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Analysis of protein profiles and peptides during in vitro gastrointestinal digestion of four Chinese dry-cured hams
Journal Pre-proof Analysis of protein profiles and peptides during in vitro gastrointestinal digestion of four Chinese dry-cured hams Shui Jiang, Dong Xia, Danni Zhang, Gaole Chen, Yuan Liu PII:
S0023-6438(19)31223-X
DOI:
https://doi.org/10.1016/j.lwt.2019.108881
Reference:
YFSTL 108881
To appear in:
LWT - Food Science and Technology
Received Date: 31 May 2019 Revised Date:
24 October 2019
Accepted Date: 24 November 2019
Please cite this article as: Jiang, S., Xia, D., Zhang, D., Chen, G., Liu, Y., Analysis of protein profiles and peptides during in vitro gastrointestinal digestion of four Chinese dry-cured hams, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108881. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Analysis of protein profiles and peptides during in vitro gastrointestinal
2
digestion of four Chinese dry-cured hams
3
Shui Jianga,b, Dong Xiac, Danni Zhangb, Gaole Chenb, Yuan Liua,b*
4
a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100000, China
5 6
b
Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
7 8 9 10
c
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
*Corresponding author: Tel.: +86-021-34208536; Email: [email protected] (Y. Liu)
Abstract
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In this study, the in vitro digestion with pepsin and trypsin enzymes was applied to mimic the
12
gastrointestinal digestion of dry-cured hams from different producing districts of China. The analysis of
13
digestibility and particle size revealed that the digestion characteristics of different dry-cured hams were
14
significantly different (p < 0.05). During the gel electrophoresis analysis, protein profiles were characterized by
15
analyzing 14 and 9 proteins generated during pepsin and pepsin/trypsin digestion. The relative intensities of
16
protein bands could be used to distinguish different dry-cured hams based on principal component analysis
17
(PCA) and linear discriminant analysis (LDA). According to the analysis of peptide profiles, more than 50% of
18
peptides were distributed in the molecular weight ranging from 1500 to 2500 Da, and two-step digestion had
19
little effect on peptide generation due to the cleavage site specificity of pepsin and trypsin. This study provided
20
evidences that it was possible to identify producing districts of different dry-cured hams based on digested
21
characteristics.
22
Keywords: Dry-cured ham; Meat protein; Digestibility; In vitro digestion; Peptide 1
23
1. Introduction
24
Chinese dry-cured hams are increasingly appreciated by consumers because of the rose-red color, intense
25
flavor and desirable taste which are obtained from traditional processing including salting, shaping,
26
fermentation and post-ripening (Morales, Guerrero, Claret, Guàrdia, & Gou, 2008; Yang, Ma, Qiao, Song, & Du,
27
2005). Due to the extensive land in China, the actual manufacturing processes of dry-cured hams in different
28
regions are usually modified according to the local climate and culture. The modified process methods including
29
raw pork meat, saltiness and ripening process will affect the quality of dry-cured hams (Echegaray, Gómez,
30
Barba, Franco, Estévez, Carballo, Marszałek, & Lorenzo, 2018; Cilla, Martínez, Guerrero, Guàrdia, Arnau,
31
Altarriba, & Roncales, 2006; Wang, Xu, Zhang, Li, Lin, & Ma, 2011). Traditionally, the quality of dry-cured
32
hams are evaluated by sensory evaluation and physicochemical methods such as detections of volatile
33
compounds, colors and taste (Laureati, Buratti, Giovanelli, Corazzin, Lo Fiego, & Pagliarini, 2014;
34
Garcia-Gonzalez, Tena, Aparicio-Ruiz, & Aparicio, 2014; Lorido, Hort, Estévez, & Ventanas, 2016; Xia et al.,
35
2017). However, these physicochemical parameters could only characterize the eating quality but could not
36
reveal the nutritional quality of dry-cured hams. In the recent years, nutritional quality is becoming an
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increasingly important factor influencing food choices of consumers and processing optimization of meat
38
products (Bax et al., 2012; Zhou, et al., 2018). But at present, few data about the nutritional quality especially
39
protein bioavailability of Chinese dry-cured hams from different producing districts are available. Therefore, it
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is crucial to characterize the digestibility of different Chinese dry-cured hams in the gastrointestinal digestion.
41
Recently, the in vitro protein digestion has been developed to reveal the protein compositions of different
42
dry-cured hams (Zou et al., 2018). To simulate the gastrointestinal conditions, pepsin and trypsin are usually
43
used to enzymatically digest proteins (Li et al., 2017; Gallego, Mora, Hayes, Reig, & Toldrá, 2017). With the
2
44
application of gel electrophoresis method, protein profiles of many foods could be analyzed by the in vitro
45
digestion (Sanchon et al., 2018; Luo, Taylor, Nebl, Ng, & Bennett, 2018). In the abovementioned researches, the
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protein profiles are qualitatively and quantitatively analyzed by the molecular weight and band intensities of
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proteins in the gel electrophoresis analysis (Li et al., 2017; Wen et al., 2015a). However, in the most of these
48
studies, the protein profiles are only exhibited via a gel electrophoresis figure or a Table. To adequately use the
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protein profiles, the differences among dry-cured hams could be visualized based on multivariate statistical
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methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) (Jiang, Wang,
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Wang, & Cheng, 2017). The nutritional quality of meat was not only associated with protein profiles but also
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with peptide profiles (Escudero, Sentandreu, & Toldrá, 2010). In recent years, mass spectrum methods have
53
been developed to identify the peptides of digestion products obtained from different foods (Escudero, et al.,
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2010; Montowska, Rao, Alexander, Tucker, & Barrett, 2014). By comparing with existing sequence database,
55
the specific functional bioactivity of newly found peptides could be predicted. And this efficient method has
56
been used to mine new functional peptides from different food sources (Mora, Gallego, & Toldrá, 2018; Tu et al.,
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2018). However, few researches were conducted to study the peptide size and sequences during the digestion of
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dry-cured hams from different producing districts of China.
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The main objectives of this study were to characterize the protein profiles and peptide products of four
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dry-cured hams from different producing districts of China by the in vitro digestion combined with mass
61
spectrum methods.
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2. Materials and methods
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2.1 Reagents and instruments
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Pepsin from porcine gastric mucosa (P7125) with enzyme activity larger than 400 units/mg, and trypsin 3
65
from porcine pancreas (T7409) with the enzyme activity larger than 1645 units/mg were obtained from
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Sigma-Aldrich, St. Louis, Mo., U.S.A. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo
67
Scientific, Rockford, Ill., U.S.A. 4-12% Criterion™ XT Bis-Tris Protein Gel (3450125), XT Sample Buffer
68
(1610791), XT MES Running Buffer (1610789), and Precision Plus Protein™ Dual Xtra Prestained Protein
69
Standards (1610377) were obtained from Bio-Rad, Hercules, Calif., U.S.A. Amicon Ultra-0.5 Centrifugal Filter
70
Unit (UFC501008) and ZipTip with 0.6 µL C18 pipette tips (ZTC18S096) were obtained from Merck Millipore,
71
Tullagreen, Ireland. All other regents and chemicals were analytical grade and were used directly without
72
purification unless otherwise stated.
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High-speed refrigerated centrifuge (Avanti J-C, Beckman Coulter Company, U.S.A.), electronic analytical
74
balance (CPA2245T25, Sartorius Company, Germany), multi-mode microplate reader (Molecular Devices
75
Company, U.S.A.), LCG freezing dryer (Christ Company, Germany), medium 6 vertical electrophoresis system
76
(Bio-Rad Company, U.S.A.), pH meter (Fiveeasy, Mettler Toledo Company), particle size analyzer (Malvern
77
3000, Malvern Company, U.K.), homogenizer (T25, IKA, Germany), liquid chromatography mass spectrometry
78
(LC-MS) (Nano LC-LTQ-Orbitrap, Thermo Fisher Company, U.S.A.) were used.
79
2.2 Samples preparation
80
In this study, four kinds of dry-cured hams were provided by companies from different producing districts
81
of China, i.e., Xuanwei (XW), Sanchuan (SC), Nuodeng (ND) and Saba (SB). These dry-cured hams were
82
processed via the traditional method including salting, sun-drying shaping, ripening and post-ripening. A total
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24 hams (6 hams for each category) were used in the whole experiment. Biceps femoris portion of hams were
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cut into cubes (1 cm × 1 cm × 1 cm), and were then ground for 10 s by a pulverizer (4500 rpm) with liquid
85
nitrogen. Sample particles were stored in -20 °C refrigerator until further analysis.
4
86
2.3 In vitro digestion
87
In this study, the in vitro digestion was conducted by using both pepsin and trypsin to simulate human
88
gastrointestinal digestion of dry-cured hams. Before the in vitro digestion, the ham sample (1.0 g) was put into 4
89
mL of PBS (0.01 mol/L Na2HPO4-NaH2PO4, pH 7.0) and the homogenizer worked in the ice-bath environment.
