Degradation and antioxidant activities of peptides and zinc-peptide complexes during in vitro gastrointestinal digestion

Article abstract of DOI:10.1016/j.foodchem.2014.10.066

The degradation characteristics of three peptides (Ser-Met, Asn-Cys-Ser, and glutathione) and their zinc-peptide complexes were studied using a two-stage in vitro digestion model. Enzyme-resistant peptides and zinc-peptide complexes, antioxidant activities, and free amino acids released by digestive enzymes, were measured in this study. The results revealed that the three peptides and their zinc-peptide complexes were resistant to pepsin but not to pancreatin. Pancreatin can partly hydrolyse both peptides and zinc-peptide complexes, but more than half of them remaining in their original form after gastrointestinal digestion. The coordination of zinc improved the enzymatic resistance of the peptide due to lower solubility of complexes and affected the hydrolytic site of pepsin and pancreatin. Zinc-Asn-Cys-Ser, which is highly resistant to enzymatic hydrolysis and maintains Zn in a soluble form, may have potential to improve Zn bioavailability.

Full text of DOI:10.1016/j.foodchem.2014.10.066

Food Chemistry 173 (2015) 733–740
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Degradation and antioxidant activities of peptides and zinc–peptide
complexes during in vitro gastrointestinal digestion
a
a,b,
⇑
a
a
Chan Wang , Bo Li
, Bo Wang , Ningning Xie
a
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
Key Laboratory of Functional Dairy, Ministry of Education, Beijing 100083, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2013
Received in revised form 19 September
The degradation characteristics of three peptides (Ser-Met, Asn-Cys-Ser, and glutathione) and their zinc–
peptide complexes were studied using a two-stage in vitro digestion model. Enzyme-resistant peptides
and zinc–peptide complexes, antioxidant activities, and free amino acids released by digestive enzymes,
were measured in this study. The results revealed that the three peptides and their zinc–peptide
complexes were resistant to pepsin but not to pancreatin. Pancreatin can partly hydrolyse both peptides
and zinc–peptide complexes, but more than half of them remaining in their original form after gastroin-
testinal digestion. The coordination of zinc improved the enzymatic resistance of the peptide due to
lower solubility of complexes and affected the hydrolytic site of pepsin and pancreatin. Zinc–Asn-Cys-
Ser, which is highly resistant to enzymatic hydrolysis and maintains Zn in a soluble form, may have
potential to improve Zn bioavailability.
2
014
Accepted 9 October 2014
Available online 18 October 2014
Keywords:
Antioxidant peptide
Zinc–peptide complexes
Gastrointestinal digestion
Zinc supplement
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
and improve the bioavailability of Zn (Harzer & Kauer, 1982;
Miquel & Farré, 2007).
Zinc (Zn), a trace element that is essential for health, is the sec-
ond most abundant inorganic micronutrient in the body
Cummings & Kovacic, 2009; Hambidge & Krebs, 2007). Several
Researchers reported that metal-chelating peptides increase
mineral bioavailability either by maintaining the mineral in a sol-
uble form or by increasing its absorption through carrier-mediated
processes (Chaud et al., 2002; Miquel & Farré, 2007). The positive
effects of these peptides on the absorption of iron (Fe) (Pérès
et al., 1999) and Zn (Miquel & Farré, 2007) have been reported both
in vivo and in vitro. Wang, Zhou, Tong, and Mao (2010) reported
that yak milk casein hydrolysate (YCH) could bind with Zn ions
and form complexes making Zn more soluble under simulated
intestinal conditions, and YCH–Zn complexes may have potential
to improve Zn bioavailability. The findings of Pérès et al. (1999)
revealed that Fe bound to b-CN (1–25), a peptide obtained from
the enzymatic hydrolysis of b-casein, improves Fe bioavailability
in rats. However, an in vitro digestion assay cannot release dialyz-
able Fe from Fe-b-CN (1–25) complexes (Aît-Oukhatar et al., 2000).
