α-lactalbumin ( α-LA) might increase its antioxidant potential after hydrolysis. In particular, low molecular weight (LMW) peptides showed greater antioxidant capacity. Different hydrolysis conditions with Alcalase enzyme were optimized with a composite central design and surface methodology. Sample obtained after 0.1% (w/w enzyme:substrate), 60 min hydrolysis, ultrafiltrated with membranes of 3 kDa (named 4 LMW), showed the greatest antioxidant values: 1.574 ± 0.060 and 1.636 ± 0.076 μmolTE/mg of protein for ABTS and ORAC-FL, respectively. Sample 4 LMW produced mild ACE inhibition capacity, 22% related to Captopril. 4 LMW was submitted to in vitro gastrointestinal conditions using α-amylase, pepsin, pancreatin and bile-extract; its antioxidant capacity was enhanced by the shorter peptides released, confirmed by SE-HPLC. Antioxidant capacity of digested 4 LMW sample (D 4 LMW) was 1.743 ± 0.086 and 2.542 ± 0.245 μmolTE/mg of protein for ABTS and ORAC-FL, respectively, showing improvement on bioaccessibility. Intestinal cells viability was higher for D 4 LMW.
Antioxidants are substances present in foods that decrease the negative effects of reactive oxygen and nitrogen species which are produced under oxidative stress conditions [
Several studies have reported the antioxidant capacity of milk and its protein fractions (whey, caseins, lactoferrin, albumin) as well as peptides [
Hypertension is defined as a sustained elevated arterial pressure which is associated with an increased risk of developing heart disease [
As it has already been mentioned, enzymatic hydrolysis represents a wide mechanism used for improving food bioactive properties by obtaining low molecular weight peptides. Proteins are one of the most sensible bioactive molecules to gastrointestinal tract conditions [
The gastrointestinal tract is formed by mouth, stomach, small intestine and colon. The first region (the mouth) is where food interacts with saliva which is a complex aqueous fluid of neutral pH and polymers, salts, buffers and digestive enzymes such as amylase. Moreover, foods also interact with tongue, teeth, palate, cheeks and throat [
Hollebeeck et al. [
In a previous study [
Protein isolate of α-lactalbumin (Biopure-lactoalbuminTM) was provided by Davisco Food International Inc. (Le Sueur, MN, USA). Alcalase was provided by Novozymes Biopharma US Inc (AlcalaseÒ 2.4 L, Proteinase from Bacillus Licheniformis, Subtilisin A). Buffer salts Na2HPO4 (Mallinckrodt) and NaH2PO4 came from J. T. Baker. Folin reagent was purchased from Sigma Aldrich (St. Louis, MO). For antioxidant assays: 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-acid (Trolox), fluorescein (FL) disodium salt and 2,20-azobis(2-methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma Aldrich (St. Louis, MO), and potassium persulphate was from J. T. Baker. Histidil-hipuril-leucine (HHL) was purchased from Sigma Aldrich (St. Louis, MO) for performing angiotensin converting enzyme (ACE)-inhibition assay. To carry out digestion studies, α-amylase, pepsin and pancreatin came from Sigma Aldrich (St. Louis, MO).
To carry out cell studies, High-glucose Dulbecco’s modified Eagle medium (DMEM) with L-glutamine and pyruvate (Phenol red-DMEM), High-glucose Dulbecco’s modified Eagle medium without L-glutamine neither pyruvate (Phenol red-free DMEM), Dulbecco’s phosphate-buffered saline (DPBS) ± Ca2+ and Mg2+, Hank’s Balanced Salt Solution (HBSS), penicillin-streptomycin mixture, MEM non-essential amino acid and foetal bovine serum (FBS) were purchased from Life Technologies (Villebon-sur-Yvette, France). For cell metabolic activity determination (MTT assay), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma.
