Whey is a by-product of the cheese making process, containing a lot of whey proteins
such as α-lactalbumin, β-lactoglobulin, immunoglobulin and serum
albumin (Chen et al., 2019; Xia et al., 2021). Whey also has the
characteristics of high protein efficiency ratio, high biological value and high
utilization rate (Da Silva et al., 2017). Whey
protein contains higher content of branched-chain amino acids (BCAA) than other
protein sources, which is crucial for tissue growth and repair (Kerasioti et al., 2019). Goat whey has
remarkable antioxidant property at both the molecular and tissue levels because of
its ability to neutralize numerous free radicals (Kerasioti et al., 2014). Compared with the milk from cow, buffalo, sheep
and camel, the whey protein from goat milk contains the highest levels of proline
(53±1.30 mg/g) and eight essential amino acids (Rafiq et al., 2015). However, 47% of the whey is
directly discharged around the world, and approximately 1.3 million tons of goat
whey is discarded or used as animal feeds every year, causing serious environmental
problems (Garay et al., 2021; Papademas and Kotsaki, 2019). Therefore, the
high-value reuse of whey has become an urgent problem to be solved. The value of
whey can be enhanced by hydrolysis, a process that can mitigate the sensitivity of
whey protein by reducing the size of peptides (Bordenave et al., 2000). In addition, BCAA in whey protein hydrolytes
have been shown to be able to protect skin cells from inflammation, and low
molecular weight peptides usually show strong antioxidant activity after protein
hydrolysis (Da Silva et al., 2017; Ranathunga et al., 2006). Goat whey peptides
possess biological properties such as immunomodulation, antimicrobial and
antihypertension (Osman et al., 2016). Based
on the above, high Fischer’s ratio (F ratio) oligopeptides can be prepared
from goat whey to increase the value of goat whey. High F ratio oligopeptides refers
to the active peptides with the BCAA to aromatic amino acid (AAA) ratio of over 20,
and molecular weight (Mw) of 200–1,000 Da, physiological property of it in
relieving phenylketonuria and anti-fatigue have been reported (Li et al., 2019). Up to now, the raw materials for the
preparation of high F ratio oligopeptides are mostly plant-derived proteins, such as
soy and flaxseed protein (Salma et al., 2022;
Yan et al., 2021). For animal-derived
proteins, only cow casein was reported in 2004 (Pedroche et al., 2004). As an ideal source both in terms of BCAA content
and the activity of goat whey peptides, using whey protein for the preparation of
high F ratio oligopeptides has not been reported.In this study, goat whey was hydrolyzed to obtain high F ratio oligopeptides through
two-step hydrolysis of pepsin and flavourzyme. The optimal conditions for the
double-enzyme stepwise hydrolysis of whey protein and the conditions for activated
carbon to adsorb into AAA were discussed. The Mw distribution of high
Fischer’s ratio oligopeptides from goat whey (HFO) was defined, and the
antioxidant effects of HFO were evaluated by performing various antioxidant assays
after the pre-treatment of HFO both in vivo and in
vitro. This study not only expanded the high-value processing and
utilization of goat whey, but also was of great significance for the diversification
and advanced development as well as application of HFO products.
Materials and Methods
Collection and preparation of goat whey samples
Goat milk samples were collected from healthy adult dairy goats raised in AoTe
Goat Farm Qingdao Shandong Province, China, and transported to the laboratory by
cold chain. After two 10-minute centrifugation processes at 4°C,
2,149×g, the pH value of goat milk was adjusted to 4.1 with 10%
acetic acid. Then milk samples were stored at 4°C for 30 min until casein
precipitated, and then centrifuged at 2,149×g for 10 min at 4°C to
obtain the supernatant, namely the goat whey. After that, the goat whey was
filtered with a dialysis bag of 3,500 Da to remove the small molecules, such as
salt and lactose. Then the milk whey was freeze-dried to obtain goat whey powder
for the enzymatic hydrolysis in the next step.
