Enhanced Protein Digestibility and Amino Acid Profile of a Novel Legume (Inga paterno) Seed Flours: Evaluation of Proximal Composition Changes by Sprouting

Abstract

The nutritional value of Inga paterno seeds remains largely unexplored. Given the global protein deficiency, underutilized legumes like I. paterno could serve as alternative protein sources. This study evaluated the effect of sprouting on the composition, protein digestibility (PD) as soluble protein (SP), amino acid profile, free amino acids by UHPLC, and nutritional indicators of I. paterno seed flour. Seeds were sprouted for 0, 2, 4, 6, 8, or 10 days, then dried, milled, and analyzed. The seeds reached 100% sprouting after six days. Sprouting led to a 54.36% decrease in protein content but a 109% increase in the lipid fraction by day six. PD doubled after 8–10 days of sprouting. Additionally, total amino acid content significantly increased and the chemical score of majority essential amino acids tripled. After in vitro digestion, sprouted flour released higher amounts of free amino acids, particularly aspartic acid (from 9.10 ± 0.18 to 19.65 ± 0.97 mg/L), histidine (from 33.48 ± 0.61 to 46.29 ± 2.34 mg/L), alanine (from 16.32 ± 0.40 to 23.74 ± 0.07 mg/L), and lysine (from no detected to 7.12 ± 0.36 m/L). These findings suggest that sprouted I. paterno seeds could be a valuable, digestible protein source with enhanced nutritional quality, making them a promising ingredient for the food industry.

1. Introduction

The consumption of legumes plays a crucial role in global nutrition due to their adaptability to diverse climates, short growth cycles, and potential for crop intensification [1]. Their nutritional value (high-quality composition, rich in protein, fiber, vitamins, and minerals) has driven increased interest in their consumption, particularly in species like lentils, peas, and soybeans [1]. Among leguminous trees, the Inga genus includes various underutilized species distributed from Mexico to Argentina [2]. One such species, Inga paterno (commonly known as jiniquil), is found from Puebla and Guerrero (Mexico) to Costa Rica, typically growing at altitudes between 100 and 1100 m above sea level [3]. Each pod produces 13 to 40 fresh seeds, which are often discarded despite their high protein content (20.42 g per 100 g dry weight) [4]. While the cottony sarcotesta is commonly consumed, the seeds are often discarded or boiled in a few cases for their consumption. Given the limitations associated with the digestibility of legume proteins, it is essential to explore environmentally friendly methods like sprouting to enhance their nutritional value.

Sprouting is a process independent of sunlight or soil, during which seeds absorb water to help the biological processes and develop an embryonic axis [5]. This process consists of three stages: (1) rapid self-priming, where metabolic activity resumes upon water absorption; (2) slow hydration, marked by embryo growth, biochemical transformations in polysaccharides and proteins, and increased hydrogen–potassium ATPase activity to facilitate water uptake; and (3) radicle emergence, during which macromolecules like starch and proteins are broken down into simpler components [6,7].

Sprouting has shown beneficial changes in different legume seeds by modifying their nutritional composition through enzymatic hydrolysis and the synthesis of new compounds. For example, in red kidney bean (Phaseolus vulgaris L.) sprouted milk, the soluble protein content increased to 33.65% after 30 h of treatment [8]. Rodriguez et al. [7] reported that sprouted peas, beans, and lentils increase the availability of lysine (from 41.00 ± 1.27 to 268.06 ± 14.41 mg/16 g of N) and furisine (from 21.47 ± 0.39 to 68.28 ± 1.55 mg/16 g of N). Additionally, sprouted cowpea (Vigna unguicullata) exhibited a crude protein content of up to 19.15% after sprouting [9]. Sprouting can also enhance starch digestibility, modify fiber composition, increase isoflavone content and bioaccessibility, and alter lipid profiles [10,11].

