Antioxidant potential and amino acid profile of ultrafiltration derived peptide fractions of spent hen meat protein hydrolysate

Abstract

Spent hen meat is considered as a category of waste generated by the poultry sector which can lead to serious environmental concerns if not disposed and utilized properly. In this work, spent hen meat was hydrolysed by 2% Flavourzyme (6.5 pH, 55 °C) followed by ultrafiltration to produce three peptide fractions with molecular weights > 10 kDa, 5–10 kDa and < 5 kDa. These fractions were evaluated for antioxidant potential, SDS PAGE and amino acid profile. The SDS PAGE profile demonstrated bands in the low molecular weight (< 10 kDa) region. Peptide fractions of < 5 kDa exhibited highest antioxidant activity and, essential as well as hydrophobic amino acid composition than whole hydrolysate and other peptide fractions. Incorporation of the identified hydrolysate fraction in food could improve its shelf stability while serving as a preventive component against human degenerative diseases.

Introduction

In today’s lifestyle, the production of reactive oxygen species (ROS) may cause damage to the membrane, cells and DNA thereby increasing the risk of chronic diseases like cardiovascular diseases, Alzheimer’s, Parkinson’s and cancer (Nwachukwu and Aluko 2019). These ROS are formed by oxidation of food compounds which can be scavenged by synthetic antioxidants like propyl gallate (PG) and tertiary butylhydroquinone (TBHQ). Natural antioxidants are preferred over synthetic antioxidants due the potential health risks of the latter (Shahidi and Zhong 2015). Bioactive peptides gained worldwide attention recently due to their ability to exhibit wide range of activities like antioxidant, antihypertensive, anticancer and immunomodulatory both in vitro and in vivo.

Spent hens are considered waste by the poultry industry since after losing their egg laying capacity they are not even preferred for their meat, due to increase in collagen cross-linkages with their age leading to hard and chewy texture. However, this meat is rich in protein and may be utilized after hydrolysis that can find applications in the food sector (Kumar et al. 2021c). The developed spent hen protein hydrolysate (SPH) can be subjected to ultrafiltration resulting in low molecular weight peptide fractions. There is dearth of literature on the production and quantitative analysis of bioactive peptides from spent hen protein hydrolysate. Hence to utilize the spent hen meat and to understand better its peptide fractions, amino acid profiling and antioxidant potential was investigated in this work.

Materials and methods

Sample preparation

Spent hen meat (deboned) was procured from a nearby layer farm in frozen form and stored at − 80 °C until use. For experimentation, 3–4 frozen blocks of meat weighing 45–50 g were kept overnight at 4 °C. Meat blocks were then minced using a bench top meat mincer (Sanco, Indonesia) and manually defatted with hexane (1:10; meat:hexane) thrice to remove all the fat. Defatted meat was mixed with distilled water (1:2 w/v) and homogenized using a tissue homogenizer (Polytron, Kinematica AG, Switzerland) at 7000×g for 10 min.

Preparation of SPH and ultrafiltered SPH fractions

Homogenized meat slurry was heated to 55 °C and its pH was adjusted to 6.5 using 0.1 N sodium hydroxide. Flavourzyme® 500 L (endopeptidase and exopeptidase from Aspergillus oryzae) was purchased from Sigma-Aldrich (India) and used at 2% concentration as previously optimized (Kumar et al. 2021a). After hydrolysis, the enzyme was inactivated by heating the mixture to 85 °C for 20 min. The hydrolysate slurry was cooled to room temperature and centrifuged at 6000×g for 10 min. Resulting supernatant was separated from the sediment and used for further processing. Whole hydrolysate was transferred to ultrafiltration tubes (Vivaspin turbo 4, Sartorius, India) with 10 kDa molecular weight (MW) cutoff membrane followed by centrifugation at 4000×g for 10 min. The retentate produced contained more than 10 kDa peptides and was labelled as F1. The permeate was then ultrafiltered through 5 kDa MW cutoff membrane. The resulting permeate (F3) had < 5 kDa peptides while the retentate (F2) had peptides ranging from 5 to 10 kDa (Fig. 1A). These fractions were then stored at 4 °C till further analysis.

Fig. 1.

