Whey Protein Fermentation with Aspergillus niger: Source of Antioxidant Peptides

Abstract

Aspergillus niger is a filamentous fungus that through its proteolytic activity, as a result of its proteases, hydrolyzes whey proteins into smaller peptides. These peptides are characterized by antioxidant properties due to the presence of specific amino acids, such as histidine, tyrosine, tryptophan, cysteine, and methionine, which have been shown to have antioxidant effects. Considering the above, peptide extracts derived from the fermentation of a lactic serum substrate with Aspergillus niger were obtained, which were partially purified by precipitation with ZnSO4/acetone; subsequently, the antioxidant capacity was evaluated by spectrophotometric techniques as 2,2-azinobis-3ethyl benzothiazole-6-sulfonic acid (ABTS▪+), diphenylpicrylhydrazyl (DPPH▪), in 96-well microplates, these analyses showed that these extracts have an antioxidant activity higher than 50%; likewise, the amount of thiol groups (-SH) was determined to be higher than 29 nmol/μL and the superoxide dismutase activity (SOD) with values above 0.010 SOD units/mL. For this reason, it is proposed that they can be studied in the future as substances within a food supplementation or in the therapeutic field.

1. Introduction

Peptides are low molecular weight protein fragments, consisting of ∼2 to 20 amino acid residues [1, 2, 3], some of which exhibit physiological effects, beneficial to humans, which is why they are called bioactive peptides [4]. These are obtained mainly by hydrolysis of precursor proteins during fermentation processes with exogenous enzymes [5] from plant, bacterial or fungal sources [6], or through their expression and secretion during metabolic processes [7].

Fungal sources have aroused special interest because they exhibit a number of exogenous enzymes with applicability at pharmacological and industrial level [8]; among the most widely used fungal species is the genus Aspergillus, whose species Aspergillus oryzae [9], Aspergillus flavipes and Aspergillus niger [10], when used as an enzymatic source gave way to obtain peptides with antihypertensive [11], antioxidant [12], antidiabetic [13], antimicrobial [9], and antioxidant [14] bioactivities, among others.

Likewise, among the sources of precursor proteins used as substrates by fungal enzymatic sources are various kinds of milk and their derivatives [15], gelatin [11], grains such as lentils [13] and soy [16], and even eggs [17]. It should be noted that of these protein sources, milk is the most consumed worldwide [18] and its proteins have different biological and nutritional properties [19], and make it a source of bioactive peptides, which are released from precursor proteins, such as α-lactalbumin (α-LA), β-lactoglobulin (β-LG), caseins (CN), immunoglobulins (Ig), lactoferrin (LF), peptide-protein fractions, phosphoglycoproteins and minor serum proteins (transferrin and serum albumin) [20], during processes already mentioned such as fermentation, chemical hydrolysis or enzymatic hydrolysis [21].

Products derived from fermentation processes have been relevant in people’s diets because their nutritional properties are enhanced, thanks to the fact that microorganisms synthesize vitamins, minerals, and bioactive peptides [22, 23], among others, which are beneficial to human health. This is why the evaluation of fermentation processes such as milk fermentation with Aspergillus niger arouses interest.

On the other hand, worldwide interest has increased in topics related to conditions caused by oxidative stress since this is related to the development and onset of various human diseases [24, 25], including atherosclerosis [26], Alzheimer’s disease [27] and cancer [28], among others.

To analyze the previous problem, a solution was found by evaluating the antioxidant activity through the DPPH^▪, ABTS^▪+ methodologies, evaluation of thiol groups, and evaluation of superoxide dismutase activity; all these analyses were performed by spectrophotometric techniques in 96-well plates, in the presence of peptide extracts obtained during the fermentation of lactic serum with Aspergillus niger.

2. Methodology

2.1 Obtaining the inoculum

The inoculum of A. niger CMPUJH002, provided by the collection of microorganisms of the Universidad Pontifica Javeriana, was obtained by spiking on potato dextrose agar (PDA) at 37°C for 7 days. After its use, it was preserved by the method of spore suspension in glycerol of the laboratory of the biotechnology research group — natural products of the Universidad Tecnologica de Pereira (GB-PN, UTP).