90
All samples were separately homogenized for 2 × 30 s at 9600 rpm and 2 × 30 s at 13400 rpm with 30 s interval
91
among each homogenization. The sample solution was then adjusted to pH 2.0 ± 0.1 by adding 1 mol/L HCl
92
solution to simulate the gastric juice environment. Pepsin was added into the acidic solution to guarantee a
93
concentration of 0.032 g/mL, and 1 mL pepsin premix solution was added into sample solution. Then, the
94
mixture was incubated at 37 °C for 2 h with continuous shaking. After the reaction, the pepsin enzyme was
95
inactivated by adding 1 mol/L NaOH to adjust the pH to 7.5 ± 0.1. After the pepsin digestion, 1 mL 0.024 g/mL
96
trypsin solution was added into the solution, and trypsin digestion was maintained at 37 °C for 2 h with
97
continuous shaking. After digestion of pepsin and trypsin, the sample solution was heated in the boiling water
98
bath for 5 min to inactivate pepsin and trypsin enzymes. The digestion solution (1 mL) of pepsin and
99
pepsin/trypsin were deproteinized by adding 3 mL ethanol, and were then kept at 4 °C for 12 h. The
100
centrifugation was performed for 20 min on conditions of 10000 × g and 4 °C. The supernatants and precipitates
101
got from centrifugation were used for peptides determination and electrophoresis analysis, respectively.
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In this study, the digestibility was calculated to evaluate the digestion degree of protein when added pepsin
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or pepsin/trypsin. During the digestion procedure, 3.0 g sample was taken out from each kind of dry-cured hams
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and was processed according to the digestion method described above. The protein content before and after
105
digestion procedure were detected by BCA protein assay kit. The in vitro digestibility was calculated by using
106
the equation as follows:
DT = 107
WO − WD × 100% WO 5
(1)
108
Where DT represented the digestibility of protein when added different enzymes, WO and WD respectively
109
represented protein contents of samples before and after the digestion procedure.
110
2.4 Particle size measurements
111
To further investigate the influence of different enzyme digestions on dry-cured ham samples, the particle
112
size of homogenized products was measured. In this study, particle sizes of products from different sample
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solutions (i.e., untreated samples, pepsin digestion, and pepsin/trypsin digestion) were measured by the particle
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size analyzer (Malvern 3000) on the working conditions of the nonspherical type, relative refractive index (1.54),
115
absorptivity (0.001), and density (1 g/cm3).
116
2.5 Protein profiles based on the gel electrophoresis
117
In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to
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characterize the protein profiles of samples before and after in vitro digestion. After digestion of pepsin and
119
pepsin/trypsin, the precipitates obtained from centrifugation of sample solutions were used for the
120
electrophoresis analysis. Before the electrophoresis analysis, the protein sample solutions were adjusted to the
121
concentration of 1.5 µg/µL by adding XT sample buffer. Then, the solution was heated in 90 °C water bath for
122
20 min. 15 µL of each sample buffer was run in 4-12% Bis-Tris Protein Gel for 2 h at 120 V of voltage and 4 °C
123
of temperature, and six replicates were performed. The band intensities of protein profiles were quantified with
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the Quantity One software (Bio-Rad, U.S.A.). The relative intensity of each band was calculated by comparing
125
with 100 kDa band in the calibration marker lane. The relative band intensity was calculated by the following
126
equation:
127
128
RBI =
Intband Int100 kDa
(2)
Where the RBI is relative band intensity of target band, the Intband and Int100kDa are Gauss trace values of 6
129
target band and standard band (100kDa) extracted by Quantity One software.
130
2.6 Determination of peptide sequences in digestion products
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The supernatant obtained from centrifugation of digestion sample solutions was used for peptide sequences
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determination by using LC-MS. The supernatant was concentrated and ultra-filtered to purify macromolecule (>
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10 KDa). The concentrated solutions were further filtered in C18 pre-column (2 cm × 200 µm × 5 µm), and then
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were run in C18 analytical column (2 cm × 100 µm × 3 µm). Peptides were separated by step-gradient elution in
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mobile phase A (0.2% formic acid solution) and mobile B (0.2% formic acid in 60% acetonitrile solution) at a
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constant flow rate 300 nL/min. The gradient elution program was as follows: 0-12 min (97% A and 3% B),
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12-100 min (72% A and 28% B), 100-122 min (45% A and 55% B), 122-144 min (2% A and 98% B), 144-160
138
min (97% A and 3% B). MS conditions were as follows: 300-1800 scan ranges and 200 min scan time.
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For qualitative analysis of MS spectrum, the Proteome Discoverer (Thermo Fisher Scientific, Palo Alto,
140
CA, U.S.A.) were applied to identify peptides combined with Swiss-Prot database (Sus scrofa). Additionally,
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Venn diagrams were used to determine the similarity of peptides obtained from different dry-cured hams.
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Afterward, the bioactivity of identified peptides was determined by using PeptideRanker which was supplied by
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University College Dublin (http://bioware.ucd.ie/). Finally, the specific functional bioactivity of peptides was
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predicted by comparing with PepBank database.
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2.7 Statistical analysis
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In this study, PCA and LDA were performed by Statistical Product and Service Solutions 22.0 version
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(International Business Machines Corporation, U.S.A.). Differences among different samples on protein profiles
148
were analyzed by one-way analysis of variance (ANOVA) followed by Turkey’s test and considered
149
significantly at p < 0.05. The measured results were expressed as means ± standard deviation (SD) over six
150
independent experiments. 7
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3. Results and discussion
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3.1 The in vitro digestibility
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The in vitro digestibility results were shown in Fig. 1. The one-way ANOVA result reveled that there were
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statistically significant differences among digestibility values of different samples. After pepsin digestion, the
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digestibility of XW, SC, ND and SB were 55.25 ± 7.20%, 47.61 ± 5.94%, 46.09 ± 3.95% and 36.27 ± 6.31%,
156
respectively. After being further treated by pepsin/trypsin, proteins of XW, SC, ND and SB were adequately
157
digested and the digestibility were increased to 77.74 ± 2.87%,70.19 ± 3.34%, 67.41 ± 2.82% and 66.08 ±
158
1.91%, respectively.
159
Zhou et al. (2019) revealed that lower salt contents effected release rates of cathepsin B and cathepsin L
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which may be responsible for the intense degradation of proteins during the ripening. According to the
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processing standard of China (GB/T 18357-2008, product of geographical indication-Xuanwei ham), high salt
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contents (12.5%) in XW hams could provide a protection of proteins during the post-ripening, which resulted in
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a high initial protein contents (Huang, Ge, & Huang, 2010; Yang et al., 2005). After being treated by pepsin and
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pepsin/trypsin, the proteins were enzymatically digested to a greater extent, and the losses of protein contents
165
were relatively larger than other samples. Therefore, XW hams had the largest digestibility after pepsin and
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pepsin/trypsin digestion. Compared with other samples, SB hams were processed with low-salt technic which
167
resulted in the rapid degradation of proteins during ripening. Therefore, the initial protein contents of SB hams
168
were relatively low which resulted in a low digestibility.
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3.2 Analysis of particle sizes
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The particle sizes of different dry-cured hams were measured to analyze the changes of digestibility, and
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the results of particle sizes were listed in Table 1. According to the ANOVA analysis, the particle sizes of
8
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untreated XW hams were significantly different from others. The pork resources used for XW dry-cured hams
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were different from others (GB/T 18357-2008), and the meat quality of growing pigs could be influenced by
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animal breed, dietary source and growing environment (Doti, Suárez-Belloch, Latorre, Guada, & Fondevila,
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2014). Additionally, the XW hams were aged for a longer time (more than 10 months) which resulted in a larger
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particle sizes of materials (Degnes, Kvitvang, Haslene-Hox, & Aasen, 2017).
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As shown in Table 1, after pepsin digestion, there was no significant difference among hams. For XW
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hams, it could be calculated that the decreasing rates of large-size particles (Dx (90)) and middle size particles (Dx
179
(50)
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middle size particles (Dx (50)) were decreased by 38.7% and 37.1% after trypsin digestion. The results indicated
181
that the large-size particles (> 310 µm) were mainly digested (27.5%) during the pepsin digestion. The
182
middle-size particles (> 190 µm) could be digested by trypsin digestion (38.7%). The particle size decreasing
183
rates of XW hams were the largest among four samples, which was in accordance with digestibility analysis.
184
The reason might be that pepsin enzyme was suitable for digesting large-size particles (Li et al., 2017).
185
Moreover, the trypsin cleavages were more consistent when treated different ham samples (Wen et al., 2015a).
186
Therefore, XW hams were more suitable for consumers with weak function of intestinal digestion.
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3.3 Analysis of SDS-PAGE results
) were respective 27.5% and 12.6% after the pepsin digestion. Afterward, the large-size particles (Dx (90)) and
188
The result of SDS-PAGE was shown in Fig. 2. It could be concluded from different gel lanes that there
189
were significant differences among SDS-PAGE profiles. For qualitative analysis of protein profiles, the
190
molecular weight of each protein was determined based on the standard marker lane. In this study, the relative
191
band intensity was calculated by comparing with 100 kDa band in the calibration marker lane, and the result
192
was shown in Table 2. Total 14 bands were marked and selected as characteristic bands of four dry-cured hams
193
before the digestion. The protein bands distribution of dry-cured hams before digestion was similar to those of 9
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pork products in the published article (Li et al., 2017). Moreover, there were lots of proteins with high molecular
195
weight (37-75 kDa) existed in the profiles of four dry-cured hams especially in ND hams. The ANOVA analysis
196
results revealed that the relative intensities of bands (> 50 kDa) were significantly different among different
197
hams.