Optimization of enzymatic hydrolysis conditions and the degree of hydrolysis
(DH)
The two-step enzymatic reaction was performed in a beaker containing a mixture of
100 mL of distilled water and 5 g of goat whey powder. Pepsin hydrolyses were
carried out under different conditions where five values for each indicator of
time, temperature, pH, and pepsin were chosen. Then the resulting hydrolysate
was further hydrolyzed with flavourzyme under the same varied conditions as
pepsin. The details of the conditions were shown in Table 1. An orthogonal L9 (34) test design was
used to investigate the optimal hydrolysis condition of each enzyme. Four
controllable variables, including time (A), temperature (B), pH (C), and the
amount of pepsin added (D) were selected for optimization. And three independent
trials were conducted at the optimum of enzymatic treatments. The DH of the
hydrolysate was determined with the pH-stat method, which was calculated
according to the following Eq.
(1) (Mat et al., 2020).
Table 1.
Enzymatic hydrolysis conditions
Optimal conditions
Variable
Quantitative
Time (h)
2-6
Add 1.25% (w/w) pepsin, pH=2,
45°C
Temperature (°C)
40-60
Add 1.25% (w/w) pepsin, pH=2, 2
h
pH
1-3
Add 1.25% (w/w) pepsin, 45°C,
2 h
The amount of pepsin added [%,
(w/w)]
0.75-1.75
pH=2, 45°C, 2 h
Time (h)
2-6
Add 1% (w/w) flavourzyme, pH=6.5,
45°C
Temperature (°C)
40-60
Add 0.75% (w/w) flavourzyme, pH=7, 3
h
pH
6-8
Add 1% (w/w) flavourzyme,
45°C, 3 h
The amount of flavourzyme added [%,
(w/w)]
0.25-1.25
pH=7, 3 h, 45°C
where V is the volume of added HCl (mL), N is
the normality of the acid, M is the mass of proteins (g),
h is the total number of peptide bonds in
whey proteins (8.8 meqv/g), and α is the
mean degree of dissociation of the carboxylic groups produced, which was
calculated using the following Eq.
(2).
Optimization of conditions for aromatic amino acid (AAA) adsorption with
activated carbon
The activated carbon (200 mesh, powder) was used to adsorb AAA in the hydrolysate
solution of goat milk whey (Cermakova et al.,
2017). The optimum adsorption condition was explored by evaluating
the adsorption of the hydrolysate under varying time, pH, and carbon to liquid
ratio conditions, the details of which are shown in Table 2. The F ratio of the hydrolysate was measured
according to the method of Li et al.
(2019). An orthogonal L9 (33) test design was used to
investigate the optimal adsorption condition of active carbon. Three
controllable variables, including time (A), pH (B) and carbon to liquid ratio
(C), were selected for optimization. And three independent trials were conducted
at the optimum of adsorption. The absorbance of the hydrolysate was measured at
280 nm and 220 nm using the ultraviolet spectrophotometer. F ratio was defined
according to the following Eq.
(3), where OD280 and OD220 refer to the
absorbance of the hydrolysate at 280 nm and 220 nm, respectively.
Table 2.
Adsorption conditions
Optimal conditions
Variable
Quantitative
Time (h)
0.5, 1, 2, 3, 4
pH=3, carbon to liquid
ratio=1:5
pH
1, 2, 3, 4, 5
Carbon to liquid ratio=1:10, 3
h
Carbon to liquid ratio
1:3, 1:4, 1:5, 1:6, 1:7
pH=3, 3 h
Determination of amino acids and molecular weight (Mw)
The amount of amino acids was determined with a method modified slightly
according to the study of Song et al.
(2020). The oligopeptide solution was thoroughly mixed with 6 M HCl,
where nitrogen was blown for three times, then the obtained substance was
hydrolyzed with acid for 22–24 h at 110°C. After that, the
hydrolysate was filtered with a double filter paper and diluted with ultrapure
water. The filtrate was then spin-evaporated at 50°C to remove HCl and
filtered with a 0.22 μm microfiltration membrane filter. The amino acid
content in the oligopeptide was determined with an automatic amino acid analyzer
(Hitachi High-Tech Science Corporation, Tokyo, Japan).The Mw distribution of the oligopeptide was determined by gel permeation
chromatography (PL_GPC50, Polymer Laboratories, Church Stretton, UK) with
two PL aqua gel-OH mixed 8 μm (Agilent, Santa Clara, CA, USA) columns at
30°C, and R1 detector (Waters Corporation, Milford, MA, USA). The mobile
phase was 0.1 M sodium nitrate and 500 ppm sodium azide aqueous solution.