It has been reported that Fe bound to b-CN (1–25) may have a dif-
ferent intestinal absorption mechanism to that of Fe salts, e.g., Fe
bound to casein phosphopeptides is absorbed mainly by endocyto-
sis (Pérès et al., 1999).
(
conditions, including growths retardation, hypogonadism,
impaired immune system, and neurological dysfunctions, are
attributed to Zn deficiency (Maret & Krezel, 2007). Zn deficiency
appears to be common in developing countries, particularly in
infants, pre-schoolers, pregnant and lactating women, and older
adults (Maret & Sandstead, 2006; Wegmüller, Tay, Zeder, Brni c´ , &
Hurrell, 2014). Intestinal Zn absorption depends on the amount
of mineral consumed and its bioavailability. Supplemental Zn
exists in several forms including mineral salts, metal-chelating
agents, and zinc-binding proteins or peptides (Hurrell, 2002). How-
ever, mineral salts have poor bioavailability due to the inhibitory
effects of dietary components such as tannins, phytate, and dietary
fibre (Fredlund, Isaksson, Rossander-Hulthén, Almgren,
&
Sandberg, 2006; Sandberg, 2002). On the other hand, soluble,
low-molecular weight organic compound, such as some amino
acids, polypeptides and organic acids, can act as Zn-binding ligands
Preformed Zn complexes may prevent Zn precipitation and
minimise interactions with other metals and mineral inhibitors.
Peptides are considered to be effective Zn binding ligands espe-
cially those isolated from protein hydrolysates, e.g., chickpea pro-
tein (Torres-Fuentes, Alaiz, & Vioque, 2011), casein (Harzer &
⇑
Corresponding author at: P.O. Box 294, Qinghua East Road 17, Haidian District,
Beijing 100083, China. Tel./fax: +86 10 62738988.
E-mail address: libo@cau.edu.cn (B. Li).
http://dx.doi.org/10.1016/j.foodchem.2014.10.066
0
308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

7
34
C. Wang et al. / Food Chemistry 173 (2015) 733–740
Kauer, 1982; Wang et al., 2010) and sesame protein (Wang, Li, &
Ao, 2012). However, the structures of these peptides, which con-
tain metal-binding loops require further characterisation. For
enhanced mineral bioavailability, it is crucial that metal-chelating
peptides should be partially or fully resistant to the hydrolytic
action of digestive enzymes (Miquel & Farré, 2007). However,
few studies have focused on the enzymatic resistance of peptides
and Zn–peptide complexes.
Sesame (Sesamum indicum L.), which grows widely in mainland
China, is an important oilseed crop. Sesame cake, which is an inex-
pensive oil byproduct, is extensively used as animal feed and fertil-
izer. Sesame protein is rich in Glu, Asp, Arg, and Leu (Johnson,
Suleiman, & Lusas, 1979). Several studies have reported that ses-
ame protein hydrolysate (SPH) has antihypertensive properties
Alting, Meijer, and Van-Beresteijn (1997) with a slight modification
(Ruiz, Ramos, & Recio, 2004). The samples were diluted to 10 mg/
ml with 0.01 mol/l HCl (pH 2.0). Pepsin (enzyme to substrate ratio
1:50, w/w) was added and the mixture was incubated in a shaking
platform for 2 h at 37 °C. The pH was first adjusted to 5.3 with
3
0.9 mol/l NaHCO and subsequently to pH 7.5 with 2 mol/l NaOH.
Pancreatin (enzyme to substrate ratio 1:25, w/w) was added and
the mixture was incubated in a water bath for 4 h at 37 °C under
constant stirring.