198
After pepsin digestion, the proteins with high molecular weight were enzymatically digested to small
199
molecular weight proteins. As shown in Table 2, 9 bands (10-50 kDa) were marked as characteristic bands of
200
four dry-cured hams. During the pepsin digestion, most high molecular weight proteins were hydrolyzed to
201
small molecular weight proteins. For instance, the proteins such as 99 kDa, 72 kDa, 41 kDa and 39 kDa were
202
digested and the relative intensities of some proteins such as 143 kDa, 55 kDa, 47 kDa and 25 kDa decreased
203
prominently. Additionally, small molecular weight proteins such as 12 kDa and 9 kDa occurred and the relative
204
intensities increased significantly. After the pepsin/trypsin digestion, almost all the proteins with high molecular
205
weight were hydrolyzed and only protein fragments with small molecular weight could be detected by the
206
electrophoresis. Additionally, there was no significant difference on particle size (Table 1) among hams.
207
Therefore, comparing to pepsin, there were more cleavage sites in proteins for trypsin, which resulted in an
208
adequate digestion (Erickson & Kim, 1990).
209
To further analyze the difference of samples, PCA and LDA were applied to visualize the data space of
210
protein profiles and to qualitatively discriminate different samples. The relative intensities of 14 bands and 9
211
bands before and after pepsin digestion were selected as features to do PCA and LDA analysis, and the results
212
were shown in Fig. 3. According to SDS-PAGE profiles of proteins (Fig. 2), almost no protein bands could be
213
detected by the electrophoresis after pepsin/trypsin digestion. Therefore, there was no features (relative
214
intensities) could be used for PCA and LDA visualization, which was the reason that only PCA and LDA results
215
based on protein profiles before and after pepsin digestion were presented in Fig. 3. Three-dimensional plots of
10
216
PCA and LDA were shown in Fig. 3(a) and (b). The first three PCs were calculated to characterize the data
217
space of protein profiles and 56.6% information of the variance was contained in the score plot. In the PCA plot,
218
all sample groups overlapped to some extent and could not be distinguished from each other. As shown in Fig.
219
3(b), the first three function scores could present almost 100% useful information of original protein profiles. In
220
LDA plot, sample groups of XW, ND, SC and SB could be separated to different districts and there were clear
221
boundaries among each group.
222
According to the results of protein profiles before and after pepsin digestion, the relative intensities of
223
some original characteristic bands were changed significantly and some new protein bands were produced.
224
Subsequently, the relative intensities of these protein bands mentioned above were used to do PCA and LDA,
225
and the results were shown in Fig. 3(c) and (d). Total 61.9% useful information of the variance were represented
226
by the first three PCs. In the PCA plot, all the sample groups were mixed into one and could not be separated
227
from each other, which was in accordance with the analysis results of protein profiles. LDA was then applied to
228
analyze protein profiles, and all information contained in original data could be expressed by the first three
229
function scores, as shown in Fig. 3(d). In the LDA plot, sample groups of XW, ND and SC could be
230
distinguished clearly from each other. However, sample group of SB was close to that of ND, which could be
231
explained by that relative intensities of almost all protein bands obtained from SB and ND were very similar.
232
The reason might be that SB and ND hams were aged for same time in a similar weather of moderate rainfall.
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Furthermore, the internal quality of dry-cured hams such as protein profiles were significantly influenced by the
234
aging time and pork resources (Paolella et al., 2015; Wang et al., 2018). According to the results of PCA and
235
LDA, the protein profiles could be used to qualitatively and quantitatively analyze different dry-cured hams.
236
3.4 Peptide profiling of dry-cured hams
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In this study, the digestion sample solutions obtained from different dry-cured hams were analyzed by 11
238
using LC-MS to determine the peptide sequence, and the mass spectrums of different hams were listed in
239
Supplementary information (Fig. S1). According to the LC-MS results, the numbers of peptides collected from
240
XW, SC, ND and SB samples after pepsin digestion were 117, 105, 126 and 118, respectively. After trypsin
241
digestion, the numbers of obtained peptides were respectively increased to 331, 366, 336 and 293, the result was
242
shown in Fig. 4. More proteins were digested to fragments during trypsin digestion, which was in accordance
243
with the result of published article in which dry-cured hams were analyzed (Li et al., 2017). However, the
244
numbers of peptides in pepsin digestion and pepsin/trypsin digestion were significantly different from fresh pork
245
(Wen et al., 2015b). The reason might be that salting and post-ripening changed the internal quality of meat.
246
These peptides were then sequenced and Venn diagrams were performed. The Venn diagrams of peptides
247
obtained from different dry-cured hams were shown in Fig. 5. Among these sequenced peptides, 48 and 171
248
peptides were common for different dry-cured hams after pepsin and pepsin/trypsin digestion. Escudero (2010)
249
reported that peptides detected in vitro digested fresh pork were obtained from muscle protein including actin,
250
myosin and kinase. The molecular weight of peptides could be mainly divided into three groups (i.e., 800 to
251
1500 Da, 1500 to 2500 Da and 2500 to 3500 Da), and the result was shown in Fig. 6. The most of peptides (>
252
50%) belonged to 1500 to 2500 Da, and peptide numbers of XW, SC, ND and SB were 58, 55, 68 and 65. After
253
digestion of pepsin/trypsin, there were 200, 198, 199 and 171 peptides distributed into 1500 to 2500 Da, and
254
occupied 60.42%, 54.37%, 58.92% and 58.36% of total peptides, respectively. As shown in Fig. 5(a), total 185
255
peptides with different sequences were identified in the Venn diagram of pepsin digestion, and 48 common
256
peptides (14, 13, 15 and 15 peptides were from XW, SC, ND and SB) were identified. As shown in Fig. 5(b),
257
total 453 peptides with different sequences were identified after pepsin/trypsin digestion, and the number of
258
specific peptides from XW, SC, ND and SB were 25, 57, 20 and 21, respectively.
259
Subsequently, peptides were matched with porcine muscle proteins against the Swiss-Prot database (Sus
12
260
scrofa). In this study, most peptides were mainly identified as fragment from several myofibrillar proteins such
261
as tropomyosin, actin, titin and myosin, and several sarcoplasmic proteins such as glyceraldehyde-3-phosphate
262
dehydrogenase, phosphorylase, creatine kinase, 6-phosphofructokinase. The protein profiles of different hams
263
were in accordance with those of cooked pork due to that biceps femoris was used in this study (Wen et al.,
264
2015a; Zou et al., 2018). The bioactivity of obtained peptides were determined by comparing with Swiss-Prot
265
database and PepBank database. In XW, SC, ND and SB dry-cured hams, 25, 24, 26 and 27 functional peptides
266
were found and showed the antioxidant bioactivity. Additionally, one peptide (MNVKHWPWMKL) obtained
267
from four ham samples after pepsin digestion might have the bioactivity angiotensin-converting enzyme
268
inhibitor (ACEI). In this study, the ACEI peptides were obtained from the pepsin digestion of actin and myosin,
269
and the bioactivity could be affected by processing and storage conditions (Mora et al., 2018). After trypsin
270
digestion, another 42 new functional peptides were generated, which indicated that original peptides were
271
constantly digested and new peptides generated. This corresponded to the analysis of SDS-PAGE that almost all
272
of the bands were digested after pepsin/trypsin digestion. The result suggest that dry-cured hams could be
273
resources of natural peptides which had positive effect on health (Gallego, Mora, Hayes, Reig, & Toldrá, 2019).
274
Moreover, it was found that 8.4-17.3% of total peptide numbers were identified as fragment obtained from
275
myosin-1.The location of identified peptides after pepsin and pepsin/trypsin digestion were shown as an
276
example in Supplementary information (Table S1 and Table S2). According to the location of peptides on the
277
myosin-1 proteins, it could be concluded that the cleavage sites of pepsin and trypsin were different. For the
278
pepsin digestion, it could be found that peptides generated in pepsin digestion with a wide range of cleavage
279
sites such as Leu, Phe, Arg and Lys. After further trypsin digestion, some new peptides were generated with the
280
carbon-terminal cleavage sites as Lys and Ary. The cleavage site specificity of pepsin and trypsin was in
281
accordance with the summary by the European Molecular Biology Laboratory (https://www.ebi.ac.uk/) and
13
282
published articles (Alves et al., 2007; Furlong, Mauger, Strimpler, Liu, Morris, & Edwards, 2002). Although
283
protein formations of dry-cured hams could be changed due to different producing districts, generated peptides
284
during the in vitro digestion changed little because of the cleavage site specificity of pepsin and trypsin. In
285
summary, dry-cured hams from different producing districts had significant effect on in vitro digestibility of
286
proteins but little effect on peptide generation during the pepsin and pepsin/trypsin digestion.