Optimization of hydrolysis process was carried out as in Fernández-Fernández et al. [
Yi = β0 + β1r + β2t + β1,2r × t + ε (1)
where β0 is the regression coefficient for the intercept point; β1 and β2 are the linear regression coefficients; β1,2 is the regression coefficient for the interaction between the independent variables (factors r and t); and ε is the variable error. Model parameters were calculated with Statgraphic Plus version 5.1 program by multiple linear regression (MLR).
Enzymatic hydrolysis reaction was performed using Alcalase enzyme (1.158 mg/mL, enzymatic activity ≥ 2.4 AU/g) with 8% (w/V) of protein isolate (α-LA) in phosphate buffer solution 100 mM pH 7 and was incubated in a water bath at 30˚C with agitation of 150 rpm. After incubation time (as in Section 2.2), reaction was stopped by heating at 100˚C for 10 minutes. Ultrafiltration was carried out using an Amicon membrane (cut-off of 3 kDa, Merk Millipore) to separate peptides of low molecular weight (LMW) from high molecular weight (HMW). For each hydrolysate, two separate fractions were obtained: LMW and HMW fractions. From each of the seven samples, two fractions were obtained resulting in 14 samples (7 LMW and 7 HMW). Samples were frozen, lyophilized and stored at −20˚C for subsequent analysis.
Protein content determination was performed by Lowry method [
SE-HPLC analysis was performed as described by Molina Ortiz [
Selected sample with greater antioxidant capacity, sample 4 LMW was submitted to in vitro digestion (D 4 LMW) by the gastrointestinal digestion simulation model described by Hollebeeck et al. [
Antioxidant capacity was determined by electron transfer (ET) and hydrogen atom transfer (HAT) methods ABTS and ORAC-FL, respectively. ABTS was performed based on the method described by Re et al. [
% Inhibition = A control − A antioxidant A control (2)
where Acontrol is the absorbance of 3 mL of ABTS in buffer with 30 μL of buffer and Aantioxidant is the absorbance of 3 mL of ABTS in the same phosphate buffer with 30 μL of Trolox or sample.
ORAC-FL was performed as described by Ou et al. [
AUC = 1 + ∑ i = 1 i = 104 f i / f 0 (3)
where f0 is the fluorescence at 10 minutes of incubation at 37˚C and fi is the fluorescence measured every minute, for 104 minutes. Curves of Fluorescence vs Time were normalized to the curve of the blank calculating the net AUC as the difference between AUCantioxidant (Trolox or sample) minus AUCblank. Trolox calibration curve (net AUCTrolox vsnTE (μmol TE, Trolox equivalents)) was constructed in order to calculate samples antioxidant capacity (μmol TE/mg of protein). Besides punctual measurements, IC50 values were obtained by constructing the curve nTE vs [Protein] (mg/mL) to obtain a logarithmic function from which it could be calculated the concentration of protein correspondent to 50% inhibition of peroxyl radicals.
Antihypertensive activity was determined as described by Cushman and Cheung [
% ACE Inhibition = 100 ∗ [ 1 − ( A S − A 0 S A max − A 0 max ) ] (4)
where As is the absorbance of sample with ACE, A0s is the absorbance of sample without ACE, Amax is the absorbance in the absence of sample and A0max is the absorbance without sample and ACE.
Cell metabolic activity (or cell viability; MTT assay) of TC7-cells was determined after 1 h incubation in the presence of HBSS (control), the hydrolysate or the in vitro digestion of the hydrolysate that presented greater antioxidant capacity, sample 4 LMW and its digestion (D 4 LMW), respectively.
