.27 ± 0.05	81.77 ± 1.63	0.61 ± 0.18	8.16 ± 0.32
Control60	60	8.64 ± 0.37	0.45 ± 0.07	82.15 ± 0.39	0.78 ± 0.02	7.95 ± 0.03
A	60	17.25 ± 0.76 *	0.42 ± 0.12	74.74 ± 0.60 *	1.72 ± 0.00 *	7.57 ± 0.04 *
B	50	13.25 ± 0.37 *	0.55 ± 0.03 *	77.04 ± 0.90 *	1.85 ± 0.10 *	6.98 ± 0.11 *
C	50	14.86 ± 1.10 *	0.76 ± 0.03 *	74.82 ± 1.90 *	1.39 ± 0.01 *	7.31 ± 0.19
D	60	20.97 ± 0.01 *	0.66 ± 0.07	69.62 ± 0.28 *	1.75 ± 0.02 *	7.26 ± 0.15 *
E	60	10.88 ± 0.75	0.57 ± 0.10	79.39 ± 1.45	1.84 ± 0.02 *	8.15 ± 0.21
F	50	12.78 ± 0.41 *	0.27 ± 0.08	78.43 ± 0.38	1.31 ± 0.01 *	7.20 ± 0.04

* Indicates statistical differences (t-student p < 0.05), NA is not applicable for the sample, gum rate condition was specified in Table 1. A T-Student test was conducted for sample vs. control at the same temperature.

. On the values of protein of semolina, the values were similar to those reported in [28]. Also, similar values of lipids, carbohydrates, and humidity were found; however, the levels of ash were low. For the pea protein isolate, the protein value was higher in the present experiment in comparison to the values reported in [13]; however, this behavior could be related to the pea cultivar and method of the obtention of protein isolate. For mesquite flour, Campos et al. [29] used P. Laviegata and reported values like those found in the present work (protein and ash 11.90 and 3.50%, respectively).

The results of pasta samples showed higher values of protein in comparison to the spaghetti control, independent of the drying temperature except for formulation E, which presents the lowest PPI content (5%). Trypsin inhibitors were not detected in the spaghetti samples.

Culinary properties

The culinary properties of the experimental samples of pasta were analyzed through a linear generalized model (LGM) (p < 0.05). The optimization was conducted using the desirability concept (the values range from 0 to 1, the closer to 1, the greater the desirability). The effect of the temperature on the optimal cooking time (OCT) is shown in Figure 1a (50 °C) and Figure 1b (60 °C), they showed the behavior of the desirability of the optimal cooking time (OCT) with respect to the partial substitution of PPI and mesquite flour. As expected, higher desirability OCT was obtained in pasta made with semolina; however, the partial substitution of semolina (20%, maximum), could achieve acceptable results, independent of the temperature and gums ratio.

Swelling index (SI)

The behavior of swelling index is shown in Figure 2a (50 °C) and Figure 2b (60 °C). As can be seen, the optimum value of the swelling index (SI) (with respect to desirability) was found at the central point in a range of semolina between 75 and 85%, regardless of the temperature and ratio of gums used.

Texture Profile Analysis

The optimum values of the adhesiveness correspond to the highest value of semolina; a negative relationship between semolina and the mesquite flour and PPI can be observed in Figure 3a (50 °C) and Figure 3b (60 °C). However, the use of up to a 20% substitution of semolina shows acceptable results (desirability > 0.8).

The optimum values of elasticity correspond to high values of desirability, Figure 4a (50 °C) and Figure 4b (60 °C), a value closer to 1 can be observed when the amount of semolina in the system decreases and the mesquite flour content increases; also, the most important factor is the presence of the hydrocolloids, independent of their concentration, thus the optimum value of elasticity was obtained at 70% of semolina and the use of hydrocolloids.

Color change

Results of the optimization of the color change (ΔΕ) are shown in Figure 5a (50 °C) and Figure 5b (60 °C). High desirability was observed with high levels of semolina. While high contents, especially of mesquite flour affect the color change, this may be mainly due to the contribution of ash and the contribution of color by mesquite flour and PPI [30].

The results for soluble protein (

Table 3. Soluble protein content in the spaghetti pasta samples.