287
4. Conclusions
288
The protein profiles of dry-cured hams from different producing districts were significantly different and
289
would affect the protein digestion in the gastrointestinal digestion. In this study, the in vitro digestion with
290
pepsin and trypsin enzymes was applied to simulate the gastrointestinal digestion procedure. The analysis of
291
digestibility and particle size revealed that the digestion characteristics of dry-cured hams were different, and
292
XW hams were more suitable for consumers with weak function of intestinal digestion. The SDS-PAGE data
293
could be used to distinguish different dry-cured hams based on PCA and LDA. According to the analysis of
294
peptide profiles, most peptides were identified as fragment of myosin-1 protein, and two-step digestion had little
295
effect on peptide generation due to the cleavage site specificity of pepsin and trypsin. This study provided
296
evidences that it was possible to identify producing districts of different dry-cured hams based on digested
297
characteristics. In the subsequent work, more efforts should be focused on bioactivity identification of peptides
298
in digested products.
299
Acknowledgements
300
This work was funded by Beijing Advanced Innovation Center for Food Nutrition and Human Health and
301
Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), National 14
302
Natural Science Foundation of China (Grant no. 31540087), and the National Key Research and Development
303
Program of China (2016YFD0400803, 2016YFD0401501).
304
Conflicts of interest
305
The authors declare that they have no conflicts of interest.
306
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18
Captions of Tables and Figures Table 1 Particle sizes of four dry-cured hams with different treatments. Table 2 Band intensities of proteins before and after pepsin digestion.
Figure 1 Digestibility in vitro of four dry-cured hams treated with pepsin and pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Different letters on the same samples indicate statistically significant difference among the values (p < 0.05). Figure 2 SDS-PAGE profiles of total proteins obtained from four dry-cured hams with different treatments: (a) before digestion, (b) after digestion with pepsin, (c) after digestion with pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 3 Results of PCA and LDA: (a) PCA and (b) LDA plots based on SDS-PAGE data before digestion; (c) PCA and (d) LDA plots based on SDS-PAGE data after pepsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 4 Total numbers of identified peptides from four dry-cured hams. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 5 Venn diagrams of peptides obtained from different dry-cured hams: (a) In vitro digestion with pepsin; (b) In vitro digestion with pepsin followed by trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 6 Peptide numbers on the basis of molecular weights from four dry-cured hams. (a) Pepsin digestion; (b) Pepsin/trypsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. 19
Table 1 Particle sizes of four dry-cured hams with different treatments.
Samples
Dx (10)/µm
Dx (50) /µm
Dx (90) /µm
D [4,3] /µm
D [3,2] /µm
XW
9.26±2.19 a
121.83±5.91 a
427.50±35.06 a
174.67±10.39 a
28.62±4.01 a
SC
9.21±1.41 a
106.83±5.42 b
348.50±28.67 b
147.00±8.49 b
29.50±2.77 a
ND
8.76±1.37 a
101.27±13.84 b
374.67±25.26 b
150.00±21.38 b
26.73±3.22 ab
SB
6.90±0.78 b
103.67±7.52 b
386.00±13.87 ab
151.00±3.16 b
23.67±2.39 b
XW
10.92±2.90 a
106.43±9.07 a
310.00±41.59 a
138.00±13.65 a
29.60±5.85 a
SC
7.77±1.05 a
88.47±10.59 b
258.67±33.22 a
118.33±14.39 a
23.63±3.48 a
ND
10.75±3.25a
90.22±13.84 b
272.33±44.37 a
120.10±18.44 a
28.80±5.88 a
SB
8.58±2.45 a
97.13±12.98 ab
297.17±43.77 a
128.50±17.00 a
24.63±3.62 a
XW
6.05±0.62 b
67.00±11.40 ab
190.17±19.77 a
83.70±10.96 a
18.40±2.59 b
SC
7.08±0.88 a
71.78±4.76 a
184.00±9.59 a
88.82±5.32 a
24.93±3.98 a
ND
5.90±0.31 b
57.02±10.45 ab
171.67±15.65 a
74.70±8.51 a
17.65±1.71 b
SB
5.75±0.29 b
53.73±14.11 b
191.17±14.79a
77.32±17.21 a
16.17±1.29 b
Untreated
Pepsin digestion
Pepsin + trypsin digestion
Values were expressed as means (n = 6) ± SD. SD: standard deviation. XW, SC, ND and SB were samples from Xuanwei,
Sanchuan, Nuodeng and Saba, respectively. Means with different superscripts (a, b) in the same column were statistically different
(p < 0.05).
20
Table 2 Band intensities of proteins before and after pepsin digestion. Band intensities (mean ± SD)
Molecular weight Bands (kDa)
XW
ND
SC
SB
Untreated 1
143.13
0.27 ± 0.09ab
0.16 ± 0.12ab
0.10 ± 0.07b
0.34 ± 0.18a
2
99.05
0.07 ± 0.03bc
0.03 ± 0.01c
0.19 ± 0.07a
0.17 ± 0.12ab
3
87.30
0.60 ± 0.39a
0.62 ± 0.25a
0.44 ± 0.24a
0.59 ± 0.51a
4
72.07
0.81 ± 0.40a
0.54 ± 0.20ab
0.29 ± 0.25b
0.38 ± 0.22ab
5
66.46
0.23 ± 0.16a
0.41 ± 0.20ab
0.75 ± 0.30a
0.51 ± 0.21ab
6
55.09
0.42 ± 0.15a
0.84 ± 0.52a
0.44 ± 0.05a
0.4 0± 0.21a
7
46.92
0.80 ± 0.26a
0.45 ± 0.23b
0.34 ± 0.09b
0.46 ± 0.10b
8
41.04
0.74 ± 0.52a
0.14 ± 0.13b
0.37 ± 0.29ab
0.22 ± 0.17ab
9
37.96
1.86 ± 1.04a
2.19 ± 0.46a
1.78 ± 1.09a
1.6 0± 0.74a
10
34.75
1.31 ± 0.40a
0.28 ± 0.23b
1.08 ± 1.02ab
1.33 ± 0.36a
11
32.66
0.49 ± 0.19a
0.49 ± 0.16a
0.20 ± 0.18ab
nd
12
27.75
0.21 ± 0.09a
0.18 ± 0.10a
0.54 ± 0.81a
0.14 ± 0.04a
13
25.50
1.55 ± 0.70a
0.50 ± 0.29a
1.57 ± 0.90a
1.77 ± 1.07a
14
18.11
1.01 ± 0.58a
0.51 ± 0.39ab
0.32 ± 0.13b
0.63 ± 0.10ab
1
144.14
0.14 ± 0.09a
0.18 ± 0.13a
0.22 ± 0.08a
0.17 ± 0.10a
2
87.31
0.85 ± 0.52a
0.47 ± 0.45a
0.54 ± 0.87a
0.67 ± 0.64a
3
66.12
0.16 ± 0.09a
0.12 ± 0.09a
0.68 ± 0.30a
0.41 ± 0.69a
4
53.81
0.25 ± 0.10a
0.15 ± 0.05a
0.42 ± 0.51a
0.10 ± 0.07a
5
47.58
0.13 ± 0.09a
0.33 ± 0.34a
0.22 ± 0.18a
0.10 ± 0.08a
6
30.10
0.20 ± 0.10a
0.94 ± 1.31a
0.40 ± 0.22a
0.4 0± 0.33a
7
24.12
0.86 ± 0.41a
0.48 ± 0.35a
0.88 ± 0.28a
0.60 ± 0.47a
8
11.56
0.57 ± 0.52a
0.59 ± 0.71a
1.11 ± 1.71a
0.62 ± 0.67a
9
8.98
1.65 ± 1.11a
1.10 ± 1.08a
2.01 ± 0.95a
0.95 ± 0.83a
Pepsin digestion
Values were expressed as means (n = 6) ± SD. SD: standard deviation. nd: not detected. XW, SC, ND and SB were samples from
Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Means with different superscripts (a, b, c) in the same row were statistically
different (p < 0.05).
21
Fig. 1 Digestibility in vitro of four dry-cured hams treated with pepsin and pepsin + trypsin. XW, SC, ND and SB were samples
from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Different letters on the same samples indicate statistically significant
difference among the values (p < 0.05).
22
Fig. 2 SDS-PAGE profiles of total proteins obtained from four dry-cured hams with different treatments: (a) before digestion, (b)
after digestion with pepsin, (c) after digestion with pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan,
Nuodeng and Saba, respectively.
23
Fig. 3 Results of PCA and LDA: (a) PCA and (b) LDA plots based on SDS-PAGE data before digestion; (c) PCA and (d) LDA
plots based on SDS-PAGE data after pepsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and
Saba, respectively.
24
Fig. 4 Total numbers of identified peptides from four dry-cured hams. XW, SC, ND and SB were samples from Xuanwei, Sanchuan,
Nuodeng and Saba, respectively.
25
Fig. 5 Venn diagrams of peptides obtained from different dry-cured hams: (a) In vitro digestion with pepsin; (b) In vitro digestion
with pepsin followed by trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively.
26
Fig. 6 Peptide numbers on the basis of molecular weights from four dry-cured hams. (a) Pepsin digestion; (b) Pepsin/trypsin
digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively.
