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Ice crystal growth inhibition by peptides from fish gelatin hydrolysate
Accepted Manuscript Ice Crystal Growth Inhibition by Peptides from Fish Gelatin Hydrolysate
Srinivasan Damodaran, ShaoYun Wang PII:
S0268-005X(16)30621-X
DOI:
10.1016/j.foodhyd.2017.03.029
Reference:
FOOHYD 3840
To appear in:
Food Hydrocolloids
Received Date:
19 October 2016
Revised Date:
21 March 2017
Accepted Date:
21 March 2017
Please cite this article as: Srinivasan Damodaran, ShaoYun Wang, Ice Crystal Growth Inhibition by Peptides from Fish Gelatin Hydrolysate, Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd. 2017.03.029
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Graphical Abstract:
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Highlights:
Peptides in the molecular weight range of 1000 – 2000 Da in fish gelatin hydrolysate were able to inhibit ice crystal growth in an ice cream mix and in 23% sucrose solution.
Among these, cationic peptides were much more effective than anionic/neutral peptides.
The amino acid sequences of two of these fish gelatin peptides have been determined.
Electrostatic, hydrogen bonding, and hydrophobic forces might be involved in a concerted manner in the binding process.
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Ice Crystal Growth Inhibition by Peptides from Fish Gelatin
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Hydrolysate
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Srinivasan Damodaran* and ShaoYun Wang Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706
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* Corresponding author.
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Tel.: +1 608 263 2012; Fax: +1 608 262 6872.
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E-mail address: [email protected] (S. Damodaran).
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Abstract
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Ice crystal growth inhibition in an ice cream mix matrix and in sucrose solutions by
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peptides derived from alcalase (also known as subtilisin) hydrolyzed fish gelatin was
23
investigated. Hydrolysis of fish gelatin at an optimum hydrolysis condition (i.e. 20% w/w
24
gelatin solution treated with Alcalase at an enzyme-to-substrate ratio of 0.176 Anson units/g
25
gelatin at pH 9.0 for 25 min at 45 oC) released peptides with maximum ice crystal growth
26
inhibition activity.
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exchange chromatography resulted in isolation of a cationic peptide fraction containing two
28
prominent peptides having 1850.82 Da and 2036.88 Da molecular masses.
29
fraction had the highest ice crystal growth inhibition activity. The amino acid sequences of
30
these peptides showed no sequence similarity other than that they both contained –GTPG-
31
and –GPP(OH)G- motifs and 3 to 5 hydroxyl containing amino acid residues. The results of
32
this study supported the hypothesis that short collagen/gelatin polypeptides in the molecular
33
mass range of 1000 to 2500 Da, regardless of their source, would have the ability to inhibit
34
ice crystal growth in frozen systems. The results also suggested that the mechanism of ice
35
crystal growth inhibition by gelatin peptides might involve three steps, namely, initial
36
nonspecific electrostatic interaction of cationic peptides with the negatively charged ice
37
surface, followed by structural realignment to optimally hydrogen bond with the oxygen-
38
oxygen lattice on the ice surface, and stabilization of the electrostatic and hydrogen bonding
39
in the peptide – ice crystal complex by a partial nonpolar environment created by neighboring
40
hydrophobic residues of the peptide.
Fractionation of gelatin hydrolysate using size exclusion and ion
2
This cationic
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Keywords: Fish gelatin hydrolysate; antifreeze peptides; ice crystal growth inhibition; ice
43
structuring peptides.
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3
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1. Introduction
47 48
Ice crystal growth during cold storage is a quality issue in frozen foods. In nature,
49
organisms that have adapted to sub-freezing temperatures, such as fishes, arthropods, winter
50
plants, bacteria, and fungi, produce antifreeze proteins (AFP) (Clarke, Buckley, & Lindner,
51
2002; Du, Liu, & Hew, 2003; Kristiansen et al., 2005; Kontogiorgos, Regand, Yada, & Goff,
52
2007; Pentelute et al., 2008; Knight, Cheng, & DeVries, 1991; Sidebottom et al., 2000;
53
Worrall et al., 1998; Graham, & Davies, 2005; Wierzbicki et al., 2007; Graether et al., 2000).
54
The AFPs depress the freezing point of water from 1 oC
55
depending on their living habitat (Graham, Liou, Walker, Davies, 1997; Graham, & Davies,
56
2005), but they do not alter the melting point of ice, which remains at 0 oC. This thermal
57
hysteresis is a direct evidence that the APFs depress freezing point of water via a non-
58
collegative mechanism. However, it has been observed that some plant-derived AFPs do not
59
exhibit thermal hysteresis, but cause ice recrystallization inhibition or ice restructuring (RI or
60
ISP) (Sidebottom et al., 2000; Worrall et al., 1998).
(fish AFP) to 5 oC (insect AFP)
61
These natural AFP and ISP proteins may be used in frozen foods and in pharmaceutical
62
products to retard ice crystal growth in order to reduce freezing-induced damage and to
63
extend their shelf life (Griffith, & Ewart, 1995; Adapa et al., 2000; Hartel, 2001; Regend &
64
Goff, 2002); however, their limited availability and the economics have restricted their
65
extensive use in foods.
66
Several studies have reported that hydrocolloids, such as xanthan gum, locust bean gum,
67
carrageenan, alginate, etc., decreased the rate of recrystallization of ice in ice cream and in
68
concentrated sucrose solutions (Regand & Goff, 2002; Adapa et al., 2000; Kaminska4
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Dworznicka et al., 2015). Some of these hydrocolloids have synergistic effect on inhibition of
70
ice recrystallization by fish antifreeze proteins (Gaukel, Leiter, & Spieb, 2014).
71
Enzymatically and non-enzymatically hydrolyzed kappa-carrageenan have been shown to
72
inhibit ice recrystallization in sorbet and concentrated sucrose solutions better than intact
73
kappa-carrageenan (Kaminska-Dworznicka, Skrzypczak, & Gondek, 2016; Kaminska-
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Dworznicka et al., 2015). However, whether or not this inhibitory effect of hydrocolloids on
75
ice recrystallization is related to increase of viscosity or to specific interaction of
76
hydrocolloids with the ice surface has not been satisfactorily resolved.
77
An examination of the structural attributes of natural AFPs and ISPs shows that they are
78
structurally diverse with regard to molecular mass, amino acid composition and sequence,
79
etc.
80
containing groups geometrically spaced in a two-dimensional array that mimics the spacing
81
of oxygen atoms in the prism face of hexagonal ice (Pentelute et al., 2008; Liou, Tocilj,
82
Davies, & Jia, 2000; Graether, & Sykes, 2004). This flat surface is thought to bind to the
83
prism face of ice via hydrogen bonding, while the other structural elements (i.e. neighboring
84
hydrophobic residues) of the protein create a nonpolar environment wherein the above
85
hydrogen bonding interactions are stabilized (Liou, Tocilj, Davies, & Jia, 2000; Wen, &
86
Laursen, 1992; Knight, Driggers, & DeVries, 1993; Zhang, & Laursen, 1998; Graether, &
87
Sykes, 2004). However, because of lack of direct crystallographic data on the AFP-ice
88
complex, the validity of the hydrogen-bonding lattice match model remains controversial
89
(Antson et al., 2001; Jia & Davies, 2014).
90
interactions have been proposed (Sonnichsen et al., 1996).
However, they all invariably contain a flat ice-binding region with polar oxygen-
Alternative mechanisms involving hydrophobic
5
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Recently, we have hypothesized that if the oxygen-oxygen spacing mimicry in the flat
92
hydrophilic surface with that of the oxygen-oxygen geometry in hexagonal ice is the primary
93
requirement for ice binding, then any polypeptide that can dynamically adapt its backbone
94
conformation as it approached an ice surface and aligned its oxygen containing groups with
95
that of ice surface should also be able to bind and inhibit ice crystal growth in a manner
96
similar to antifreeze proteins (Damodaran, 2007; Wang, & Damodaran, 2009). The protein
97
that fits this requirement is peptides derived from collagen/gelatin, which has highly flexible
98
polypeptide chain with a repeat sequence of – Gly – Xaa – Yaa - (where Xaa is mostly
99
proline or hydroxyproline and Yaa is any other amino acid residue). In support of this
100
hypothesis, recently we had shown that peptides in the molecular weight range of 1000 –
101
3000 Da derived from bovine skin gelatin hydrolysate inhibited growth of ice crystals in an
102
ice cream mix (Damodaran, 2007; Wang, & Damodaran, 2009; Wang, Agyare, &
103
Damodaran, 2009). Molecular dynamics simulations of a model gelatin fragment, viz., Gly-
104
Pro-Ala-Gly, also have demonstrated the mode of binding of this peptide to the prism face of
105
ice nuclei and inhibition of ice crystal growth under super-cooled conditions (Kim,
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Damodaran, & Yethiraj, 2009).
107
Collagen/gelatin from different species, specifically mammalian versus marine species,
108
differ significantly in their amino acid composition/sequence and other physicochemical
109
properties (Lin, & Liu, 2006; Bae et al., 2008; Nagai, & Suzyki, 2000).
110
collagen contains significantly lower hydroxyproline content than mammalian collagens.