2.2 Lactic fermentation

To obtain the peptide extracts, the methodology proposed by Channe & Shewale [29] was used, where lactic serum (enriched and prepared medium) was prepared and sterilized (Table 1), as a substrate for A. niger, in a 1 L Erlenmeyer, covered with vinipelt plastic. Fermentation was carried out over a period of 8 days, with 400 mL of substrate and 1.6 cm² of inoculum of A. niger grown on PDA agar after 7 days of growth at 37°C. The fermentation process was carried out with the following conditions pH 4.5, temperature 30°C, and an agitation of 71.76 ± 1.25 rpm, leaving a headspace of 60%. This assay was assembled in quadruplicate, coding each extract with an F indicating fermentation and a letter D followed by a number indicating the day it was collected.

Component	Concentration
Meat peptone	20 g/L
Starch	20 g/L
Glucose	5 g/L
Powdered milk	10 g/L
CaCl2.2H2O	1 g/L
MgSO4.7H2O	0.3 g/L
FeSO4.7H2O	5 mg/L
MnSO4.1H2O	1.56 mg/L
ZnSO4.7H2O	5.34 mg/L
COCl2.6H2O	2 mg/L

Table 1.

Composition of lactic whey fermentation medium enriched with Aspergillus niger taken from Channe & Shewale [29].

2.3 Partial purification of peptide extracts

The extracts collected daily were added 10% trichloroacetic acid [30], in equal proportion with respect to the sample (1, 1), in order to precipitate the large proteins and eliminate contaminants, then the precipitation of low molecular weight proteins was carried out by adding 50 mM ZnSO4/Acetone 80% to the sample in a ratio of 0.25:0.25:0.5 with respect to the sample, then it was placed in a refrigerator at 2°C for 24 hours for subsequent centrifugation at 5000 rpm at 3°C [31].

2.4 Antioxidant activity against DPPH^▪ radicals

A solution of DPPH^▪ 20 mg/L in methanol was prepared, and 100 μL of this solution was added to each of the wells containing 25 μL of the extract to be evaluated; it was left to react for 30 minutes in darkness and after this time the absorbance was read at 517 nm [25] using the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer. This analysis was carried out in quadruplicate.

2.5 ABTS^▪+ antioxidant activity

A solution of ABTS 3.5 mM and potassium persulfate 1.25 mM dissolved in H2O was prepared (this mixture was made at least 12 hours before its use), after this time the absorbance of the ABTS▪ + solution was adjusted to 0.7+/−0.02 units at 732 nm with ethanol; 194 μL of this solution was transferred to the well containing 6 μL of the extract to be evaluated, and it was left to react for 30 minutes in darkness to read its absorbance at 732 nm, in the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer. This analysis was carried out in quadruplicate [32].

From the absorbances obtained, the percentage of antioxidant activity is determined with the following equation described by Perez and coworkers [25, 32].

$$\%\text{Antioxidant Activity}=\left[\frac{A_{\mathit{\text{control}}\left(-\right)}-\left(A_{\mathit{\text{extract}}}-A_{\mathit{\text{white extract}}}\right)}{A_{\mathit{\text{control}}}}\right]x100$$E1

Where,

Aextract: Absorbance of the extracts.

Ablanco extracto: Absorbance of the blank of the extracts.

Acontrol (−): Absorbance of the negative control.

2.6 Content analysis of thiol groups (-SH)

For the quantification of thiols, the reaction of the samples with Ellmann’s reagent (DNTB (5,5’-Dithiobis(2-nitrobenzoic acid)) was carried out, taking 95 μL of sample with 30 μL of Na2HPO4 buffer, 0.5 M pH 7.0, with 125 μL DNTB at 10 nM incubating for 15 minutes and reading their absorbances at 412 nm, on Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer equipment; the calibration curve was prepared at different concentrations using glutathione enzyme, Na2HPO4 buffer and sulfoanilic acid 20%(m/v), 125 μL of each standard was taken and analyzing as samples [33].

2.7 Quantification of the enzyme superoxide dismutase (SOD)

The method used for the determination of superoxide dismutase was based on the protocol carried out by Betancur and Mosquera [33], where a hydroxylamine calibration curve is constructed that interacts with a xanthine/xanthine oxidase system as a source of generation of a superoxide anion flux that oxidizes hydroxylamine to nitrite for a subsequent measurement of nitrite concentration by UV/visible spectrometry.

