Food Chemistry 230 (2017) 58–67

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidant potential of edible mushroom (Agaricus bisporus) protein

hydrolysates and their ultrafiltration fractions
Benard Muinde Kimatu a,c, Liyan Zhao a, Yuan Biao a, Gaoxing Ma a, Wenjian Yang b, Fei Pei b, Qiuhui Hu a,⇑
a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
b
College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210023, PR China
c
Department of Dairy and Food Science and Technology, Egerton University, Egerton, Kenya

a r t i c l e i n f o a b s t r a c t

Article history: Mushroom protein isolate (MPI) from Agaricus bisporus was hydrolyzed using Alcalase, Pancreatin,
Received 13 November 2016 Flavourzyme, Alcalase-Pancreatin and Alcalase-Flavourzyme. The obtained hydrolysates (MPHs) were
Received in revised form 28 February 2017 ultrafiltered to generate peptide fractions (UFs) of molecular sizes (<1, 1–3, 3–5 and 5–10 kDa). The elec-
Accepted 6 March 2017
trophoretic profile results indicated that the enzymatic systems were efficient in hydrolyzing the MPI
Available online 8 March 2017
into low molecular weight peptides. Hydrolysate yields of >57% and protein recoveries of >43% were
obtained. Effective concentration that scavenged 50% (EC50) of DPPH radicals was similar for the MPHs
Keywords:
while inhibition against linoleic acid oxidation was strongest (66.49%) for Alcalase-Flavourzyme hydro-
Agaricus bisporus
Protein hydrolysates
lysate on day 5 of incubation. UFs exhibited a concentration-dependent FRAP, with the highest activity
Peptide fractions for fractions from Alcalase and Pancreatin recorded in 1–3 kDa. The antioxidant activities of MPHs and
Electrophoretic profile their UFs suggested that they could be potential bioactive ingredients for use in the formulation of func-
Antioxidant activity tional foods as well as natural antioxidants in lipid food systems.
Ó 2017 Published by Elsevier Ltd.

1. Introduction under strict regulation due to their potential health risks associ-
ated with long-term use (Ndhlala, Moyo, & Van Staden, 2010).
Free radicals are constantly generated through the normal Thus, natural antioxidants from food protein-derived bioactive
physiological reactions in humans for diverse functions like signal- peptides has been a focus of attention of numerous studies. These
ing roles and providing defense against infections (Dhaval, Yadav, bioactive peptides can be released from the native protein mole-
& Purwar, 2016). However, when produced in excess, the normal cule through gastrointestinal digestion, in vitro chemically, enzy-
cellular physiological functioning is altered due to protein malfor- matic hydrolysis and fermentation (Boschin, Scigliuolo, Resta, &
mation, DNA mutation, oxidation of membrane phospholipids and Arnoldi, 2014; Onuh, Girgih, Aluko, & Aliani, 2014).
modification of the low density lipoproteins (Alaiz, Beppu, Ohishi, Numerous studies have reported antioxidant properties of
& Kikugawa, 1994; Lee, Koo, & Min, 2004). This leads to several enzymatic hydrolysates from various protein sources; chicken skin
neurodegenerative disorders, diabetes, arthritis, atherosclerosis, (Onuh et al., 2014), bambara groundnut (Arise et al., 2016), black
cancer, Alzheimer’s disease, Parkinson’s disease and ageing prob- bean (Evangelho et al., 2017) and corn (Jin, Liu, Zheng, Wang, &
lems (Halliwell, 1994). Moreover, free radical-mediated lipid per- He, 2016). The antioxidant activities of these hydrolysates have
oxidation in food products is a major global problem in the food been reported to depend on enzyme specificity, molecular weight,
industry (Sarmadi & Ismail, 2010). When the endogenous protec- the degree of hydrolysis and amino acid composition (Alashi et al.,
tion system under certain circumstances fails, oxidative stress sets 2014; He, Girgih, Malomo, Ju, & Aluko, 2013).
in resulting into an unstable cellular state (Maritim, Sanders, & Agaricus bisporus (white button mushroom) is an edible fungus
Watkins, 2003). In food systems, synthetic antioxidants including and the world’s leading cultivated mushroom with yields account-
butylated hydroxyl toluene (BHT), butylated hydroxyl anisole ing for 70% of the total edible fungi (Kalac, 2013). Its worldwide
(BHA), tertiary butyl hydroquinone (TBHQ) and propyl gallate consumption is attributed to its delicious taste and flavour in addi-
(PG) have been used to prevent lipid peroxidation (Girgih et al., tion to being a rich source of nutrients including proteins, essential
2015). However, the use of synthetic antioxidants has been put amino acids, minerals and vitamin B (Liu, Jia, Kan, & Jin, 2013).
Several bioactivities from A. bisporus have been reported including
⇑ Corresponding author. ACE inhibitory activity (Lau, Abdullah, Shuib, & Aminudin, 2014),
E-mail address: qiuhuihu@njau.edu.cn (Q. Hu). hypoglycaemic (Mao, Mao, & Meng, 2013) antioxidant and

http://dx.doi.org/10.1016/j.foodchem.2017.03.030
0308-8146/Ó 2017 Published by Elsevier Ltd.
 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67 59