 A Ultrafiltration of whole spent hen protein hydrolysate through 10 kDa and 5 kDa MW cutoff membranes for generation of peptide fractions F1 (> 10 kDa), F2 (5–10 kDa) and F3 (< 5 kDa); B Antioxidant profile of whole hydrolysate (WH) and different peptide fractions as analyzed by DPPH, FICA and FRAP. Bars with different small case letters (a, b, c) differ significantly at p ≤ 0.05; C SDS- PAGE profile of WH and peptide fractions against a broad range MW (2–250 kDa) marker (M)

Antioxidant activity

DPPH radical scavenging activity

The DPPH radical scavenging activity (DPPH RSA) was estimated according to the procedure of Tang et al. (2013), with slight modifications. Briefly, the hydrolysate fractions (500 µL, 5 mg/mL) and ethanolic DPPH (500 µL, 0.1 mM) were mixed and vortexed for 5 min. Samples were then incubated in dark for 30 min and the absorbance was measured at 517 nm and RSA was determined using Eq. (1)

$$\text{DPPH RSA}\left(\%\right)=\left(1,-,\frac{A_1-A_2}{A_0}\right)\times 100$$ 1

where $A_1$ = sample + ethanolic DPPH, $A_2$ = sample + ethanol, $A_0$ = ethanol + distilled water

Ferrous ion chelating activity

Ferrous ion chelating activity (FICA) was estimated according to the method of Tarafdar et al. (2021) with minor modifications. Ferrous sulfate (0.5 mL, 0.2 mM) was added to the hydrolysate fractions (0.25 mL) and the reaction was initiated by ferrozine (0.1 mM, 0.5 mL) addition followed by vortexing for 1 min. Sample mixture was incubated for 10 min at 30 °C and centrifuged at 8000×g for 5 min. Absorbance of the mixture was measured at 562 nm and chelating activity was calculated using Eq. (2).

$$\text{Chelating activity}\left(\%\right)=\frac{A_0-A_1}{A_0}\times 100$$ 2

where $A_0$ = absorbance of blank; $A_1$ = absorbance of sample

Ferric reducing antioxidant power

The ferric reducing-antioxidant power (FRAP) assay was conducted according to the method reported by Kumar et al. (2021b). Fresh FRAP reagent (300 mM acetate buffer [pH 3.6] + 20 mM ferric chloride + 10 mM TPTZ prepared in 40 mM HCl) was prepared in the ratio of 10:1:1 and incubated at 37 °C. Reagent volume of 900 µL was taken and mixed with 100 µL of hydrolysate and incubated again at 37 °C for 40 min. The absorbance was recorded at 593 nm. Trolox used for preparing the standard curve.

Amino acid profiling

Amino acid composition of spent hen meat, SPH and its fractions were estimated according to the method of Kurozawa et al. (2008) with minor modifications. Samples were digested for 20 h with 6 N HCl/0.1% phenol at 110 °C. After digestion, samples were derivatized by adding 20 µL of reagent mixture containing ethanol:water:triethylamine:phenyisothiocyanate (7:1:1:1, v/v) followed by vortexing and incubation at room temperature for 20 min. Amino acids were quantified using HPLC equipped with reverse phase column (Novapack C18, 4 μm, Waters, Milford, MA).

Electrophoretic profile by SDS PAGE

Molecular weight distribution of the SPH and its fractions was determined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) according to the method of Laemmli (1970). Samples were diluted by Tris–HCl buffer (0.125 mol/L Tris–HCl, 4% SDS, 20% v/v Glycerol, 0.2 mol/L DTT, 0.02% bromophenol blue, and pH 6.8) and boiled for 2 min to cleave all non-covalent bonds. Casting was done with 15% resolving gel and 5% stacking gel. Electrophoretic separation was done in SE300 miniVE integrated vertical protein electrophoresis and blotting unit assembly (Hoefer, Holliston, MA) using power supply of 120 V. A molecular weight marker containing protein bands in the range of 2–250 KDa (Precision Plus Protein Dual Xtra standard, Bio-Rad Laboratories Inc., Hercules, CA) was used.

Statistical analysis

All experiments were performed in triplicates except for amino acid profiling. Results of the analysis were expressed as mean ± standard deviation. Analysis of variance (ANOVA) was performed and the Duncan’s post hoc multiple range test was applied at p ≤ 0.05 in SPSS v.26 software (IBM, USA).