111
Also, the thermal denaturation temperature of fish collagen is lower than that of bovine skin
112
collagen (Lin, & Liu, 2006). Because of these differences, the physicochemical properties of
6
For example, fish
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peptides released from fish collagen/gelatin under similar enzymatic digestion conditions
114
would be different, and hence their ice crystal growth inhibiting properties also might be
115
different from those from mammalian collagen/gelatin hydrolysate.
116
The objective of this study was to determine if, despite significant differences between
117
physicochemical properties of fish versus mammalian collagen/gelatin, Alcalase hydrolysis
118
liberated ice crystal growth inhibiting peptides from fish gelatin.
119
human population do not consume mammalian gelatin for religious reasons, and fish skin and
120
bones are underutilized byproducts of the fish processing industry, elucidation of the
121
conditions under which Alcalase hydrolysis of fish collagen/gelatin produces ice crystal
122
growth inhibiting peptides and molecular characterization of such peptides will expand the
123
utilization of this important biomass.
Since certain segments of
124 125
2. Materials and methods
126 127
2.1. Materials
128 129
Ice cream mix was obtained from the dairy plant of the Department of Food Science at
130
the University of Wisconsin-Madison. The total non-fat solids content of the ice cream mix
131
(milk solids + sucrose) was 27% (w/w) and its pH was 6.6.
132
ice cream mix was diluted 15 wt% with water to compensate for inclusion of up to 4% gelatin
133
hydrolysate as nonfat solids.
134
mix which was stored at -20 °C in 2 mL aliquots in cryo-vials and all experiments were
In all experiments, the original
All experiments were conducted on a single batch of ice cream
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carried out on this single batch by using one vial at a time.
136
Dr. Joe Regenstein at the Department of Food Science at Cornell University, Ithaca, NY,
137
kindly provided a commercial fish gelatin, prepared by enzymatic hydrolysis of fish skin of a
138
carp species. Alcalase (EC 3.4.21.14) in a solution form (2.64 Anson units/g solution) was
139
purchased from Sigma Chemicals Co. (St. Louis, MO, U.S.A). Sephadex G-50 was from
140
Fisher Scientific Co. (Fairlawn, NJ, U.S.A), and Sulfopropyl-Sephadex C-25 (SP-Sephadex)
141
was from Sigma Chemicals Co. (St. Louis, MO, U.S.A).
142 143
2.2. Fish gelatin hydrolysis and fractionation
144 145
Alcalase hydrolysis of fish gelatin was performed as described previously for bovine
146
gelatin hydrolysis (Wang, & Damodaran, 2009; Wang, Agyare, & Damodaran, 2009). The
147
optimum pH for Alcalase activity ranges from pH 7.5 at 50 oC to pH 10 at 65 oC.
148
in preliminary experiments, fish gelatin hydrolysates produced at pH 9.0 and 45 oC using an
149
enzyme-to-substrate ratio of 1:15 (w/w) were found to be more effective in inhibiting ice
150
recrystallization than those obtained at pH 7.0. These conditions were used in the present
151
study to produce fish gelatin hydrolysates.
152
of fish gelatin in deionized water at pH 9.0 were incubated at 45 oC in a water bath until
153
gelatin completely dissolved into a solution. Alcalase was added at an enzyme-to-substrate
154
ratio of 0.176 Anson units/g gelatin (which was equivalent to 1 g of the enzyme solution to
155
15 g of gelatin).
156
stopped by incubating the solutions for 10 min in boiling water.
However,
Briefly, 50 mL aliquots of a 20% (w/w) solution
Hydrolysis was carried out for 10, 15, 20, 25, and 30 min intervals and
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Fractionation of fish gelatin hydrolysate on Sephadex G-50 and Sulfopropyl-Sephadex
158
was performed as described previously (Wang, & Damodaran, 2009; Wang, Agyare, &
159
Damodaran, 2009).
160 161
2.3.
Mass spectrometry
162 163
The molecular weight distribution of peptide fractions was analyzed by Matrix-Assisted
164
Laser Desorption IonizationTime of Flight (MALDITOF) mass spectrometry (Applied
165
Biosystems, Foster City, CA, U.S.A).
166
growth inhibiting peptides were determined by LC/MS/MS mass spectrometry at the
167
Biotechnology Center of the University of Wisconsin-Madison.
The amino acid sequences of two of the ice crystal
168 169
2.4.
Ice crystal growth inhibition activity determination
170 171
Ice crystal growth in ice cream mix and in 23% sucrose solution was studied using a cold
172
stage (Model HCS302, Instec Scientific Instruments Ltd., Boulder, CO, U.S.A) mounted on a
173
Nikon Eclipse microscope (E200, Nikon Inc., Japan) as described previously (Damodaran,
174
2007; Wang, & Damodaran, 2009; Wang, Agyare, & Damodaran, 2009). Images of ice
175
crystal growth were captured and analyzed using IMAGE-PRO PLUS software (Media
176
Cybernetics, Silver Spring, MD, U.S.A).
177
cream or sucrose solution on a microscope slide covered with a cover slip was mounted on
178
the thermal stage of the microscope and subjected to the following time-temperature
179
program: The sample was cooled rapidly to -40 °C at the rate of 40 °C/min, held for 5 min at
180
that temperature. The temperature was then raised to -14 °C at the rate of 1°C/min and then
In a typical experiment, a small drop (5 μL) of ice
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ACCEPTED MANUSCRIPT 181
cycled 7 times between -14 and -12 °C at a rate of 1 cycle/3 min.
Images of the sample at -
182
40 oC and immediately after 7 cycles at -14 and -12 °C were captured at a magnification of
183
220X. The IMAGE-PRO PLUS software was capable of automatically scanning and
184
determining the average two-dimensional size of the crystals in the captured image.
185
Typically, triplicate runs were made on each sample and the ice crystal size was determined
186
as average of the triplicate.
187 188
2.5.
Statistical analysis
189 190
Ice crystal growth experiments on each hydrolysate sample were done in triplicate. The
191
ice crystal sizes are reported as average ± standard deviation of the triplicate measurement.
192
When significant treatment effects (P 0.05) were found, their means were separated by
193
Duncan’s multiple range tests using SAS statistical package.
194 195
3.
Results
196
Figure 1A shows elution profiles of fish gelatin hydrolysates on a Sephadex G-50
197
column. As expected, the elution profile shifted towards lower molecular weight as the
198
hydrolysis time was increased from 10 min to 30 min.
199
of hydrolysis time on the ice structuring activity of the hydrolysates. At 4wt% level, the 25
200
min hydrolysate produced peptides with maximum ice crystal growth inhibition activity.
201
Previously, it has been reported that in the case of bovine gelatin the optimum hydrolysis
202
time with Alcalase under similar experimental conditions was 30 min (Wang & Damodaran,
203
2009). The difference of 5 min between bovine and fish gelatin might be due to differences in 10
Figures 1B and 1C show the effect
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amino acid composition/sequence and other physicochemical properties that might make fish
205
gelatin more susceptible than bovine gelatin to Alcalase hydrolysis.
206
in Figures 1A-C indicate that the size distribution of peptides in fish gelatin hydrolysate
207
played a crucial role in the ice structuring activity.
Nevertheless, the data
208
The MALDI-TOF mass spectrum of peptides in the total fish gelatin hydrolysate
209
produced under the optimum hydrolysis condition (i.e., 20% w/w gelatin solution hydrolyzed
210
with Alcalase at an enzyme-to-substrate ratio of 1:15 at pH 9.0, 45 oC for 25 min) is shown in
211
Figure 2.
212
To elucidate which peptides within the molecular mass range were greatly responsible for the
213
ice structuring activity, the eluted fractions from the 25 min hydrolysate on Sephadex G-50
214
column were pooled into three fractions as shown in Figure 3A and they were lyophilized.
215
The ice structuring activity of these three fractions at 4% (w/w) level in ice cream mix after 7
216
thermal cycles between -14 and -12 oC is shown in Figure 3B, and the average size of ice
217
crystals produced in the ice cream mix is presented in Table 1. Among these three fractions,
218
fraction 2 (from the middle portion of the elution profile in Figure 3A) exhibited the best ice
219
structuring activity with an average ice crystal size of 4.6 0.5 µm. Although the average ice
220
crystal size in ice cream mix containing 4% total fish gelatin hydrolysate (6.4 0.5 µm) was
221
statistically larger than that containing fraction 2 (4.6 0.5 µm), its activity was better than
222
fraction 1 (18.2 0.9 µm), fraction 3 (9.3 1.2 m), and the control ice cream (22.2 1.2
223
µm).
224
hydrolysate (6.4 0.5 µm) was mostly derived from peptides in fractions 2 and 3.
225
The molecular weight distribution of the peptides ranged from 800 to 3300 Da.
Thus, the data in Table 1 tentatively suggest that the ice structuring activity of total
Shown in Figure 4 is the MALDI-TOF mass spectrum of fraction 2.
11
Although faction 2
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contained numerous peptides, the most dominant ones were the peptides corresponding to
227
molecular mass of 852.45, 1182.52, 1410.65, 1850.83, 2036.88, 2080.03, 2728.18, and
228
3320.49 Da. Fraction 2 was further fractionated on SP-Sephadex C-25 cation-exchange
229
column, first using 20 mM phosphate buffer (pH 7.0) to elute the anionic and neutral peptides
230
(SP1 fraction) and then with a 0 – 0. 5 M NaCl gradient in 0.02 M phosphate buffer (pH 7.0)
231
to elute the adsorbed cationic peptides (SP2 fraction) (Figure 5A). The SP1 and SP2
232
fractions were pooled separately and dialyzed for 48 h at 4 °C against water using a 500
233
molecular weight cut-off membrane to remove all salts, and lyophilized. The ice structuring
234
activity of these dialyzed and lyophilized SP1 and SP2 fractions are shown in Figure 5B and
235
the average ice crystal size distributions after 7 thermal cycles are presented in Table 2.
