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Keywords:

  • Biological activities;
  • functional properties;
  • hard-to-cook;
  • hydrolysates

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard-to-cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I-converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase-Flavourzyme (AF) and pepsin–pancreatin (Pep-Pan) at 90 min ACE inhibition expressed as IC50 values were 4.5 and 6.5 mg protein per mL with AF and Pep-Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm mg−1 sample with AF and 6.4 mm mg−1 sample with Pep-Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Enzymatic hydrolysis of proteins is a promising option for modifying the biological and functional properties of food proteins. Production of enzymatic protein hydrolysates has been developed considerably in recent years. Protein hydrolysates can be classified according to their degree of hydrolysis. Those with a low degree of hydrolysis (<10%) frequently have better functional properties than the original source proteins and are widely used as food ingredients (Vioque et al., 2000). Extensive protein hydrolysates, that is, those with a high degree of hydrolysis (>10%), are used as protein supplements or as ingredients in special medical diets (Frokjear, 1994). Recent research has focused on the properties of food protein-derived peptides, their biological activities and potential health benefits. Peptides extracted from partial enzymatic hydrolysates of food proteins can provide specific health benefits such as antihypertensive and antioxidant effects (Je et al., 2004). Inadequate postharvest handling and storage under high temperature (>25 °C) and relative humidity (>65%) conditions produce the hard-to-cook (HTC) defect in beans. This defect is the result of physical and chemical changes at the intercellular level during storage. When cooked, HTC beans absorb sufficient water but fail to soften, requiring longer cooking time and consequently greater energy used in preparation. These in turn compromise nutritional quality and reduce marketability (Nyakuni et al., 2008). A large portion of the beans consumed in Mexico exhibit some degree of hardening because the available infrastructure is insufficient for the storage of the 6.5 million tons produced annually (SAGARPA, 2000). The present study objective was to evaluate the angiotensin I-converting enzyme inhibitory and antioxidant capacities and some functional properties of protein hydrolysates from HTC Phaseolus vulgaris.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Seeds and chemicals

Common bean (Phaseolus vulgaris L.) samples were obtained from the 2011 harvest in Yucatán, México. The variety used was black Jamapa one of the most consumed in the south-east of México. Beans were cleaned to remove extraneous matter and then packaged in gunny bags. The beans were stored under ambient conditions (23–27 °C and 63–74% RH) for 6 months. All chemicals were reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Cooking time and degree of hardness

These parameters were measured following official Mexican methods (NMX-FF-038-SCFI-2002,2002). For cooking time, a 300 g of beans was previously soaked in water for 12 h and boiled. Cooking time was based on the time after which thirty seeds were soft when pressed between the thumb and index finger. Texture was measured by placing thirty previously cooked beans in an Instron model 4411 universal machine (Instron, Norwood, MA, USA) at a compression speed of 10 mm min−1, using an 8.0-mm-diameter probe and a 5-kg-load cell.

Protein concentrate

Seeds were processed in a disc mill (model 4-E Quaker; Mill Straub Co., Philadelphia, PA, USA) to produce flour. The flour was then sifted through 4.76 and 2.38 mm screens and the hulls removed with a fluidising air bed. The sifted flour was then milled in a Cyclotec mill (Tecator, Hoganas, Sweden) until passing through a 0.841-mm screen. The protein extraction was done using a wet fractionation method (Betancur-Ancona et al., 2004). Briefly, 1 kg batch of bean flour was suspended in distilled water at a 1:10 (w/v) ratio, pH was adjusted at eleven and the suspension left to soak under constant agitation for 1 h. The suspension was then passed through a 0.177-mm screen to separate the fibre from the protein- and starch-containing liquid portion. The suspension left to sediment for 30 min to discard the starch. The pH of the protein solution was adjusted to 4.3 and then was centrifuged, the liquid discarded and the precipitate freeze-dried at −47 °C and 13 × 10−3 bar (FreeZone 4.5; Labconco. Kansas City, MO, USA).