Spaghetti Samples	Drying Temperature (°C)	Phosphate Buffer + NaCl Solubility (%)	Phosphate Buffer + Urea Solubility (%)	Phosphate Buffer + Urea + DTT Solubility (%)
Control50	50	4.91 ± 0.18	9.26 ± 0.03	11.54 ± 0.53
Control60	60	3.71 ± 0.10	5.71 ± 0.21	9.17 ± 0.01
A	60	7.93 ± 0.12 *	5.94 ± 0.4 *	7.38 ± 0.52 *
B	50	8.66 ± 0.10 *	8.46 ± 0.7 *	9.30 ± 0.09
C	50	6.32 ± 0.20 *	6.15 ± 0.47	6.90 ± 0.32 *
D	60	4.48 ± 0.13	4.76 ± 0.1 *	8.21 ± 0.04 *
E	60	12.98 ± 0.1 *	11.4 ± 0.5 *	14.28 ± 0.27 *
F	50	6.41 ± 0.06 *	7.73 ± 0.0 *	11.91 ± 0.04 *

* Indicates statistical differences (t-test, p < 0.05, sample vs. control spaghetti at the same temperature). Gum conditions are indicated in Table 1.

) of spaghetti pasta with partial substitution of semolina indicated that the pasta dried at 50 °C present a higher solubility than the control sample (medium 1), while the pasta dried at 60 °C present the same behavior as the control, except for formulation D (70,20,10,1). In the second medium, samples dried at 50 °C increased the soluble protein content, except in formulation C, which may be due to the amount of semolina and differs from formulation F in the hydrocolloid content.

Raman spectroscopy

Disulfide bridges region (490–550 cm⁻¹)

The formulations dried at 50 °C are shown in Figure 6, it is observed that the pasta control showed a t-g-g-g conformation like sample B (70, 10, 20) and F (85, 7.5, 7.5), while formulation C (80, 10, 10) showed a g-g-g-g conformation, suggesting the presence of intramolecular disulfide bridges [31].

Raman spectra of the samples dried at 60 °C are shown in Figure 7; the pasta control proteins showed a g-g-g-g conformation, while in partial substituted pasta A (70, 15, 15), D (70, 20, 10) and E (70, 5, 25) the conformation was t-g-g-g.

Amide-III

The pasta samples dried at 50 °C are shown in Figure 8, in which the pasta control in the region amide III shows bands at 1261 cm⁻¹ and 1350 cm⁻¹, which is related to the presence of random coils and α-helices, while formulation B presents β-lamellae and random coils, while formulations C and F, present β-lamellae and α-helices [32,33]. Regarding the formulations dried at 60 °C, results are shown in Figure 9, from this it is possible to see that the spaghetti control shows bands at 1255 and 1344 cm⁻¹, which has been reported to correspond to secondary structures of the random spiral and α-helix type [32].

Amide I region

The Amide I region (1570–1720 cm⁻¹) samples dried at 50 °C are shown in Figure 10, where the control pasta dried at 50 °C presents aggregate structures of the β-hydrated lamella by the presence of a band at 1585 while the band at 1648 cm⁻¹ corresponds to α helices. Sample B presents aggregated structures in the form of pseudolamines by the presence of the band at 1625 cm⁻¹ and regular structures in the form of β-structures with twists without H-bonds by the bands at 1677 cm⁻¹; formulation C presents protein aggregates by the band at 1602 cm⁻¹, also, the band at 1631 cm⁻¹ may correspond to pseudolamines or β-sheets, although the band at 1666 cm⁻¹ and the combination with the band at 1631 cm⁻¹ may be indicative of β-structures with twists joined by hydrogen bonds. Finally, the band at 1689 cm⁻¹ corresponds to an antiparallel sheet joined by hydrogen bonds. Formulation F presents a band at 1625 which is indicative of the presence of pseudolamines [32,33].

The pasta control dried at 60 °C (Figure 11), shows protein aggregates characterized by the presence of a band at 1608 cm⁻¹, regular α-helix type structures, whereby temperature influences the arrangement of the secondary structure of the amide I region [32,33]. Samples A and D showed protein aggregates in β-hydrated lamellae conformation characterized by the presence of bands at 1597 and 1585 cm⁻¹, respectively; also, together with sample E, they showed random spiral structures and antiparallel lamellae joined by hydrogen bonds.

Confocal Laser Microscopy

The microstructures of the different spaghetti samples dried at 50 and 60 °C are shown in Figure 12 (S50, S60, A–F). The different experimental conditions are described in Table 1. From the images, it is possible to observe the gluten network which is stained reddish-orange due to the action of rhodamine and the starch granules wrapped in green due to fluorescein. It was observed that the pasta control dried at 60 °C presents better uniformity in the image, for which the temperature plays an important role in the correct formation of the gluten network.