27
Graphical Abstract
28
Highlights Using in vitro digestion of four types dry-cured hams with pepsin and trypsin PCA and LDA can distinguish different dry-cured hams based on protein profiles Digestibility and particle size of different hams are significantly different Peptides of in vitro digestion products are distributed in ranges of 1500 to 2500 Da Two-step digestion with pepsin and trypsin had little effect on peptide generation
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
S0023-6438(19)31223-X
DOI:
https://doi.org/10.1016/j.lwt.2019.108881
Reference:
YFSTL 108881
To appear in:
LWT - Food Science and Technology
Received Date: 31 May 2019 Revised Date:
24 October 2019
Accepted Date: 24 November 2019
Please cite this article as: Jiang, S., Xia, D., Zhang, D., Chen, G., Liu, Y., Analysis of protein profiles and peptides during in vitro gastrointestinal digestion of four Chinese dry-cured hams, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108881. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Analysis of protein profiles and peptides during in vitro gastrointestinal
2
digestion of four Chinese dry-cured hams
3
Shui Jianga,b, Dong Xiac, Danni Zhangb, Gaole Chenb, Yuan Liua,b*
4
a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100000, China
5 6
b
Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
7 8 9 10
c
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
*Corresponding author: Tel.: +86-021-34208536; Email: [email protected] (Y. Liu)
Abstract
11
In this study, the in vitro digestion with pepsin and trypsin enzymes was applied to mimic the
12
gastrointestinal digestion of dry-cured hams from different producing districts of China. The analysis of
13
digestibility and particle size revealed that the digestion characteristics of different dry-cured hams were
14
significantly different (p < 0.05). During the gel electrophoresis analysis, protein profiles were characterized by
15
analyzing 14 and 9 proteins generated during pepsin and pepsin/trypsin digestion. The relative intensities of
16
protein bands could be used to distinguish different dry-cured hams based on principal component analysis
17
(PCA) and linear discriminant analysis (LDA). According to the analysis of peptide profiles, more than 50% of
18
peptides were distributed in the molecular weight ranging from 1500 to 2500 Da, and two-step digestion had
19
little effect on peptide generation due to the cleavage site specificity of pepsin and trypsin. This study provided
20
evidences that it was possible to identify producing districts of different dry-cured hams based on digested
21
characteristics.
22
Keywords: Dry-cured ham; Meat protein; Digestibility; In vitro digestion; Peptide 1
23
1. Introduction
24
Chinese dry-cured hams are increasingly appreciated by consumers because of the rose-red color, intense
25
flavor and desirable taste which are obtained from traditional processing including salting, shaping,
26
fermentation and post-ripening (Morales, Guerrero, Claret, Guàrdia, & Gou, 2008; Yang, Ma, Qiao, Song, & Du,
27
2005). Due to the extensive land in China, the actual manufacturing processes of dry-cured hams in different
28
regions are usually modified according to the local climate and culture. The modified process methods including
29
raw pork meat, saltiness and ripening process will affect the quality of dry-cured hams (Echegaray, Gómez,
30
Barba, Franco, Estévez, Carballo, Marszałek, & Lorenzo, 2018; Cilla, Martínez, Guerrero, Guàrdia, Arnau,
31
Altarriba, & Roncales, 2006; Wang, Xu, Zhang, Li, Lin, & Ma, 2011). Traditionally, the quality of dry-cured
32
hams are evaluated by sensory evaluation and physicochemical methods such as detections of volatile
33
compounds, colors and taste (Laureati, Buratti, Giovanelli, Corazzin, Lo Fiego, & Pagliarini, 2014;
34
Garcia-Gonzalez, Tena, Aparicio-Ruiz, & Aparicio, 2014; Lorido, Hort, Estévez, & Ventanas, 2016; Xia et al.,
35
2017). However, these physicochemical parameters could only characterize the eating quality but could not
36
reveal the nutritional quality of dry-cured hams. In the recent years, nutritional quality is becoming an
37
increasingly important factor influencing food choices of consumers and processing optimization of meat
38
products (Bax et al., 2012; Zhou, et al., 2018). But at present, few data about the nutritional quality especially
39
protein bioavailability of Chinese dry-cured hams from different producing districts are available. Therefore, it
40
is crucial to characterize the digestibility of different Chinese dry-cured hams in the gastrointestinal digestion.
41
Recently, the in vitro protein digestion has been developed to reveal the protein compositions of different
42
dry-cured hams (Zou et al., 2018). To simulate the gastrointestinal conditions, pepsin and trypsin are usually
43
used to enzymatically digest proteins (Li et al., 2017; Gallego, Mora, Hayes, Reig, & Toldrá, 2017). With the
2
44
application of gel electrophoresis method, protein profiles of many foods could be analyzed by the in vitro
45
digestion (Sanchon et al., 2018; Luo, Taylor, Nebl, Ng, & Bennett, 2018). In the abovementioned researches, the
46
protein profiles are qualitatively and quantitatively analyzed by the molecular weight and band intensities of
47
proteins in the gel electrophoresis analysis (Li et al., 2017; Wen et al., 2015a). However, in the most of these
48
studies, the protein profiles are only exhibited via a gel electrophoresis figure or a Table. To adequately use the
49
protein profiles, the differences among dry-cured hams could be visualized based on multivariate statistical
50
methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) (Jiang, Wang,
51
Wang, & Cheng, 2017). The nutritional quality of meat was not only associated with protein profiles but also
52
with peptide profiles (Escudero, Sentandreu, & Toldrá, 2010). In recent years, mass spectrum methods have
53
been developed to identify the peptides of digestion products obtained from different foods (Escudero, et al.,
54
2010; Montowska, Rao, Alexander, Tucker, & Barrett, 2014). By comparing with existing sequence database,
55
the specific functional bioactivity of newly found peptides could be predicted. And this efficient method has
56
been used to mine new functional peptides from different food sources (Mora, Gallego, & Toldrá, 2018; Tu et al.,
57
2018). However, few researches were conducted to study the peptide size and sequences during the digestion of
58
dry-cured hams from different producing districts of China.
59
The main objectives of this study were to characterize the protein profiles and peptide products of four
60
dry-cured hams from different producing districts of China by the in vitro digestion combined with mass
61
spectrum methods.
62
2. Materials and methods
63
2.1 Reagents and instruments
64
Pepsin from porcine gastric mucosa (P7125) with enzyme activity larger than 400 units/mg, and trypsin 3
65
from porcine pancreas (T7409) with the enzyme activity larger than 1645 units/mg were obtained from
66
Sigma-Aldrich, St. Louis, Mo., U.S.A. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo
67
Scientific, Rockford, Ill., U.S.A. 4-12% Criterion™ XT Bis-Tris Protein Gel (3450125), XT Sample Buffer
68
(1610791), XT MES Running Buffer (1610789), and Precision Plus Protein™ Dual Xtra Prestained Protein
69
Standards (1610377) were obtained from Bio-Rad, Hercules, Calif., U.S.A. Amicon Ultra-0.5 Centrifugal Filter
70
Unit (UFC501008) and ZipTip with 0.6 µL C18 pipette tips (ZTC18S096) were obtained from Merck Millipore,
71
Tullagreen, Ireland. All other regents and chemicals were analytical grade and were used directly without
72
purification unless otherwise stated.
73
High-speed refrigerated centrifuge (Avanti J-C, Beckman Coulter Company, U.S.A.), electronic analytical
74
balance (CPA2245T25, Sartorius Company, Germany), multi-mode microplate reader (Molecular Devices
75
Company, U.S.A.), LCG freezing dryer (Christ Company, Germany), medium 6 vertical electrophoresis system
76
(Bio-Rad Company, U.S.A.), pH meter (Fiveeasy, Mettler Toledo Company), particle size analyzer (Malvern
77
3000, Malvern Company, U.K.), homogenizer (T25, IKA, Germany), liquid chromatography mass spectrometry
78
(LC-MS) (Nano LC-LTQ-Orbitrap, Thermo Fisher Company, U.S.A.) were used.
79
2.2 Samples preparation
80
In this study, four kinds of dry-cured hams were provided by companies from different producing districts
81
of China, i.e., Xuanwei (XW), Sanchuan (SC), Nuodeng (ND) and Saba (SB). These dry-cured hams were
82
processed via the traditional method including salting, sun-drying shaping, ripening and post-ripening. A total
83
24 hams (6 hams for each category) were used in the whole experiment. Biceps femoris portion of hams were
84
cut into cubes (1 cm × 1 cm × 1 cm), and were then ground for 10 s by a pulverizer (4500 rpm) with liquid
85
nitrogen. Sample particles were stored in -20 °C refrigerator until further analysis.
4
86
2.3 In vitro digestion
87
In this study, the in vitro digestion was conducted by using both pepsin and trypsin to simulate human
88
gastrointestinal digestion of dry-cured hams. Before the in vitro digestion, the ham sample (1.0 g) was put into 4
89
mL of PBS (0.01 mol/L Na2HPO4-NaH2PO4, pH 7.0) and the homogenizer worked in the ice-bath environment.