Enzymatic hydrolysis

Hydrolysis was done using Alcalase® (pH 8.0 and temperature 50 °C); Flavourzyme® (pH 7.0 and temperature 50 °C); pepsin from porcine gastric mucosa (pH 2.0 and temperature 37 °C); pancreatin from porcine pancreas (pH 7.5 and temperature 37 °C); and sequential Alcalase®-Flavourzyme® and pepsin–pancreatin systems. The Alcalase®, Flavourzyme® and sequential Alcalase®-Flavourzyme® enzymatic system were applied according to (Pedroche et al., 2002). Hydrolysis with pepsin, pancreatin and the sequential pepsin–pancreatin system was applied according to Megías et al. (2004). Reaction times were 15, 45, 90 and 120 min. The reaction was stopped by heating to 80 °C for 20 min, followed by centrifuging at 1317 g for 12 min (Mistral 3000i; MSE, Leicestershire, England) to remove the insoluble portion.

Degree of hydrolysis

Degree of hydrolysis (DH) was calculated by determining free amino groups with o-phthaldialdehyde (Nielsen et al., 2001): DH = h/htot * 100; where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolysed bonds. The htot factor is dependent on raw material amino acid composition. In this case for hard-to-cook protein isolated was htot = 7.66 mmol g−1 of protein.

Biological activities of protein hydrolysates

ACE inhibitory activity

Angiotensin I-converting enzyme inhibitory activity in the hydrolysates was analysed following Hayakari & Kondo (1978). This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5 mL incubation mixture containing 40 μmol potassium phosphate buffer (pH 8.3), 300 μmol sodium chloride, 40 μmol 3% HHL in potassium phosphate buffer (pH 8.3) and 100 mU mL−1 ACE. ACE inhibitory activity was quantified by a regression analysis of ACE inhibitory activity (%) versus peptide concentration and defined as an IC50 value, that is, the peptide concentration (mg protein per mL) required to produce 50% ACE inhibition.

Radical scavenging activity

The radical scavenging activity (RSA) was determined according to the method described by Pukalskas et al. (2002), using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS). The ABTS●+ radical cation was produced by reacting with potassium persulfate according. The percentage decrease in absorbance at 734 nm was calculated and plotted as a function of the antioxidant concentration of Trolox for the standard reference data. To calculate the Trolox equivalent antioxidant capacity (TEAC), the slope of the absorbance inhibition percentage vs. antioxidant concentration plot was divided by the slope of the Trolox plot.

Functional properties of protein hydrolysates

Nitrogen solubility

Nitrogen solubility (NS) was determined following the procedure of Were et al. (1997). A total of 125 mg of each hydrolysate was dispersed in 25 mL distilled water. The pH was adjusted to 2, 4, 6, 8 and 10 with 0.1 m NaOH or 0.1 m HCl. The solutions were stirred for 30 min at room temperature and centrifuged at 4320 g for 30 min. The supernatant was analysed for nitrogen using AOAC method 954.01 (AOAC, 1997). NS was calculated as follows: NS (%) = (Supernatant nitrogen concentration/Sample nitrogen concentration) × 100.

Emulsifying capacity and emulsion stability

Emulsifying capacity was measured by an oil titration method (Chau et al., 1997). A total of 300 mg hydrolysate sample was dissolved in 300 mL distilled water with 3.0% NaCl. The pH was adjusted to 2, 4, 6, 8 and 10. The solutions were placed in a blender beaker. A burette containing 100% pure corn oil was placed above the beaker. A pair of electrodes connected to a multimeter was fixed to the beaker to measure the electrical resistance (ohms) of the emulsion. Emulsifying capacity (EC) was expressed in mL of oil/mg of protein in the dispersion and calculated as follows:% EC = (mL oil expended in test sample/mL dispersion used) × 100. Emulsion stability (ES) was determined according to Dagorn-Scaviner et al. (1986). Briefly, 10 mL 100% pure corn oil was added to 30 mL of protein (3 mg mL−1) with 3.0% NaCl. The pH was adjusted to 5.5 and the solution homogenised by blending for 30 s. Volume (mL) of the aqueous phase was determined at 30 s, 5, 30 and 120 min.