PEG/PEO was prepared in the mobile phase as a reference standard for determining
Mw distribution of the oligopeptide. The oligopeptide solutions were filtered
with a 0.45 μm filter before sampling and analysis. The relative Mw and
Mw distribution were calculated with the software according to the peak time of
the oligopeptide solution.
The DPPH scavenging activity of the HFO was determined according to the method
described by Duan et al. (2014). HFO
solution was freeze-dried afterwards to obtain HFO powder. 2 mL solutions with
different HFO concentrations were added respectively to 2.0 mL ethanol with DPPH
(0.1 mM). The mixture was left in dark for 30 min. The absorbances by the HFO
solutions were measured using the spectrophotometer (UV756, Shanghai Yoke
Instrument, Shanghai, China) at 517 nm. The DPPH RSA was calculated using the
following Eq. (4).In the formula, A0 is the DPPH absorbance by distilled water, and A is
the DPPH absorbance by the HFO solution. Analyses of all samples were run in
triplicate and averaged.
The ABTS free radicals assay was conducted according to the method reported by
Li et al. (2012). 0.3 mL solutions
with different HFO concentrations were added to 1.2 mL of
ABTSo+ solution. After incubation for 30 min, the
absorbance levels at 734 nm were recorded using the spectrophotometer (UV756,
Shanghai Yoke Instrument). An equivalent volume with ascorbic acid concentration
was used as the control. The initial absorbance at 734 nm was controlled to
0.70±0.02. The ABTS radical scavenging effect was calculated using the
following Eq. (5).In the formula, A0 is the absorbance at 734 nm with ascorbic acid
solution, while A is the absorbance at 734 nm by the HFO solution. Analyses of
all samples were run in triplicate and averaged.
Hydroxyl radical scavenging activity (RSA)
The hydroxyl RSA was determined according to the method described by Tang and Huang (2018). 1 mL solutions with
different HFO concentrations were put into a test tube and then 1 mL
9×10–3 M FeSO4 solution, 1 mL
9×10–3 M salicylic acid ethanol (70%)
solution and 1 mL 9×10–3 M H2O2
were added. After reaction in a water bath for 30 minutes at 37°C, the
ultraviolet absorbance of the mixture was measured at 510 nm. Ascorbic acid was
used as a control. The scavenging effect of hydroxyl radical was calculated
using the following Eq. (6).In the formula, A0 is the absorbance of the mixture by the ascorbic
acid solution, and A is the absorbance of the mixture by the HFO solution.
Analyses of all samples were run in triplicate and averaged.
Animals and treatment
Six-week-old male Kunming mice (20±2 g) from Jinan Pengyue Laboratory
Animal Breeding (Jinan, China) were kept in polypropylene cages and were
maintained at 25±2°C and 55±10% relative humidity
with equal 12-h light/dark cycles. All animals were on a standard laboratory
feed and water ad libitum. All procedures were approved by the
Animal Welfare Ethics Committee of the Shandong Academy of Sciences.After adaptive feeding for 7 days, the mice were randomly divided into 5 groups.
The grouping and administration are as follows (n=6): normal control (NC;
Physiological Saline), model group (MG; Physiological Saline), positive control
(PC; 10 mg/mL Silymarin) and two groups treated with HFO at a dosage of 300
mg/mL (HDG, high dose group) and 75 mg/mL (LDG, low dose group), respectively.
All groups were administered with 0.2 mL of Physiological Saline, Silymarin or
HFO with gastrointestinal administration for 10 consecutive days. Except for the
NC group, all other groups were injected intraperitoneally (i.p.) with
CCl4 (7 mL/kg BW, 0.2% in peanut oil) 2 hours after the
last gastrointestinal administration, and mice in the NC group were injected
with an equivalent volume of peanut oil (i.p.). All the mice were sacrificed
after fasting and continuous water for 24 h. Blood was taken from the eyeballs
and the serum was separated.