To perform the analysis, sample preparation was done with the addition of 150 μL KH2PO4 buffer, pH 7.8, 40 μL of deionized water, 15 μL of xanthine, 15 μL hydroxylamine hydrochloride 1 mM, and 75 μL xanthine oxidase (0.2 mg protein/mL); the standards for the curve were prepared with phosphate buffer, water, xanthine, xanthine oxidase, hydroxylamine chloride, and the enzyme (SOD), and the system was left to react for 20 minutes in the dark, Then the reaction was stopped in an ice bath to add 100 μL of 19 mM sulfanilic acid and 7 mM α-naphthylamine, after which 100 μL of sample or standard was added, incubated for 20 minutes and its absorbance was measured at 529 nm in the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer.

2.8 Protein profile of peptide extracts by denaturing electrophoresis

Electrophoresis was carried out according to Bio-Rad guidelines under denaturing conditions. Separation gels were 15% SDS-polyacrylamide (30% bis-acrylamide, 1.5 M Tris-HCl pH 8.8, 10% ammonium persulfate, 0.04% tetramethylethylenediamine (TEMED), 10% SDS, and distilled water to final volume) and 5% SDS-polyacrylamide stacking gels (30% bis-acrylamide, 1 M Tris-HCl pH 6.8, 10% ammonium persulfate, 0.1% TEMED, 10% sodium dodecyl sulfate (SDS), and distilled water. The solutions were cast in mini protean (Bio-Rad) with the addition of ammonium persulfate and TEMED to polymerize the gels (1 mm thick).

The gels were loaded with 5% concentration samples, which were diluted in 1:1 reducing buffer (Tris HCl (63 mM), glycerol (10%), SDS (2%), bromophenol blue (0.0025%) pH 6.8), heated at 85°C for 2 to 5 minutes; the electrophoresis was performed at room temperature (24 and 27°C) using a constant current of 35 mA at 120 V for approximately 5 h. Once the run was completed, the gels were washed two times with deionized water and stained with a 0.1% Coomassie brilliant blue R-250 solution with 40% methanol and 10% acetic acid for 12 h with gentle agitation and then destained 2 times for 20 minutes with a 25% ethanol and 8% acetic acid solution [34].

2.9 Statistical analysis

For the statistical treatment of the data obtained for antioxidant and antibacterial activity, a one-way ANOVA analysis by replicates with a confidence level of 95% was carried out. All statistical analysis was carried out using GraphPad Prism 8.4.3 software.

3. Results and discussion

3.1 Antioxidant evaluation by means of the DPPH-radical

The DPPH- decolorization methodology carried out allowed establishing that the extracts evaluated have an antioxidant capacity that inhibits the DPPH radical by more than 50%; however, these values are lower than the percentage of antioxidant capacity of the hydroquinone positive control (Figure 1). Likewise, these results are lower, compared to other similar works, where the antioxidant activity on DPPH- presented by peptides derived from proteases of Aspergillus oryzae and Aspergillus flavipes species in milk, exceeded ∼90% [9], through the same method, since the method of obtaining the peptides was carried out from the already purified fungal proteases, which leads to their obtaining in a targeted manner, without competition from other enzymes and without the generation of secondary reactions due to the use of the complete metabolism of the microorganism [35].

Figure 1.

One-way ANOVA of the determination of antioxidant activity (%AA) by DPPH^▪ of fermentations (F) 1, 2, 3, and 4 with their respective days (D) 1, 2, 3, 4, 5, 6, and 7. A) Fermentation 1, b) fermentation 2, c) fermentation 3, and d) fermentation 4. Equal symbol (a; b; c) indicates that there are no significant differences, and equal sign indicates that there are significant differences (P < 0.05).

The results are attributed to the activity coming from the phenolic compounds, which are mainly responsible for the antioxidant activity, being one of the reasons why it is not a recommended technique for samples of biological origin [36], which is why the evaluation of ABTS− + cation radical decolorization was carried out as a complementary technique to contrast the results.

3.2 ABTS^▪+ antioxidant evaluation

In this method, the evaluation of antioxidant activity exceeds 90% in almost all cases and can be considered a good antioxidant because the activity significantly exceeds the positive control with equal concentration (1000 ppm) (Figure 2).