Foaming capacity and foam stability

Foaming capacity (FC) and foam stability (FS) were measured according to Chau et al. (1997). Initially, 100 mL of a suspension containing 1.5% hydrolysate was prepared, and pH adjusted to 2, 4, 6, 8 and 10. This suspension was stirred at low speed in a blender for 5 min, transferred to a 250-mL-graduated test tube and foam volume recorded at 30 s. FC was expressed as the percentage increase in foam volume after 30 s, and FS was expressed as the foam volume remaining after 5, 30 and 120 min.

Experimental design

All results were analysed using descriptive statistics with a central tendency and dispersion measures. One-way anovas were run to evaluate protein concentrate hydrolysis data, biological and functional properties. A LSD multiple range test was applied to determine differences between treatments according to (Montgomery, 2006) and processed with the Statgraphics Plus version 5.1 software (Statistical Graphics Corp., Madrid, Spain).

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Cooking time and degree of hardness

The beans required 35 min more cooking time (90 min) than freshly harvested beans (≤55 min). Preparation of legumes for eating involves hydration and cooking. The cooking process comprises at least two phases: an initial phase that follows first order kinetics characterised by the middle lamella breakdown and cell separation, and a second phase with starch gelatinisation inside the cells as the predominant feature (Yousif et al., 2007). The cooking quality of beans is related to postharvest handling and storage conditions. Beans stored at high temperatures hydrate unevenly during cooking, resulting in uneven bean softening and to the presence of hard-to-cook beans (HTC). HTC beans fail to soften enough to be eaten after cooking for a reasonable time. Bean texture (15 N) exceeded the acceptable value (≤5 N) (NMX-FF-038-SCFI-2002,2002). A number of factors are responsible for the HTC defect in beans, formation of insoluble pectinates due to phytic acid hydrolysis and release of divalent cations (Ca2+ and Mg2+), which associate with the pectin in the middle lamella, rendering it insoluble (Morales De León et al., 2007). Loss of solubility causes cotyledon cell clustering, reduced water penetration into cells, and reduced starch gelatinisation, producing the overall effect of hardness and graininess (Yousif et al., 2007). The presence of HTC during processing of P. vulgaris Jamapa black beans results in cooked beans of poor nutritional value and sensory (textural) quality.

Degree of hydrolysis (DH%)

Progress of the hydrolysis was monitored by measuring the extent of proteolytic degradation through the DH% according to the o-phthaldialdehyde reaction with free amino groups (Fig. 1). According to Sathivel et al. (2003), after an initial rapid phase of hydrolysis, the rate of hydrolysis tends to decrease, entering a stationary phase. In this study, there was an initial rapid rate of hydrolysis followed by a decreasing rate of hydrolysis. The yield of hydrolysate was not calculated. However, the data from Fig. 1 provide a preliminary estimate of the amount of protein that can be hydrolysed.

image

Figure 1. Degree of hydrolysis as a function of time for hard-to-cook Phaseolus vulgaris protein concentrates. (a) Hydrolyzed with Alcalase (A), Flavourzyme (F) and a sequential Alcalase–Flavourzyme system (AF). (b) Hydrolyzed with pepsin (Pep), pancreatin (Pan) and a sequential pepsin-pancreatin (Pep-Pan) system. Results are the means of three replicates.