antimicrobial (Smolskaitė, Venskutonis, & Talou, 2015). However, water in a 250 mL glass beaker that was placed on a magnetic stir-
no reports have been made on the antioxidant potential of A. bis- ring hot plate equipped with an external temperature probe and a
porus protein hydrolysate(s). In this study, A. bisporus protein iso- pH electrode for temperature and pH control, respectively. The
late was hydrolyzed using single (Alcalase, Pancreatin and mixture was heated to the appropriate temperature and the pH
Flavourzyme) and sequential (Alcalase-Pancreatin and Alcalase- adjusted using 1 M NaOH and allowed to equilibrate for 30 min.
Flavourzyme) enzymatic processes. The aim was to generate Enzymatic hydrolysis was initiated by addition of each enzyme
mushroom protein hydrolysates (MPHs), fractionate the hydroly- at an enzyme/substrate ratio of 2.5% (w/w, protein basis). Hydrol-
sates into peptides of various molecular weights and evaluate the ysis was carried out at the stated conditions (Table 1) maintaining
potential antioxidant activity of these samples using different constant pH of the reaction mixture by the pH-stat technique with
in vitro antioxidant evaluation systems. 0.5 M NaOH. For sequential hydrolysis, Alcalase was used first,
reaction terminated after 2 h by immersing the digest in boiling
2. Material and methods water for 10 min, cooling to the appropriate temperature and
adjusting the pH for the subsequent enzyme (Pancreatin or Fla-
2.1. Materials vourzyme) and allowing to equilibrate for 30 min. Hydrolysis with
the second enzyme was done for 2 h, maintaining temperature and
Fresh fruiting bodies of A. bisporus were purchased from a local pH constant for each enzyme. Alkaline consumption over the 4 h
market (Nanjing, China). Proteases Alcalase 2.4U/g and Flavour- hydrolysis period was recorded after every 15 min. To terminate
zyme 500MG were from Solarbio Life Sciences (Beijing, China). the hydrolysis, pH was adjusted to 7.0 with 1 M NaOH or 1 M
Pancreatin 4.6x106U/g was bought from Ryon Biological Technol- HCl and the digest immediately immersed in boiling water for
ogy (Shanghai, China). Reduced L-glutathione (GSH), ultrafiltration 15 min. After cooling to room temperature, the undigested pro-
cassettes with 1, 3, 5, and 10 kDa molecular weight cut-off teins were precipitated by centrifugation at 21,000g for 20 min at
(MWCO) sizes were procured from Millipore Corp. (Billerica, MA, 4 °C. The supernatants were lyophilized as mushroom protein
USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 30% and 40% Acry- hydrolysates (MPHs) powders (AH, PH, FH, APH and AFH for Alca-
lamide/Bis were obtained from Sigma-Aldrich (St. Louis, MO, lase, Pancreatin, Flavourzyme, Alcalase-Pancreatin and Alcalase-
USA) Ferrozine iron reagent, linoleic acid, and Tricine were from Flavourzyme hydrolysates, respectively). MPHs were stored at
Macklin Biochemical (Shanghai, China). Other reagents used were
20 °C. The protein content of the lyophilized MPHs was deter-
of analytical grade. mined as described in section 2.2.1. MPH percent yields and pro-
tein recovery were determined as follows:
2.2. Sample Preparation  
weight of hydrolysate
MPH yield ð%Þ ¼  100
The fruiting bodies were washed, sliced and immediately freeze weight of MPI used for hydrolysis
dried after which they were ground to pass through an 80-mesh
MPH protein recov ery ð%Þ
standard screen. The powder was packed in air-tight plastic bags  
and stored at
20 °C. weight of protein in hydrolysate
¼  100
weight of protein in MPI used for hydrolysis
2.2.1. Protein extraction
Protein extraction was done according to Jeurink, Noguera, 2.2.3. Ultrafiltration of the hydrolysates
Savelkoul, and Wichers (2008) with modifications. The mushroom A 0.3% solution (w/v) of MPHs was prepared in distilled water.
powder was added to 5% ice-cold acetic acid solution (1:15, w/v) in The solution was ultrafiltered by sequentially passing it through
the presence of 2-mercaptoethanol (0.1%) and stirred for 3 h. The Pellicon 2 Mini cassettes with MWCO of 1, 3, 5, and 10 kDa. The
slurry was centrifuged at 15,500g for 15 min at 4 °C, supernatant retentate from 1 kDa was passed through 3 kDa; retentate from
filtered with Whatman filter paper No. 5, and the solubilized pro- 3 kDa was passed through 5 kDa whose retentate was finally
teins precipitated overnight (12 h) at 4 °C by the addition of passed through the 10 kDa cassette. Permeates from each cassette
ammonium sulphate to 75% saturation. The mixture was cen- were collected as ultrafiltered peptide fractions (UFs) with molec-
trifuged at 25,000g for 20 min at 4 °C and pellets re-dissolved in ular weight (MW) distributions of <1, 1–3, 3–5 and 5–10 kDa,
a small volume of distilled water. Re-dissolved extract was respectively. The UFs were concentrated by rotary evaporation
desalted by dialyzing (10 kDa) against distilled water for 24 h at (45 °C), freeze dried and stored at
20 °C. The protein content of
4 °C. The dialysate was lyophilized to produce mushroom protein the UFs was determined as described in section 2.2.1 while the
isolate (MPI) powder which was stored at
20 °C. Protein content UF yields and protein recovery were calculated as follows:
of MPI was determined by the modified Lowry method (Markwell,  
Haas, Bieber, & Tolbert, 1978) with bovine serum albumin used as a weight of UF
UF yield ð%Þ ¼  100
standard. weight of respectiv e hydrolysate
 
weight of protein in UF
2.2.2. Preparation of mushroom protein isolate hydrolysates (MPH) UF protein recov ery ð%Þ ¼
The MPI was hydrolyzed separately with Alcalase, Flavourzyme weight of protein in respectiv e hydrolysate
and Pancreatin at their optimum hydrolysis conditions (Table 1).  100
MPI (5% w/v, protein content basis) was dispersed in deionized

Table 1
Hydrolysis conditions for the various enzymatic hydrolyses.