236
The results indicate that at pH 7.0 the cationic peptides fraction (SP2) was more effective
237
(Figure 5B, panel C) than the anionic peptides fractions (SP1) (Figure 5B, panel B) in
238
inhibiting ice recrystallization. The average size of ice crystals in SP2-containing ice cream
239
mix was 2.5 ± 0.7 m compared to 9.5 ± 0.9 m in SP1 containing samples and 22.1 ± 1.2
240
m in control ice cream mix. It is interesting to note that at pH 4.0 both SP1 and SP2
241
fractions were equally effective in their ice structuring activity with an average ice crystal
242
size of 4.8 ± 0.8 and 4.0 ± 0.6 m, respectively (Table 2). The increase in ice crystal
243
inhibition activity of the SP1 fraction at pH 4 is likely due to charge reversion from
244
anionic/neutral at pH 7 to cationic state at pH 4. These results indicate that, in addition to size
245
distribution, the charge characteristics of gelatin peptides also play a critical role in their ice
246
structuring activity.
247
The MALDI-TOF mass spectrum of the SP2 fraction is shown in Figure 6.
12
It
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contained 1182.52 Da, 1850.82 Da, and 2036.88 Da peptides as the prominent ones along
249
with several other peptides, suggesting that these peptides could be responsible for the ice
250
crystal inhibiting activity of SP2. Significantly, the SP2 peptides were able to retard ice
251
crystal growth in ice cream mix even after 25 thermal cycling between -14 and -12 oC
252
(Figure 7).
253
9.8 1.3 µm after 25 cycles, whereas under these conditions the average ice crystal size in
254
the control ice cream mix grew from 22.1 1.2 µm to about 46.6 2.9 µm (Table 3).
255
Previously, experimental evidences were provided to show that ice crystal inhibition by
256
peptides derived from bovine gelatin was not due to any non-specific effects, such as
257
alterations in viscosity (Wang & Damodaran, 2009).
258
SP2 fraction might arise from specific interaction of the peptides in this fraction, e.g. 1850.82
259
Da and 2036.88 Da peptides, with the ice surface.
The average ice crystal size increased from 2.5 ± 0.7 m after 7 cycles to about
Thus, the ice structuring activity of the
260
The fraction 2 and the SP2 fraction inhibited ice crystal growth in 23% sucrose solution
261
as well (Figure 8). The inhibition was more pronounced with the SP2 fraction than with
262
fraction 2, which was also the case in the ice cream mix (Table 4), indicating that the
263
composition of the medium did not have any effect. Given that the 1850.82 Da and 2036.88
264
Da peptides were present both in fraction 2 and in the SP2 fraction (Figures 4 and 6), the
265
differences in the effectiveness of these two fractions might be due to differences in relative
266
amounts of these two peptides in these fractions.
267
To understand the structure-function relationship of the 1850.82 Da and 2036.88 Da
268
peptides with respect to their ice crystal inhibiting properties, the amino acid sequence of
269
these peptides were determined using LC/MS/MS mass spectrometry.
13
The amino acid
ACCEPTED MANUSCRIPT 270
sequences of these peptides are shown in Table 5.
It should be noted that whereas
271
mammalian collagen/gelatin strictly contains –Gly – X – Y – repeat motif, this does not
272
appear to be the case in fish collagen/gelatin as evidenced from the presence of a –Gly – X –
273
Gly – motif in both the 1850.82 Da and the 2036.88 Da peptides (Table 5). In addition, fish
274
collagen/gelatin has -hydroxyasparagine (N(OH)), which, to our knowledge, is not found in
275
mammalian collagen/gelatin. However, -hydroxyaspartate and -hydroxyasparagine are
276
found in the human cytoskeletal ankyrin family of proteins (Yang et al., 2011). The
277
calculated net charge of these peptides was +1.4 and +0.4, respectively.
278
peptide has three side chain OH groups, whereas the 2036.88 Da peptide contains five side
279
chain OH groups.
280
interactions with the ice surface.
The 1850.82 Da
These OH groups may potentially be involved in hydrogen bonding
281 282
4.
Discussion
283 284
The results presented here clearly demonstrate that despite wide differences in the
285
physicochemical properties, such as amino acid composition and sequence, peptides derived
286
from alcalase hydrolysis of fish collagen/gelatin were able to inhibit ice crystal growth in a
287
manner similar to peptides derived from bovine collagen/gelatin hydrolysate reported
288
previously (Damodaran, 2007).
289
the molecular range of 1000 – 2500 Da, corresponding to about 10 to 25 amino acid residues,
290
were more effective than the others, suggesting that gelatin peptides in this molecular weight
291
range possessed the structural properties needed for binding to ice crystals. This indicates that
The following are evident in both cases: First, peptides in
14
ACCEPTED MANUSCRIPT 292
binding of gelatin peptides to ice surface is inexplicably linked to the unique amino acid
293
sequence pattern, i.e. the –Gly – X – Y – (where X is often either Pro or hydroxyproline
294
residue and Y is any other residue) repeat motif, which is found in all collagens, including
295
mammalian and fish collagen/gelatin.
296
the abundance of Gly residues (and Pro residues as well), the functional similarity of both
297
bovine and fish gelatin peptides vis-à-vis ice crystal growth inhibition, supports the
298
previously proposed hypothesis (Damodaran, 2007; Wang, & Damodaran, 2009) that
299
polypeptides that can dynamically reconfigure its backbone conformation to align their
300
oxygen-containing groups to match with the oxygen-oxygen geometry of hexagonal ice
301
lattice should be able to bind to ice surface and inhibit its growth.
Since gelatin peptides are highly flexible owing to
302
Second, in both bovine (Damodaran 2007; Wang, & Damodaran, 2009) and fish gelatin
303
peptides cases, cationic peptides (the SP2 fraction) were much more effective than either
304
neutral or anionic peptides (the SPI fraction) in inhibiting ice crystal growth. This indicates
305
that in addition to the molecular weight and flexibility requirements, the efficient binding of
306
gelatin peptides to ice crystal surface requires the peptide to be cationic. This is obvious from
307
the fact that while the cationic SP2 fraction was more effective than the anionic/neutral
308
fraction SP1 at pH 7, they both were equally effective at pH 4.0 (where protonation of
309
carboxyl groups (pKa ≈ 4.6) would make the SP1 peptides positively charged) in inhibiting
310
ice crystal growth (Table 2).
311
This positive charge requirement implicitly suggests involvement of electrostatic
312
interaction in the peptide – ice surface binding process. In order for this to be true, the ice
313
surface ought to be negatively charged. As a matter of fact, electrokinetic and potentiometric
15
ACCEPTED MANUSCRIPT 314
measurements have shown that the surface potential of the ice-water interface swings from a
315
positive potential at pH < 3.5 to a negative potential at pH > 3.5 (Kallay & Cakara, 2000;
316
Kallay, Cop, Chibowski, & Holysz, 2003; Cop & Kallay, 2004). This pH-dependent
317
reversible surface potential of the ice-water interface is due to protonation and deprotonation
318
of amphoteric surface OH groups on the ice surface. The surface potential of ice-water
319
interface at pH > 6 is typically in the range of -200 to -300 mV at 0 oC (Kallay, Cop,
320
Chibowski & Holysz, 2003; Cop & Kallay, 2004).
321
property of cationic peptides fraction (SP2) at pH 7.0 and both SP1 and SP2 fractions at pH
322
4.0 is partly due to attractive electrostatic interaction of the peptides with the negatively
323
charged ice surface at these pHs. Interestingly, several antifreeze proteins from fish (type III)
324
and insects are also cationic at neutral pH, with isoelectric pH in the range of 7.1 – 9.8 (Xu et
325
al., 2008; Kristiansen et al., 2005; Hossain, 2012), suggesting that attractive electrostatic
326
interaction between the ice surface and antifreeze proteins and peptides might be an initial
327
step in the binding process. However, it should be recognized that electrostatic interaction is a
328
nonspecific interaction and it alone might possess the ability to block ice crystal growth,
329
which is a structurally very specific process. It is conceivable, however, that this nonspecific
330
electrostatic interaction might initially drive the cationic gelatin peptides (and AFPs) toward
331
the ice surface, and facilitate specific hydrogen bonding of the structurally complimentary
332
region on the AFP (e.g. the flat surface of AFP) with the binding site on the ice surface.
333
the case of gelatin peptides this may involve dynamic alignment of the oxygen-containing
334
groups of gelatin peptides, facilitated by their molecular flexibility, with the oxygen-oxygen
335
geometry of the ice lattice.
Thus, the better ice growth inhibition
In
Once formed, these hydrogen bonds might be further stabilized
16
ACCEPTED MANUSCRIPT 336
by partial nonpolar environment created by hydrophobic residues in other structural elements
337
of peptides/proteins. Thus, electrostatic, hydrogen bonding, and hydrophobic forces might
338
involved in a concerted manner in the binding of gelatin peptides as well as AFPs to the ice
339
surface.