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The lowest DH in the HTC bean hydrolysates was produced at 15 min with Flavourzyme (F15, 8.9%) and with pancreatin (Pan15, 5.0%). The highest DH values were produced with the sequential systems: 33.29% for Alcalase–Flavourzyme and 28.47% for pepsin–pancreatin. With both sequential systems, the amount of hydrolysates produced at 90 min and 120 was statistically equal, it was observed that prolonging the reaction time did not produce any significant improvement in the DH. Although hydrolysates obtained at 90 min require less time and energy to obtain a maximum proteolysis. According to Gómez-Ruiz et al. (2006), hydrolysates that showed high values of proteolysis also showed high ACE inhibitory activity. Therefore, the hydrolysates produced with the sequential systems at 90 min were used in the evaluation of biological properties. On the contrary, functional properties were evaluated using the hydrolysates obtained at 15 min with Flavourzyme and pancreatin that showed the lowest values of DH%. According to Vioque et al. (2000), limited protein hydrolysis may improve functional properties of the original material. But, above a certain DH%, these properties disappear as a consequence of the smaller peptide size. Thus, depending on the substrate and enzyme used, hydrolysates between 1% and 10% DH on average possess better functional properties than the original proteins.

Biological properties of protein hydrolysates

ACE inhibitory activity

The hydrolysate concentration required to produce 50% inhibition of ACE (IC50) was used as an activity indicator. This indicator is expressed as mg protein/mL, with smaller values indicating greater ACE inhibiting power. The nonhydrolysed HTC Phaseolus vulgaris protein concentrate showed no inhibitory activity on ACE. ACE inhibitory activity was generated from the HTC P. vulgaris protein after enzymatic hydrolysis. The IC50 value for AF at 90 min was 4.5 mg mL−1 and that for Pep-Pan was 6.5 mg mL−1. These are much higher IC50 values than that reported for enzymatic hydrolysates from different protein sources (IC50 = 0.2–2.47 mg mL−1) with antihypertensive activity in spontaneously hypertensive rats (Hong et al., 2005). The IC50 values for the AF and Pep-Pan are outside this range because they require large quantities to inhibit 50% of ACE activity. IC50 values also indicated that the ability of the hydrolysate to inhibit the ACE cannot be attributed entirely to the degree of hydrolysis but rather may be provided by the amino acid composition of peptides present in the hydrolysates, which in turn depends on the proteolytic activity of the enzymatic system employed (Tsai et al., 2008). Hydrolysis is necessary to release ACE peptides from an inactive form within the sequence of HTC P. vulgaris protein. On the other hand, both hydrolysates have good solubility in water allowing their incorporation into food matrices to generate physiologically functional foods for preventing hypertension as well as for therapeutic purposes.

ABTS●+ decolorisation assay

The antioxidant activity of both HTC P. vulgaris hydrolysates was evaluated by the ABTS●+ method and calculated as Trolox equivalent antioxidant capacity (TEAC) (mm per mg sample). Results showed that hydrolysate obtained at 90 min with Alcalase–Flavourzyme was the most active with a TEAC of 8.1 mm mg−1 sample. For the hydrolysate obtained at 90 min with Pepsin–Pancreatin the TEAC value was 6.4 mm mg−1 sample. These results indicated that enzymatic hydrolysis released antioxidant peptides that were inactive when still embedded in the intact protein and revealed an antioxidant capacity similar to that of Trolox, that is, a concentration of 8.1 mm mg−1 sample of HTC P. vulgaris hydrolysate obtained with Alcalase–Flavourzyme was able to scavenge the same amount of radicals as a solution with the same concentration of Trolox. Some amino acids were widely believed to be direct radical scavengers due to their special side chain groups, such as His (imidazole group), Trp (indolic group) and Tyr (phenolic group) (Chen et al., 1995). These groups may act as hydrogen donors. Additionally, Met is prone to oxidation of Met sulfoxide, and Cys donates hydrogen sulphide (Hernández-Ledesma et al., 2005). Aromatic amino acids (Tyr and Phe) are considered to be effective radical scavengers because they can easily donate protons to electron deficient radicals while simultaneously maintaining their stability via resonance structures (Rajapakse et al., 2005). A study on the antioxidant activity of different amino acids assessed by the ABTS●+ assay, reported that Cys was the most active amino acid followed by Trp, Tyr and His. The remaining amino acids analysed did not exhibit antioxidant activity by the ABTS●+ method (Aliaga & Lissi, 2000). It can be considered that the antioxidant activity depends on the primary structure of the peptides present in the hydrolysates; this is related to the enzymatic system used for the hydrolysis. Usually intestinal enzymes such as pepsin and pancreatin generate a large amount of proteins and oligopeptides. While enzymes of bacterial and fungal origin such as Alcalase and Flavourzyme generate a large amount of small peptides and free amino acids, which exhibit greater activity. According to Gómez-Ruiz et al. (2006), the accessibility to the oxidant–antioxidant test systems is greater for small peptides and amino acids than for large peptides and proteins.