90
All samples were separately homogenized for 2 × 30 s at 9600 rpm and 2 × 30 s at 13400 rpm with 30 s interval
91
among each homogenization. The sample solution was then adjusted to pH 2.0 ± 0.1 by adding 1 mol/L HCl
92
solution to simulate the gastric juice environment. Pepsin was added into the acidic solution to guarantee a
93
concentration of 0.032 g/mL, and 1 mL pepsin premix solution was added into sample solution. Then, the
94
mixture was incubated at 37 °C for 2 h with continuous shaking. After the reaction, the pepsin enzyme was
95
inactivated by adding 1 mol/L NaOH to adjust the pH to 7.5 ± 0.1. After the pepsin digestion, 1 mL 0.024 g/mL
96
trypsin solution was added into the solution, and trypsin digestion was maintained at 37 °C for 2 h with
97
continuous shaking. After digestion of pepsin and trypsin, the sample solution was heated in the boiling water
98
bath for 5 min to inactivate pepsin and trypsin enzymes. The digestion solution (1 mL) of pepsin and
99
pepsin/trypsin were deproteinized by adding 3 mL ethanol, and were then kept at 4 °C for 12 h. The
100
centrifugation was performed for 20 min on conditions of 10000 × g and 4 °C. The supernatants and precipitates
101
got from centrifugation were used for peptides determination and electrophoresis analysis, respectively.
102
In this study, the digestibility was calculated to evaluate the digestion degree of protein when added pepsin
103
or pepsin/trypsin. During the digestion procedure, 3.0 g sample was taken out from each kind of dry-cured hams
104
and was processed according to the digestion method described above. The protein content before and after
105
digestion procedure were detected by BCA protein assay kit. The in vitro digestibility was calculated by using
106
the equation as follows:
DT = 107
WO − WD × 100% WO 5
(1)
108
Where DT represented the digestibility of protein when added different enzymes, WO and WD respectively
109
represented protein contents of samples before and after the digestion procedure.
110
2.4 Particle size measurements
111
To further investigate the influence of different enzyme digestions on dry-cured ham samples, the particle
112
size of homogenized products was measured. In this study, particle sizes of products from different sample
113
solutions (i.e., untreated samples, pepsin digestion, and pepsin/trypsin digestion) were measured by the particle
114
size analyzer (Malvern 3000) on the working conditions of the nonspherical type, relative refractive index (1.54),
115
absorptivity (0.001), and density (1 g/cm3).
116
2.5 Protein profiles based on the gel electrophoresis
117
In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to
118
characterize the protein profiles of samples before and after in vitro digestion. After digestion of pepsin and
119
pepsin/trypsin, the precipitates obtained from centrifugation of sample solutions were used for the
120
electrophoresis analysis. Before the electrophoresis analysis, the protein sample solutions were adjusted to the
121
concentration of 1.5 µg/µL by adding XT sample buffer. Then, the solution was heated in 90 °C water bath for
122
20 min. 15 µL of each sample buffer was run in 4-12% Bis-Tris Protein Gel for 2 h at 120 V of voltage and 4 °C
123
of temperature, and six replicates were performed. The band intensities of protein profiles were quantified with
124
the Quantity One software (Bio-Rad, U.S.A.). The relative intensity of each band was calculated by comparing
125
with 100 kDa band in the calibration marker lane. The relative band intensity was calculated by the following
126
equation:
127
128
RBI =
Intband Int100 kDa
(2)
Where the RBI is relative band intensity of target band, the Intband and Int100kDa are Gauss trace values of 6
129
target band and standard band (100kDa) extracted by Quantity One software.
130
2.6 Determination of peptide sequences in digestion products
131
The supernatant obtained from centrifugation of digestion sample solutions was used for peptide sequences
132
determination by using LC-MS. The supernatant was concentrated and ultra-filtered to purify macromolecule (>
133
10 KDa). The concentrated solutions were further filtered in C18 pre-column (2 cm × 200 µm × 5 µm), and then
134
were run in C18 analytical column (2 cm × 100 µm × 3 µm). Peptides were separated by step-gradient elution in
135
mobile phase A (0.2% formic acid solution) and mobile B (0.2% formic acid in 60% acetonitrile solution) at a
136
constant flow rate 300 nL/min. The gradient elution program was as follows: 0-12 min (97% A and 3% B),
137
12-100 min (72% A and 28% B), 100-122 min (45% A and 55% B), 122-144 min (2% A and 98% B), 144-160
138
min (97% A and 3% B). MS conditions were as follows: 300-1800 scan ranges and 200 min scan time.
139
For qualitative analysis of MS spectrum, the Proteome Discoverer (Thermo Fisher Scientific, Palo Alto,
140
CA, U.S.A.) were applied to identify peptides combined with Swiss-Prot database (Sus scrofa). Additionally,
141
Venn diagrams were used to determine the similarity of peptides obtained from different dry-cured hams.
142
Afterward, the bioactivity of identified peptides was determined by using PeptideRanker which was supplied by
143
University College Dublin (http://bioware.ucd.ie/). Finally, the specific functional bioactivity of peptides was
144
predicted by comparing with PepBank database.
145
2.7 Statistical analysis
146
In this study, PCA and LDA were performed by Statistical Product and Service Solutions 22.0 version
147
(International Business Machines Corporation, U.S.A.). Differences among different samples on protein profiles
148
were analyzed by one-way analysis of variance (ANOVA) followed by Turkey’s test and considered
149
significantly at p < 0.05. The measured results were expressed as means ± standard deviation (SD) over six
150
independent experiments. 7
151
3. Results and discussion
152
3.1 The in vitro digestibility
153
The in vitro digestibility results were shown in Fig. 1. The one-way ANOVA result reveled that there were
154
statistically significant differences among digestibility values of different samples. After pepsin digestion, the
155
digestibility of XW, SC, ND and SB were 55.25 ± 7.20%, 47.61 ± 5.94%, 46.09 ± 3.95% and 36.27 ± 6.31%,
156
respectively. After being further treated by pepsin/trypsin, proteins of XW, SC, ND and SB were adequately
157
digested and the digestibility were increased to 77.74 ± 2.87%,70.19 ± 3.34%, 67.41 ± 2.82% and 66.08 ±
158
1.91%, respectively.
159
Zhou et al. (2019) revealed that lower salt contents effected release rates of cathepsin B and cathepsin L
160
which may be responsible for the intense degradation of proteins during the ripening. According to the
161
processing standard of China (GB/T 18357-2008, product of geographical indication-Xuanwei ham), high salt
162
contents (12.5%) in XW hams could provide a protection of proteins during the post-ripening, which resulted in
163
a high initial protein contents (Huang, Ge, & Huang, 2010; Yang et al., 2005). After being treated by pepsin and
164
pepsin/trypsin, the proteins were enzymatically digested to a greater extent, and the losses of protein contents
165
were relatively larger than other samples. Therefore, XW hams had the largest digestibility after pepsin and
166
pepsin/trypsin digestion. Compared with other samples, SB hams were processed with low-salt technic which
167
resulted in the rapid degradation of proteins during ripening. Therefore, the initial protein contents of SB hams
168
were relatively low which resulted in a low digestibility.
169
3.2 Analysis of particle sizes
170
The particle sizes of different dry-cured hams were measured to analyze the changes of digestibility, and
171
the results of particle sizes were listed in Table 1. According to the ANOVA analysis, the particle sizes of
8
172
untreated XW hams were significantly different from others. The pork resources used for XW dry-cured hams
173
were different from others (GB/T 18357-2008), and the meat quality of growing pigs could be influenced by
174
animal breed, dietary source and growing environment (Doti, Suárez-Belloch, Latorre, Guada, & Fondevila,
175
2014). Additionally, the XW hams were aged for a longer time (more than 10 months) which resulted in a larger
176
particle sizes of materials (Degnes, Kvitvang, Haslene-Hox, & Aasen, 2017).
177
As shown in Table 1, after pepsin digestion, there was no significant difference among hams. For XW
178
hams, it could be calculated that the decreasing rates of large-size particles (Dx (90)) and middle size particles (Dx
179
(50)
180
middle size particles (Dx (50)) were decreased by 38.7% and 37.1% after trypsin digestion. The results indicated
181
that the large-size particles (> 310 µm) were mainly digested (27.5%) during the pepsin digestion. The
182
middle-size particles (> 190 µm) could be digested by trypsin digestion (38.7%). The particle size decreasing
183
rates of XW hams were the largest among four samples, which was in accordance with digestibility analysis.
184
The reason might be that pepsin enzyme was suitable for digesting large-size particles (Li et al., 2017).
185
Moreover, the trypsin cleavages were more consistent when treated different ham samples (Wen et al., 2015a).
186
Therefore, XW hams were more suitable for consumers with weak function of intestinal digestion.
187
3.3 Analysis of SDS-PAGE results
) were respective 27.5% and 12.6% after the pepsin digestion. Afterward, the large-size particles (Dx (90)) and
188
The result of SDS-PAGE was shown in Fig. 2. It could be concluded from different gel lanes that there
189
were significant differences among SDS-PAGE profiles. For qualitative analysis of protein profiles, the
190
molecular weight of each protein was determined based on the standard marker lane. In this study, the relative
191
band intensity was calculated by comparing with 100 kDa band in the calibration marker lane, and the result
192
was shown in Table 2. Total 14 bands were marked and selected as characteristic bands of four dry-cured hams
193
before the digestion. The protein bands distribution of dry-cured hams before digestion was similar to those of 9
194
pork products in the published article (Li et al., 2017). Moreover, there were lots of proteins with high molecular
195
weight (37-75 kDa) existed in the profiles of four dry-cured hams especially in ND hams. The ANOVA analysis
196
results revealed that the relative intensities of bands (> 50 kDa) were significantly different among different
197
hams.