340
Although there is no amino acid sequence similarity, the two peptides from the SP2
341
fraction (Table 5) show some structural similarities: Both contain –GTPG- and –GPP(OH)G-
342
motifs.
343
groups might be involved in hydrogen bonding with the oxygen-oxygen lattice on the ice
344
surface.
345
gelatin peptide, Gly-Pro-Ala-Gly, was able to retard ice crystal growth in a supercooled
346
system at 260 K, and the mechanism involved hydrogen bonding between the ice surface and
347
the carbonyl groups of the peptide (Kim, Damodaran, & Yethiraj, 2009). A similar
348
mechanism also might be possible in the case of the cationic peptides listed in Table 5.
In addition, they contain 3 to 5 hydroxyl-containing residues.
These hydroxyl
Previously, using molecular dynamics simulation we had shown that a model
349
Although the SP2 fraction was the most effective in retarding ice crystal growth in ice
350
cream mix as well as in concentrated sucrose solutions, for all practical purposes the total fish
351
gelatin hydrolysate itself was quite effective at 4% (w/w) level in retarding ice crystal growth
352
in ice cream mix (Table 1).
353 354
5.
Conclusion
355 356
The results of this study supported the original hypothesis (Damodaran, 2007) that
357
short collagen/gelatin polypeptides in the molecular mass range of 1000 to 2500 Da,
17
ACCEPTED MANUSCRIPT 358
regardless of their source, would have the ability to inhibit ice crystal growth in frozen
359
systems. This is due to their ability to dynamically change their conformation as they
360
approach the ice surface and bind to the surface. The results also suggested that the
361
mechanism of ice crystal growth inhibition by gelatin peptides might involve three steps,
362
namely, initial nonspecific electrostatic interaction with the negatively charged ice surface,
363
followed by structural rearrangement to optimally hydrogen bond with the oxygen-oxygen
364
lattice on the ice surface, and stabilization of the electrostatic and hydrogen bonding
365
interaction in the peptide – ice crystal complex by a partial nonpolar environment created by
366
neighboring hydrophobic residues of the peptide. A better understanding of this peptide – ice
367
surface interaction may lead to rationale designing of peptide cryoprotectants with greater
368
antifreeze activity.
369 370
ACKNOWLEGEMENT The authors are grateful to Professor Joe Regenstein at Cornell University for
371 372
donating fish gelatin.
This material is based upon work supported by the National Institute
373
of Food and Agriculture, United States Department of Agriculture Grant No. 2006-35503-
374
16998).
375
18
ACCEPTED MANUSCRIPT 377
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Xu, H., Perumal, S., Zhao, X., Du, N., Liu, X.Y, Jia, Z, & Lu, R. (2008). Interfacial
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Schofield, C.J. (2011). Asparagine and aspartate hydroxylation of the cytoskeletal
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Biological Chemistry, 286, 7648-7660.
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Zhang, W., & Laursen, R.A. (1998). Structure-function relationship in a type I antifreeze
483
polypeptide – The role of threonine methyl and hydroxyl group in antifreeze activity.
484
Journal of Biological Chemistry, 273, 34806-34812.
485 486
23
ACCEPTED MANUSCRIPT 488 489 490
Table 1. Influence of fish gelatin peptide fractions on ice crystal growth in an ice cream mix. Fraction
491 492 493 494
Cycles
Diameter (μm)
15% diluted ICM (Control)
7
22.2 1.2A
15% diluted ICM + 4% Total Gelatin Hydrolysate
7
6.4 ± 0.5C
15% diluted ICM + 4% Fraction 1
7
18.2 0.9A
15% diluted ICM + 4% Fraction 2
7
4.6 0.5D
15% diluted ICM + 4% Fraction 3
7
9.3 1.2B
Values expressed as mean SD. A-EValues with different letters in the same column differ significantly (P < 0.05). ICM = Ice cream mix, Fraction 1= Fraction 1 from Sephadex G-50 column, Fraction 2 = Fraction 2 from Sephadex G-50 column, Fraction 3 = Fraction 3 from Sephadex G-50 column.
24
ACCEPTED MANUSCRIPT 496 497 498
Table 2. Influence of SPI and SP2 gelatin peptide fractions on average Ice Crystal size in an ice cream mix at different pH.
499 500
Peptide Fraction
501 502 503
Cycles
Diameter (μm)
15% diluted ICM (Control)
7
22.2 1.2A
15% diluted ICM + 4% SP1 at pH4
7
4.8 0.8C
15% diluted ICM + 4% SP2 at pH4
7
4.1 0.6C
15% diluted ICM + 4% SP1 at pH7
7
9.5 0.9B
15% diluted ICM + 4% SP2 at pH7
7
2.5 0.8D
Values expressed as mean SD. A-EValues with different letters in the same column differ significantly (P < 0.05). ICM = Ice cream mix, SP1= Fraction SP1 from SP-Sephadex C25 column, and SP2 = Fraction SP2 from SP-Sephadex C25 column.
25
ACCEPTED MANUSCRIPT 505 506 507 508
Table 3.
Mean Ice-Crystal size in an Ice Cream Mix treated with SP2 peptide fraction after two thermal cycling conditions at pH 7.0.
509 510 511
512 513 514
Fraction Control Control Sample of SP2 Sample of SP2 Values expressed as mean SD. significantly different (P < 0.05).
Cycles Diameter (μm ) 7 22.2±1.2A 25 46.6±2.9B 7 2.5 0.8C 25 9.8± 1.3D A,BValues with the same letters in the same column are not
26
ACCEPTED MANUSCRIPT 516 517 518 519 520
Table 4:
Effect of Fraction 2 and SP2 fish gelatin peptides on ice crystal growth in
sucrose solutions.
521 522
Fraction
Cycles
Diameter (μm)
23% Sucrose solution (control)
7
24.3 1.7A
27% Sucrose solution
7
22.0 1.2A
23% Sucrose solution + 4% Fraction 2
7
7.6 0.9B
23% Sucrose solution + 4% SP2
7
4.4 0.6C
523 524
Values expressed as mean SD.
525
significantly (P < 0.05).
526
Fraction SP2 from SP-Sephadex C25 column at pH 7.
A-CValues
with different letters in the same column differ
Fraction 2 = Fraction 2 from Sephadex G-50 column, and SP2 =
27
ACCEPTED MANUSCRIPT 528
Table 5:
Amino acid sequences of ice structuring peptides from the SP2 fraction.
529
530
Peptide molecular weight
Amino acid sequencea
Net charge at pH 7b
1850.82 Da
KDGTPGQFGP(OH)PGAPGKGN(OH)H
+1.4
2036.88 Da
NEGTPGTGPAGPP(OH)GFHTPK(OH)W
+0.4
aP
(OH)
is hydroxproline, K(OH) is hydroxylysine, and N(OH) is 3-hydroxyasparagine.
531
bEstimated
532
terminal (7.8), and C-terminal (3.5).
using the pKa values of amino acid residues K (10.2), H (7.0), E (4.6), D (4.6), N-
533
28
ACCEPTED MANUSCRIPT 535
Figure Legends
536
Figure 1:
537
Sephadex G-50 column. Hydrolysis was done at an alcalase-to-gelatin ratio of 1:15 (w/w) at
538
pH 9.0, 45 C for different hydrolysis times (10, 15, 20, 25, and 30 min).
539
gelatin hydrolysates on ice crystal growth in an ice cream mix after 7 thermal cycles between
540
-14 and -12 C at the rate of 3 oC/min: (a) ice cream mix alone (control); (bf) ice cream mix
541
+ 4 wt% gelatin hydrolysate obtained at 10, 15, 20, 25, and 30 min hydrolysis time,
542
respectively.
543
hydrolysis time.
544
Figure 2:
545
hydrolysis time.
546
Figure 3:
547
hydrolysate obtained under optimum hydrolysis conditions, i.e., 20% (w/w) fish gelatin in
548
deionized water at pH 9.0 and 45 oC treated with alcalase at an enzyme-to-substrate ratio of
549
1:15 (which was equivalent to 0.176 Anson units/g gelatin) for 25 min.
550
correspond to fractions between the vertical dotted lines that were pooled together.
551
Effects of fraction 1, 2 and 3 (from A) on ice crystal growth in an ice cream mix after 7
552
thermal cycles at -14 to -12 C. (a) Ice cream mix (control); (bd), ice cream mix + 4 wt% of
553
fraction 1, fraction 2, and fraction 3, respectively.
554
Figure 4:
555
Figure 5:
556
exchanger: The column was eluted with 20 mM phosphate buffer (pH 7.0) for the first 100
(A) Elution profiles of alcalase-hydrolyzed fish gelatin hydrolysates on a
(B) Effect of
(C) The size distribution of ice crystals formed in B, plotted as a function of
MALDI-TOF mass spectrum of gelatin hydrolysate obtained at 25 min
(A) Sephadex G-50 size exclusion chromatographic profile of fish gelatin
Fractions 1, 2, and 3 (B)
MALDI-TOF mass spectrum of fraction 2. (A) Elution profile of fraction 2 on Sulfopropyl-Sephadex C-25 cation
29
ACCEPTED MANUSCRIPT 557
mL, followed by elution with 0 – 0.5 M NaCl gradient in 20 mM phosphate buffer (pH 7.0).
558
SP1 and SP2 refer to anionic and cationic peptide sub-fractions, which were pooled
559
separately and lyophilized after exhaustive dialysis against water using a 500 Da cut-off
560
membrane.