Biochemistry experiments
The blood was collected and centrifuged at 3,500 r/min for 10 min, then the
supernatant was taken. Malondialdehyde (MDA), superoxide dismutase (SOD) and
glutathione peroxidase (GSH-Px) levels were measured on an automatic biochemical
analyzer (7500, Hitachi, Tokyo, Japan) for the antioxidant activity in
vitro on an automatic biochemical analyzer (7500, Hitachi),
according to the instructions of assay kits (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China).
Data processing and analysis
IBM SPSS Statistics 23 software is used for the analysis of the experimental data
by using analysis of variance (ANOVA) and the difference analysis of the
significance at the 0.05 level between means were determined by Duncan’s
multiple range test. The analysis of K and R (the result of extreme analysis) in
orthogonal analysis and all the charts were processed using Office Excel
2019.
Results and Discussion
Optimization of enzymolysis conditions
Taking F ratio and DH as indicators, the hydrolysis conditions such as reaction
time, the amount of enzyme, temperature and pH were optimized as illustrated in
Fig. 1 (pepsin) and Fig. 2 (flavourzyme). When the pH,
temperature and amount of enzyme were constant, DH showed a gradual increase. In
contrast, F ratio showed an initial increase followed by a decline with the
prolongation of hydrolysis time (Figs. 1A
and 2A). However, changes to both F ratio
and DH were insignificant. This is because the enzyme is more active at the
beginning of hydrolysis, and with the progress of hydrolysis, the amount of
enzyme and its combination with substrate diminished. The increase of AAA in
hydrolyzed liquid resulted in the drop of F ratio. The DH value increased and
reached saturation finally due to the increase in the number of active sites of
enzymes in the hydrolysis system, and the F ratio reached its maximum at the
addition of 1% (w/w) pepsin (Fig.
1B) and 0.5% (w/w) flavourzyme (Fig.
2B). As seen from Figs. 1C and
2C, both DH and F ratio showed a
tendency of increasing first and then decreasing with the rise of temperature.
Pepsin had the highest DH and F ratio at 50°C, while flavourzyme showed
maximum values at 45°C. When the range of pH change is not large, the
activity of enzymes is affected by the dissociation of the relevant chemical
groups at the active site (Li et al.,
2019). Both the DH and F ratio of pepsin hydrolysis occur at pH 2
(Fig. 1D) and the maximum ratio of
flavourzyme DH and F ratio at pH 7 (Fig.
2D). In view of orthogonal analysis (Tables 3 and 4), the range
analysis revealed that the influential extent of the four factors to DH for
pepsin and flavourzyme were C>B>D>A, and the optimal
conditions were A2B2C2D2 and A2B1C2D1, respectively. The DH of pepsin obtained
in three parallel experiments under optimal conditions were 15.93%,
15.52%, and 15.77%, with an average of 15.74%, and that of
flavourzyme were 46.64%, 49.86%, and 47.53%, with an
average of 48.01%. Based on the above results, the enzymatic conditions
in this study were selected as follows. Pepsin (1%, w/w) was used to
hydrolyze the goat whey at pH 2 and 50°C for 2 h. Afterwards, flavourzyme
(0.5%, w/w) was used to hydrolyze the former hydrolysates at pH 7 and
45°C for 3 h.
Fig. 1.
Effects of different factors on enzymolysis by pepsin.
A–E, a–e Different letters indicate a
significant difference (p<0.05) among the different enzymatic
hydrolysis conditions and the error bars represent the SD. F ratio,
Fischer’s ratio; DH, degree of hydrolysis.
Fig. 2.
Effects of different factors on enzymolysis by flavourzyme.
A–D, a–d Different letters indicate a
significant difference (p<0.05) among the different enzymatic
hydrolysis conditions and the error bars represent the SD. F ratio,
Fischer’s ratio; DH, degree of hydrolysis.
Table 3.
The range analysis of pepsin on DH obtained from the L9 (34)
orthogonal experiment
No.
(A)
(B)
(C)
(D)
DH (%)
1
1
1
1
1
8.80
2
1
2
2
2
15.64
3
1
3
3
3
6.39
4
2
1
2
3
11.75
5
2
2
3
1
10.93
6
2
3
1
2
12.09
7
3
1
3
2
8.30
8
3
2
1
3
11.90
9
3
3
2
1
9.75
K1
10.28
9.62
10.93
9.83
K2
11.59
12.82
12.38
12.01
K3
9.98
9.41
8.54
10.01
Best level
A2
B2
C2
D2
R
1.61
3.41
3.84
2.18
R order
C>B>D>A
Four controllable variables, including time (A), temperature (B), pH
(C), and the amount of pepsin added (D) were selected for
optimization.