198
After pepsin digestion, the proteins with high molecular weight were enzymatically digested to small
199
molecular weight proteins. As shown in Table 2, 9 bands (10-50 kDa) were marked as characteristic bands of
200
four dry-cured hams. During the pepsin digestion, most high molecular weight proteins were hydrolyzed to
201
small molecular weight proteins. For instance, the proteins such as 99 kDa, 72 kDa, 41 kDa and 39 kDa were
202
digested and the relative intensities of some proteins such as 143 kDa, 55 kDa, 47 kDa and 25 kDa decreased
203
prominently. Additionally, small molecular weight proteins such as 12 kDa and 9 kDa occurred and the relative
204
intensities increased significantly. After the pepsin/trypsin digestion, almost all the proteins with high molecular
205
weight were hydrolyzed and only protein fragments with small molecular weight could be detected by the
206
electrophoresis. Additionally, there was no significant difference on particle size (Table 1) among hams.
207
Therefore, comparing to pepsin, there were more cleavage sites in proteins for trypsin, which resulted in an
208
adequate digestion (Erickson & Kim, 1990).
209
To further analyze the difference of samples, PCA and LDA were applied to visualize the data space of
210
protein profiles and to qualitatively discriminate different samples. The relative intensities of 14 bands and 9
211
bands before and after pepsin digestion were selected as features to do PCA and LDA analysis, and the results
212
were shown in Fig. 3. According to SDS-PAGE profiles of proteins (Fig. 2), almost no protein bands could be
213
detected by the electrophoresis after pepsin/trypsin digestion. Therefore, there was no features (relative
214
intensities) could be used for PCA and LDA visualization, which was the reason that only PCA and LDA results
215
based on protein profiles before and after pepsin digestion were presented in Fig. 3. Three-dimensional plots of
10
216
PCA and LDA were shown in Fig. 3(a) and (b). The first three PCs were calculated to characterize the data
217
space of protein profiles and 56.6% information of the variance was contained in the score plot. In the PCA plot,
218
all sample groups overlapped to some extent and could not be distinguished from each other. As shown in Fig.
219
3(b), the first three function scores could present almost 100% useful information of original protein profiles. In
220
LDA plot, sample groups of XW, ND, SC and SB could be separated to different districts and there were clear
221
boundaries among each group.
222
According to the results of protein profiles before and after pepsin digestion, the relative intensities of
223
some original characteristic bands were changed significantly and some new protein bands were produced.
224
Subsequently, the relative intensities of these protein bands mentioned above were used to do PCA and LDA,
225
and the results were shown in Fig. 3(c) and (d). Total 61.9% useful information of the variance were represented
226
by the first three PCs. In the PCA plot, all the sample groups were mixed into one and could not be separated
227
from each other, which was in accordance with the analysis results of protein profiles. LDA was then applied to
228
analyze protein profiles, and all information contained in original data could be expressed by the first three
229
function scores, as shown in Fig. 3(d). In the LDA plot, sample groups of XW, ND and SC could be
230
distinguished clearly from each other. However, sample group of SB was close to that of ND, which could be
231
explained by that relative intensities of almost all protein bands obtained from SB and ND were very similar.
232
The reason might be that SB and ND hams were aged for same time in a similar weather of moderate rainfall.
233
Furthermore, the internal quality of dry-cured hams such as protein profiles were significantly influenced by the
234
aging time and pork resources (Paolella et al., 2015; Wang et al., 2018). According to the results of PCA and
235
LDA, the protein profiles could be used to qualitatively and quantitatively analyze different dry-cured hams.
236
3.4 Peptide profiling of dry-cured hams
237
In this study, the digestion sample solutions obtained from different dry-cured hams were analyzed by 11
238
using LC-MS to determine the peptide sequence, and the mass spectrums of different hams were listed in
239
Supplementary information (Fig. S1). According to the LC-MS results, the numbers of peptides collected from
240
XW, SC, ND and SB samples after pepsin digestion were 117, 105, 126 and 118, respectively. After trypsin
241
digestion, the numbers of obtained peptides were respectively increased to 331, 366, 336 and 293, the result was
242
shown in Fig. 4. More proteins were digested to fragments during trypsin digestion, which was in accordance
243
with the result of published article in which dry-cured hams were analyzed (Li et al., 2017). However, the
244
numbers of peptides in pepsin digestion and pepsin/trypsin digestion were significantly different from fresh pork
245
(Wen et al., 2015b). The reason might be that salting and post-ripening changed the internal quality of meat.
246
These peptides were then sequenced and Venn diagrams were performed. The Venn diagrams of peptides
247
obtained from different dry-cured hams were shown in Fig. 5. Among these sequenced peptides, 48 and 171
248
peptides were common for different dry-cured hams after pepsin and pepsin/trypsin digestion. Escudero (2010)
249
reported that peptides detected in vitro digested fresh pork were obtained from muscle protein including actin,
250
myosin and kinase. The molecular weight of peptides could be mainly divided into three groups (i.e., 800 to
251
1500 Da, 1500 to 2500 Da and 2500 to 3500 Da), and the result was shown in Fig. 6. The most of peptides (>
252
50%) belonged to 1500 to 2500 Da, and peptide numbers of XW, SC, ND and SB were 58, 55, 68 and 65. After
253
digestion of pepsin/trypsin, there were 200, 198, 199 and 171 peptides distributed into 1500 to 2500 Da, and
254
occupied 60.42%, 54.37%, 58.92% and 58.36% of total peptides, respectively. As shown in Fig. 5(a), total 185
255
peptides with different sequences were identified in the Venn diagram of pepsin digestion, and 48 common
256
peptides (14, 13, 15 and 15 peptides were from XW, SC, ND and SB) were identified. As shown in Fig. 5(b),
257
total 453 peptides with different sequences were identified after pepsin/trypsin digestion, and the number of
258
specific peptides from XW, SC, ND and SB were 25, 57, 20 and 21, respectively.
259
Subsequently, peptides were matched with porcine muscle proteins against the Swiss-Prot database (Sus
12
260
scrofa). In this study, most peptides were mainly identified as fragment from several myofibrillar proteins such
261
as tropomyosin, actin, titin and myosin, and several sarcoplasmic proteins such as glyceraldehyde-3-phosphate
262
dehydrogenase, phosphorylase, creatine kinase, 6-phosphofructokinase. The protein profiles of different hams
263
were in accordance with those of cooked pork due to that biceps femoris was used in this study (Wen et al.,
264
2015a; Zou et al., 2018). The bioactivity of obtained peptides were determined by comparing with Swiss-Prot
265
database and PepBank database. In XW, SC, ND and SB dry-cured hams, 25, 24, 26 and 27 functional peptides
266
were found and showed the antioxidant bioactivity. Additionally, one peptide (MNVKHWPWMKL) obtained
267
from four ham samples after pepsin digestion might have the bioactivity angiotensin-converting enzyme
268
inhibitor (ACEI). In this study, the ACEI peptides were obtained from the pepsin digestion of actin and myosin,
269
and the bioactivity could be affected by processing and storage conditions (Mora et al., 2018). After trypsin
270
digestion, another 42 new functional peptides were generated, which indicated that original peptides were
271
constantly digested and new peptides generated. This corresponded to the analysis of SDS-PAGE that almost all
272
of the bands were digested after pepsin/trypsin digestion. The result suggest that dry-cured hams could be
273
resources of natural peptides which had positive effect on health (Gallego, Mora, Hayes, Reig, & Toldrá, 2019).
274
Moreover, it was found that 8.4-17.3% of total peptide numbers were identified as fragment obtained from
275
myosin-1.The location of identified peptides after pepsin and pepsin/trypsin digestion were shown as an
276
example in Supplementary information (Table S1 and Table S2). According to the location of peptides on the
277
myosin-1 proteins, it could be concluded that the cleavage sites of pepsin and trypsin were different. For the
278
pepsin digestion, it could be found that peptides generated in pepsin digestion with a wide range of cleavage
279
sites such as Leu, Phe, Arg and Lys. After further trypsin digestion, some new peptides were generated with the
280
carbon-terminal cleavage sites as Lys and Ary. The cleavage site specificity of pepsin and trypsin was in
281
accordance with the summary by the European Molecular Biology Laboratory (https://www.ebi.ac.uk/) and
13
282
published articles (Alves et al., 2007; Furlong, Mauger, Strimpler, Liu, Morris, & Edwards, 2002). Although
283
protein formations of dry-cured hams could be changed due to different producing districts, generated peptides
284
during the in vitro digestion changed little because of the cleavage site specificity of pepsin and trypsin. In
285
summary, dry-cured hams from different producing districts had significant effect on in vitro digestibility of
286
proteins but little effect on peptide generation during the pepsin and pepsin/trypsin digestion.
287
4. Conclusions
288
The protein profiles of dry-cured hams from different producing districts were significantly different and
289
would affect the protein digestion in the gastrointestinal digestion. In this study, the in vitro digestion with
290
pepsin and trypsin enzymes was applied to simulate the gastrointestinal digestion procedure. The analysis of
291
digestibility and particle size revealed that the digestion characteristics of dry-cured hams were different, and
292
XW hams were more suitable for consumers with weak function of intestinal digestion. The SDS-PAGE data
293
could be used to distinguish different dry-cured hams based on PCA and LDA. According to the analysis of
294
peptide profiles, most peptides were identified as fragment of myosin-1 protein, and two-step digestion had little
295
effect on peptide generation due to the cleavage site specificity of pepsin and trypsin. This study provided
296
evidences that it was possible to identify producing districts of different dry-cured hams based on digested
297
characteristics. In the subsequent work, more efforts should be focused on bioactivity identification of peptides
298
in digested products.