To investigate the changes in the peptides and zinc–peptide
complexes during the in vitro digestion, aliquots of the digests were
removed at 0, 0.5, 1, 2 (hydrolysis with pepsin), 4, and 6 h (hydroly-
sis with pancreatin). To terminate the enzymatic digestion, the ali-
quots were submerged in boiling water for 10 min and allowed to
cool to room temperature. The digests were centrifuged at 8000g
for 10 min. The supernatants (containing peptides and Zn–peptide
complexes) were collected, adjusted to pH 7, and stored at ꢁ20 °C.
(Nakano et al., 2006). Our previous study revealed that SPH has
both zinc chelating abilities and antioxidant properties (Wang
et al., 2012). In that study, six Zn chelating peptides were isolated
from SPH. In this study, two of these metal-chelating peptides,
which have high Zn chelating and antioxidant properties, i.e.,
Ser-Met (SM) and Asn-Cys-Ser (NCS), were used as models for
investigating the degradation of peptide ligands and their zinc
complexes in the gastrointestinal tract. Reduced glutathione
2.4. Content analysis of the digests
2.4.1. Content analysis of enzyme-resistant peptides and Zn–peptide
complexes
Enzyme-resistant peptides and Zn–peptide complexes were
analysed in SHIMADZU LC-15C equipped with an HPLC column
m, Agilent Technolo-
(
GSH) constitutes another zinc ligand with powerful antioxidant
and metal chelating properties (Zhao, Ruan, & Li, 2011). Therefore,
the objective of this study were to (a) investigate the resistance of
peptides and Zn–peptide complexes to in vitro digestion conditions
and (b) evaluate whether the coordination of Zn affects the resis-
tance of these peptides to enzymatic hydrolysis.
(
ZORBAX SB-C18, 4.6 mm i.d. ꢂ 250 mm, 5
l
gies, USA). A gradient elution was performed with A (0.01% formic
acid in water) and B (acetonitrile) at 0.3 ml/min: 5% B, 0–5 min;
6
0% B, 25 min; and 5% B, 40 min. The sample (20
ll) was monitored
at 215 nm.
2
. Materials and methods
2
.4.2. Determination of free amino groups
The concentration of free amino groups was determined by the
TNBS method as reported by Xie, Wang, Ao, and Li (2013). Digests
(10 l) were mixed with 100 l of potassium borate (0.1 mol/l) and
40 l of TNBS (1.2 mg/ml) in a 96-well plate and incubated in the
2
.1. Materials and reagents
SM (Ser-Met) and NCS (Asn-Cys-Ser) were synthesized by Apep-
l
l
l
tide Co (Shanghai, China). The purity of the synthesized peptides
was more than 95%. Enzymes (pepsin and pancreatin), chemicals
and reagents, including
ox, fluorescein sodium salt, dithizone and 2,2 -azino-bis(3-ethyl-
benzothiazoline-6-sulphonic acid) diammonium salt (ABTS), were
purchased from Sigma–Aldrich (St. Louis, MO, USA), Other chemi-
cals and reagents, which were analytical grade, were purchased
from Beijing Chemical Co. (Beijing, China).
dark for 1 h at 37 °C. Absorbance was measured at 405 nm in a
micro plate reader (Thermo Multiskan MK3). Different concentra-
L
-glutathione reduced, triethylamine, Trol-
0
tions of glycine (0–1
curve. The contents of free amino groups in the digest samples
were expressed as glycine amino equivalents ( mol/l), based on
the equation of glycine standard curve generated.
lmol/l) were used to generate the standard
l
2.4.3. Free amino acid analysis
2.2. Synthesis of Zn–peptide complexes and identification by ESI-MS
The free amino acids in the digests were analysed by HPLC
according to the method described by Vasanits and Molnár-Perl
(1999) with some modifications. A pre-column derivatization
method (phenylthiocarbamyl, PITC) was used. Amino acid standard
solutions were used as standard and the contents of free amino
acids were expressed as concentration with mg/ml.