Alcalase Pancreatin Flavourzyme Alcalase-Pancreatin Alcalase-Flavourzyme

Temp (°C) 50 37 50 50 then 37 50
pH 9.0 7.5 7.0 9.0 then 7.5 9.0 then 7.0
Time (h) 4 4 4 2 then 2 2 then 2
E/S ratio (w/w) 2.5/100 2.5/100 2.5/100 2.5/100 2.5/100
Substrate concentration (w/v) 5/100 5/100 5/100 5/100 5/100
60 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67

2.3. Determination of the degree of hydrolysis of DPPH radical in anhydrous (99.7%) ethanol was prepared. A
2 mL aliquot of this solution was added to 2 mL of sample solutions
Degree of hydrolysis (DH%) was measured by the pH-stat tech- at concentrations 0–2 mg/mL and vortexed for 10 s. The mixture
nique as described by Zhao et al. (2012) and calculated as: was incubated at room temperature in the dark for 30 min. Absor-
bance values for the control (Ac) and the samples (As) were read at
h B  Nb 1 1
DHð%Þ ¼  100 ¼    100 517 nm. The control comprised of water in place of the sample
htot MP a htot while GSH was used as a positive control. The percent DRSA was
where B = the amount (mL) of NaOH consumed to keep the pH con- calculated as:
stant during the hydrolysis; Nb = the normality of the base (NaOH);  
Ac
As
MP is the mass (g) of protein (N  6.25); and a = the average degree DRSAð%Þ ¼  100
Ac
of dissociation of the a-NH2 groups released during hydrolysis (see
below); the total number of peptide bonds (htot) in the protein sub- The concentration of sample that caused a 50% (EC50) reduction
strate (meqv/g protein) was assumed to be 8meqv/g for mushroom in DRSA was calculated from a non-linear regression plot of per-
protein (Adler-Nissen, Eriksen, & Olsen, 1983). centage scavenging activity versus sample concentration.
The degree of dissociation;
a ¼ 1þ10pH
pK where pK = average pK-value of the a-amino groups
pH
pK
10
2.6.2. Metal (Fe2+) chelating activity
liberated during hydrolysis (7.1 for Alcalase and Flavourzyme; 7.3 The Fe2+-chelating activity of the MPHs and their UF was deter-
for Pancreatin (Adler-Nissen et al., 1983). mined using a method described by Memarpoor-Yazdi, Mahaki,
and Zare-Zardini (2013). Sample solutions were dissolved in water
2.4. SDS-PAGE electrophoresis to a final concentration of 0.2–2.0 mg/mL. An aliquot of 200 ml of
sample was mixed with 10 ml of FeCl2 (2 mM) and 600 mL of double
2.4.1. Tris-glycine gel electrophoresis distilled water. Subsequently, 20 mL of ferrozine solution (5 mM)
The electrophoretic profile of MPI and the MPHs was deter- was added to the mixture, followed by vigorous mixing for
mined on a 5% stacking and 16.5% resolving gel using 30% Acry- 2 min. The mixture was then kept for 10 min at room temperature
lamide/Bis solution (29:1) according to Laemmli (1970) with after which the absorbance of the sample (As) and the control (Ac)
modifications. Samples were dissolved in deionized water were read at 562 nm. Ethylenediaminetetraacetic acid (EDTA) was
(20 mg/mL) and filtered through 0.45 mm membranes. Sample ali- used as a positive control. The percent chelating activity was calcu-
quots (20 mL) were mixed with 4 mL of 5X loading buffer (Beyotime lated as:
Biotechnology) containing SDS, b-mercaptoethanol, glycerol and  
Ac
As
bromophenol blue. The mixture was heated in a boiling water bath Metal chelating activ ityð%Þ ¼  100
Ac
for 5 min, vortexed for 30 s and allowed to cool to room tempera-
ture. Aliquots of 10 mL of the samples were loaded onto the gel. A EC50 of the sample that reduced chelating activity by 50% was
mixture of proteins (14.4 kDa–116 kDa, Solarbio Sciences, Beijing, calculated from a non-linear regression plot of percentage activity
China) was used as a broad range MW marker. Electrophoresis versus sample concentration.
was run on a Mini-Protean II Cell. The resolved protein bands were
stained by immersing the gels in a solution containing 45% metha- 2.6.3. Ferric reducing antioxidant power (FRAP)
nol, 10% glacial acetic acid and 0.25% Coomassie Brilliant Blue R- The reducing power of the MPHs and UFs was determined
250 for 1.5 h. To visualize the bands, the gels were destained in a according to He et al. (2013). Briefly, samples were dissolved in
solution containing 45% methanol and 10% glacial acetic acid until 0.2 M sodium phosphate buffer at pH 6.6 to a concentration of 1,
they were clearly visible. 2, and 3 mg/mL. An aliquot of 250 mL sample was mixed with
250 mL of buffer and 250 mL of 1% (w/v) potassium ferricyanide
2.4.2. Tris-tricine gel electrophoresis solution. The blank reaction consisted of double distilled water
To resolve peptides below 10 kDa, Tris-Tricine SDS-PAGE was instead of sample and GSH (1 mg/mL) was used as a positive con-
used following the method of (Schägger, 2006). A 16.5% resolving trol. The resulting mixture was heated to 50 °C and incubated for
gel using 40% Acrylamide/Bis solution (19:1) and including 6 M 20 min. After incubation, 250 mL of 10% (w/v) of aqueous TCA
urea was prepared and overlaid with 4% stacking gel. The samples was added. Thereafter, 250 mL of peptide/TCA mixture was com-
were prepared, electrophoresed, stained and destained as bined with 50 mL of 0.1% (w/v) FeCl3 and 200 mL of double distilled
described in section 2.4.1 and the MW marker used was water and allowed to stand at room temperature for 10 min. The
3.313 kDa–20.1 kDa (Tiandz Inc, China). solution was then centrifuged at 1000g for 10 min at 25 °C and
the supernatant’s absorbance read at 700 nm.
2.5. Amino acid composition analysis
2.6.4. Inhibition of linoleic acid oxidation
The amino acid composition of the samples were determined The ability to inhibit the activity of lipid peroxidation was mea-
according to Girgih, Udenigwe, Li, Adebiyi, and Aluko, 2011 using sured as described by Alashi et al. (2014). Samples or standard
a HPLC system after hydrolysis with 6 M HCl. Cysteine and compounds (GSH and vitamin C (Vc)) were dissolved in 0.1 M
methionine contents were determined after performic acid oxida- sodium phosphate buffer (pH 7.0) to a final concentration of
tion while the tryptophan content was determined after alkaline 1 mg/mL. A 1 mL aliquot of 50 mM linoleic acid (dissolved in
hydrolysis. 99.7% ethanol) was added to 1.5 mL sample and blank (buffer).
The mixtures were incubated at 60 °C in the dark for 7 days. The
2.6. Determination of antioxidant properties degree of oxidation was evaluated by measuring the ferric thio-
cyanate values according to the method described by Hwang,
2.6.1. DPPH radical scavenging activity Chen, Luo, and Chiang (2016) at intervals of 24 h as follows: The
The DPPH radical scavenging activity (DRSA) of MPHs and UFs sample solution (100 mL) incubated in the linoleic acid model sys-
were measured using a method described by Zhang, Li, Miao, and tem was transferred into reaction tube to which 4.7 mL of 75% (v/v)
Jiang (2011) with slight modifications. Briefly, a 0.1 mM solution ethanol, 0.1 mL of 30% (w/v) ammonium thiocyanate and 0.1 mL of
 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67 61