TC7-cells (passage 42 - 47) were routinely grown according to Benzaria et al. [
Samples were prepared in HBSS at concentrations of 1, 5 and 10 mg/mL protein. For the hydrolysate (4 LMW) and its gastro-intestinal digestion (D 4 LMW), 26.35 and 61.26 mg of dry powder were weighted for 1 mL of HBSS, corresponding to 20 mg/mL of protein for both samples (75.9% and 32.65% of protein, respectively). A volume of 500 μL sample solutions were deposed on the cells and incubated for 1 h at 37˚C and 8% CO2, 100% RH. After incubation, the apical TC7-culture media in the cell-wells were taken out and MTT assay was performed by adding 500 µL MTT reagent (0.15 mg/mL in FBS-free phenol red-free DMEM) to TC7-cells [
Amounts of 100 µL lysate were then transferred into 96-well plates to measure FormazanÒ absorbance at 570 nm in a microplate reader, after half diluting with DMSO. Viability percentage was calculated by taking the absorbance value of the control (HBSS) as 100%.
All the measurements were determined at least in triplicate. Results were expressed as mean values ± standard deviation. One-way analysis of variance (ANOVA) and pos-hoc Tukey test was applied to determine significant differences between values (p < 0.05). Statistical analysis was done using Infostat v. 2015 and Statgraphic Plus v. 5.1 programs.
Conditions of hydrolysis and characteristics of hydrolysates are shown in
Protein content and hydrolysis percentage were determined in the low molecular weight (LMW) fractions of samples 1 to 7 with the exception of sample 3, where hydrolysis conditions led to gelation. Gelification probably took place be due to a combination of factors such as: lower enzyme to protein ratio; lower hydrolysis rate; time and temperature of hydrolysis. For this reasons results of sample 3 are not included in
Sample | Factors | % Protein | % Hydrolysis | |
---|---|---|---|---|
r | t | |||
1 LMW | 0.0050 | 0 | 56.0a | 0a |
2 LMW | 0.1000 | 0 | 59.0a | 3a |
3 LMW | 0.0050 | 60 | - | - |
4 LMW | 0.1000 | 60 | 75.9c | 56b |
5 LMW | 0.0525 | 30 | 70.2b,c | 61b |
6 LMW | 0.0525 | 30 | 63.4a,b | 65b |
7 LMW | 0.0525 | 30 | 85.7d | 56b |
Results are expressed as the means ± SD (n = 6). ANOVA analysis was made by column using Tukey test. Means in the same column with different letters state significant differences (p < 0.05).
5, 6 and 7 stating that short peptides resulting from hydrolysis were separated correctly by ultrafiltration.
Other authors [
ABTS and ORAC-FL assays were the selected methods for evaluating the relationship between the factors enzyme:substrate ratio and time of reaction with the antioxidant capacity. The coefficients obtained from multiple linear regression analyses are shown in
Terms | ABTS value (μmol Trolox/mg protein) | ORAC-FL value (μmol Trolox/mg protein) |
---|---|---|
Constant | 0.2196 | 0.6622 |
Enzyme:substrate ratio (w/w) (r) | - | 3.9581 |
Time (minutes) (t) | 0.0424 | 0.0104 |
R × t | −0.1985 | - |
R2 | 0.9339 | 0.8810 |
p | 0.0000 | 0.0000 |
r: enzyme:substrate ratio; t: time; R2: regression coefficient; p: p-value for the unfit of the model (coefficients were p < 0.01).