DH, degree of hydrolysis.
Table 4.
The range analysis of flavourzyme on DH obtained from the L9 (34)
orthogonal experiment
No.
(A)
(B)
(C)
(D)
DH (%)
1
1
1
1
1
42.64
2
1
2
2
2
40.98
3
1
3
3
3
32.47
4
2
1
2
3
46.76
5
2
2
3
1
34.65
6
2
3
1
2
35.34
7
3
1
3
2
35.96
8
3
2
1
3
33.56
9
3
3
2
1
43.45
K1
38.70
41.79
37.18
40.25
K2
38.92
36.4
43.73
37.43
K3
37.66
37.09
34.36
37.60
Best level
A2
B1
C2
D1
R
1.26
5.39
9.37
2.65
R order
C>B>D>A
Four controllable variables, including time (A), temperature (B), pH
(C), and the amount of flavourzyme added (D) were selected for
optimization.
DH, degree of hydrolysis.
Effects of different factors on enzymolysis by pepsin.
A–E, a–e Different letters indicate a
significant difference (p<0.05) among the different enzymatic
hydrolysis conditions and the error bars represent the SD. F ratio,
Fischer’s ratio; DH, degree of hydrolysis.
Effects of different factors on enzymolysis by flavourzyme.
A–D, a–d Different letters indicate a
significant difference (p<0.05) among the different enzymatic
hydrolysis conditions and the error bars represent the SD. F ratio,
Fischer’s ratio; DH, degree of hydrolysis.Four controllable variables, including time (A), temperature (B), pH
(C), and the amount of pepsin added (D) were selected for
optimization.DH, degree of hydrolysis.Four controllable variables, including time (A), temperature (B), pH
(C), and the amount of flavourzyme added (D) were selected for
optimization.DH, degree of hydrolysis.
Optimization of aromatic amino acid (AAA) adsorption with activated
carbon
Activated carbon is preferred for AAA adsorption because the benzene ring in AAA
is mediated in combination with the graphite microcrystal structure of the
activated carbon surface (Majidi and Karami,
2015; Rahmani et al., 2016).
As can be seen from Fig. 3A, with the
extension of adsorption time, the F ratio of the solution showed the tendency of
rises followed by drops, and the F ratio peaked at 2 h of adsorption. The
adsorption with activated carbon is better under the acidic conditions, for
hydrolytes have a lower solubility at acidic pH, which can facilitate the
efficient adsorption of AAA onto activated carbon (Jiang et al., 2017; Li et
al., 2019). The results in this study showed that the maximum F ratio
was reached at pH 2 (Fig. 3B). The addition
of active carbon also affected the F ratio, according to Fig. 3C. The carbon to liquid ratio of 1:5 was shown to be
the most ideal. Under the condition of constant pH and duration, the adsorption
of active carbons onto AAA may be inadequate when the amount of active carbon is
not enough. As the activation of excessive carbon can cause BCAA to be adsorbed,
both cases would result in a decrease in F ratio. The results of the orthogonal
analysis are shown in Table 5, the range
analysis revealed that the influential extent of the three factors to F ratio
for active carbon was B>A>C and the optimal conditions were
A2B2C2. And the F ratio of the solution obtained from three parallel experiments
under optimal conditions were 24.43, 27.35, and 27.18, with an average of 26.32.
To sum up, the optimized condition is pH 2 and carbon to liquid ratio of 1:5 for
2 h.
Fig. 3.
Effects of different factors on adsorption of AAA by activated
carbon.
a–d Different letters indicate a significant difference
(p<0.05) among different conditions and the error bars represent
the SD. F ratio, Fischer’s ratio; AAA, aromatic amino acid.
Table 5.
The range analysis of active carbon on F ratio obtained from the L9
(33) orthogonal experiment
No.