0.02 M FeCl2 dissolved in 1 M HCl was added. After shaking and the values reported in this study. This could probably be due to the
incubating at room temperature for 3 min, the absorbance of the difference in the protein composition of the substrate. Overall, the
sample (As) and blank (Ab) was read at 500 nm. An increase in high yields (>84%) and high PRs (>60%), except for Flavourzyme,
absorbance value implied an increase in the level of linoleic acid may be a potential incentive for the use of these enzymes in com-
oxidation. mercial generation of MPI hydrolysates for both functional foods
The percent inhibition of linoleic acid oxidation was calculated and nutraceuticals applications.
as: For the UFs, both the yield and PR increased significantly
  (p < 0.05) from <1 kDa to 1–3 kDa. In all the enzymatic hydrolyses,
As
Inhibition of linoleic acid ð%Þ ¼ 1
 100 the 1–3 kDa had significantly (p < 0.05) higher yields and PRs than
Ab the other fractions for each enzyme, with Flavourzyme having the
highest values. However, >78% yield and >80% PR, for all the
2.7. Statistical analysis enzyme hydrolyses was low MW (<3 kDa). This suggests high effi-
ciency of the enzymes used to hydrolyze the MPI into smaller-sized
All experiments were run in triplicate and the data subjected to peptides which may lead to increased bioactivity.
one-way analysis of variance (ANOVA) using Statistical Analysis
Software, PC-SAS V9.1 (SAS Institute, Cary, North Carolina, USA). 3.2. Degree of hydrolysis
Treatment means separation was done by Fisher’s Least Significant
Difference at a = 0.05. DH% is usually used as a proteolysis-monitoring parameter to
ascertain the extent to which peptide bonds have been broken
down to release short-chain peptides. The results for DH% (Fig. 1)
3. Results and discussion
were characterized by a high rate for the first 30 min in all the
enzymatic processes except for Flavourzyme where the initial rate
3.1. Hydrolysate yields and protein recovery
exhibited a gentle increase for a longer period (1.5 h). This initial
high rate is indicative of maximum cleavage of peptide bonds
The MPI subjected to the enzymatic processes had a protein
(Shahidi, Han, & Synowiecki, 1995) while the slowing down as
content of 75.16%. Percentage yield and protein recovery (PR) of
the hydrolysates and their UFs are shown in Table 2. Percent yield
is an indicator of the efficiency of enzyme hydrolysis process
because a higher yield of peptides is the expected outcome for Alcacase Pancreatin Flavourzyme
Alcalase-Pancreatin Alcalase-Flavourzyme
increased protein breakdown (Girgih et al., 2011). The yield will 20
also help gauge the potential viability of the protein hydrolysate
for commercial application. MPH yields obtained for all the enzy- 18

matic hydrolyses were 57%, with Alcalase-Pancreatin yield being 16

the highest at 95%. Alcalase, Alcalase-Flavourzyme and Pancreatin
yields were 93.2, 87.2 and 84.2%, respectively. These values corre- 14
% Degree of hydrolysis

late well with the extent of DH%, an observation in congruent with

12
previous reports by Girgih et al. (2011) and Zhao et al. (2012).
These high yields are indicative of the susceptibility of MPI to enzy- 10
matic hydrolysis and that it could be hydrolyzed into shorter pep-
tide products. PR is also a parameter that can be used to determine 8
the efficacy of the hydrolysis process (Girgih et al., 2011). The low-
6
est and highest percentage PR values, (43.8 and 81.9%), were
recorded for Flavourzyme and Alcalase hydrolysis, respectively. 4
The use of Flavourzyme or Pancreatin (both have endo- and exo-
protease activity), either alone or in sequence with Alcalase, 2
resulted in significantly (p < 0.05) lower recovery values than the
0
value for Alcalase hydrolysis alone (Table 2). This could be due to 0 30 60 90 120 150 180 210 240
excessive generation of free amino acids which were not measured
Time (min)
by the method used for protein content determination. PR in
hydrolysates is not commonly reported in literature, however Fig. 1. Degree of hydrolysis (DH%) of MPI catalysed by Alcalase, Pancreatin,
(Alashi et al., 2014) reported 66.8 and 55.2% for Alcalase and Pan- Flavourzyme, Alcalase-Pancreatin and Alcalase-Flavourzyme. Values are presented
creatin, respectively, for canola hydrolysates, which are lower than as means ± SD, (n = 3).

Table 2
Protein recovery (PR) and yields of mushroom protein isolate hydrolysates (MPHs) and ultrafiltration fractions. Mean ± standard deviation (n = 3); means followed by different
lower case letters in the same column are significantly different (p < 0.05); means for the same parameter followed by different upper case letters in the same row are significantly
different (p < 0.05).