The Zn–peptide complexes were prepared by mixing peptide
with ZnSO
was added drop-wise to the peptide solution (5 ml, 50% ethanol,
final concentration 0.016 mol/l) before 100 l triethylamine was
4
4 2
in solution. ZnSO ꢀ7H O (50.6 mg in 6 ml 50% ethanol)
l
also added. After constant stirring for 1 h at room temperature,
the solution was centrifuged at 8000g for 15 min. The resulting
precipitate was repeatedly washed with 80% ethanol and freeze-
dried for 24 h. The Zn chelating properties of SM, NCS, and GSH
were evaluated by EDTA complexometric titration. The Zn–peptide
complexes were purified with dithizone and ninhydrin.
Zn–SM and Zn–NCS were identified by ESI-MS (microTOF-Q II,
Bruker Daltonics Inc.) with the following conditions, an ESI voltage
2
of 3.5 kV, a nebulizer of 7.25 psi, a N flow rate of 5.01 l/min, a
2
2
.5. Antioxidant activity assays
.5.1. ABTSÅ+ radical scavenging assay
Å+
The ABTS radical scavenging activities of the digests were
assessed using the method reported by Wang and Xiong (2005)
and You, Zhao, Regenstein, and Ren (2010). An ABTS stock solu-
tion (2.45 mmol/l potassium persulfate and 7 mmol/l ABTS ) was
incubated in the dark for 14 h at room temperature and was
diluted with 0.2 mol/l PBS (pH 7.4) to achieve an absorbance of
Å+
Å+
temperature of 350 °C, a mass range of 50–1000, and a positive full
ion monitoring mode.
0
.70 ± 0.02 at 734 nm. Trolox was used for the generation of the
standard curve. Trolox (40
1.0, 1.5, 2.0, and 2.5 mmol/l) was added to 4 ml diluted ABTS solu-
l
l) of different concentrations (0.5,
Å+
2.3. In vitro digestion
tion, mixed for 30 s, and allowed to stand in the dark for 6 min.
Å+
Peptides and Zn–peptide complexes were enzymatically
Digests (40 ll of 5.0 mg/ml) were mixed with 4 ml diluted ABTS
digested with pepsin and pancreatin according to the method of
solution. The absorbance of the Trolox standards and digests was

C. Wang et al. / Food Chemistry 173 (2015) 733–740
735
measured at 734 nm. ABTSÅ+ radical scavenging activity was
expressed as Trolox equivalent antioxidant capacity (TEAC, g/l).
its multiple five-membered rings; therefore, EDTA is commonly
used as a standard solution for ligand titration. According to the
results, NCS had the highest Zn-chelating ability (60.77%), followed
by GSH (40.08%), and SM (30.10%). These results were in agree-
ments with our previous findings (Wang et al., 2012). In terms of
2.5.2. ORAC assay
The oxygen radical absorbance capacity (ORAC) assay was per-
Å+
formed using fluorescein (FL) as the probe (Dávalos, Gómez-
Cordovés, & Bartolomé, 2004). Black 96-well microplates and a
Tecan Infinite M200 microplate reader equipped with Tecan i-
Control software were used. Fluorescence measurements were
performed at 37 °C. The excitation and emission wavelengths were
ABTS radical scavenging activity, GSH had the highest antioxidant
property, followed by NCS and SM. Several peptides are multi-func-
tional with both metal-chelating and antioxidant properties. Metals
are capable of producing reactive oxygen species, that damage bio-
molecules. Wang et al. (2009) reported an excessive amount of Zn
ions induces oxidative stress in rapeseed (Brassica napus) seedlings.
Chelating peptides may also exhibit antioxidant activities not only
by preventing this pro-oxidant effect (Torres-Fuentes et al., 2011)
but also by exhibiting free radical-scavenging abilities.