Functional properties of protein hydrolysates

Nitrogen solubility

Solubility is one of the most important functional properties of protein and protein hydrolysates. Many of the other functional properties such as emulsification and foaming are affected by solubility. The solubility for hydrolysates F15 and Pan15 at pH ranging from 2.0 to 10.0 is shown in Fig. 2.

image

Figure 2. Nitrogen solubility in water at different pH values of hard-to-cook Phaseolus vulgaris protein hydrolysates produced with Flavourzyme (F15) and pancreatin (Pan15) at 15 min. Protein solubility is expressed as a percentage of soluble nitrogen at a given pH value. Results are the means of three replicates.

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The Pan15 hydrolysate was less soluble (52.9% at pH 2, 59.9% at pH 10) than the F15 hydrolysate (65.8% at pH 2, 70.23% at pH 10). The solubility of both hydrolysates was due to cleavage of proteins into smaller peptides that usually have increased solubility. These results were consistent with those reported by Kristinsson & Rasco (2000), which showed that the increase in hydrolysates solubility was due to the reduction in the molecular size and the hydrophobic character of proteins, exposing more polar groups to the aqueous environment. Solubility in the F15 and Pan15 hydrolysates suggests potential applications in formulated food systems such as improving appearance and creating a smooth mouth-feel.

Emulsifying capacity and emulsion stability

Interactions between proteins and lipids are common in many food systems, and thus, the ability of proteins to form stable emulsions is important. An increase in the number of peptide molecules and exposed hydrophobic amino acid residues due to hydrolysis of proteins would contribute to an improvement in the formation of emulsions. Both hydrolysates exhibited high emulsifying activity at pH 2–10, although Pan15 (5.0% DH) had an overall higher EC than F15 (8.9% DH). Apparently, EC decreased as DH increased (Fig. 3).

image

Figure 3. Emulsifying capacity at different pH values of hard-to-cook Phaseolus vulgaris protein hydrolysates produced with Flavourzyme (F15) and pancreatin (Pan15) at 15 min. Results are the means of three replicates. Different letters in the same row indicate statistical difference (P < 0.05).

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ES was higher in Pan15 than in F15 at different times (5–120 min) (Table 1). This higher ES in Pan15 is essentially due to increased hydrolysate hydrophobicity and consequently lower solubility (Fig. 3). There is a direct relationship between surface activity and peptide length. Smaller peptides often have reduced emulsifying properties; indeed, a minimum peptide length of about twenty residues is required to provide good emulsifying and interfacial properties (Souissi et al., 2007). Thus, considering these emulsifying properties, HTC P. vulgaris protein hydrolysates could be used as ingredients in emulsion-based food formulations such as salad dressing and mayonnaise.

Table 1. Emulsion and foam stability at different pH values (2–10) of hard-to-cook Phaseolus vulgaris protein hydrolysates produced with Flavourzyme (F15) and pancreatin (Pan15) at 15 min
HydrolysateF15Pan15
pH246810246810
  1. Data represent the mean of three replicates.