There are different protein nutritional quality indicators. One of them is the biological value (BV); BV is a measure of how much dietary nitrogen, a key component of proteins, is absorbed and utilized by the body. It is determined by comparing the amount of nitrogen consumed with the amount excreted in feces [12]. Other methods to assess protein quality include the protein efficiency ratio (PER), which measures weight gain in animals fed the protein [13], and the Protein Digestibility Corrected Amino Acid Score (PDCAAS), which considers both the amino acid profile and digestibility [14]. While these methods are expensive and require animal model or fecal material, in vitro methods offer a cost-effective, ethically sound, reproducible approach to studying digestion, and valuable tools for screening proteins [15,16]. Additionally, UHPLC provides a highly efficient and sensitive method for the qualitative and quantitative analysis of amino acids [17]. These amino acid profiles can be used in combination with mathematical models to predict protein nutritional quality indicators like BV and PER [18], with the advantages of reduced costs, minimized environmental impact, and highlighting areas requiring further research [19]. The data about different nutritional parameters like protein content, protein digestibility, or amino acid profile of I. paterno seeds are null and no previous studies have investigated the effect of the different stages of sprouting on the proximal composition of their seeds. This work aims to evaluate the effect of sprouting time on the bromatological composition, protein digestibility, amino acid profile, and the estimation of nutritional parameters of I. paterno seed flour.

2. Materials and Methods

2.1. Materials

The chemicals and reagents used throughout this study were of HPLC or analytical grade. Pepsin from porcine gastric mucosa, α-amylase from porcine pancreas, copper sulphate, mercury oxide, methyl red, monobasic sodium phosphate, dibasic sodium phosphate, disodium phosphate (Na₂HPO₄), sodium tetraborate (Na₂B₄O₇), calcium chloride, sodium chloride, water methanol, and acetonitrile were purchased from Sigma Aldrich (St. Louis, MI, USA). Sodium hydroxide (NaOH), methanol, hexane, sulfuric acid, and hydrochloric acid were obtained from J.T. Baker (Radnor, PA, USA).

2.2. Sprouting Treatment

I. paterno pods at consumption maturity (Figure 1a) of the November 2022 harvest from the region of Tochimilco in the Puebla state of Mexico (north latitude 18°50′–19°02′ and west longitude 97°18′–97°26′) were used. The pods were disinfected with a 0.1% hypochlorite solution for 5 min. The pod, sarcotesta, and seeds were separated. Per experiment, 50 seeds were placed in a 0.1% hypochlorite solution for 1 min and then rinsed with drinking water. The seeds were deposited in sprouting trays (Figure 1b) and covered with wet cotton. Then, samples were kept at 23 °C with a relative humidity of 78%. The experiments were run in triplicate for 0, 2, 4, 6, 8, or 10 days. After that, the seeds were dried for 32 h at 35 °C (Food Dehydrator, Excalibur, Sacramento, CA USA). The flours were obtained (Figure 1d) by grinding the seeds in an electric grain mill (Moongiantgo, Hangzhou, China). All the samples were stored in bags at −4 °C for further analysis [20].

The sprouting percentage of the 50 seeds was obtained as the measure of the number of sprouted seeds related to the total number of seeds used and the radicle size was determined with a Vernier caliper (Kynup, Longgang, Shenzhen, China) every two days [21].

2.3. Proximate Composition Analysis

The crude protein (method 920.87), crude fat (method 950.54), ash (method 945.39), and moisture (method 925.09) contents were determined using standard AOAC methods [22], and the carbohydrate content was determined by difference.

2.4. In Vitro Protein Digestibility

An amount of 3 g of each flour was mixed with 100 mL of artificial saliva (phosphate buffer pH 6.9, 0.04% NaCl, 0.04% CaCl₂), and 30 mg α-amylase from porcine pancreas was added. The samples were stirred at 200 rpm for 5 min at 37 °C. The pH was adjusted to 2 with 1 N HCl and 45 mg of pepsin from porcine gastric mucosa was incorporated. The samples were stirred for 1 h at 37 °C and 200 rpm in a multipoint stirrer (Cimarec I, Thermo Scientific, Waltham, MA, USA) in a thermal heating bath with recirculation (Haake SC100 Thermo Scientific, Waltham, MA, USA) [23].