Protease MPHs < 1 kDa 1-3 kDa 3-5 kDa 5-10 kDa
Yield (%) PR (%) Yield (%) PR (%) Yield (%) PR (%) Yield (%) PR (%) Yield (%) PR (%)
a a abB aB cA cdA aC aC aC
Alcalase 93.20 ± 1.60 81.94 ± 3.54 31.85 ± 2.66 27.32 ± 2.22 46.41 ± 3.45 53.5 ± 4.94 3.67 ± 0.61 3.62 ± .053 2.44 ± 0.18 2.93 ± 0.21aC
Pancreatin 84.19 ± 1.48c 68.35 ± 3.47b 37.94 ± 3.77aB 25.93 ± 2.94aB 51.15 ± 2.19bcA 61.26 ± 1.87bA 2.58 ± 0.95aC 2.27 ± 0.88bC 2.16 ± 1.39aC 2.28 ± 1.49aC
Flavourzyme 57.07 ± 1.85d 43.77 ± 2.08c 24.99 ± 1.60cB 13.48 ± 0.72bB 59.66 ± 3.11aA 74.08 ± 5.88aA 2.86 ± 1.09aC 2.41 ± 0.93bC 2.60 ± 0.76aC 3.37 ± 0.97aC
Alcalase- 95.04 ± 1.28a 61.80 ± 3.44b 34.91 ± 5.73abB 29.96 ± 4.33aB 49.94 ± 3.35bcA 51.42 ± 4.10 dA 2.65 ± 0.70aC 2.51 ± 0.66bC 2.08 ± 0.83aC 2.50 ± 0.99aC
Pancreatin
Alcalase- 87.18 ± 1.01b 62.32 ± 6.23b 31.35 ± 1.38bcB 29.61 ± 1.21aB 53.25 ± 0.51bA 58.93 ± 1.12bcA 3.38 ± 0.48aC 4.20 ± 0.52aC 1.77 ± 0.22aC 2.26 ± 0.30aC
Flavourzyme
62 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67

the hydrolysis progresses has been attributed to the formation of disappearance of the bands. This implied that MPI was more prone
reaction products which become substrate competitors, decrease to these two enzymatic processes than it was to Alcalase and
in concentration of peptide bonds available for hydrolysis, enzyme Alcalase-Flavourzyme hydrolysis. This is further attested in the Tri-
inhibition and enzyme deactivation (Ketnawa, Benjakul, Martínez- cine SDS-PAGE (Fig. 2B) where the generated low MW peptides
Alvarez, & Rawdkuen, 2017). For the sequential enzymatic treat- appear as smear bands whose intensity is lighter for APH and PH
ment, an abrupt increase in the DH% rate was observed immedi- than for AFH and AH hydrolysis. The addition of Flavourzyme or
ately after the addition of the second enzyme and the rate was Pancreatin to the Alcalase pre-digested hydrolysate resulted in
higher upon addition of Pancreatin than Flavourzyme. This is sug- an additional reduction of the intensity of both the prominent pro-
gestive of more susceptible catalytic sites being available for Pan- tein band and the lower MW smear bands (Fig. 2A) suggesting that
creatin than for Flavourzyme hydrolysis. Since Pancreatin and pre-digestion of MPI with Alcalase exposed more hydrolytic sites
Flavourzyme have both endo- and exo-peptidase activity, pre- that were susceptible to Flavourzyme and Pancreatin hydrolysis.
digestion with Alcalase would be expected to increase the N- Flavourzyme hydrolysis appears to be the least effective in
terminal sites thereby facilitating more hydrolysis of MPI (Kou hydrolyzing MPI. Although it extensively depolymerized both the
et al., 2013). However, only a slight change in DH% was observed high and low MW proteins in MPI to lower MW peptides, the
when Flavourzyme was added after pre-digestion with Alcalase prominent protein band appears to be more resistant to Flavour-
and this ended with a lower DH% (16.13%) than when Alcalase zyme as evidenced by the reduction in its intensity to a lesser
was used alone (18.2%). This suggests that more peptide bonds extent (Fig. 2A). The susceptibility of the MPI to hydrolysis by these
were cleaved by Alcalase in the last 2 h of hydrolysis when Alcalase enzymatic processes is an indication of their potential application
was used alone than were by Flavourzyme in the Alcalase pre- to generate bioactive peptides for use as functional foods and
digested hydrolysate. This was unexpected since a combination nutraceuticals.
of two enzymes is thought to yield a more extensively hydrolyzed
product than using one enzyme alone as reported by García-
3.4. Amino acid composition of MPI and MPHs
Moreno et al. (2014) and Shi, Kovacs-Nolan, Jiang, Tsao, and Mine
(2014). We therefore infer that the Alcalase pre-digestion of MPI
Amino acid composition in addition to structure and hydropho-
did not expose more Flavourzyme-susceptible bonds. DH% is influ-
bicity, has been reported to influence the antioxidant activity of
enced by the cleavage patterns and specificities of the enzyme
peptides (Sarmadi & Ismail, 2010; Zhao et al., 2012). A summary
used, substrate source, substrate/enzyme ratio as well as duration
of the amino acid composition of the MPI and the hydrolysates is
of hydrolysis (García-Moreno et al., 2014; Ketnawa et al., 2017).
presented in Table 3. The hydrolysates were characterized by high
levels of Asx and Glx, i.e. negatively charged amino acids (NCAA),
3.3. SDS-PAGE electrophoresis in addition to Ala, Leu and Lys. NCAA confer antioxidant activity
to peptides owing to the abundance of their electrons that can be
The results of the polypeptide MW distribution in MPI and donated to quench free radicals (He et al., 2013). Cys was the lim-
MPHs are presented in Fig. 2. High MW polypeptide and low MW iting amino acid as it was not detected in both the MPI and the
polypeptide bands in the MPI were well resolved in the Glycine hydrolysates while Met, the other sulphur containing amino acid,
SDS-PAGE (Fig. 2A) and Tricine SDS-PAGE (Fig. 2B), respectively. was present in amounts (1.49% for APH and 1.17% for FH) compa-
The results indicate that the unhydrolyzed MPI consisted of molec- rable to rapeseed hydrolysates (He et al., 2013). Met has the ability
ular weight bands ranging from approximately 10–116 kDa and to donate its sulphur hydrogen and therefore is considered as an
above (Fig. 2A). Within this range, there were at least three major efficient radical scavenger. APH, AH and PH had significantly
protein bands at 15 kDa, 21 kDa and 40 kDa among which the higher (p < 0.05) AAA at 5.75, 5.72 and 5.62%, respectively, com-
21 kDa was the most prominent. All the enzymatic processes pared to PH and FH values of 5.25 and 4.91%, respectively. AAA
used hydrolyzed the MPI into smaller peptides, though at varying have been credited to enhance the potency of radical scavenging
efficiencies. No differences were observed in the APH and PH elec- activity by donating protons to stabilize electron-deficient radicals
trophoretic profiles where extensive hydrolysis led to the complete while retaining their own stability through resonance structures