antioxidant capacity with time, presenting a maximum for the conditions of sample 4 LWM (0.1000 w/w and 60 minutes). Sample 4 LMW antioxidant value was 1.574 ± 0.060 and 1.636 ± 0.076 μmol TE/mg of protein, compared to α-LA 0.191 ± 0.007 and 0.159 ± 0.011 μmol TE/mg of protein, for ABTS and ORAC-FL antioxidant capacity, respectively. Sample 4 LMW had 8.2 and 10.2 times more antioxidant capacity for ABTS and ORAC-FL, respectively, than α-LA. This sample values of antioxidant capacity differed greatly from other samples, showing increased antioxidant power. In our previous work, sample 4 presented 1.015 ± 0.042 and 1.495 ± 0.114 μmol TE/mg of protein in which case sample 4 LMW shows 1.55 and 1.09 times more antioxidant capacity than sample 4 (ABTS and ORAC-FL values, respectively). These values were not surprisingly higher than those of sample 4 for ORAC-FL value but for ABTS value antioxidant capacity increased, meaning separation by ultrafiltration seems to favor the concentration of peptides with ET mechanism for the neutralization of radicals. In addition, IC50 values were obtained for sample 4 LMW (0.805 ± 0.035 and 0.065 ± 0.004 mg/mL of protein for ABTS and ORAC-FL, respectively) and α-LA (15.732 ± 0.256 and 0.223 ± 0.014 mg/mL of protein for ABTS and ORAC-FL, respectively). With these values 19.5 and 3.4 times more α-LA than sample 4 LWM to neutralize 50% of ABTS and peroxyl radicals, respectively. The latter confirms that sample 4 LWM had a higher antioxidant capacity which increases with the percentage of hydrolysis [
efficient than other enzymes, attaining powerful antioxidant hydrolysates in less time of reaction and strengthening antioxidant capacity by obtaining short peptides through ultrafiltration.
Regarding antihypertensive properties measured in vitro, sample 4 LMW presented 22% of ACE inhibition percentage with respect to Captopril. This percentage is similar to the obtained for sample 4 without ultrafiltration, as reported by Fernández-Fernández et al. [
As described in section2.5, in vitro digestion of α-LA (D α-LA, control), sample 4 (D 4) and 4 LMW (D 4 LMW) were performed in order to evaluate the effect of digestion conditions on antioxidant capacity. Characterization of each digestion was performed by determining protein content and percentage of hydrolysis by HPLC analysis (as described in Section 2.4), and antioxidant capacity determination by ABTS and ORAC-FL assays described in Section 2.6. Percentages of protein content were 50.98% ± 1.80%, 41.48% ± 0.52% and 32.65% ± 1.79%, for the digestion of α-LA, sample 4 and 4 LMW, respectively. Hydrolysis percentages of the digestion of α-LA, sample 4 and 4 LMW were 35.59% ± 0.22%, 41.72% ± 0.13% and 80.89% ± 0.44%, respectively (
According to the results obtained, it can be said that the increasing percentage of protein content of in vitro digestions resulted in diminished hydrolysis percentage. Hydrolysis percentage of the samples tends to increase with digestion related to samples without digestion (
As to antioxidant capacity, α-LA had an ABTS value of 0.191 ± 0.007 µmol TE/mg of protein and for its in vitro digestion 0.619 ± 0.023 µmol TE/mg of protein increasing 3.2 times fold the antioxidant capacity (
For ORAC-FL assay, α-LA presented values of 0.156 ± 0.011 µmol TE/mg of protein without in vitro digestion and 1.819 ± 0.141 µmol TE/mg of protein for
Sample | % Hydrolysis | |
---|---|---|
Non-digested | Digested | |
α-LA | 0 | 35.59 |
4 | 31.20 | 41.72 |
4 LMW | 56.30 | 80.89 |
the in vitro digestion, showing a huge improvement on antioxidant capacity due to in vitro digestion (11.6 times fold). For sample 4, antioxidant capacity increased 1.4 times fold from 1.495 ± 0.114 (non-digested) to 2.046 ± 0.113 µmol TE/mg of protein after in vitro digestion showing less improvement than for α-LA. Similar results were observed for sample 4 LMW which presented a value of 1.636 ± 0.076 and for its in vitro digestion 2.542 ± 0.245 µmol TE/mg of protein (1.5 times fold).