The digestibility of the protein was calculated as the soluble protein (SP) content measured with a Total Protein Kit, Micro Lowry, Peterson’s Modification (Sigma-Aldrich Co., St. Louis, MI, USA). The sample collection was performed after the buccal digestion (D₂) and gastric digestion (D₃), whereas the fresh sample was considered as time 0 (D₁). The liquid samples were centrifugated at 9000× g for 5 min at 25 °C. Then, 50 μL of a deoxycholate solution (0.15%) was added to 500 μL of each supernatant, homogenized, and kept in the dark for 10 min, after which 50 μL of trichloroacetic acid (72%) was added and the samples were centrifugated at 9000× g for 5 min at 25 °C. The samples were decanted, and the pellets were resuspended in 500 μL of Lowry’s reagent and 500 μL of water and kept in the dark for 20 min, followed by the addition of Folin Ciocalteau reagent (250 μL) and left for another 30 min in the dark. In a 96-well microplate, 200 µL of each sample was transferred. The absorbance was measured using a UV–Vis Multiskan Sky Microplate (Thermo Fischer Scientific, Walthman, MA, USA) spectrophotometer at 720 nm. A standard curve bovine serum albumin (0–400 µg/mL) was used [24].

2.5. Free Amino Acid Profile

The samples with 10 mg of N were digested for 24 h in a 6 M HCl solution (400 μL) at 100 °C, followed by filtration and dilution with distilled water (500 μL). The samples were concentrated at 5 μL until vacuum conditions were met. The residues were resuspended in diethyl ethoxy methylene malonate for a pre-column derivation for the primary amino acids with OPA and for secondary amino acids (Pro) with FMOC. To determine the free amino acid profile, an Agilent 1290 Infinity II UHPLC (Palo Alto, CA, USA) with a diode array detector and an autosampler/injector was used. Compound separation was achieved using a ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) (Palo Alto, CA, USA). The mobile phases comprised 10 mM Na₂HPO₄: 10 mM Na₂B₄O₇ buffer at pH 8.4 for phase A and a solution of acetonitrile: methanol: water (45:45:10 v:v:v) for phase B. An injection volume of 1 μL was used through a 1.5 mL/min flow rate at a column constant temperature of 40 °C. The processes started with 98% A at 0.35 min, 0% A at 13.5 min, and 98% A at 16 min for a total run time of 16 min. The measurement was carried out at 262 and 338 nm. Norvaline (Nva) was used as an internal standard and an amino acid mix calibration curve (Sigma Aldrich, St. Louis, MI, USA) for the quantification [25].

2.6. Estimation of Nutritional Parameters

The amino acid profile of every flour sample was used for the prediction of different nutritional parameters. The amino acid score or chemical score (CS) was determined in relation to the essential amino acid according to Equation (1) [18].

CS (%) = (AAP/AAR) × 100,

(1)

where AAP is the milligrams of amino acid in 1 g of test protein and AAR is the milligrams of amino acid in 1 g of reference protein or FAO requirement pattern [14].

Prediction of the protein efficiency ratios (PERs) was calculated as stated in Equations (2)–(4) [18].

PER₁ = −1.816 + 0.435 × Met + 0.78 × Leu + 0.211 × Hys − 0.944 × Tyr,

(2)

PER₂ = −0.468 + 0.454 × Leu − 0.105 × TyrPER₁,

(3)

PER₃ = −0.684 + 0.456 × Leu − 0.047 × ProPER₂,

(4)

where Met, Leu, Hys, Tyr, and Pro are the percentages of methionine, leucine, histidine, tyrosine, and proline, respectively.

2.7. Statistical Analysis

The experimental treatments were performed in a completely randomized design. The results are expressed as mean ± standard deviation and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (p < 0.05) and principal component analysis using Minitab v. 20.3.2021 statistical software (Minitab Inc., State College, PA, USA).