Fig. 2. Electrophoretic patterns of MPI and its resultant hydrolysates from various enzymatic processes on 16.5% polyacrylamide gels; A – Tris-glycine gel electrophoresis, B –
Tris-tricine gel electrophoresis. AFH: Alcalase followed by Flavourzyme hydrolysate, APH: Alcalase followed by Pancreatin hydrolysate, FH: Flavourzyme hydrolysate, PH:
Pancreatin hydrolysate, AH: Alcalase hydrolysate, MPI: Mushroom protein isolate, M: Molecular weight marker.
 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67 63

Table 3
Percent amino acid composition of mushroom protein isolate (MPI) and hydrolysates. Mean ± standard deviation (n = 3); means followed by different letters in the same row are
significantly different (p < 0.05). Alcalase hydrolysate (AH), Pancreatin hydrolysate (PH), Flavourzyme hydrolysate (FH), Alcalase followed by Pancreatin hydrolysate (APH) and
Alcalase followed by Flavourzyme hydrolysate (AFH).

Amino acid MPI AH PH FH APH AFH

Asx 6.15 ± 0.12d 6.43 ± 0.12c 6.21 ± 0.11 cd 6.72 ± 0.12b 7.11 ± 0.15a 5.47 ± 0.10e
Thr* 3.65 ± 0.07e 4.02 ± 0.09c 3.85 ± 0.09d 4.24 ± 0.06b 4.45 ± 0.10a 3.34 ± 0.06f
Ser 3.49 ± 0.07e 3.82 ± 0.06c 3.65 ± 0.07d 3.95 ± 0.08b 4.19 ± 0.06a 3.15 ± 0.06f
Glx 11.84 ± 0.16d 12.67 ± 0.17c 12.53 ± 0.21d 13.57 ± 0.22b 14.46 ± 0.20a 10.87 ± 0.17f
Gly 3.33 ± 0.08d 3.63 ± 0.08b 3.48 ± 0.08c 3.70 ± 0.10b 3.95 ± 0.09a 2.98 ± 0.04f
Ala 5.37 ± 0.09d 5.70 ± 0.12c 5.54 ± 0.08 cd 6.04 ± 0.13b 6.43 ± 0.11a 4.80 ± 0.09e
Cys n.d n.d n.d n.d n.d n.d
Val* 3.84 ± 0.08d 4.23 ± 0.10c 4.19 ± 0.06c 4.47 ± 0.08b 4.77 ± 0.08a 3.63 ± 0.07e
Met* 1.30 ± 0.07d 1.27 ± 0.04d 1.33 ± 0.06c 1.37 ± 0.04b 1.49 ± 0.06a 1.17 ± 0.04e
Ile* 2.62 ± 0.06d 3.02 ± 0.06c 3.03 ± 0.07c 3.22 ± 0.06b 3.48 ± 0.06a 2.55 ± 0.07d
Leu* 4.33 ± 0.11e 5.05 ± 0.13c 4.86 ± 0.012d 5.36 ± 0.11b 5.66 ± 0.10a 4.20 ± 0.10e
Tyr 0.94 ± 0.03d 1.65 ± 0.06a 1.33 ± 0.06c 1.49 ± 0.06b 1.49 ± 0.04b 1.28 ± 0.04c
Phe* 2.34 ± 0.07e 2.90 ± 0.07b 2.73 ± 0.07c 3.00 ± 0.08ab 3.09 ± 0.05a 2.47 ± 0.06d
Lys* 4.58 ± 0.11c 4.74 ± 0.10b 4.52 ± 0.08c 4.81 ± 0.09b 5.15 ± 0.10a 3.96 ± 0.07d
His* 1.78b ± 0.06c 1.99 ± 0.05a 1.82 ± 0.04b 2.01 ± 0.05a 2.03 ± 0.04a 1.70 ± 0.03c
Arg 3.23 ± 0.07d 3.86 ± 0.07b 3.68 ± 0.09c 3.99 ± 0.07b 4.23 ± 0.07a 3.21 ± 0.08d
Pro 2.66 ± 0.06a 2.39 ± 0.06c 2.23 ± 0.05d 2.54 ± 0.05b 2.62 ± 0.05a 2.10 ± 0.05e
Trp* 1.23 ± 0.04a 1.18 ± 0.04abc 1.20 ± 0.05ab 1.13 ± 0.04c 1.17 ± 0.04abc 1.16 ± 0.03bc
HAA 23.40 ± 0.54e 26.19 ± 0.61c 25.23 ± 0.54d 27.49 ± 0.22b 29.02 ± 0.56a 22.20 ± 0.50f
PCAA 9.59 ± 0.23e 10.59 ± 0.21c 10.02 ± 0.20d 10.81 ± 0.16b 11.42 ± 0.21a 8.87 ± 0.17f
NCAA 18.00 ± 0.27 e 19.10 ± 0.29c 18.75 ± 0.31d 20.29 ± 0.23b 21.57 ± 0.35a 16.34 ± 0.26f
AAA 4.51 ± 0.13d 5.72 ± 0.17a 5.25 ± 0.17b 5.62 ± 0.18a 5.75 ± 0.15a 4.91 ± 0.13c
TEAA 25.35 ± 0.62e 28.86 ± 0.66 c 27.65 ± 0.61d 29.98 ± 0.61b 31.61 ± 0.65a 24.31 ± 0.52f

Hydrophobic amino acids (HAA) – Ala, Val, Ile, Leu, Tyr, Phe, Try, Pro, Met and Cys.
Positively charged amino acids (PCAA) – Arg, His and Lys.
Negatively charged amino acids (NCAA) – Asx (asparagine + aspartic acid), and Glx (glutamine + glutamic acid).
Aromatic amino acids (AAA) – Tyr, Phe and Trp.
n.d – not detected.
*
Total essential amino acids – (TEAA).