4
85 nm and 535 nm, respectively. A fluorescein stock solution
(
(
1.17 mmol/l) was prepared with 75 mmol/l phosphate buffer
pH 7.4) and stored at 4 °C for 4 weeks. The fluorescein working
solution, AAPH, and Trolox solution were prepared fresh each
day. The digests (20 l) and fluorescein working solutions (120
of 117 nmol/l) were added to each well of the 96-well microplate,
which was incubated for 12 min at 37 °C. AAPH solution (60 l of
0 mmol/l) was then added rapidly to each well using multichan-
l
ll
3
.1.2. Mass spectra of Zn–SM and Zn–NCS
The COO of the peptide backbone, and the amino (NH) and car-
ꢁ
l
bonyl (CO) groups of the peptide bond play an important role in
the formation of metal ion complexes (Katsoulakou et al., 2009).
The hydroxyl group of Ser and the sulfhydryl group of Cys in SM
and NCS may participate in the coordination between peptides
and Zn (Wang et al., 2012).
The complexes between Zn and SM (Zn–SM) and between Zn and
NCS (Zn–NCS) were identified by electrospray ionisation-mass spec-
trometry. Fig. 1 shows that Zn–SM and Zn–NCS were synthesized at
4
nel pipet. The 96-well plate was transferred to the microplate
reader, automatically shaken for 2 s and measured every minute
for 120 min. The blank contained phosphate buffer instead of
digest. Six calibration solutions using Trolox standards (0, 10, 30,
4
0, 70, and 80
lmol/l) were used.
Fluorescence measurements were normalised to the blank
curve. From the normalised curves, the area under the fluorescence
decay curve (AUC) was calculated by the following equation,
a 1:1 ratio. The mass spectrum of Zn–SM revealed that H
2
O was
involved in the coordination between Zn and SM. The MS spectrum
i¼120
+
X
of the charged ion with m/z at 337 was [SM ꢁ 2H
2
O ꢁ Zn] ; the
AUC ¼ 1 þ
fi=f
0
+
charged ions with m/z at 237 and 259.1 were [SM + H] and
i¼1
+
+
+
[
SM ꢁ Na] , respectively. Similarly, [NCS ꢁ Zn + H] , [NCS + H] ,
+
0 i
where f is the initial fluorescence reading at 0 min and f is the fluo-
rescence reading at time i. The net AUC corresponding to the digest
and [NCS ꢁ Na] were in the MS spectrum of Zn–NCS.
was calculated by the following equation,
3.2. Stability of peptides and Zn–peptide complexes during in vitro
digestion
net AUC ¼ AUCantioxidant ꢁ AUCblank
The regression equations between net AUC and trolox standard
were determined; the ORAC values of the digests were expressed
To evaluate and compare the resistance of the peptides and Zn–
peptide complexes to digestive enzymes a two-stage enzymatic
hydrolysis was performed. Pepsin was used to simulate gastric
digestion, whereas pancreatin was used to simulate intestinal con-
ditions. The digests were analysed by HPLC. The amount of intact
peptides, Zn–peptide complexes, and free amino acids released
during the in vitro digestion were used to assess the enzymatic
resistance of the peptides and Zn–peptide complexes.
as
.6. Statistical analysis
All experiments were conducted in triplicate. Data were
lmol/l trolox equivalents (lmol/l TE).
2
expressed as means ± standard deviation (SD). The data were ana-
lysed by ANOVA and Duncan’s mew multiple range test using SPSS
1
8.0 (SPSS Inc, Chicago, IL, USA). Statistical significance was set at
3
.2.1. HPLC profile of peptides and Zn–peptide complexes
The results revealed that there was a significant difference
p < 0.05.
between peptides and Zn–peptide complexes in terms of their sol-
ubility. Peptides of SM, NCS, and GSH were readily soluble in both
gastric and intestinal digests whereas Zn–peptide complexes were
extremely difficult to dissolve in such environment. According to
the HPLC results, the solubility of Zn–NCS was higher than that
of Zn–SM and Zn–GSH. The soluble portion of Zn–NCS in the diges-
tive supernatant can be detected by HPLC, but those of Zn–SM and
Zn–GSH are difficult to detect given the same conditions.