3. Results

3.1. Radicle Growth

I. paterno seeds exhibited a high germination rate, reaching 93.00 ± 1.41% by the 2nd day and 96.00 ± 2.83% by the 4th day, reaching 100% by the 6th day. Radicle growth progressed as follows: 9.0 ± 3.3 mm (day 2), 36.7 ± 11.2 mm (day 4), 46.2 ± 15.7 mm (day 6), 48.2 ± 14.4 mm (day 8), and 49.7 ± 9.1 mm (day 10) (Figure 2). Significant differences (p < 0.05) were observed up to day 6, after which growth stabilized.

3.2. Chemical Composition Changes

Protein content significantly decreased (p < 0.05) from 23.23 ± 0.17 to 10.60 ± 0.72 g/100 g dry matter (dm) between days 6 and 10 of sprouting (

Table 1. Composition modification of flours of sprouted I. paterno seeds.

Sprouting Time (Days)	Crude Fat *	Crude Protein *	Ash *	Carbohydrates *
0	0.45 ± 0.00 D	23.23 ± 0.17 C	2.71 ± 0.00 A	67.81 ± 0.68 D
2	1.01 ± 0.04 C	17.46 ± 0.48 C	2.72 ± 0.02 A	69.80 ± 0.96 D
4	1.16 ± 0.05 B	18.45 ± 0.33 C	2.79 ± 0.27 A	67.44 ± 1.01 CD
6	1.25 ± 0.05 B	12.34 ± 0.67 B	2.50 ± 0.13 A	73.19 ± 0.22 AB
8	1.34 ± 0.05 A	10.27 ± 0.94 B	2.57 ± 0.07 A	76.00 ± 0.58 A
10	1.54 ± 0.04 A	10.60 ± 0.72 A	2.74 ± 0.07 A	71.94 ± 1.15 BC

Different capital letters in the same column indicate a significant difference (p < 0.05) between sprouting time. * g/100 g of dry matter.

). Additionally, crude fat content ranged from 0.45% to 1.54%, peaking on days 8 (1.34 ± 0.05%) and 10 (1.54 ± 0.04%), and ash content remained relatively stable from 2.50 to 2.79 g/100 g dm (p > 0.05).

3.3. Flour Amino Acid Profile and Nutritional Parameters

Sprouting increased total amino acid (TAA) content from 8.46 ± 0.10 g/100 g dm (non-sprouted) to 9.42 ± 0.09 g/100 g dm (day 6) and 9.31 ± 0.001 g/100 g dm (day 10), a 12% increase (Figure 3a). Essential amino acids (TEAA) rose from 2.33 ± 0.10 to 2.76 ± 0.01 g/100 g dm, and non-essential amino acids (TNEA) increased from 3.76 ± 0.001 to 4.12 ± 0.02 g/100 g dm.

The most abundant amino acids were arginine (1.39–1.72 g/100 g dm), aspartic acid (1.78–2.14 g/100 g dm), and glutamic acid (1.18–1.28 g/100 g dm) (Figure 3a). Serine, threonine, lysine, and proline were also present in notable amounts (0.34–0.66 g/100 g dm). Asparagine and methionine were not detected, while cysteine was only found in non-sprouted flour (0.04 ± 0.001 g/100 g dm).

A significant increase (p < 0.05) was observed in aspartic acid and glycine after 6 days of sprouting, though levels declined by day 10. Serine, phenylalanine, tyrosine, and valine significantly increased throughout sprouting. The TEAA/TAA ratio improved (p < 0.05) from 27.56 ± 0.25% (non-sprouted) to 29.60 ± 0.15% (day 10).

Moreover, the nutritional indicators of the seed flours were positively affected by the sprouting process. The protein efficiency ratios (PER₁, PER₂, and PER₃) were particularly influenced. PER₁ and PER₂ increased (p < 0.05) from 1.76 ± 0.02 to 2.07 ± 0.01 and from 1.39 ± 0.02 to 1.46 ± 0.01, respectively, while PER₃ decreased due to increased tryptophan content.