Result and discussion

Antioxidant potential of whole hydrolysate and its fractions

DPPH RSA

The DPPH assay is used widely to access the antioxidant capacity of compounds by analysing their ability of donating hydrogen. In this study, all fractions (F1, F2, F3) showed higher scavenging activity than the parent hydrolysate (WH) (Fig. 1B). The DPPH scavenging activity of F3 (49.96 ± 0.72%) was found to be the highest (p < 0.05) followed by F2 (42.28 ± 0.71%) and F1 (39.35 ± 0.98%). These results revealed that the hydrolysate fractions of lower molecular weight (MW) had higher capability to stabilize free radical as compared to the higher MW fractions. This means DPPH RSA of peptide fractions is MW dependent. These results are in agreement with the findings of Girgih et al. (2011) in hemp seed hydrolysate fractions and Hwang et al. (2010) in peanut protein hydrolysate fractions. Akinyede et al. (2021) also observed that low MW peptide fractions of nutmeg possessed higher DPPH activity than high MW fractions. The unfractioned hydrolysate (WH) exhibited significantly lower DPPH scavenging ability (37.21 ± 0.59%) as compared to peptide fractions. Interestingly, the amount of hydrophobic amino acids were higher in < 5 kDa followed by 5–10 kDa and > 10 kDa peptide fraction. The strong association of DPPH RSA with hydrophobicity was observed. Previous literatures also reported about this association (Zaky et al. 2020; Pownell at al., 2011). Scavenging ability of protein hydrolysates depend on the factors like specificity of enzymes used in hydrolysis, amino acid composition of hydrolysates, MW of peptides and DPPH assay conditions (Girgih et al. 2011).

Ferrous ion chelating activity

The ferrous ion chelating activity of bioactive peptides is characterized by its ability to reduce the absorbance of ferrozine-Fe²⁺ complex. Ferrous ions can catalyse the reaction to form hydroxyl ions from superoxide anions that can trigger tissue damage (Girgih et al. 2011). Ferrous ion chelating potential of peptide fractions was found to be significantly higher (p < 0.05) than the unfractioned hydrolysate. F3 (46.15 ± 2.05%) showed highest chelating activity followed by F2 (39.31 ± 1.56%) and F1 (34.01 ± 1.29%). Results of this study were similar to the findings of Phongthai et al. (2018), who stated that peptides ranging from 3 to 5 kDa showed highest MICA. The results of present study are in contrast to the findings of Girgih et al. (2011) as they showed that metal ion chelation increased with increase in MW of peptides. Current study supports evidence from the previous report (Akinyede et al. 2021) that unfractioned hydrolysate showed lower chelating activity as compared to the peptide fractions. It was also observed that high percentage of histidine could be responsible for higher FICA. Megias et al. (2008) reported that chelating activity does not only depend on size of peptide but also on the amount of specific amino acids like histidine since it contains imidazole ring which exhibits strong ferrous ion chelation. Hence, the peptide fractions specifically F3 and F2 can be used to enhance the shelf stability of foods by lowering the metal ion dependent lipid oxidation.

Ferric reducing antioxidant power

FRAP measures the ability of hydrolysate/peptide fractions to donate electron/ hydrogen to reduce Fe³⁺ (ferric) to Fe²⁺ (ferrous) ion. Previous studies had showed that there is direct relation of reducing power with antioxidant activity (Tarafdar et al. 2021). Significant difference was observed in FRAP values of F1, F2, F3 fractions and WH (Fig. 1B). The highest FRAP value was observed for F3 fraction (1.33 ± 0.02 mM aEAC/g) while unfractioned hydrolysate (0.90 ± 0.02 mM TEAC/g) showed the lowest value. Results revealed that reducing power decreased with increase in size of peptides F3 > F2 > F1 > WH. In contrast, it was reported by Girgih et al. (2011) that low MW hemp protein hydrolysate fractions exhibited lower reducing power. Higher hydrophobicity could be another possible reason for higher FRAP of F3 and F2 peptide fractions.

Amino acid profiling

Bioactivity of peptides depends on the amino acid composition and molecular mass (Lassoud et al. 2015). Glutamic acid was the predominant amino acid in all the samples which is consistent with reports on nutmeg hydrolysate (Akinyede et al. 2021), soybean protein hydrolysate (Rayaprolu et al. 2015), followed by lysine, leucine and valine (Table 1). The results indicated that amino acid content of WH was very similar to spent hen meat except for some amino acids such as glutamic acid, serine, leucine, lysine, tryptophan and valine which were present in higher quantities. Ultrafiltered peptide fractions had higher essential amino acid content as compared to WH and spent hen meat. F3 exhibited highest amino acid content (55.60%) followed by F2 (53.30%), WH (52.23%) and F1 (49.82%). F3 (< 5 kDa) fraction also showed the presence of higher hydrophobic amino acids (HAA) and antioxidant amino acids (AAA) which confirmed the results indicating its high antioxidant potential (Fig. 1B). Moreover, histidine and tryptophan which contain imidazole and indolic group, respectively, was also found to be higher in the F3 and F2 fractions, which could be another possible reason of their high antioxidant activity since these groups act as hydrogen donors. Similar findings have been reported by Aderinola et al. (2021) for moringa olifera seed protein hydrolysate. Branched chain amino acids (BCAA) in the F3 fraction (24.18%) were found to be higher than the unfractioned hydrolysate (22.97%) and other peptide fractions. BCAA of F3 was also higher than that reported in previous studies on nutmeg hydrolysate fraction (19.05%) and hemp protein fraction (14.75–16.03%) (Akinyede et al. 2021; Malomo and Aluko 2015). It is also noteworthy that the essential amino acid quantified for the peptide fractions were well above the minimum recommended amino acid level outlined by WHO/FAO. Overall, low MW fractions produced in this study were found to have enhanced bioactivity which could be utilized by incorporating these fractions for the development of functional foods.