561
cream mix after 7 thermal cycles at -14 to -12 C: (a) Ice cream mix (control); (b) ice cream
562
mix + 4 wt% SP1 fraction; (c) ice cream mix + 4 wt% SP2 fraction.
563
Figure 6. MALDI-TOF mass spectrum of the SP2 fraction.
(B) Effect of SP1 and SP2 peptide fractions on ice crystal growth in an ice
564
Figure 7:
Effect of the SP2 fraction on ice crystal growth in an ice cream mix after 7 and 25
565
thermal cycles at -14 to -12 C. (A) and (C) control ice creams after 7 and 25 cycles
566
respectively; (B) and (D) ice cream + 4 wt% SP2 after 7 and 25 cycles, respectively.
567
Figure 8:
568
solution after 7 thermal cycles at -14 to -12C.
569
sucrose solution + 4 wt% fraction 2; (c) 23% sucrose solution + 4 wt% SP2 fraction.
Effects of fraction 2 and the SP2 fraction on ice crystal growth in 23% sucrose
570 571 572 573 574 575
30
(a) 23% sucrose solution (control); (b) 23%
ACCEPTED MANUSCRIPT 577
Figure 1A:
578
Figure 1 B:
579 580 581 582 583 584 585 586
(A)
(B)
(D)
(E)
(C)
587 588 589 590 591 592 593 594 595 596
31
(F)
ACCEPTED MANUSCRIPT 598
Figure 1C:
599 600
25
602 603 604
Ice crystal size (µm)
601
20
15
10
5
0 0
10
20
Hydrolysis Time (min)
32
30
ACCEPTED MANUSCRIPT 606
Figure 2:
607
608 609
33
ACCEPTED MANUSCRIPT Figure 3:
Fraction 1
613
30
614
A
Absorbance at 225nm
25
615 616 617
Fraction 2
612
Fraction 3
611
20 15 10
618
5
619
0 150
200
250
620
300
350
400
450
500
550
Elution volume (mL)
621 622 623 624
B
625 626 627
(a)
(b)
(c)
34
(d)
ACCEPTED MANUSCRIPT 629
Figure 4: MALDI of Fraction 2
630
631 632 633 634 635 636 637
35
ACCEPTED MANUSCRIPT 639
Figure 5A:
640
6
0.50 0.40 0.35
4
0.30 3
0.25 0.20
2
0.15 0.10
1
0.05 0
0.00 0
50
100
150
200
Elution Volume (mL)
641
Figure 5B
642
(A)
(B) 36
(C)
NaCl Concentration (M)
Absorbance at 225nm
0.45 5
ACCEPTED MANUSCRIPT 643
Figure 6:
644
645 646 647 648 649 650 651 652
37
ACCEPTED MANUSCRIPT 654
Figure 7: A,B 7cyles; C, D, 25 cycles; SP2
655 656 657
Figure 8:
658 659 660 661 662 663 664
(a)
(b)
665 666 667
38
(c)
Srinivasan Damodaran, ShaoYun Wang PII:
S0268-005X(16)30621-X
DOI:
10.1016/j.foodhyd.2017.03.029
Reference:
FOOHYD 3840
To appear in:
Food Hydrocolloids
Received Date:
19 October 2016
Revised Date:
21 March 2017
Accepted Date:
21 March 2017
Please cite this article as: Srinivasan Damodaran, ShaoYun Wang, Ice Crystal Growth Inhibition by Peptides from Fish Gelatin Hydrolysate, Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd. 2017.03.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT
Graphical Abstract:
ACCEPTED MANUSCRIPT
Highlights:
Peptides in the molecular weight range of 1000 – 2000 Da in fish gelatin hydrolysate were able to inhibit ice crystal growth in an ice cream mix and in 23% sucrose solution.
Among these, cationic peptides were much more effective than anionic/neutral peptides.
The amino acid sequences of two of these fish gelatin peptides have been determined.
Electrostatic, hydrogen bonding, and hydrophobic forces might be involved in a concerted manner in the binding process.
ACCEPTED MANUSCRIPT
1
Ice Crystal Growth Inhibition by Peptides from Fish Gelatin
2
Hydrolysate
3 4 5 6 7 8 9 10
Srinivasan Damodaran* and ShaoYun Wang Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706
11 12
* Corresponding author.
13
Tel.: +1 608 263 2012; Fax: +1 608 262 6872.
14
E-mail address: [email protected] (S. Damodaran).
15 16 17
1
ACCEPTED MANUSCRIPT 19 20
Abstract
21
Ice crystal growth inhibition in an ice cream mix matrix and in sucrose solutions by
22
peptides derived from alcalase (also known as subtilisin) hydrolyzed fish gelatin was
23
investigated. Hydrolysis of fish gelatin at an optimum hydrolysis condition (i.e. 20% w/w
24
gelatin solution treated with Alcalase at an enzyme-to-substrate ratio of 0.176 Anson units/g
25
gelatin at pH 9.0 for 25 min at 45 oC) released peptides with maximum ice crystal growth
26
inhibition activity.
27
exchange chromatography resulted in isolation of a cationic peptide fraction containing two
28
prominent peptides having 1850.82 Da and 2036.88 Da molecular masses.
29
fraction had the highest ice crystal growth inhibition activity. The amino acid sequences of
30
these peptides showed no sequence similarity other than that they both contained –GTPG-
31
and –GPP(OH)G- motifs and 3 to 5 hydroxyl containing amino acid residues. The results of
32
this study supported the hypothesis that short collagen/gelatin polypeptides in the molecular
33
mass range of 1000 to 2500 Da, regardless of their source, would have the ability to inhibit
34
ice crystal growth in frozen systems. The results also suggested that the mechanism of ice
35
crystal growth inhibition by gelatin peptides might involve three steps, namely, initial
36
nonspecific electrostatic interaction of cationic peptides with the negatively charged ice
37
surface, followed by structural realignment to optimally hydrogen bond with the oxygen-
38
oxygen lattice on the ice surface, and stabilization of the electrostatic and hydrogen bonding
39
in the peptide – ice crystal complex by a partial nonpolar environment created by neighboring
40
hydrophobic residues of the peptide.
Fractionation of gelatin hydrolysate using size exclusion and ion
2
This cationic
ACCEPTED MANUSCRIPT 41 42
Keywords: Fish gelatin hydrolysate; antifreeze peptides; ice crystal growth inhibition; ice
43
structuring peptides.
44
3
ACCEPTED MANUSCRIPT 46
1. Introduction
47 48
Ice crystal growth during cold storage is a quality issue in frozen foods. In nature,
49
organisms that have adapted to sub-freezing temperatures, such as fishes, arthropods, winter
50
plants, bacteria, and fungi, produce antifreeze proteins (AFP) (Clarke, Buckley, & Lindner,
51
2002; Du, Liu, & Hew, 2003; Kristiansen et al., 2005; Kontogiorgos, Regand, Yada, & Goff,
52
2007; Pentelute et al., 2008; Knight, Cheng, & DeVries, 1991; Sidebottom et al., 2000;
53
Worrall et al., 1998; Graham, & Davies, 2005; Wierzbicki et al., 2007; Graether et al., 2000).
54
The AFPs depress the freezing point of water from 1 oC
55
depending on their living habitat (Graham, Liou, Walker, Davies, 1997; Graham, & Davies,
56
2005), but they do not alter the melting point of ice, which remains at 0 oC. This thermal
57
hysteresis is a direct evidence that the APFs depress freezing point of water via a non-
58
collegative mechanism. However, it has been observed that some plant-derived AFPs do not
59
exhibit thermal hysteresis, but cause ice recrystallization inhibition or ice restructuring (RI or
60
ISP) (Sidebottom et al., 2000; Worrall et al., 1998).
(fish AFP) to 5 oC (insect AFP)
61
These natural AFP and ISP proteins may be used in frozen foods and in pharmaceutical
62
products to retard ice crystal growth in order to reduce freezing-induced damage and to
63
extend their shelf life (Griffith, & Ewart, 1995; Adapa et al., 2000; Hartel, 2001; Regend &
64
Goff, 2002); however, their limited availability and the economics have restricted their
65
extensive use in foods.
66
Several studies have reported that hydrocolloids, such as xanthan gum, locust bean gum,
67
carrageenan, alginate, etc., decreased the rate of recrystallization of ice in ice cream and in
68
concentrated sucrose solutions (Regand & Goff, 2002; Adapa et al., 2000; Kaminska4
ACCEPTED MANUSCRIPT 69
Dworznicka et al., 2015). Some of these hydrocolloids have synergistic effect on inhibition of
70
ice recrystallization by fish antifreeze proteins (Gaukel, Leiter, & Spieb, 2014).
71
Enzymatically and non-enzymatically hydrolyzed kappa-carrageenan have been shown to
72
inhibit ice recrystallization in sorbet and concentrated sucrose solutions better than intact
73
kappa-carrageenan (Kaminska-Dworznicka, Skrzypczak, & Gondek, 2016; Kaminska-
74
Dworznicka et al., 2015). However, whether or not this inhibitory effect of hydrocolloids on
75
ice recrystallization is related to increase of viscosity or to specific interaction of
76
hydrocolloids with the ice surface has not been satisfactorily resolved.
77
An examination of the structural attributes of natural AFPs and ISPs shows that they are
78
structurally diverse with regard to molecular mass, amino acid composition and sequence,
79
etc.