299
Acknowledgements
300
This work was funded by Beijing Advanced Innovation Center for Food Nutrition and Human Health and
301
Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), National 14
302
Natural Science Foundation of China (Grant no. 31540087), and the National Key Research and Development
303
Program of China (2016YFD0400803, 2016YFD0401501).
304
Conflicts of interest
305
The authors declare that they have no conflicts of interest.
306
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Captions of Tables and Figures Table 1 Particle sizes of four dry-cured hams with different treatments. Table 2 Band intensities of proteins before and after pepsin digestion.
Figure 1 Digestibility in vitro of four dry-cured hams treated with pepsin and pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Different letters on the same samples indicate statistically significant difference among the values (p < 0.05). Figure 2 SDS-PAGE profiles of total proteins obtained from four dry-cured hams with different treatments: (a) before digestion, (b) after digestion with pepsin, (c) after digestion with pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 3 Results of PCA and LDA: (a) PCA and (b) LDA plots based on SDS-PAGE data before digestion; (c) PCA and (d) LDA plots based on SDS-PAGE data after pepsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 4 Total numbers of identified peptides from four dry-cured hams. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 5 Venn diagrams of peptides obtained from different dry-cured hams: (a) In vitro digestion with pepsin; (b) In vitro digestion with pepsin followed by trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Figure 6 Peptide numbers on the basis of molecular weights from four dry-cured hams. (a) Pepsin digestion; (b) Pepsin/trypsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. 19
Table 1 Particle sizes of four dry-cured hams with different treatments.
Samples
Dx (10)/µm
Dx (50) /µm
Dx (90) /µm
D [4,3] /µm
D [3,2] /µm
XW
9.26±2.19 a
121.83±5.91 a
427.50±35.06 a
174.67±10.39 a
28.62±4.01 a
SC
9.21±1.41 a
106.83±5.42 b
348.50±28.67 b
147.00±8.49 b
29.50±2.77 a
ND
8.76±1.37 a
101.27±13.84 b
374.67±25.26 b
150.00±21.38 b
26.73±3.22 ab
SB
6.90±0.78 b
103.67±7.52 b
386.00±13.87 ab
151.00±3.16 b
23.67±2.39 b
XW
10.92±2.90 a
106.43±9.07 a
310.00±41.59 a
138.00±13.65 a
29.60±5.85 a
SC
7.77±1.05 a
88.47±10.59 b
258.67±33.22 a
118.33±14.39 a
23.63±3.48 a
ND
10.75±3.25a
90.22±13.84 b
272.33±44.37 a
120.10±18.44 a
28.80±5.88 a
SB
8.58±2.45 a
97.13±12.98 ab
297.17±43.77 a
128.50±17.00 a
24.63±3.62 a
XW
6.05±0.62 b
67.00±11.40 ab
190.17±19.77 a
83.70±10.96 a
18.40±2.59 b
SC
7.08±0.88 a
71.78±4.76 a
184.00±9.59 a
88.82±5.32 a
24.93±3.98 a
ND
5.90±0.31 b
57.02±10.45 ab
171.67±15.65 a
74.70±8.51 a
17.65±1.71 b
SB
5.75±0.29 b
53.73±14.11 b
191.17±14.79a
77.32±17.21 a
16.17±1.29 b
Untreated
Pepsin digestion
Pepsin + trypsin digestion
Values were expressed as means (n = 6) ± SD. SD: standard deviation. XW, SC, ND and SB were samples from Xuanwei,
Sanchuan, Nuodeng and Saba, respectively. Means with different superscripts (a, b) in the same column were statistically different
(p < 0.05).
20
Table 2 Band intensities of proteins before and after pepsin digestion. Band intensities (mean ± SD)
Molecular weight Bands (kDa)
XW
ND
SC
SB
Untreated 1
143.13
0.27 ± 0.09ab
0.16 ± 0.12ab
0.10 ± 0.07b
0.34 ± 0.18a
2
99.05
0.07 ± 0.03bc
0.03 ± 0.01c
0.19 ± 0.07a
0.17 ± 0.12ab
3
87.30
0.60 ± 0.39a
0.62 ± 0.25a
0.44 ± 0.24a
0.59 ± 0.51a
4
72.07
0.81 ± 0.40a
0.54 ± 0.20ab
0.29 ± 0.25b
0.38 ± 0.22ab
5
66.46
0.23 ± 0.16a
0.41 ± 0.20ab
0.75 ± 0.30a
0.51 ± 0.21ab
6
55.09
0.42 ± 0.15a
0.84 ± 0.52a
0.44 ± 0.05a
0.4 0± 0.21a
7
46.92
0.80 ± 0.26a
0.45 ± 0.23b
0.34 ± 0.09b
0.46 ± 0.10b
8
41.04
0.74 ± 0.52a
0.14 ± 0.13b
0.37 ± 0.29ab
0.22 ± 0.17ab
9
37.96
1.86 ± 1.04a
2.19 ± 0.46a
1.78 ± 1.09a
1.6 0± 0.74a
10
34.75
1.31 ± 0.40a
0.28 ± 0.23b
1.08 ± 1.02ab
1.33 ± 0.36a
11
32.66
0.49 ± 0.19a
0.49 ± 0.16a
0.20 ± 0.18ab
nd
12
27.75
0.21 ± 0.09a
0.18 ± 0.10a
0.54 ± 0.81a
0.14 ± 0.04a
13
25.50
1.55 ± 0.70a
0.50 ± 0.29a
1.57 ± 0.90a
1.77 ± 1.07a
14
18.11
1.01 ± 0.58a
0.51 ± 0.39ab
0.32 ± 0.13b
0.63 ± 0.10ab
1
144.14
0.14 ± 0.09a
0.18 ± 0.13a
0.22 ± 0.08a
0.17 ± 0.10a
2
87.31
0.85 ± 0.52a
0.47 ± 0.45a
0.54 ± 0.87a
0.67 ± 0.64a
3
66.12
0.16 ± 0.09a
0.12 ± 0.09a
0.68 ± 0.30a
0.41 ± 0.69a
4
53.81
0.25 ± 0.10a
0.15 ± 0.05a
0.42 ± 0.51a
0.10 ± 0.07a
5
47.58
0.13 ± 0.09a
0.33 ± 0.34a
0.22 ± 0.18a
0.10 ± 0.08a
6
30.10
0.20 ± 0.10a
0.94 ± 1.31a
0.40 ± 0.22a
0.4 0± 0.33a
7
24.12
0.86 ± 0.41a
0.48 ± 0.35a
0.88 ± 0.28a
0.60 ± 0.47a
8
11.56
0.57 ± 0.52a
0.59 ± 0.71a
1.11 ± 1.71a
0.62 ± 0.67a
9
8.98
1.65 ± 1.11a
1.10 ± 1.08a
2.01 ± 0.95a
0.95 ± 0.83a
Pepsin digestion
Values were expressed as means (n = 6) ± SD. SD: standard deviation. nd: not detected. XW, SC, ND and SB were samples from
Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Means with different superscripts (a, b, c) in the same row were statistically
different (p < 0.05).
21
Fig. 1 Digestibility in vitro of four dry-cured hams treated with pepsin and pepsin + trypsin. XW, SC, ND and SB were samples
from Xuanwei, Sanchuan, Nuodeng and Saba, respectively. Different letters on the same samples indicate statistically significant
difference among the values (p < 0.05).
22
Fig. 2 SDS-PAGE profiles of total proteins obtained from four dry-cured hams with different treatments: (a) before digestion, (b)
after digestion with pepsin, (c) after digestion with pepsin + trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan,
Nuodeng and Saba, respectively.
23
Fig. 3 Results of PCA and LDA: (a) PCA and (b) LDA plots based on SDS-PAGE data before digestion; (c) PCA and (d) LDA
plots based on SDS-PAGE data after pepsin digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and
Saba, respectively.
24
Fig. 4 Total numbers of identified peptides from four dry-cured hams. XW, SC, ND and SB were samples from Xuanwei, Sanchuan,
Nuodeng and Saba, respectively.
25
Fig. 5 Venn diagrams of peptides obtained from different dry-cured hams: (a) In vitro digestion with pepsin; (b) In vitro digestion
with pepsin followed by trypsin. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively.
26
Fig. 6 Peptide numbers on the basis of molecular weights from four dry-cured hams. (a) Pepsin digestion; (b) Pepsin/trypsin
digestion. XW, SC, ND and SB were samples from Xuanwei, Sanchuan, Nuodeng and Saba, respectively.
27
Graphical Abstract
28
Highlights Using in vitro digestion of four types dry-cured hams with pepsin and trypsin PCA and LDA can distinguish different dry-cured hams based on protein profiles Digestibility and particle size of different hams are significantly different Peptides of in vitro digestion products are distributed in ranges of 1500 to 2500 Da Two-step digestion with pepsin and trypsin had little effect on peptide generation
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.