The SM, GSH, NCS, and Zn–NCS digests were separated and
detected by HPLC. Fig. 2 shows the HPLC profiles before digestion
and after gastric (2 h) and intestinal digestion (4 h). The results
revealed that NCS and GSH were more stable during in vitro diges-
tion than SM and Zn–NCS. SM and Zn–NCS were more likely to be
partially degraded, especially by pancreatin.
3
. Results and discussion
3
3
.1. Characteristics of Zn–peptide complexes
.1.1. Zn-chelating and antioxidant abilities of SM, NCS, and GSH
Å+
EDTA complexometric titration and ABTS radical scavenging
activity assays were used to evaluate the Zn-chelating and antiox-
idant properties, respectively, of SM, NCS, and GSH. The Zn-chelat-
ing and antioxidant properties of the peptide are shown in Table 1.
As an organic ammonia carboxy complexing agent, EDTA can
chelate metal ions in a 1:1 ratio. EDTA is highly stable because of
Table 1
Zinc chelating and antioxidant abilities of SM, NCS and GSH.
Peptides Zinc chelating abilities
%)
ABTSÅ+ radical scavenging activity
(TE g/l)
3
.2.2. Concentrations of peptides and Zn–peptide complexes during
(
in vitro digestion
SM
30.10 ± 0.02
60.77 ± 0.00
40.08 ± 0.03
0.02 ± 0.006
0.55 ± 0.012
1.03 ± 0.142
Fig. 3(A) shows the changes in SM, NCS, GSH, and Zn–NCS
concentrations during the in vitro GI digestion. Both Zn–SM and
Zn–GSH were not included in the results due to their poor solubility.
NCS
GSH

7
36
C. Wang et al. / Food Chemistry 173 (2015) 733–740
Fig. 1. The mass spectrum of Zn–SM (A) and Zn–NCS (B).
Fig. 2. RP-HPLC profiles of peptides and Zn–peptide complexes in digests. (A) SM, (B) NCS, (C) GSH, (D) Zn–NCS, and 1–3 on behalf of before digestion (0 h), after gastric
digestion (2 h), and after sequential intestinal digestion (6 h), respectively.

C. Wang et al. / Food Chemistry 173 (2015) 733–740
737
Fig. 3. (A) Concentration changes of SM (A-1), NCS (A-2), GSH (A-3) and Zn–NCS (A-4) during in vitro digestion. (B) Amount of amino groups of peptides and their Zn–peptide
complexes during in vitro digestion. (B-1) SM, (B-2) Zn–SM, (B-3) NCS, (B-4) Zn–NCS, (B-5) GSH, (B-6) Zn–GSH, respectively. The data with different lowercase letters in test
are significantly different (p < 0.05).
The concentration of GSH during the entire in vitro digestion
was relatively stable with no significant differences amongst the
different digestion periods (p > 0.05). Similar results were obtained
between NCS and SM. These two peptides resisted gastric digestion
groups is an indicator of the actual number of peptide bonds cleaved
by digestive enzymes (Spellman, McEvoy, O’Cuinn, & FitzGerald,
2003). As Zn–SM and Zn–GSH could not be detected by HPLC
because of their poor solubility, the amounts of free amino groups
from peptides and Zn–peptide complexes during in vitro digestion
were determined to reflect their stability in simulated gastrointes-
tinal conditions.
(p > 0.05). However, their concentrations significantly decreased
during intestinal digestion (p < 0.05). Similar changes also occurred
in Zn–NCS. Experiments indicated that Zn–NCS could survive gas-
tric digestion because of the buffering effect of the complex.
When the Zn–peptide complexes were adjusted to pH 2.0
with HCl, precipitates formed. During the in vitro digestion, the
pH of the digests changed from 2.0 to 6.5, which is not the opti-
mum pH of pepsin. On the other hand, NCS, SM, and GSH were
readily soluble in gastric conditions. Therefore, Zn–peptide com-
plexes can resist the action of pepsin because of their buffering
action.