Figure 2.

One-way ANOVA ABTS^▪+ of fermentations (F) 1, 2, 3, and 4 with their respective days (D) 1, 2, 3, 4, 5, 6, and 7. A) Fermentation 1, b) fermentation 2, c) fermentation 3, and d) fermentation 4. Equal symbol (a; b; c) indicates no difference means, and equal sign indicates significant differences (p < 0.05).

Although oxidative stress is generated when the oxide-reduction homeostasis state of the cell becomes unbalanced because the antioxidant and prooxidant counterparts are altered. Aerobic organisms activate their defense mechanisms such as the secretion and action of glutathione, which is a tripeptide, composed of glutamate, cysteine, and glycine, that is used at the biological level for the regulation of this oxidation, which is why it is considered the universal antioxidant, in fact, it has been attributed that the proper functioning of most other antioxidants is due to the presence of glutathione [37].

Therefore, in addition to the biological activities proposed, the determination of thiols (-SH) in the different peptide extracts was also carried out through the construction of a calibration curve as shown in Figure 3.

Figure 3.

Calibration curve for thiol (-SH) quantification.

This analysis provided the plot eq. Y = 0.03769X + 0.02248, with an r² of 0.9927 and a p < 0.05, to determine the concentration of glutathione conferring antioxidant activity to the peptides present in each sample, as synthesized in Table 2.

Concentration (nmol/μL)
Día	F1	F2	F3	F4
0	39.877 ± 0.109	38.763 ± 0.297	38/763 ± 0.074	38.505 ± 0.126
1	30.983 ± 0.040	38.0571 ± 0.082	-a	41.432 ± 0.068
2	- a	-a	30.382 ± 0.051	33.705 ± 0.135
3	32.749 ± 0.053	30.382 ± 0.022	31.294 ± 0.091	30.375 ± 0.096
4	33.576 ± 0.020	31.294 ± 0.055	30.880 ± 0.063	32.475 ± 0.152
5	35.919 ± 0.133	30.880 ± 0.0178	31.033 ± 0.044	32.695 ± 0.098
6	31.034 ± 0.059	31.033 ± 0.047	29.549 ± 0.049	32.056 ± 0.0027
7	30.870 ± 0.054	29.549 ± 0.056	38.763 ± 0.126	32.308 ± 0.077

Table 2.

SH concentration (mmol/μL) of the different peptide extracts.

- a Missing data are the result of sample loss in storage or low extraction yield.

To complement the results obtained on the antioxidant potential of peptide extracts, the evaluation of superoxide dismutases (SOD), whose main function is the defense of aerobic organisms such as yeasts and filamentous fungi, such as Aspergillus oryzae, was carried out, where through transcriptomic analysis the expression of enzymes, such as catalase, glutathione peroxidase, and superoxide dismutase, has been demonstrated to regulate the concentration of oxidizing agents, such as oxygen free radicals, especially superoxide anion radicals [9]. The results obtained from the radical uptake assessment using the superoxide dismutase model are given in Figure 4.

Figure 4.

Calibration curve for SOD determination, −log (SOD units) vs. absorbance.

It has been identified that the antioxidant capacity of milk and its derivatives are mainly due to sulfur-rich amino acids such as tyrosine and cysteine, vitamins A and E, carotenoids, and enzyme systems such as the enzyme superoxide dismutase (SOD), which are useful so that superoxide radicals (O2--), hydroxyl radicals, and peroxide radicals can be inhibited [9].

This analysis provided the equation of the graph Y = 0.3146X - 0.2602 with an r² equal to 0.9933 and a p < 0.05; to determine the concentration of SOD units present in each sample, as synthesized in Table 3, which has the corresponding calculations of the transformation from -log (SOD units) to SOD units.