Table 1.

Amino acid profiling (g/100 g protein) of spent hen hydrolysate and its peptide fractions

Amino acid	Spent hen meat	WH	> 10 kDa	5–10 kDa	< 5 kDa	WHO/ FAO
Non-essential
Alanineh	5.41	5.27	5.01	5.22	5.15	–
Arginine	5.37	5.85	5.26	5.28	5.43	–
Aspartic acid	6.15	6.55	5.28	5.22	5.23	–
Cystine	1.19	1.14	1.11	1.05	1.29	–
Glutamic acid	10.81	11.23	10.33	10.37	10.41	–
Glycine	3.51	3.37	2.99	3.27	3.33	–
Prolineh	2.33	2.33	2.08	2.31	2.37	–
Serine	3.19	3.98	3.29	3.56	3.66	–
Tyrosineha	2.94	2.87	2.18	2.55	2.75	–
Essential
Histidinea	3.67	3.19	2.84	2.95	3.03	1.50
Isoleucineh	7.94	7.58	7.17	7.12	7.42	3.00
Leucineh	6.56	8.13	7.44	8.34	8.93	5.90
Lysinea	7.55	8.41	8.34	8.18	8.48	4.50
Methionineha	5.25	4.21	3.23	5.16	5.23	2.20*
Phenylalanineh	3.35	3.27	2.87	3.36	3.34	3.00**
Threonine	4.15	4.01	3.92	4.66	4.6	2.30
Tryptophana	4.32	6.17	6.57	5.89	6.74	0.60
Valineh	5.41	7.26	7.44	7.64	7.83	3.90
Total EAA	48.2	52.23	49.82	53.3	55.6
Total AAA	23.73	24.85	23.16	24.73	26.23
Total HAA	39.19	40.92	37.42	41.7	43.02

Essential amino acids (EAA); ^hHydrophobic amino acids (HAA); ^aAntioxidant amino acids (AAA)

^#Reference FAO/WHO (2002) RDA pattern; *Methionine + cystine; **Phenylalanine + Tyrosine

Electrophoretic profile by SDS PAGE

SDS PAGE profile of SPH and its various ultrafiltered fractions (F1, F2, F3) is depicted in Fig. 1C. When compared with broad range MW marker (2–250 kDa), bands were detected at molecular weights of 11, 16, 35 and 57 kDa in WH which are associated with prolamine, globulin, albumin and glutelin fractions of protein, respectively. Similar observations were made by Zaky et al. (2020) with rice bran protein hydrolysate.

Band pattern in WH was found to be similar to F1 fraction (> 10 kDa) except for the bands lower than 10 kDa. Distinguished band at 5 kDa emerged in F2 fraction (5–10 kDa). These low molecular weight peptides could be better absorbed in the human gastro-intestinal tract resulting enhanced bioactivities (Fig. 2). However, bands in the F3 (< 5 kDa) fraction entirely disappeared that could be attributed to the formation of peptides of MW lower than the detection limit of the gel. Similar trend was observed in rice bran protein hydrolysate in low MW fractions (Zaky et al. 2020).

Fig. 2.

Detailed representation of the polypeptide cleavage and subsequent absorption in the human gastrointestinal tract (through the intestinal villi). The low molecular weight peptides absorbed via intestine travel through the blood stream to reach the cells enhancing its bioactivity

Conclusion

The study highlights that low molecular weight peptide fraction derived from spent hen meat protein could have promising applications in the health food/functional food industry. Enzyme specificity, hydrolysis conditions and amino acid composition were identified as the major drivers for the high functionality of low molecular weight peptides. However, the process economics in an industrial setting should be looked into given the low yields of ultrafiltered peptide fractions. In this regard, the utilization of whole hydrolysates could be a possible alternative while compromising some of the functional benefits, for achieving better process economics.

Author contribution

DK: Conceptualization, formal analysis, data interpretation, writing-original draft. AT: formal analysis, data interpretation, writing-reviewing and editing. SLD: data interpretation; resources, writing-reviewing and editing. SP: resources, writing-reviewing and editing. PB: supervision, resources, validation, writing-reviewing and editing.

Funding

The authors have not disclosed any funding.

Data availability

All data pertaining to this research is available within the manuscript.

Declarations

Conflict of interest

The authors have not disclosed any competing interests.