80
containing groups geometrically spaced in a two-dimensional array that mimics the spacing
81
of oxygen atoms in the prism face of hexagonal ice (Pentelute et al., 2008; Liou, Tocilj,
82
Davies, & Jia, 2000; Graether, & Sykes, 2004). This flat surface is thought to bind to the
83
prism face of ice via hydrogen bonding, while the other structural elements (i.e. neighboring
84
hydrophobic residues) of the protein create a nonpolar environment wherein the above
85
hydrogen bonding interactions are stabilized (Liou, Tocilj, Davies, & Jia, 2000; Wen, &
86
Laursen, 1992; Knight, Driggers, & DeVries, 1993; Zhang, & Laursen, 1998; Graether, &
87
Sykes, 2004). However, because of lack of direct crystallographic data on the AFP-ice
88
complex, the validity of the hydrogen-bonding lattice match model remains controversial
89
(Antson et al., 2001; Jia & Davies, 2014).
90
interactions have been proposed (Sonnichsen et al., 1996).
However, they all invariably contain a flat ice-binding region with polar oxygen-
Alternative mechanisms involving hydrophobic
5
ACCEPTED MANUSCRIPT 91
Recently, we have hypothesized that if the oxygen-oxygen spacing mimicry in the flat
92
hydrophilic surface with that of the oxygen-oxygen geometry in hexagonal ice is the primary
93
requirement for ice binding, then any polypeptide that can dynamically adapt its backbone
94
conformation as it approached an ice surface and aligned its oxygen containing groups with
95
that of ice surface should also be able to bind and inhibit ice crystal growth in a manner
96
similar to antifreeze proteins (Damodaran, 2007; Wang, & Damodaran, 2009). The protein
97
that fits this requirement is peptides derived from collagen/gelatin, which has highly flexible
98
polypeptide chain with a repeat sequence of – Gly – Xaa – Yaa - (where Xaa is mostly
99
proline or hydroxyproline and Yaa is any other amino acid residue). In support of this
100
hypothesis, recently we had shown that peptides in the molecular weight range of 1000 –
101
3000 Da derived from bovine skin gelatin hydrolysate inhibited growth of ice crystals in an
102
ice cream mix (Damodaran, 2007; Wang, & Damodaran, 2009; Wang, Agyare, &
103
Damodaran, 2009). Molecular dynamics simulations of a model gelatin fragment, viz., Gly-
104
Pro-Ala-Gly, also have demonstrated the mode of binding of this peptide to the prism face of
105
ice nuclei and inhibition of ice crystal growth under super-cooled conditions (Kim,
106
Damodaran, & Yethiraj, 2009).
107
Collagen/gelatin from different species, specifically mammalian versus marine species,
108
differ significantly in their amino acid composition/sequence and other physicochemical
109
properties (Lin, & Liu, 2006; Bae et al., 2008; Nagai, & Suzyki, 2000).
110
collagen contains significantly lower hydroxyproline content than mammalian collagens.
111
Also, the thermal denaturation temperature of fish collagen is lower than that of bovine skin
112
collagen (Lin, & Liu, 2006). Because of these differences, the physicochemical properties of
6
For example, fish
ACCEPTED MANUSCRIPT 113
peptides released from fish collagen/gelatin under similar enzymatic digestion conditions
114
would be different, and hence their ice crystal growth inhibiting properties also might be
115
different from those from mammalian collagen/gelatin hydrolysate.
116
The objective of this study was to determine if, despite significant differences between
117
physicochemical properties of fish versus mammalian collagen/gelatin, Alcalase hydrolysis
118
liberated ice crystal growth inhibiting peptides from fish gelatin.
119
human population do not consume mammalian gelatin for religious reasons, and fish skin and
120
bones are underutilized byproducts of the fish processing industry, elucidation of the
121
conditions under which Alcalase hydrolysis of fish collagen/gelatin produces ice crystal
122
growth inhibiting peptides and molecular characterization of such peptides will expand the
123
utilization of this important biomass.
Since certain segments of
124 125
2. Materials and methods
126 127
2.1. Materials
128 129
Ice cream mix was obtained from the dairy plant of the Department of Food Science at
130
the University of Wisconsin-Madison. The total non-fat solids content of the ice cream mix
131
(milk solids + sucrose) was 27% (w/w) and its pH was 6.6.
132
ice cream mix was diluted 15 wt% with water to compensate for inclusion of up to 4% gelatin
133
hydrolysate as nonfat solids.
134
mix which was stored at -20 °C in 2 mL aliquots in cryo-vials and all experiments were
In all experiments, the original
All experiments were conducted on a single batch of ice cream
7
ACCEPTED MANUSCRIPT 135
carried out on this single batch by using one vial at a time.
136
Dr. Joe Regenstein at the Department of Food Science at Cornell University, Ithaca, NY,
137
kindly provided a commercial fish gelatin, prepared by enzymatic hydrolysis of fish skin of a
138
carp species. Alcalase (EC 3.4.21.14) in a solution form (2.64 Anson units/g solution) was
139
purchased from Sigma Chemicals Co. (St. Louis, MO, U.S.A). Sephadex G-50 was from
140
Fisher Scientific Co. (Fairlawn, NJ, U.S.A), and Sulfopropyl-Sephadex C-25 (SP-Sephadex)
141
was from Sigma Chemicals Co. (St. Louis, MO, U.S.A).
142 143
2.2. Fish gelatin hydrolysis and fractionation
144 145
Alcalase hydrolysis of fish gelatin was performed as described previously for bovine
146
gelatin hydrolysis (Wang, & Damodaran, 2009; Wang, Agyare, & Damodaran, 2009). The
147
optimum pH for Alcalase activity ranges from pH 7.5 at 50 oC to pH 10 at 65 oC.
148
in preliminary experiments, fish gelatin hydrolysates produced at pH 9.0 and 45 oC using an
149
enzyme-to-substrate ratio of 1:15 (w/w) were found to be more effective in inhibiting ice
150
recrystallization than those obtained at pH 7.0. These conditions were used in the present
151
study to produce fish gelatin hydrolysates.
152
of fish gelatin in deionized water at pH 9.0 were incubated at 45 oC in a water bath until
153
gelatin completely dissolved into a solution. Alcalase was added at an enzyme-to-substrate
154
ratio of 0.176 Anson units/g gelatin (which was equivalent to 1 g of the enzyme solution to
155
15 g of gelatin).
156
stopped by incubating the solutions for 10 min in boiling water.
However,
Briefly, 50 mL aliquots of a 20% (w/w) solution
Hydrolysis was carried out for 10, 15, 20, 25, and 30 min intervals and
8
ACCEPTED MANUSCRIPT 157
Fractionation of fish gelatin hydrolysate on Sephadex G-50 and Sulfopropyl-Sephadex
158
was performed as described previously (Wang, & Damodaran, 2009; Wang, Agyare, &
159
Damodaran, 2009).
160 161
2.3.
Mass spectrometry
162 163
The molecular weight distribution of peptide fractions was analyzed by Matrix-Assisted
164
Laser Desorption IonizationTime of Flight (MALDITOF) mass spectrometry (Applied
165
Biosystems, Foster City, CA, U.S.A).
166
growth inhibiting peptides were determined by LC/MS/MS mass spectrometry at the
167
Biotechnology Center of the University of Wisconsin-Madison.
The amino acid sequences of two of the ice crystal
168 169
2.4.
Ice crystal growth inhibition activity determination
170 171
Ice crystal growth in ice cream mix and in 23% sucrose solution was studied using a cold
172
stage (Model HCS302, Instec Scientific Instruments Ltd., Boulder, CO, U.S.A) mounted on a
173
Nikon Eclipse microscope (E200, Nikon Inc., Japan) as described previously (Damodaran,
174
2007; Wang, & Damodaran, 2009; Wang, Agyare, & Damodaran, 2009). Images of ice
175
crystal growth were captured and analyzed using IMAGE-PRO PLUS software (Media
176
Cybernetics, Silver Spring, MD, U.S.A).
177
cream or sucrose solution on a microscope slide covered with a cover slip was mounted on
178
the thermal stage of the microscope and subjected to the following time-temperature
179
program: The sample was cooled rapidly to -40 °C at the rate of 40 °C/min, held for 5 min at
180
that temperature. The temperature was then raised to -14 °C at the rate of 1°C/min and then
In a typical experiment, a small drop (5 μL) of ice
9
ACCEPTED MANUSCRIPT 181
cycled 7 times between -14 and -12 °C at a rate of 1 cycle/3 min.
Images of the sample at -
182
40 oC and immediately after 7 cycles at -14 and -12 °C were captured at a magnification of
183
220X. The IMAGE-PRO PLUS software was capable of automatically scanning and
184
determining the average two-dimensional size of the crystals in the captured image.
185
Typically, triplicate runs were made on each sample and the ice crystal size was determined
186
as average of the triplicate.
187 188
2.5.
Statistical analysis
189 190
Ice crystal growth experiments on each hydrolysate sample were done in triplicate. The
191
ice crystal sizes are reported as average ± standard deviation of the triplicate measurement.
192
When significant treatment effects (P 0.05) were found, their means were separated by
193
Duncan’s multiple range tests using SAS statistical package.
194 195
3.