Additionally, the chemical score (CS) of several essential amino acids significantly improved (Figure 4). The most notable increase (p < 0.05) was in tryptophan from 248.00 ± 7.54% to 852.30 ± 2.59% after 10 days (Figure 4h). Phenylalanine plus tyrosine (Figure 4f) increased from 132.73 ± 5.60% to 454.93 ± 3.00%, and valine (Figure 4i) increased from 65.54 ± 0.01% to 179.44 ± 1.88%. CS values for histidine, isoleucine, leucine, lysine, and threonine (Figure 4a, 4b, 4c, 4d, and 4g, respectively) increased 2.5 times. Conversely, sulfur amino acids (methionine + cystine) decreased from 14.57 ± 0.00% to zero (Figure 4e).

3.4. Protein Digestibility and Free Amino Acids Release

In vitro digestibility significantly improved after 8 days of sprouting, with soluble protein (SP) increasing from 37.39 ± 3.18% (non-sprouted) to 78.53 ± 4.38% (

Table 2. Changes in protein in vitro digestibility of flours of sprouted I. paterno seeds.

Sprouting Time (Days)	D1 (% of Soluble Protein)	D2 (% of Soluble Protein)	D3 (% of Soluble Protein)
0	31.77 ± 2.1.44 Dab	29.37 ± 0.41 Db	37.44 ± 1.95 Da
2	36.44 ± 1.71 CDb	33.64 ± 1.93 Cb	45.86 ± 1.53 Ca
4	40.03 ± 1.77 BCb	39.12 ± 0.24 Bb	48.68 ± 1.24 Ca
6	40.88 ± 1.88 BCb	39.56 ± 0.89 Bb	64.12 ± 0.56 Ba
8	44.73 ± 0.13 BCDa	49.15 ± 0.40 Ab	81.74 ± 1.05 Ac
10	54.54 ± 1.72 Aa	50.30 ± 0.17 Ab	79.35 ± 1.78 Ab

Different capital letters indicate a significant difference (p < 0.05) between sprouting times and different lowercase letters indicate a significant difference (p < 0.05) between gastric digestion steps. D₁ is the soluble protein of flours without enzyme addition, D₂ is the soluble protein after buccal digestion, and D₃ is the soluble protein after gastric digestion.

). This improvement was evident from the onset of digestion (D₁) and persisted through buccal (D₂) and gastric (D₃) phases.

Free amino acid release significantly increased (p < 0.05) after 6 (183.03 ± 6.54 mg/L) and 10 days (180.47 ± 0.30 mg/L) of sprouting (Figure 3b). Aspartic acid (21.72–31.84%) and histidine (13.50–19.82%) were the most abundant. Sprouting increased aspartic acid, glutamine, histidine, and arginine levels. Serine, absent in non-sprouted flour, appeared in sprouted samples. Alanine and asparagine were elevated on day 6 but declined by day 10.

3.5. Principal Component Analysis

To understand the main characteristics produced in I. paterno seed flours by the effect of sprouting time, a principal component analysis (PCA) was conducted (Figure 5). The PCA of the amino acids profile and nutritional parameters (Figure 5a) presented a first component (PC1) that described 76.7% and second component (PC2) that corresponded to 23.3% of the variability, where the amount of lysine (0.221), glycine (0.228), serine (0.233), threonine (0.233), valine (0.233), isoleucine (0.233), glutamic acid (0.224), leucine (0.222), alanine (−0.230), and cystine (−0.219) were the main variables that contributed to PC1, and the amount of arginine (0.420) was the primarily variable of PC2. TEAA correlated positively with isoleucine, valine, threonine, and serine, while alanine and cysteine showed negative correlations. Samples from days 6 and 10 of sprouting had improved %TEAA/TAA, TEAA, PER2, and PER1.