(A)
(B)
(C)
F ratio
1
1
1
1
12.09
2
1
2
2
23.48
3
1
3
3
14.23
4
2
1
2
21.50
5
2
2
3
25.79
6
2
3
1
18.98
7
3
1
3
13.89
8
3
2
1
19.63
9
3
3
2
16.90
K1
16.60
15.83
16.90
K2
22.09
22.97
20.63
K3
16.81
16.70
17.97
Best level
A2
B2
C2
R
5.49
7.14
3.73
R order
B>A>C
Three controllable variables, including time (A), temperature (B),
and pH (C) were selected for optimization.
F ratio, Fischer's ratio.
Effects of different factors on adsorption of AAA by activated
carbon.
a–d Different letters indicate a significant difference
(p<0.05) among different conditions and the error bars represent
the SD. F ratio, Fischer’s ratio; AAA, aromatic amino acid.Three controllable variables, including time (A), temperature (B),
and pH (C) were selected for optimization.F ratio, Fischer's ratio.
Amino acid and molecular weight distribution
The amino acid profile of the HFO mixture was listed in Fig. 4A and Table 6.
The BCAA content in the solution was 101.568 ng/mL and the phenylalanine content
was only 3.752 ng/mL. BCAA accounted for 23.32% of total amino acids
while AAA accounted for only 0.86%. The F ratio was calculated at 27.070,
which is very close to the value of 26.32 calculated with absorbance values in
2.2.2. The content of Lys was 50.201 ng/mL, which can make up for the deficiency
of lysine content in edible grain food. His, Tyr, Met, and Cys have been
reported to have antioxidant activity (Xie et
al., 2008). The results showed that when the amino acids have a high
BCAA content and low AAA content, HFO mixture may have such functions as
anti-fatigue and liver protection (Kawaguchi and
Torimura, 2019; Mikulski et al.,
2015; Park et al., 2020). HFO
might be a good active ingredient to be added to the diet of patients with
Phenylketonuria to reduce the intake of phenylalanine (Lichter-Konecki and Vockley, 2019). In addition, a total of
171.736 ng/mL of human essential amino acids were identified in this mixture. It
might be reasonable to infer that HFO mixture obtained in this study was a
hydrolysate possessing many physiological functions and nutritional value.
Fig. 4.
The results of amino acid analysis and the molecular weight (Mw)
distribution.
Table 6.
Amino acid composition of HFO
Amino acid
Concentration (ng/mL)
Type of amino acid
Aspartic acid (Asp)
15.520
Threonine (Thr)
14.760
Serine (Ser)
6.521
Glutamate (Glu)
38.199
Glycine (Gly)
25.330
Alanine (Ala)
123.091
Cysteine (Cys)
27.063
Valine (Val)
48.423
BCAA
Methionine (Met)
5.207
Isoleucine (Ile)
30.114
BCAA
Leucine (Leu)
23.031
BCAA
Tyrosine (Tyr)
3.752
AAA
Phenylalanine (Phe)
0.000
AAA
Lysine (Lys)
50.201
Histidine (His)
6.542
Tryptophan (Trp)
0.000
AAA
Arginine (Arg)
17.757
BCAA
101.568
AAA
3.752
F ratio
27.070
HFO, high Fischer's ratio oligopeptides from goat whey; F ratio,
Fischer ratio; BCAA, branched-chain amino acids; AAA, aromatic amino
acid.
HFO, high Fischer's ratio oligopeptides from goat whey; F ratio,
Fischer ratio; BCAA, branched-chain amino acids; AAA, aromatic amino
acid.As illustrated in Fig. 4B, the hydrolysates
showed three peaks: Peak 1, from 17.38 to 18.83 min, with the average Mw of 790,
including about 4 to 7 amino acid residues of the medium peptide segment,
accounting for 16.73%; Peak 2, from 18.83 to 20.51 min, with the average
Mw of 236, dipeptides and tripeptides accounting for 60.98% of the total
amino acids; Peak 3, from 20.51 to 22.47 min, with the average Mw of 29, which
may be residual salts, accounting for 22.29%. Based on the result of
amino acid composition, it can be concluded that HFO were obtained from goat
whey after hydrolysis and adsorption. It is easier for small-molecular peptides
such as HFO to be absorbed by human body than proteins and free amino acids. In
addition, previous studies have reported that α-leaf protein with a Mw of
below 1,000 Da has antioxidant properties (Xie
et al., 2008), the hydrolysate with the strongest hydroxyl RSA in
soybean protein components is mainly the peptides of 0.7–0.8 kDa (Moure et al., 2006). The Mw of HFO obtained
in this study was similar to that of the peptides reported previously.