Results
196
Figure 1A shows elution profiles of fish gelatin hydrolysates on a Sephadex G-50
197
column. As expected, the elution profile shifted towards lower molecular weight as the
198
hydrolysis time was increased from 10 min to 30 min.
199
of hydrolysis time on the ice structuring activity of the hydrolysates. At 4wt% level, the 25
200
min hydrolysate produced peptides with maximum ice crystal growth inhibition activity.
201
Previously, it has been reported that in the case of bovine gelatin the optimum hydrolysis
202
time with Alcalase under similar experimental conditions was 30 min (Wang & Damodaran,
203
2009). The difference of 5 min between bovine and fish gelatin might be due to differences in 10
Figures 1B and 1C show the effect
ACCEPTED MANUSCRIPT 204
amino acid composition/sequence and other physicochemical properties that might make fish
205
gelatin more susceptible than bovine gelatin to Alcalase hydrolysis.
206
in Figures 1A-C indicate that the size distribution of peptides in fish gelatin hydrolysate
207
played a crucial role in the ice structuring activity.
Nevertheless, the data
208
The MALDI-TOF mass spectrum of peptides in the total fish gelatin hydrolysate
209
produced under the optimum hydrolysis condition (i.e., 20% w/w gelatin solution hydrolyzed
210
with Alcalase at an enzyme-to-substrate ratio of 1:15 at pH 9.0, 45 oC for 25 min) is shown in
211
Figure 2.
212
To elucidate which peptides within the molecular mass range were greatly responsible for the
213
ice structuring activity, the eluted fractions from the 25 min hydrolysate on Sephadex G-50
214
column were pooled into three fractions as shown in Figure 3A and they were lyophilized.
215
The ice structuring activity of these three fractions at 4% (w/w) level in ice cream mix after 7
216
thermal cycles between -14 and -12 oC is shown in Figure 3B, and the average size of ice
217
crystals produced in the ice cream mix is presented in Table 1. Among these three fractions,
218
fraction 2 (from the middle portion of the elution profile in Figure 3A) exhibited the best ice
219
structuring activity with an average ice crystal size of 4.6 0.5 µm. Although the average ice
220
crystal size in ice cream mix containing 4% total fish gelatin hydrolysate (6.4 0.5 µm) was
221
statistically larger than that containing fraction 2 (4.6 0.5 µm), its activity was better than
222
fraction 1 (18.2 0.9 µm), fraction 3 (9.3 1.2 m), and the control ice cream (22.2 1.2
223
µm).
224
hydrolysate (6.4 0.5 µm) was mostly derived from peptides in fractions 2 and 3.
225
The molecular weight distribution of the peptides ranged from 800 to 3300 Da.
Thus, the data in Table 1 tentatively suggest that the ice structuring activity of total
Shown in Figure 4 is the MALDI-TOF mass spectrum of fraction 2.
11
Although faction 2
ACCEPTED MANUSCRIPT 226
contained numerous peptides, the most dominant ones were the peptides corresponding to
227
molecular mass of 852.45, 1182.52, 1410.65, 1850.83, 2036.88, 2080.03, 2728.18, and
228
3320.49 Da. Fraction 2 was further fractionated on SP-Sephadex C-25 cation-exchange
229
column, first using 20 mM phosphate buffer (pH 7.0) to elute the anionic and neutral peptides
230
(SP1 fraction) and then with a 0 – 0. 5 M NaCl gradient in 0.02 M phosphate buffer (pH 7.0)
231
to elute the adsorbed cationic peptides (SP2 fraction) (Figure 5A). The SP1 and SP2
232
fractions were pooled separately and dialyzed for 48 h at 4 °C against water using a 500
233
molecular weight cut-off membrane to remove all salts, and lyophilized. The ice structuring
234
activity of these dialyzed and lyophilized SP1 and SP2 fractions are shown in Figure 5B and
235
the average ice crystal size distributions after 7 thermal cycles are presented in Table 2.
236
The results indicate that at pH 7.0 the cationic peptides fraction (SP2) was more effective
237
(Figure 5B, panel C) than the anionic peptides fractions (SP1) (Figure 5B, panel B) in
238
inhibiting ice recrystallization. The average size of ice crystals in SP2-containing ice cream
239
mix was 2.5 ± 0.7 m compared to 9.5 ± 0.9 m in SP1 containing samples and 22.1 ± 1.2
240
m in control ice cream mix. It is interesting to note that at pH 4.0 both SP1 and SP2
241
fractions were equally effective in their ice structuring activity with an average ice crystal
242
size of 4.8 ± 0.8 and 4.0 ± 0.6 m, respectively (Table 2). The increase in ice crystal
243
inhibition activity of the SP1 fraction at pH 4 is likely due to charge reversion from
244
anionic/neutral at pH 7 to cationic state at pH 4. These results indicate that, in addition to size
245
distribution, the charge characteristics of gelatin peptides also play a critical role in their ice
246
structuring activity.
247
The MALDI-TOF mass spectrum of the SP2 fraction is shown in Figure 6.
12
It
ACCEPTED MANUSCRIPT 248
contained 1182.52 Da, 1850.82 Da, and 2036.88 Da peptides as the prominent ones along
249
with several other peptides, suggesting that these peptides could be responsible for the ice
250
crystal inhibiting activity of SP2. Significantly, the SP2 peptides were able to retard ice
251
crystal growth in ice cream mix even after 25 thermal cycling between -14 and -12 oC
252
(Figure 7).
253
9.8 1.3 µm after 25 cycles, whereas under these conditions the average ice crystal size in
254
the control ice cream mix grew from 22.1 1.2 µm to about 46.6 2.9 µm (Table 3).
255
Previously, experimental evidences were provided to show that ice crystal inhibition by
256
peptides derived from bovine gelatin was not due to any non-specific effects, such as
257
alterations in viscosity (Wang & Damodaran, 2009).
258
SP2 fraction might arise from specific interaction of the peptides in this fraction, e.g. 1850.82
259
Da and 2036.88 Da peptides, with the ice surface.
The average ice crystal size increased from 2.5 ± 0.7 m after 7 cycles to about
Thus, the ice structuring activity of the
260
The fraction 2 and the SP2 fraction inhibited ice crystal growth in 23% sucrose solution
261
as well (Figure 8). The inhibition was more pronounced with the SP2 fraction than with
262
fraction 2, which was also the case in the ice cream mix (Table 4), indicating that the
263
composition of the medium did not have any effect. Given that the 1850.82 Da and 2036.88
264
Da peptides were present both in fraction 2 and in the SP2 fraction (Figures 4 and 6), the
265
differences in the effectiveness of these two fractions might be due to differences in relative
266
amounts of these two peptides in these fractions.
267
To understand the structure-function relationship of the 1850.82 Da and 2036.88 Da
268
peptides with respect to their ice crystal inhibiting properties, the amino acid sequence of
269
these peptides were determined using LC/MS/MS mass spectrometry.
13
The amino acid
ACCEPTED MANUSCRIPT 270
sequences of these peptides are shown in Table 5.
It should be noted that whereas
271
mammalian collagen/gelatin strictly contains –Gly – X – Y – repeat motif, this does not
272
appear to be the case in fish collagen/gelatin as evidenced from the presence of a –Gly – X –
273
Gly – motif in both the 1850.82 Da and the 2036.88 Da peptides (Table 5). In addition, fish
274
collagen/gelatin has -hydroxyasparagine (N(OH)), which, to our knowledge, is not found in
275
mammalian collagen/gelatin. However, -hydroxyaspartate and -hydroxyasparagine are
276
found in the human cytoskeletal ankyrin family of proteins (Yang et al., 2011). The
277
calculated net charge of these peptides was +1.4 and +0.4, respectively.
278
peptide has three side chain OH groups, whereas the 2036.88 Da peptide contains five side
279
chain OH groups.
280
interactions with the ice surface.
The 1850.82 Da
These OH groups may potentially be involved in hydrogen bonding
281 282
4.
Discussion
283 284
The results presented here clearly demonstrate that despite wide differences in the
285
physicochemical properties, such as amino acid composition and sequence, peptides derived
286
from alcalase hydrolysis of fish collagen/gelatin were able to inhibit ice crystal growth in a
287
manner similar to peptides derived from bovine collagen/gelatin hydrolysate reported
288
previously (Damodaran, 2007).
289
the molecular range of 1000 – 2500 Da, corresponding to about 10 to 25 amino acid residues,
290
were more effective than the others, suggesting that gelatin peptides in this molecular weight
291
range possessed the structural properties needed for binding to ice crystals. This indicates that
The following are evident in both cases: First, peptides in
14
ACCEPTED MANUSCRIPT 292
binding of gelatin peptides to ice surface is inexplicably linked to the unique amino acid
293
sequence pattern, i.e. the –Gly – X – Y – (where X is often either Pro or hydroxyproline
294
residue and Y is any other residue) repeat motif, which is found in all collagens, including
295
mammalian and fish collagen/gelatin.
296
the abundance of Gly residues (and Pro residues as well), the functional similarity of both
297
bovine and fish gelatin peptides vis-à-vis ice crystal growth inhibition, supports the
298
previously proposed hypothesis (Damodaran, 2007; Wang, & Damodaran, 2009) that
299
polypeptides that can dynamically reconfigure its backbone conformation to align their
300
oxygen-containing groups to match with the oxygen-oxygen geometry of hexagonal ice
301
lattice should be able to bind to ice surface and inhibit its growth.