During in vitro digestion, PC1 (78.4%) was driven by glutamine (0.376), arginine (0.376), aspartic acid (0.375), and histidine (0.359), while PC2 (21.6%) was influenced by alanine (−0.564), tyrosine (0.407), and asparagine (−0.450) (Figure 5b). Lysine release negatively correlated with serine and asparagine. Non-sprouted samples contained higher tyrosine, serine, and asparagine, while sprouted samples (day 10) showed a higher release of lysine, glutamine, arginine, and aspartic acid.

4. Discussion

This study demonstrated that I. paterno seeds exhibit a sprouting potential, particularly after the 4th day of treatment. The sprouting percentage surpassing that of soybeans (92.28 ± 2.51%), black beans (60.79 ± 4.94%), chickpeas (87.45 ± 2.67%), and lentils (92.58 ± 4.42%) [21]. Additionally, radicle growth of I. paterno seeds on day four was similar to soybeans (9.13 ± 0.12 cm), black beans (5.06 ± 0.17 cm), and lentils (5.12 ± 0.25 cm) and exceeded that of red kidney beans (1.16 ± 0.04 cm) and chickpeas (1.67 ± 0.09 cm) [21]. The rapid radicle elongation of I. paterno seeds highlights their viability for quick sprouting.

Changes in macronutrient composition during sprouting vary by species. Swieca [26] reported no significant protein changes in lentils, adzuki beans, and soybeans after sprouting, except for mung beans, where the evaluated period was limited to eight hours. In Sterculia urens, protein content declined from 24.5 ± 0.28 mg/g (day 0) to 15.38 ± 0.21 mg/g (day 9), with a sharp decrease observed on day six of sprouting [27], a trend similar to our findings. The initial protein content of non-sprouted I. paterno seeds was comparable to the 20.42 ± 0.59 g/100 g dry matter (dm) reported by Sánchez Mendoza [4].

Protein reduction during sprouting has been linked to enzymatic degradation [28] and respiratory loss [27]. DELLA proteins and phytohormones, such as abscisic acid (ABA) and gibberellin (GA), regulate sprouting through proteasome-mediated degradation pathways. Also, the accumulation or degradation of the proteins is affected by the presence or absence of light and the increase of different metabolisms during the sprouting time that produce degradation of DELLA proteins via the 26S proteasome [29]. Additionally, early-stage declines (days 1–4) are attributed to nitrogen and energy demands for metabolic activity, whereas later reductions result from protein consumption by the developing root system [30].

The lipid content in the non-sprouted and sprouted I. paterno seeds was lower than the 8.55 ± 0.49 g/100 g dm reported by Sánchez Mendoza [4]. Despite the results obtained, the variability of the data could be related to the year of harvest and processing as was described for different soybean meals [31]. Additionally, sprouting affects lipid content differently across species; common bean varieties (Lundazi, Lyambai 4-4-B, Lyambai Parent, and Carioca 38) exhibited increased lipid fractions due to carbohydrate breakdown and crude fat release [32], whereas mung beans showed lipid reductions linked to soaking loss and lipid utilization as an energy source [33].

The primary fatty acids in non-sprouted I. paterno seeds include palmitic acid (37.69 ± 0.09%), linoleic acid (31.06 ± 0.01%), α-linolenic acid (11.17 ± 0.05%), stearic acid (9.73 ± 0.17%), 7-octadecanoic acid (8.10 ± 0.45%), oleic acid (1.66 ± 0.14%), and arachidonic acid (0.59 ± 0.12%) [4]. These compounds have been associated with beneficial effects, such as LDL cholesterol reduction, prostaglandin synthesis, and cardiovascular regulation [4]. Enhancing the lipid fraction through sprouting could potentially amplify these health benefits in I. paterno flour.

Ash content in sprouted I. paterno seeds remained consistent with non-sprouted values (2.47 ± 0.04 g/100 g dm) [4] and was similar to values reported in lentil, adzuki bean, soybean, and mung bean (4.20 ± 0.09, 4.19 ± 0.13, 1.16 ± 0.096, and 3.67 ± 0.20 g/100 g of dm, respectively) [26]. Unlike other nutritional components, ash content remained stable throughout the sprouting process.