• Protein digestibility

Results about protein and starch digestibility were collected only to the “best” formulations obtained at each experimental temperature and their proper spaghetti pasta controls (samples F and A).

The results about protein digestibility are shown in

Table 4. Digestibility, availability, and percentage of protein present in the control pasta and formulations A and F.

Formulation	% Protein Cooked Pasta	Protein Digestibility	Protein Availability
Control pasta at 50 °C	11.20 ± 0.65	80.32 ± 0.25	8.99 ± 0.02
Formulation F (85, 7.5, 7.5, 1) 1	15.74 ± 0.40 *	80.77 ± 0.12	12.71 ± 0.02 *
Control pasta at 60 °C	11.04 ± 0.38	79.33 ± 0.12	8.75 ± 0.01
Formulation A (70, 15, 15, 0.5) 1	17.76 ±0.79 *	80.5 ± 0.00 *	14.29 ± 0.00 *

¹ Indicates the percentage of wheat semolina, pea protein isolate, mesquite flour, and hydrocolloid ratio, respectively. * Indicates significant statistical differences, T student, p < 0.05.

; from this, it is evident that the enrichment of pasta with mesquite flour and pea protein isolate increases both the protein percentage and protein availability in comparison to the pasta control (p < 0.05). Protein digestibility was improved in formulation A with 30% semolina substitution with pea protein and mesquite flour, mainly due to a higher percentage of pea protein and the absence of anti-nutritional compounds such as trypsin inhibitors (p < 0.05). In contrast, formulation F, with 15% substitution, showed no improvement in digestibility.

• Starch digestibility

The results for starch digestibility are shown in Figure 13. The amount of reducing sugars released in 120 min of digestion in spaghetti pasta with the addition of mesquite flour and pea protein isolate was lower than observed in spaghetti pasta control at 50 or 60 °C.

4. Discussion

• Proximate analysis

The results of pasta samples are shown in Table 2 according to the obtained results, all formulations, independent of the drying temperature, showed an increase in protein content except for formulation E, which presents the lowest PPI content (5%). Thus, the addition of PPI plays a determining role in the increase in protein and therefore in the decrease in carbohydrates; also, the partial substitution of semolina by mesquite flour and PPI increased the ash amount, this behavior could be related to the presence of minerals, mainly potassium (K), calcium (Ca), phosphorus (P) and magnesium (Mg), reported in mesquite flour and PPI [34]. Trypsin inhibitors were not detected (Data not shown) in the pasta samples, several authors mention that processes such as extrusion or cooking inhibit trypsin activity making it possible to use legume flours in the food industry [13].

The main changes in the structure of the pasta occur during the cooking process, such as starch gelatinization. When gluten dilution occurs, the rate of water penetration increases, thus decreasing cooking times, as reported by several authors [35,36,37,38]. This explains the influence of mesquite flour and PPI in decreasing cooking times and lowering the desirability function.

The SI indicates the capacity of the pasta to absorb water and consequently increase its volume during the cooking process. It is an indicator of the ability of the starch to maintain its integrity when heated. The greater the swelling capacity of starch granules, the weaker the binding forces. In some studies with pasta, a correlation has been seen between the in vitro glycemic response of pasta with the swelling index (SI), which increases elasticity and water absorption [39,40].

The results obtained for the SI also indicated that all experimental formulations showed lower SI in comparison to the pasta control, which is due to the action of the hydrocolloids since they prevent excessive swelling of the starch granules through the formation of a starch-protein-polysaccharide complex; this network prevents the starch granules from escaping into the cooking water, especially when hydrocolloids are used at low concentrations [14,19]. This would be the main reason why hydrocolloids are influencing the results of the system.

Adhesiveness is associated with changes in viscosity above the gelatinization temperature following a heating/cooling and shearing process due to swelling of the starch granules, it is an undesirable variable as it measures the product’s ability to attach to the teeth [41]. Adhesiveness is negatively correlated with OCT [21]. Elasticity is defined as the ability of a material to resist a force and return to its original shape. It has been reported that elasticity is negatively related to solids loss, that when the dough is fortified the gluten network weakens facilitating the leaching of mainly starch and minerals [19].

Color is considered an important factor in pasta quality and has been related to consumer acceptability. The characteristic color of a pasta is positively related to the content of natural colorants such as carotenoids and xanthophylls. On the other hand, ash content, protein molecular composition, and poor milling processing can produce dark spots in the material and has a negative effect on color [42].