Concentration (unidades SOD/mL)
Day	F1	F2	F3	F4
0	0.020 ± 0.010	0.030 ± 0.01	0.032 ± 0.005	0.024 ± 0.005
1	0.020 ± 0.020	0.023 ± 0.004	-a	0.035 ± 0.013
2	-a	-a	0.022 ± 0.016	0.022 ± 0.002
3	0.021 ± 0.003	0.021 ± 0.004	0.027 ± 0.004	0.028 ± 0.017
4	0.017 ± 0.007	0.010 ± 0.005	0.010 ± 0.039	0.027 ± 0.011
5	0.019 ± 0.006	0.010 ± 0.004	0.010 ± 0.016	0.032 ± 0.017
6	0.026 ± 0.008	0.010 ± 0.011	0.013 ± 0.02	0.038 ± 0.003
7	0.078 ± 0.004	0.042 ± 0.003	0.011 ± 0.005	0.027 ± 0.014

Table 3.

Concentrations in SOD units of the different peptide extracts.

-a Missing data are the result of sample loss in storage or low extraction yield.

This analysis revealed the presence of peptides with SOD-type antioxidant character; although a difference in this concentration is evident in the different fermentations, the reason for these differences cannot be discerned without other analyses, which is why the analysis support is required as a characterization of the peptide extracts.

However, taking into account reports by other authors on the antioxidant activity of raw bovine milk with respect to SOD concentration of 0.92 to 3 units/mL and low concentrations of -SH [38, 39], a decrease is evidenced by the elimination of proteins of higher molecular weight removed during partial purification, that is, concentrated proteins of low molecular weight are good antioxidant agents.

3.3 Analysis of the peptide profile of the extracts by SDS-page electrophoresis

Figure 5 shows the peptide profile exhibited by the peptides obtained, where several fragments of molecular weight above 10 KDa are evident, which led to think that there are still precursor proteins within the extracts; these proteins have already been reported in other studies [40] with their molecular weights as expressed in Table 4; therefore, the purification process was not effective in eliminating these high molecular weight proteins.

Figure 5.

Electrophoresis, Tris-glycine-SDS for polyacrylamide gel electrophoresis.

Protein	Molecular weight (KDA)	Reported activities	References
Lactoferrin	±80–76	Antiviral, antibacterianas, antivirales, antifúngicas, antiinflamatorias, antioxidantes e inmunomoduladoras	[41, 42, 43, 44]
BSA	±66	Antioxidante, antitumoral e inhibidora de la enzima convertidora de angiotensina (ECA)	[45]
Caseins*	±19-30	Antioxidante y antimicrobiana	[46]
β- Lactoglubulin	±18	Antioxidante	[47]
α- Lactoalbumina	±14	Antimicrobiana	[48]

Table 4.

Molecular weights in KDa of some important proteins in bovine milk.

^*

The hydrolyzates of these proteins exhibit the following activity.

Likewise, although the technique used does not allow the characterization of peptides of molecular weights lower than 10 KDa, it was the only one that allowed the visualization of bands of protein origin because techniques, such as SDS-triscin [7], did not allow the visualization of bands, as well as silver staining, the result of the electrophoresis can be seen in Figure 5; although theoretically it is known that the protein concentration in a sample should be greater than 0.5 mg/mL [34], the samples analyzed have a variable composition and of different concentrations.

4. Conclusions

Antioxidant bioactive peptides have become a very valuable tool in both the food and pharmacological industries due to their ability to act as antioxidants, and thus combat oxidative stress in the human body. These peptides can be obtained from proteins by fermentation processes with microorganisms and enzymes.

In this case, Aspergillus niger was used as an enzymatic source to obtain antioxidant peptides from lactic serum. The results obtained indicate that the extracts obtained from this fermentation process have a promising antioxidant activity, which makes them a very interesting nutraceutical substrate.

Importantly, the antioxidant activity of these extracts can be attributed to the presence of thiol groups and positive SOD activity. The ability to catalyze the dismutation of superoxide anion into hydrogen peroxide and molecular oxygen is a very important characteristic of these extracts as it confers them a greater capacity to combat oxidative stress in the human body.

Therefore, the results obtained indicate that the use of Aspergillus niger as an enzymatic source for obtaining antioxidant peptides from lactic serum is a very valuable tool in the food and pharmacological industry. These extracts have promising antioxidant activity and are a very interesting source of nutrients to combat oxidative stress in the human body.

Acknowledgments

To the Vice-Rectory of Research of the Universidad Tecnológica de Pereira, for financing project E9-20-3 and to the Universidad de Manizales, where the characterization of the extracts by electrophoresis was carried out.

Conflict of interest

The authors declare no conflict of interest.