Therefore, it was inferred that HFO might show good oxidation resistance due to
its small Mw.
Free radical scavenging activity (RSA) in vitro
DPPH free radical is characterized by stability at room temperature, which is
different from most other free radicals that are highly active but short-lived.
The ability of HFO to scavenge DPPH was verified using ascorbic acid as the PC,
as represented in Fig. 5A. There was an
increase in the scavenging rate of DPPH with the increase of HFO concentration.
A similar result has been reported for α-leaf protein hydrolysate (Xie et al., 2008). When the concentration
reached 1.6 mg/mL, the scavenging rate tended to stabilize, and remained at
about 77% (p>0.05), which indicated that the maximum activity of
HFO in scavenging DPPH was observed at the concentration of 1.6 mg/mL. At the
same concentration, the ascorbic acid showed a DPPH scavenging rate of over
95% (p>0.05).
Fig. 5.
Scavenging activity of HFO on DPPH, ABTS and hydroxyl free
radicals.
Means were performed for three samples. a–e Different
letters indicate a significant difference (p<0.05) among
different concentration and the error bars represent the SD. HFO, high
Fischer’s ratio oligopeptides from goat whey; DPPH,
1,1-diphenyl-2-picrylhydrazyl; ABTS,
3-ethylbenzthiazoline-6-sulphonate.
Scavenging activity of HFO on DPPH, ABTS and hydroxyl free
radicals.
Means were performed for three samples. a–e Different
letters indicate a significant difference (p<0.05) among
different concentration and the error bars represent the SD. HFO, high
Fischer’s ratio oligopeptides from goat whey; DPPH,
1,1-diphenyl-2-picrylhydrazyl; ABTS,
3-ethylbenzthiazoline-6-sulphonate.Antioxidants inhibit lipid peroxidation through blocking free radical chain
reactions and chelating metal ions that promote lipid oxidation (Zheng et al., 2016). As is shown in Fig. 5B, the RSA of ABTS free radicals
increased with the rise of HFO concentration. At the concentration of 0.5 mg/mL,
the RSA of ABTS free radicals reached 99.63%, which was the same as the
scavenging rate of ascorbic acid. It indicated that HFO had a strong ability of
scavenging ABTS free radicals, namely strong antioxidant activity. The ABTS
scavenging rate of HFO was much higher than that of the feather keratin
antioxidant peptides reported previously (Fontoura et al., 2019). In the amino acid sequencing results
obtained in this study, the concentration of Cys was 27.063 ng/mL, consistent
with the finding that Cys plays a major role in the scavenging of ABTS (Huo et al., 2020).Hydroxyl free radicals are the most reactive in comparison with other oxygen free
radicals and have been proven to be highly damaging species in free radical
pathology. It can react with almost all substances in cells and cause serious
damage (Zhang et al., 2012). Therefore,
the effective removal of hydroxyl free radicals may be a valid defense for the
living body against various diseases. As is shown in Fig. 5C, the scavenging ability of HFO solutions against
hydroxyl free radicals varied with the HFO concentration (p<0.05). When
the HFO concentration was at 0.25 mg/mL, high hydroxyl scavenging rate was
observed (92.31%), which was slightly lower than that of the same
ascorbic acid concentration (98.53%). This result was much higher than
the scavenging rate of soybean protein hydrolysate (69.75%). Soybean
protein hydrolytes with the strongest ability of scavenging hydroxyl free
radicals mainly consist of peptides from 0.7 to 0.8 kDa, which also indicated
that the antioxidant activity of HFO was correlated with the low molecular
weight. The high hydroxyl radical scavenging ability of HFO also may be due to
their susceptibility to oxidation by extremely reactive hydroxyl radicals,
enhancing its antioxidant activity and protecting the body’s ability of
preventing hydroxyl free radical damage (Moure
et al., 2006). The results of HFO about the hydroxyl RSA can provide
useful information for antioxidant activity (Kitts, 2005).