As shown in Fig. 3(B), during gastric digestion, free amino
groups in both peptides and Zn–peptide complexes increased
inconspicuously, except for NCS, where the free amino groups
increased twice within the 1–2 h gastric digestion. Following intes-
tinal digestion, the free amino groups in both peptides and Zn–
peptide complexes increased (p < 0.05).This result suggests that
the peptides (i.e., SM, NCS, and GSH) and Zn–peptide complexes
(i.e., Zn–SM, Zn–NCS, and Zn–GSH) were only slightly sensitive to
gastric conditions, they were nevertheless digested by pancreatin.
These results are consistent with the HPLC results. However, unlike
in the peptide digests, only a few free amino groups were present
in the Zn–peptide digests. This phenomenon may be attributed to
the poor solubility of Zn–peptide complexes and the fact that the
supernatant (as opposed to the precipitate) was used in the exper-
iment. The result also suggests that only the soluble fraction of the
peptides and Zn–peptide complexes can be degraded; insoluble
components cannot interact with digestive enzymes. Moreover,
the increase in free amino groups from the Zn–peptide complexes
occurred at higher rate than that from the peptides, particularly
during the intestinal digestion.
The concentrations of SM, NCS, GSH, and Zn–NCS before
digestion (0 h) were considered to be 100%. The survival rates after
gastric (2 h) and intestinal digestion (6 h) were calculated. The sur-
vival rates of SM and NCS after gastric digestion were 96.18% and
9
5.69%, respectively. This finding suggests that SM and NCS were
highly resistant to pepsin. However, the survival rates of all peptides
and Zn–peptide complexes decreased during the intestinal diges-
tion. SM had the highest reduction in survival rate, followed by
Zn–NCS and NCS. At the end of the in vitro digestion, 67.84% of SM,
8
3.61% of NCS, and 80.62% of Zn–NCS remained intact.
The degradation of peptides contributes to the release of free
amino groups during digestion. Therefore, the amount of free amino

7
38
C. Wang et al. / Food Chemistry 173 (2015) 733–740
Table 2
Concentration of free amino acids in digests during GI digestion in vitro.
ICd
NCS (mg/ml)
.35 ± 1.02
Zn–NCS (mg/ml)
0.18 ± 0.02
GSH (mg/ml)
8.52 ± 0.76
Zn–GSH (mg/ml)
–
i
8
Cys
Ser
Cys
Ser
Cys
Gly
Cys
Gly
ICd
0.015 ± 0.000c
0.312 ± 0.007c
–
–
0.016 ± 0.000c
–
–
0.141 ± 0.000b
0.014 ± 0.000b
0.019 ± 0.000a
e
b
b
b
b
b
a
CG
0.032 ± 0.001
0.416 ± 0.049
1.187 ± 0.025
0.031 ± 0.003
0.112 ± 0.003
0.195 ± 0.031
3.396 ± 0.105
0.014 ± 0.000
0.016 ± 0.000
f
0.346 ± 0.002a
0.20%
a
a
a
a
0.021 ± 0.004a
CI
0.007 ± 0.000
0.00%
3.63%
0.390 ± 0.036
0.00%
4.58%
0.023 ± 0.001
g
CCG /IC
CCI /IC
1.25%
10.48%
8.40%
53.27%
0.63%
38.20%
h
3.96%
a–c
The data with different lowercase letters in test are significantly different (p < 0.05).
IC means the initial concentration before digestion.
d
e
f
CG means the concentration after 2 h gastric digestion.
CI means the concentration after following 4 h intestinal digestion.
CCG means the concentration change of amino acids after gastric digestion.
CCI means the concentration change of amino acids after intestinal digestion.