In both cases (ABTS and ORAC-FL), samples 4 and 4 LMW did not show great improvements on antioxidant capacity which could be explained by the fact that these had already have enzymatic hydrolysis, releasing short peptides with antioxidant properties. Thus, in vitro digestion surely enhanced hydrolysis of all samples, increases being more evident for α-LA. Overall, samples with in vitro digestion showed increased hydrolysis as well as for non-digested 4 and 4 LMW (p < 0.05) with the associated improvement of antioxidant capacity making encrypted peptides more bioaccessible by in vitro digestion. These results agree with those of Adjonu et al. [
MTT assay was performed on TC7-cells after 1 h incubation in the presence of sample 4 LMW and its in vitro digestion (D 4 LMW) counterpart standing for the hydrolysates and the in vitro digestion of the hydrolysates that presented the best antioxidant capacity, respectively. Results in
Concentrations higher than 5 mg/mL may overcharge cell metabolism and diminish cell metabolic activity (data not shown). Clearly, D 4 LMW had a positive effect on cell metabolism up to concentrations of 5 mg/mL. For non-digested 4 LMW, concentrations tested seemed not to beneficiate cell metabolism. This could be explained by the fact that sample 4 LMW contain longer peptides unable to enter the intestinal cells and increase cell metabolic activity (for 1 h time of exposure). Digestion appears to enhance bioaccessibility of peptides within 4 LMW structure with the consequent increase in the bioavailability. In other research, Xie et al. [
The effect of ultrafiltration of different enzyme:substrate ratio and time conditions on antioxidant capacity of α-LAhydrolysates using Alcalase was evaluated using response surface methodology. The highest antioxidant capacity was found in the ultrafiltrated hydrolysate with 0.1% w/w for enzyme:substrate ratio and 60 minutes time of reaction (sample 4 LMW). Using this methodology, it was demonstrated that reaction time had a positive influence on antioxidant capacity measured by ABTS and ORAC-FL methods. Moreover, enzyme:substrate ratio had a positive influence on antioxidant capacity measured by ORAC-FL. Enhanced antioxidant capacity was explained by more hydrolysis (release of shorter peptides), confirmed by SE-HPLC. A pool of short peptides in sample 4 LMW could be responsible for these high antioxidant capacities. In contrast, low ACE-inhibitory activity was found in sample 4 LMW likely because hydrolysis did not release the tripeptides responsible for these activities. In vitro simulation of gastrointestinal digestion of α-LA, sample 4 and 4 LMW was found to increase hydrolysis with the association of enhanced antioxidant capacity as well as intestinal cell metabolic activity. By simulated digestion, it was shown that digestion improves bioaccessibility with consequent improved cell metabolic activity of bioactive peptides revealing hydrolysate 4 LMW as a promising functional ingredient.
The research that gives rise to the results presented in this publication received funding from the National Agency for Research and Innovation under the code POS_NAC_ 2013_1_11655, from the Fondo Vaz Ferreira funded project I/FVF2017/189 (Desarrollo de micro y nanovehículos de péptidos bioactivos como ingredientes funcionales para una alimentación saludab) and from ECOS-Sud Committee funded project U08B01 (Procedimientos Innovadores y valorización de compuestos bioactivos destinados a la industria alimentaria, con particular atención en la industria láctea). The authors would like to acknowledge Françoise Lazennec of Université de Montpellier for her technical contribution in celular studies; Paula Aphalo and María Cristina Añón of the Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) (CCT La Plata, CONICET), Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 116 (1900), La Plata, Argentina for their technical assistance; and Laboratorio de Fisicoquímica Biológica (Instituto Química Biológica, Facultad de Ciencias, UdelaR) for the loan of equipment.
The authors declare no conflicts of interest regarding the publication of this paper.
Fernández-Fernández, A.M., Dumay, E., López-Pedemonte, T. and Medrano-Fernandez, A. (2018) Bioaccessibility and Cell Metabolic Activity Studies of Antioxidant Low Molecular Weight Peptides Obtained by Ultrafiltration of α-Lactalbumin Enzymatic Hydrolysates. Food and Nutrition Sciences, 9, 1047-1065. https://doi.org/10.4236/fns.2018.99077