In the case of the amino acid profile, it varies among legumes, typically exhibiting low sulfur amino acid levels (methionine and cysteine). Chickpeas, for example, contain primary amino acids such as arginine, leucine, and lysine (0.72 ± 0.02 to 2.58 ± 0.01 g/100 g flour), with cysteine and methionine present in lower amounts (0.13 ± 0.01 to 0.26 ± 0.01 g/100 g flour) [34]. Other chickpea species present high amounts of glutamic acid (1.67–4.53 g/100 g of flour), arginine (0.48–2.60 g/100 g of flour), and phenylalanine (0.42–1.45 g/100 g of flour) [35].

Compared to I. paterno flour, mung bean flour exhibits fluctuating amino acid profiles over five days of sprouting, with individual amino acid levels rising and falling within 24 h intervals. Trends in total essential amino acids (TEAA), total conditionally essential amino acids (TCEA), and total non-essential amino acids (TNEA) were consistent with the present study, with TEAA increasing from 30.13 to 34.91 g/100 g protein, TCEA from 17.10 to 18.02 g/100 g protein, and TNEA from 27.29 to 30.02 g/100 g protein after 120 h [36].

Furthermore, the presence of phenylalanine, valine, and leucine in I. paterno seeds is particularly beneficial, as these amino acids are essential for cell division, carbon distribution, and nitrogen metabolism during sprouting. Additionally, glutamic acid and arginine play a crucial role in radicle elongation and cell division [30]. Additionally, high glutamic acid levels have been linked to anti-diabetic effects, while amino acids such as lysine, alanine, leucine, arginine, isoleucine, and phenylalanine enhance insulin secretion [37]. The % EAA/TAA values obtained in this study align with those reported in chickpea varieties (30 ± 1 and 34 ± 1%) [34].

Protein digestibility (PD) improvements following gastric digestion may be attributed to pH reduction, which enhances protein solubility, and the enzymatic action of pepsin, which facilitates peptide bond hydrolysis [24]. Furthermore, increased free amino acids during sprouting have been documented in soybean protein isolates, where glycine, L-alanine, L-histidine, L-lysine, and L-serine levels rose due to peptide transporter (PTR)-mediated oligopeptide hydrolysis [37]. Additionally, exogenous compounds such as γ-aminobutyric acid (GABA) and vigabatrin can induce hydrogen peroxide (H₂O₂) accumulation, altering nitrogen and carbon metabolism and affecting free amino acid concentrations [38].

In addition, legumes naturally contain trypsin inhibitors that impair protein digestion. Although resistant to heat treatments, studies have shown that sprouting significantly reduces or eliminates these inhibitors [28]. For example, sprouted soybeans exhibited decreased trypsin inhibitor levels, leading to improved protein digestibility due to the formation of pre-hydrolyzed proteins [39]. This reduction enhances the nutritional value of legume-based foods and may alleviate digestive issues associated with raw or undercooked legumes.

5. Conclusions

This study provides a pioneering investigation into the effects of sprouting on the macronutrient composition of Inga paterno seeds, demonstrating that germination is an effective method for enhancing the nutritional quality of its flour. Sprouting significantly increased lipid and carbohydrate content, while protein levels decreased slightly. However, despite this reduction, in vitro protein digestibility improved by over 100%, and essential amino acids such as lysine and tryptophan increased substantially. These findings suggest that germinated I. paterno seeds could serve as a valuable nutrient source and a promising ingredient for food formulations. Furthermore, the germination process is easily scalable for production.

Future research should explore the specific mechanisms driving these compositional changes, the potential health benefits of germinated I. paterno seeds, and the production of antioxidant compounds. Additionally, further studies should investigate the physicochemical, structural, and functional modifications of protein fractions to optimize their application in food formulations.