Undetected.
g
h
i
3
.2.3. Free amino acids released from peptides and Zn–peptide
complexes
Enzymes hydrolyse at specific cleavage sites in polypeptides
On the other hand, GSH and Zn–GSH were stable under gastric
conditions. This was evident by the fact that there were no signif-
icant differences in the concentration of free amino acids (Cys and
Gly) following gastric digestion. Cys and Gly increased following
the addition of pancreatin. However, the coordination of Zn had
obvious effect on the hydrolytic activity of pancreatin in Zn–NCS.
In GSH, Gly was released prior to Cys; while the same amount of
free Cys and Gly was detected in Zn–NCS following intestinal
digestion. Therefore, the coordination of Zn had a certain effect
on the cleavage sites of pepsin and pancreatin.
chains. The free amino acids were studied to determine the cleav-
age sites in the peptides and Zn–peptide complexes. The concen-
trations of amino acids in NCS, GSH, and their Zn complexes are
shown in Table 2.
In both NCS and Zn–NCS, Ser was releasedprior to Cys. The results
are consistent with those obtained by Schmelzer et al. (2007), who
reported that the C-terminal end of the peptide is easily hydrolysed
by digestive enzymes. The coordination of Zn had little effect on the
hydrolytic activity of pepsin in Zn–NCS. In NCS, a small amount of
Cys was released from NCS; however, free Cys was not detected in
Zn–NCS following gastric digestion. On the other hand, pancreatin
recognised both Cys and Ser in NCS and Zn–NCS, and free Cys and
Ser increased after intestinal digestion. These results suggest that
both NCS and Zn–NCS can be partially hydrolysed.
3.3. Changes in the antioxidant activities of peptides and Zn–peptide
complexes during in vitro digestion
In the presence of pepsin and pancreatin, substrates are hydro-
lysed into different fragments with different antioxidant activities.
Therefore, the changes in antioxidant activities were used as
Fig. 4. Changes of ORAC values (A) and ABTSÅ+ radical scavenging activity (B) of peptides and zinc–peptide complexes during in vitro digestion. The data with different
lowercase letters in test are significantly different (p < 0.05).

C. Wang et al. / Food Chemistry 173 (2015) 733–740
739
another indicator of the enzymatic resistance of peptides and Zn–
peptide complexes.
Antioxidant activity was determined by a number of different
assays. TEAC and ORAC assays are commonly used for assessing
the antioxidant capacity of food components (Clausen, Skibsted,
tides and Zn–peptide complexes were partially resistant to intesti-
nal conditions, more than half of the residual peptides and Zn–
peptide complexes remained in their intact forms. NCS is both an
excellent antioxidant and Zn chelator. Furthermore, NCS was par-
tially resistant to hydrolysis and can potentially increase Zn
absorption and bioavailability. Therefore, the Zn–NCS complex
could be an alternative to inorganic salts currently used in supple-
ments. In vivo studies are needed to confirm the results.
&
Stagsted, 2009).
The ORAC values of peptides and Zn–peptide complexes during
in vitro digestion are shown in Fig. 4(A). The changes in NCS and
GSH were similar. However, there were no significant changes in
the antioxidant activity during gastric digestion and during the
first 2 h of intestinal digestion (p > 0.05). Antioxidant activity sig-
nificantly increased in the last 2 h of intestinal digestion
Acknowledgements
This research was supported by National Natural Science Foun-
dation of China (NSFC, No. 31271846) and the Program for New
Century Excellent Talents in the University of Ministry of Educa-
tion of China (Grant No. 2010JS078).
(p < 0.05). The antioxidant activities of Zn–NCS and Zn–SM gradu-
ally increased during digestion; the ORAC values of these com-
plexes during digestion were higher than those prior to digestion
(p < 0.05). The results were more significant for Zn–SM than for
Zn–NCS. In Zn–GSH, the ORAC values were stable during the entire
digestion (p > 0.05). The ORAC values of SM declined with diges-
tion. After digestion, the activity was 15.76% lower than that before
digestion (p < 0.05).
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