  2. Different superscript letters in the same line indicate statistical difference (P < 0.05).

Emulsion stability (%)
5 min66.5c50.5a62.0b62.5b51.0a87.0b60.0a90.0c87.0b86.5b
30 min48.5c41.0a50.5d47.5c43.0b57.0c49.0a63.5d53.5b56.5c
120 min45.5c36.0a46.0c43.5b35.5a45.5a44.5a47.5b47.5b50.0c
Foam stability (%)
5 min37.5d24.0c18.0b14.0a18.5b36.5b21.0a57.0c76.5d114.0e
30 min5.0a8.0b11.0c4.0a13.0d25.0b18.0a54.0d48.5c84.0e
120 min0.01.0a4.5b1.0a6.0c20.0b16.0a44.0d30.5c51.0e
Foaming capacity and foam stability

Some food proteins are capable of forming good foams, and their capacity to form and stabilise foams depends on the type of protein, degree of denaturation, pH, temperature and whipping methods. Foaming properties for F15 and Pan15 were measured based on their whippability at pH 2.0, 4.0, 6.0, 8.0 and 10.0. Limited proteolysis improves FC, probably explaining why Pan15 (5.0% DH) had higher FC than F15 at pH 6.0, 8.0 and 10.0 (Fig. 4).

image

Figure 4. Foaming capacity at different pH values of hard-to-cook Phaseolus vulgaris protein hydrolysates produced with Flavourzyme (F15) and pancreatin (Pan15) at 15 min. Results are the means of three replicates. Different letters in the same row indicate statistical difference (P < 0.05).

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Foam stability decreased in both Pan15 and F15 at the same pH as a function of time. At different times, FS in Pan15 was higher than F15 at pH 6.0, 8.0 and 10.0 (Table 1). The poor foaming properties of F15 are probably due to its small peptide size, which would hinder formation of a stable film around the gas bubbles, and to the appearance of hydrophilic peptides during more extensive hydrolysis (Table 1). Apparently, hydrolysates F15 and Pan15 with 8.9 and 5.0 DH% are capable of improving foaming as the result of the reduction in protein size. Although the peptides with relatively small molecular weight were probably capable of forming films with the air interface, it is possible that the films were not strong enough to maintain their integrity. Proteolytic enzyme modification is an effective way to improve functional properties. Treatment of HTC P. vulgaris flour with Flavourzyme, and pancreatin produces hydrolysates with 8.9% and 5% degree of hydrolysis and enhanced functional properties, while maintaining. Hydrolysis results in increases in solubility, emulsifying activity and foaming capacity. Thus, these hydrolysates have potential uses in the production of foods such as salad dressings, ice creams, mayonnaises where functional properties are especially important.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

This study demonstrates that hydrolysates derived from HTC P. vulgaris may potentially serve as a good source of desirable quality peptides. The HTC P. vulgaris have desirable ACE inhibitory and antioxidant properties, solubility, emulsifying and foaming stability. They can potentially compete with hydrolysates and protein powders currently available in the marketplace. It can be concluded from the results that Alcalase and Flavourzyme were the most effective proteases to improve biological and functional properties. This fact is relevant for the production of protein hydrolysates because the choice of used protease determines both the biological and functional properties, which is of great importance with respect to food application of protein hydrolysates. The low cost of HTC P vulgaris as a substrate represent the revalorisation of an agricultural product with reduced acceptability and marketability that may be transformed into a highly valuable additive such as hypotensive and antioxidant protein hydrolysates.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

We gratefully appreciate the financial support of Industria Mexicana de Coca Cola and CONACYT through the project ‘Aprovechamiento tecnológico del frijol (Phaseolus vulgaris l.) endurecido para la obtención de fracciones funcionales de proteína, almidón y fibra” and “the Red Temática: Bioactividad de péptidos e hidrolizados,” funded by Programa de Mejoramiento al Profesorado-PROMEP-SEP.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References
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