(Sarmadi & Ismail, 2010). All the hydrolysates had significantly (Udenigwe, Lu, Han, Hou, & Aluko, 2009). For the sequential
higher (p < 0.05) HAA than the MPI except for AFH. The highest enzyme hydrolysis, the highest activity was recorded in the 5–
contribution to the HAA in all the hydrolysates was from Ala, Leu 10 kDa with AFH exhibiting stronger (EC50 value 0.16 mg/mL)
and Val. HAA enhance the solubility of peptides in lipids thus activity than its corresponding fraction from APH (EC50 value
increasing their interaction with free radicals or entry into target 0.24 mg/mL). This suggests that the sequential hydrolysis resulted
organs through hydrophobic associations and this has been in a hydrolysate whose highest DRSA was concentrated in the 5–
reported in various food-derived protein hydrolysates (Alashi 10 kDa. It would be reasonable to suggest that the various hydro-
et al., 2014; Najafian & Babji, 2014) The presence of all the EAA lysates and their UFs contained peptides/amino acids capable of
in the hydrolysates in comparable amounts to the reference FAO/ donating electrons that could stabilize DPPH radical and break
WHO standard (FAO and WHO, 1991), indicates their good nutri- the radical chain reaction. The DRSA of the peptides depended on
tional quality, in addition to their antioxidant bioactivity. However, the specificity of the enzyme(s) used and molecular weight, and
APH, FH and AH were better in TEAA content than PH and AFH. could also be due amino acid composition and peptide conforma-
tion (Girgih et al., 2011; Jin et al., 2016). GSH, in this report used
3.5. DPPH radical scavenging activity as a positive control, had an EC50 value of 0.02 mg/mL.

The ability of the generated MPHs and their UF to scavenge 3.6. Metal chelating activity
DPPH radical is presented in Fig. 3A. For the MPHs, the DRSA
depended on the enzyme process employed and the highest activ- Fig. 3B depicts the ability of EDTA, MPHs and their UFs to che-
ity (lowest EC50) was reported for Pancreatin (EC50 0.25 mg/mL). late Fe2+. The MPHs exhibited stronger activity (lowest EC50 val-
There was no significant difference (p > 0.05) in the DRSA of AH, ues) compared to their UFs. This may have been due to the
APH and AFH (EC50 values 0.49, 0.52 and 0.51 mg/mL, respec- synergistic effects of the constituent peptides responsible for the
tively). For each enzymatic process, the <1 kDa and 3–5 kDa dis- activities of the fractions. The activities reported for all the hydro-
played the least DRSA when compared to their respective parent lysates (EC50 values 0.96–2.17 mg/mL) are stronger than those
hydrolysates and other UFs. In each case the DRSA activity of reported by He et al. (2013) from rape seed hydrolysis by various
<1 kDa was significantly lower than that of 3–5 kDa with the proteases (EC50 values >5 mg/mL). Overall, Alcalase hydrolysate
exception of AH where in addition to <1 and 3–5 kDa fractions, displayed the strongest activity (EC50 value 0.96 mg/mL) compared
the 5–10 kDa was also lower in activity than its parent hydrolysate. to the other hydrolysates whose EC50 values ranged from 1.27 to
For the single enzyme hydrolysis, the highest potency of DRSA 2.17 mg/mL. The <1 kDa fraction from Pancreatin hydrolysis exhib-
seemed to be concentrated in the 1–3 kDa with the lowest EC50 ited the least chelating capacity (EC50 value 15.02 mg/mL) which
value amongst these fractions being that from PH (0.13 mg/mL). was significantly (p < 0.05) lower than similar fractions from the
This contrasted previous reports which showed that <1 kDa frac- other hydrolysates. The 1–3 kDa fractions from all the hydroly-
tions tended to have the highest DSRA activity (Arise et al., 2016; sates displayed the strongest activity compared to the other pep-
He et al., 2013). However, it was in agreement with findings tide fractions. This suggests an abundance of negatively charged
reported for Alcalase-hydrolyzed flaxseed hydrolysate fraction peptides in this fraction. Of these, the Alcalase 1–3 kDa had the
64 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67

Fig. 3. The effective concentration (EC50) values for DPPH radical scavenging activity (A) and metal chelating capacities (B) of mushroom protein isolate hydrolysates and
their ultrafiltration peptide fractions prepared by Alcalase, Pancreatin, Flavourzyme, Alcalase-Pancreatin and Alcalase-Flavourzyme. Data are presented as mean ± SEM, n = 3.
Bars with different alphabets have values that are significantly different (p < 0.05).

strongest activity (EC50 value 1.2 mg/mL).The high MW fractions 3.8 mg/mL). It has been postulated that the strong activity of
3–5 and 5–10 kDa from APH and AFH showed a stronger activity long-chain peptide could be attributed to the synergistic effects
compared to similar fractions from PH and FH. This implied that of higher number of amino acid units (He et al., 2013; Shi et al.,
the pre-digestion of the MPI with Alcalase before Pancreatin and 2014). EDTA, however, compared to all the tested samples had
Flavourzyme hydrolysis resulted in high MW peptides with significantly (p < 0.05) better Fe2+ chelating capacity (EC50 value
improved metal chelating properties than when the two enzymes of 0.01 mg/mL) while GSH did not show any measurable activity.
are used alone. The activities for 5–10 kDa from APH and AFH From these results, the demonstrated metal chelating properties
(EC50 values 2.95 and 2.96 mg/mL, respectively) are >4-fold that of the hydrolysates and the 1–3 kDa fractions could make
reported by Shi et al. (2014) for a similar fraction from Alcalase- them potential candidates for use as agents to preserve lipid foods
Protease P hydrolysis (IC50 value 13.11 mg/mL) of eggshell mem- by chelating pro-oxidative metal ions in addition to reducing
brane protein but comparable to that reported by He et al. cellular components damage resulting from metal ion-catalyzed
(2013) from Alcalase hydrolysis of rapeseed protein (EC50 value oxidation.
 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67 65