The increase in solubility has been related to the formation of a weak network because of weak disaggregation phenomena [24]. Although it could be hypothesized that the higher the percentage of solubility in the third medium, the greater the formation of disulfide bridges, the reality is that protein aggregates could be forming and other types of bonds are being formed, thus, that which gives the structure of the pasta are the hydrocolloids.

The determination of soluble protein is an important indicator of pasta stability with respect to hydrophobic interactions of disulfide bonds and aggregate stabilization. Legume proteins are more prone to denaturation compared to gluten proteins; consequently, they may not form as strong and stable protein networks [43] with proteins dissolving otherwise insoluble proteins. The increased solubility in the third medium is due to the action of dithiothreitol (DTT) which breaks disulfide bridges by reducing S-S bonds to free thiol groups.

The disulfide bridges region is important since it is related to the maintenance of the tertiary structure of the protein, which is related to the maintenance of the gluten network, it can be divided into three; the g-g-g zone corresponds to 510 ± 5 cm⁻¹, the t-g-g rotamer zone of 525 ± 5 cm⁻¹, and the 545 ± 5 cm⁻¹ zone of the t-g-t rotamers [44]. Thus, formulation C with 70% semolina, 10% pea protein, 10% mesquite flour, and 0.5% guar gum/xanthan gum, dried at 50 °C, presents greater stability in the formation of disulfide bridges. While the rest of the samples presented a similar behavior showing t-g-g type rotamers, this conformation is the second most stable and the one found in the control pasta dried at 50 °C.

In general, the obtained results indicate that mesquite flour, pea protein isolate and hydrocolloids, change the structural conformation of the amide III region, while the absence of α-helices is a sign of hydrogen bond breakage and their transformation into other types of secondary structures such as β-sheets and β-turns. However, sample E, (70% semolina, 5% PPI, 25% mesquite flour, 1% hydrocolloids, dried at 60 °C), showed the behavior of amide III more like the pasta control. Also, the behavior of the structure of α-helices in the pasta substituted with PPI and mesquite flour was modified by the breaking of H-bonds, modifying the secondary structure of the proteins present in the gluten.

In general, samples dried at 60 °C present a more homogeneous structure and it is evident that in all the micrographs there is a predominance of starch, so hydrocolloids are playing a crucial role in the structure of the dough. These results coincide with those analyzed in the amide I and amide III regions of the pastas where smaller displacements were observed in contrast to the pasta samples dried at lower temperatures.

The gluten structure depends on multiple factors such as semolina processing, lamination procedure plays an important role since insufficient lamination times will result in a lack of cohesion and compactness. Excessive rolling times will result in the destruction of the protein network and starch granules [45]. Lamination causes proteins and starch granules to be evenly distributed throughout the dough and during baking, proteins separate from starch granules and begin to aggregate, resulting in clumping of starch granules [46]. The above could explain the results of the Raman micrographs and soluble protein where the dilution of the gluten matrix by the action of mesquite flour and pea protein causes conformational changes increasing the protein aggregates and therefore the decrease in the secondary structures of the α helix and β-sheets typical of pasta.

The increase in protein availability has been related to the use of legume proteins, similar results to those found in the present work have been reported [47,48]. However, there is a lack of knowledge about the mechanism related to this behavior.

About starch digestibility, several semolina substitutions have been made in pasta where it has been observed that various factors such as temperature affect digestibility since it forms a stronger protein network, in addition to the above, particle size plays an important role since it is inversely related to starch digestibility, due to the fact that grain milling increases starch susceptibility by altering plant tissue and damaging starch granules [49]. Related research has shown that the addition of dietary fibers such as hydrocolloids disrupts the protein matrix of gluten by intensifying separation with respect to starch. However, such soluble fibers reduce the degree of starch hydrolysis by interfering with swelling by encapsulating the starch which affects the final texture of the dough [50].

5. Conclusions

Partial substitution of semolina with pea protein and mesquite flour in the preparation of pasta modifies the secondary structure of gluten proteins, altering the techno-functional properties of the pasta, however the use of xanthan and guar gums, as well as galactomannans present in mesquite flour, play a crucial role in the development of the structure and techno-functional properties of pasta, contributing to the cohesion and stability of the pasta during processing and cooking. Also, the use of pea protein isolate and mesquite flour increases protein content which inherently increases the nutritional value of the product. This makes pasta a healthier and more nutritious option for consumers, offering a final product that combines nutritional benefits with good technological functionality.