Antioxidant activity of high Fischer’s ratio oligopeptides from goat
whey (HFO) in vivo
Reactive oxygen species (ROS) are usually produced upon liver injury induced by
CCl4, resulting in lipid peroxidation and oxidative stress (Yuan et al., 2005; Zelko et al., 2002). So in this study, CCl4 was
selected to induce oxidative stress in mice (Fig.
6). Compared with the NC group, there was significant increase in MDA
level in the MG (p<0.05; Fig. 6A),
which indicated that oxidative stress was generated in mice after
CCl4 induction and this model can be used to evaluate the
antioxidant capacity of HFO in vivo. Compared with the MG
group, HDG and LDG group showed significant declines in the level of MDA
(p<0.05). As a product of membrane reaction between free radicals and
unsaturated fatty acids, MDA reflects the sensitivity of lipid peroxidation and
the degree of cell damage (Cemek et al.,
2010; Meng et al., 2019). The
results of this study showed that the intervention of HFO could delay the
accumulation of lipid peroxides in vivo. Oxidative damage
induced by CCl4 can not only cause changes in MDA, but also detect
changes in oxidative stress parameters such as SOD and GSH-Px (Hamid et al., 2017; Lin et al., 2008). Antioxidant enzymes (SOD and GSH-Px) can
convert peroxides into small or harmless substances (Zhang et al., 2003). The antioxidant enzymes are considered
the main defense agent against oxidative damage of biological macromolecules,
inhibiting the chain reaction of lipid peroxidation by effectively removing the
production of ROS (Liu et al., 2018). As
is shown in Fig. 6B, a decrease in SOD and
GSH-Px was observed in MG group, indicating that excessive ROS may inhibit the
development of antioxidant enzymes. Compared with MG group, there were
significant increases in SOD and GSH-Px for HDG group and SOD for LDG
(p<0.05), but GSH-Px of LDG showed no significant differences from that
of MG. This observation may be due to the scavenging impact on free radicals by
antioxidant enzymes. The results showed that HFO could alleviate oxidative
stress induced by CCl4. This may be related to the amino acid
composition of HFO and the molecular weight distribution. Leu and Ala could
release protons easily to electron-deficient free radicals, exhibiting excellent
free radical scavenging ability and metal ion chelation ability. Moreover,
oligopeptides can be easily absorbed by the intestine to exert an excellent
antioxidant effect in the body (Jiang et al.,
2020; Li et al., 2008; Wu et al., 2013). A research has shown that
oral administration of small peptides can improve the antioxidant activity in
ultraviolet-induced photoaging mice, which is consistent with the results of
this study.
Fig. 6.
Effect of HFO on the MDA SOD and GSH-Px level in mice.
A, B, a, b Different letters indicate a significant difference
(p<0.05) among different group and the error bars represent the
SD. HFO, high Fischer’s ratio oligopeptides from goat whey; MDA,
malondialdehyde; NC, normal control; MG, model group; PC, positive
control; HDG, high dose group; LDG, low dose group; SOD, superoxide
dismutase; GSH-Px, glutathione peroxidase.
Effect of HFO on the MDA SOD and GSH-Px level in mice.
A, B, a, b Different letters indicate a significant difference
(p<0.05) among different group and the error bars represent the
SD. HFO, high Fischer’s ratio oligopeptides from goat whey; MDA,
malondialdehyde; NC, normal control; MG, model group; PC, positive
control; HDG, high dose group; LDG, low dose group; SOD, superoxide
dismutase; GSH-Px, glutathione peroxidase.
Conclusion
In this study, goat whey was used to prepare high F ratio oligopeptides with a F
ratio of 27.070, and the optimized enzymatic hydrolysis and adsorption condition was
determined. The HFO have a high BCAA concentration and low molecular weight of less
than 1,000 Da. Free radical scavenging results showed that HFO has the ability of
scavenging DPPH, ABTS and hydroxyl free radicals, and the characteristic of
concentration-dependence. Animal experiment shows that HFO could reduce the MDA
produced by oxidative stress in mice and elevate the level of SOD and GSH-Px
in vivo. This work can not only offer an important basis for
the high-value processing of goat whey, but also for the diversification of HFO
sources, expanding the source of antioxidant peptides, while providing a theoretical
basis for the development of HFO for special medical foods.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.