3.7. Ferric reducing power activity (FRAP) power within the range of the tested concentrations. There
was a significant difference (p < 0.05) among FRAP values of
Fig. 4A-C shows the reducing power of the MPHs and UFs at the hydrolysates with PH exhibiting the highest values of 0.16
three different concentrations. All the hydrolysates and their and 0.41 at 1 and 3 mg/mL, respectively. FRAP values for the
UFs exhibited a concentration-dependent increase in reducing sequential hydrolysis were not significantly (p > 0.05) different
from that obtained when Alcalase was used alone. This implied
that predigesting the MPI with Alcalase before Pancreatin or Fla-
vourzyme did not significantly alter the FRAP activity. AH and
FH EC50 values at a concentration 3 mg/mL (0.24 and 0.31,
respectively) were higher than those obtained by Tang, Wang,
and Yang (2009) from hemp protein hydrolysates (0.22 and
0.23, respectively) with similar proteases. The different FRAP
abilities of these hydrolysates could be attributed to different
amino acid side chain groups with electron-dense areas becom-
ing exposed as the hydrolysis progresses in addition to peptides
and free amino acids released acting as an extra source of elec-
trons and protons to maintain the reduction potential (Borawska,
Darewicz, Vegarud, & Minkiewicz, 2016). For the UFs, <1 kDa
fractions were the least active at the tested concentration ranges
for each enzyme process with APH showing the lowest absor-
bance of 0.048 and 0.1 at 1 and 3 mg/mL, respectively. The high-
est FRAP was achieved with 1–3 kDa from PH with an
absorbance of 0.62 at 3 mg/ml (Fig. 4C). UFs below 5 kDa when
Alcalase and Pancreatin were used singly had higher FRAP than
when the two enzymes were used in sequence. Flavourzyme’s
5–10 kDa displayed the highest FRAP ability within this MW
range. The presence of fractions 1–3 and 5–10 kDa in FH, APH
and AFH with significantly (p < 0.05) higher activities may have
contributed to the higher activity of these hydrolysates. From
our results, it can be inferred that differences in enzyme speci-
ficity during the hydrolysis of the MPI may have led to peptides
with varying levels of electron or hydrogen donating abilities. At
a concentration of 1 mg/mL, the GSH had a significantly
(p < 0.05) higher FRAP ability (absorbance value 2.23) compared
to all MPHs and their UFs.

Sample Inhibition (%)

Day 3 Day 5
Vc 97.81±1.38a 94.33±1.01b
1.4 GSH 82.74±0.52g 74.40±1.44h
MPI 89.03±2.59de 39.71±0.72m
AH 88.57±1.60def 37.70±1.30m
PH 86.96±0.99ef 48.28±1.45l
1.2 FH 86.59±1.20f 52.05±1.23k
APH 91.40±2.19c 62.36±0.55j
AFH 90.63±1.78cd 66.49±0.46i

1.0
Absorbance at 500 nm

0.8

0.6

0.4

0.2

0.0
0 1 2 3 4 5 6 7 8
Incubation time (days)
CONTROL GSH APH AFH AH
PH FH VC MPI

Fig. 5. Inhibition of linoleic acid oxidation by mushroom protein isolate (MPI),

Fig. 4. Ferric reducing antioxidant power (FRAP) of the MPI hydrolysates and their mushroom protein hydrolysates (Alcalase-AH, Pancreatin-PH, Flavourzyme-FH,
ultrafiltration fractions prepared by Alcalase, Pancreatin, Flavourzyme, Alcalase- Alcalase followed by Pancreatin-APH and Alcalase followed by Flavourzyme-AFH),
Pancreatin and Alcalase-Flavourzyme at different concentrations compared to glutathione (GSH) and Vitamin C (Vc) at a concentration of 1 mg/mL and measured
reduced glutathione (GSH); (A) 1 mg/mL, (B) 2 mg/mL and (C) 3 mg/mL. GSH was over 7 days. Insert is the percentage inhibition of the samples when compared to
tested at 1 mg/mL. Data are presented as mean ± SEM, n = 3. Bars (mean ± SEM) the uninhibited control at days 3 and 5 of incubation. Mean values (n = 3) with
with different letters have mean values that are significantly different (p < 0.05). different letters are significantly different (p < 0.05).
66 B.M. Kimatu et al. / Food Chemistry 230 (2017) 58–67

3.8. Inhibition of linoleic acid oxidation when compared to the other MPHs, a fact further affirmed by the
metal chelating activity tests. Based on these results, the MPHs
Fig. 5 presents the results of inhibition of linoleic acid peroxida- and their UFs could be potential candidates for use as bioactive
tion by GSH, Vc and MPHs. The uninhibited (control) reaction ingredients in functional foods and nutraceuticals to ameliorate
showed a consistent increase in absorbance reaching a peak of oxidative stress effects in addition to finding application in the
1.22 absorbance units on day 3 followed by a rapid decrease for food industry as alternative natural antioxidants. Overall, the Pan-
the rest of the incubation period. This rapid increase in absorbance creatin 1–3 kDa fraction seems to have the best antioxidant activ-
is indicative of a fast autoxidation of linoleic acid in the absence of ity and its further purification and characterization, including
any inhibitors. The rapid decline in absorbance has been attributed in vivo activity, is underway.
to the decomposition of primary oxidation products, mainly
hydroxyperoxides, in addition to the depletion of the linoleic acid Conflict of interest
itself subsequently limiting the production of the oxidation prod-
ucts as the incubation time progresses (Chen, Zhao, Zhao, Cong, & None.
Bao, 2007). Vc consistently inhibited oxidation of linoleic acid as
evidenced by the low absorbance throughout the entire incubation Acknowledgement
period. GSH exhibited a better inhibition activity than the hydroly-
sates during the first two days of incubation as indicated by its low We acknowledge the financial support from China Agricultural
absorbance values. However, this activity appears to be lost after Research System (CARS-24) and the Priority Academic Programme
day 2 as shown by the increase in absorbance and is indicative of Development of Jiangsu Higher Education Institutions (PAPD).
rapid autoxidation of the linoleic acid. The decreased ability by
GSH to sustain inhibition of lipid oxidation in this test system for References
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