Since gelatin peptides are highly flexible owing to
302
Second, in both bovine (Damodaran 2007; Wang, & Damodaran, 2009) and fish gelatin
303
peptides cases, cationic peptides (the SP2 fraction) were much more effective than either
304
neutral or anionic peptides (the SPI fraction) in inhibiting ice crystal growth. This indicates
305
that in addition to the molecular weight and flexibility requirements, the efficient binding of
306
gelatin peptides to ice crystal surface requires the peptide to be cationic. This is obvious from
307
the fact that while the cationic SP2 fraction was more effective than the anionic/neutral
308
fraction SP1 at pH 7, they both were equally effective at pH 4.0 (where protonation of
309
carboxyl groups (pKa ≈ 4.6) would make the SP1 peptides positively charged) in inhibiting
310
ice crystal growth (Table 2).
311
This positive charge requirement implicitly suggests involvement of electrostatic
312
interaction in the peptide – ice surface binding process. In order for this to be true, the ice
313
surface ought to be negatively charged. As a matter of fact, electrokinetic and potentiometric
15
ACCEPTED MANUSCRIPT 314
measurements have shown that the surface potential of the ice-water interface swings from a
315
positive potential at pH < 3.5 to a negative potential at pH > 3.5 (Kallay & Cakara, 2000;
316
Kallay, Cop, Chibowski, & Holysz, 2003; Cop & Kallay, 2004). This pH-dependent
317
reversible surface potential of the ice-water interface is due to protonation and deprotonation
318
of amphoteric surface OH groups on the ice surface. The surface potential of ice-water
319
interface at pH > 6 is typically in the range of -200 to -300 mV at 0 oC (Kallay, Cop,
320
Chibowski & Holysz, 2003; Cop & Kallay, 2004).
321
property of cationic peptides fraction (SP2) at pH 7.0 and both SP1 and SP2 fractions at pH
322
4.0 is partly due to attractive electrostatic interaction of the peptides with the negatively
323
charged ice surface at these pHs. Interestingly, several antifreeze proteins from fish (type III)
324
and insects are also cationic at neutral pH, with isoelectric pH in the range of 7.1 – 9.8 (Xu et
325
al., 2008; Kristiansen et al., 2005; Hossain, 2012), suggesting that attractive electrostatic
326
interaction between the ice surface and antifreeze proteins and peptides might be an initial
327
step in the binding process. However, it should be recognized that electrostatic interaction is a
328
nonspecific interaction and it alone might possess the ability to block ice crystal growth,
329
which is a structurally very specific process. It is conceivable, however, that this nonspecific
330
electrostatic interaction might initially drive the cationic gelatin peptides (and AFPs) toward
331
the ice surface, and facilitate specific hydrogen bonding of the structurally complimentary
332
region on the AFP (e.g. the flat surface of AFP) with the binding site on the ice surface.
333
the case of gelatin peptides this may involve dynamic alignment of the oxygen-containing
334
groups of gelatin peptides, facilitated by their molecular flexibility, with the oxygen-oxygen
335
geometry of the ice lattice.
Thus, the better ice growth inhibition
In
Once formed, these hydrogen bonds might be further stabilized
16
ACCEPTED MANUSCRIPT 336
by partial nonpolar environment created by hydrophobic residues in other structural elements
337
of peptides/proteins. Thus, electrostatic, hydrogen bonding, and hydrophobic forces might
338
involved in a concerted manner in the binding of gelatin peptides as well as AFPs to the ice
339
surface.
340
Although there is no amino acid sequence similarity, the two peptides from the SP2
341
fraction (Table 5) show some structural similarities: Both contain –GTPG- and –GPP(OH)G-
342
motifs.
343
groups might be involved in hydrogen bonding with the oxygen-oxygen lattice on the ice
344
surface.
345
gelatin peptide, Gly-Pro-Ala-Gly, was able to retard ice crystal growth in a supercooled
346
system at 260 K, and the mechanism involved hydrogen bonding between the ice surface and
347
the carbonyl groups of the peptide (Kim, Damodaran, & Yethiraj, 2009). A similar
348
mechanism also might be possible in the case of the cationic peptides listed in Table 5.
In addition, they contain 3 to 5 hydroxyl-containing residues.
These hydroxyl
Previously, using molecular dynamics simulation we had shown that a model
349
Although the SP2 fraction was the most effective in retarding ice crystal growth in ice
350
cream mix as well as in concentrated sucrose solutions, for all practical purposes the total fish
351
gelatin hydrolysate itself was quite effective at 4% (w/w) level in retarding ice crystal growth
352
in ice cream mix (Table 1).
353 354
5.
Conclusion
355 356
The results of this study supported the original hypothesis (Damodaran, 2007) that
357
short collagen/gelatin polypeptides in the molecular mass range of 1000 to 2500 Da,
17
ACCEPTED MANUSCRIPT 358
regardless of their source, would have the ability to inhibit ice crystal growth in frozen
359
systems. This is due to their ability to dynamically change their conformation as they
360
approach the ice surface and bind to the surface. The results also suggested that the
361
mechanism of ice crystal growth inhibition by gelatin peptides might involve three steps,
362
namely, initial nonspecific electrostatic interaction with the negatively charged ice surface,
363
followed by structural rearrangement to optimally hydrogen bond with the oxygen-oxygen
364
lattice on the ice surface, and stabilization of the electrostatic and hydrogen bonding
365
interaction in the peptide – ice crystal complex by a partial nonpolar environment created by
366
neighboring hydrophobic residues of the peptide. A better understanding of this peptide – ice
367
surface interaction may lead to rationale designing of peptide cryoprotectants with greater
368
antifreeze activity.
369 370
ACKNOWLEGEMENT The authors are grateful to Professor Joe Regenstein at Cornell University for
371 372
donating fish gelatin.
This material is based upon work supported by the National Institute
373
of Food and Agriculture, United States Department of Agriculture Grant No. 2006-35503-
374
16998).
375
18
ACCEPTED MANUSCRIPT 377
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484
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485 486
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ACCEPTED MANUSCRIPT 488 489 490
Table 1. Influence of fish gelatin peptide fractions on ice crystal growth in an ice cream mix. Fraction
491 492 493 494
Cycles
Diameter (μm)
15% diluted ICM (Control)
7
22.2 1.2A
15% diluted ICM + 4% Total Gelatin Hydrolysate
7
6.4 ± 0.5C
15% diluted ICM + 4% Fraction 1
7
18.2 0.9A
15% diluted ICM + 4% Fraction 2
7
4.6 0.5D
15% diluted ICM + 4% Fraction 3
7
9.3 1.2B
Values expressed as mean SD. A-EValues with different letters in the same column differ significantly (P < 0.05). ICM = Ice cream mix, Fraction 1= Fraction 1 from Sephadex G-50 column, Fraction 2 = Fraction 2 from Sephadex G-50 column, Fraction 3 = Fraction 3 from Sephadex G-50 column.
24
ACCEPTED MANUSCRIPT 496 497 498
Table 2. Influence of SPI and SP2 gelatin peptide fractions on average Ice Crystal size in an ice cream mix at different pH.
499 500
Peptide Fraction
501 502 503
Cycles
Diameter (μm)
15% diluted ICM (Control)
7
22.2 1.2A
15% diluted ICM + 4% SP1 at pH4
7
4.8 0.8C
15% diluted ICM + 4% SP2 at pH4
7
4.1 0.6C
15% diluted ICM + 4% SP1 at pH7
7
9.5 0.9B
15% diluted ICM + 4% SP2 at pH7
7
2.5 0.8D
Values expressed as mean SD. A-EValues with different letters in the same column differ significantly (P < 0.05). ICM = Ice cream mix, SP1= Fraction SP1 from SP-Sephadex C25 column, and SP2 = Fraction SP2 from SP-Sephadex C25 column.
25
ACCEPTED MANUSCRIPT 505 506 507 508
Table 3.
Mean Ice-Crystal size in an Ice Cream Mix treated with SP2 peptide fraction after two thermal cycling conditions at pH 7.0.
509 510 511
512 513 514
Fraction Control Control Sample of SP2 Sample of SP2 Values expressed as mean SD. significantly different (P < 0.05).
Cycles Diameter (μm ) 7 22.2±1.2A 25 46.6±2.9B 7 2.5 0.8C 25 9.8± 1.3D A,BValues with the same letters in the same column are not
26
ACCEPTED MANUSCRIPT 516 517 518 519 520
Table 4:
Effect of Fraction 2 and SP2 fish gelatin peptides on ice crystal growth in
sucrose solutions.
521 522
Fraction
Cycles
Diameter (μm)
23% Sucrose solution (control)
7
24.3 1.7A
27% Sucrose solution
7
22.0 1.2A
23% Sucrose solution + 4% Fraction 2
7
7.6 0.9B
23% Sucrose solution + 4% SP2
7
4.4 0.6C
523 524
Values expressed as mean SD.
525
significantly (P < 0.05).
526
Fraction SP2 from SP-Sephadex C25 column at pH 7.
A-CValues
with different letters in the same column differ
Fraction 2 = Fraction 2 from Sephadex G-50 column, and SP2 =
27
ACCEPTED MANUSCRIPT 528
Table 5:
Amino acid sequences of ice structuring peptides from the SP2 fraction.
529
530
Peptide molecular weight
Amino acid sequencea
Net charge at pH 7b
1850.82 Da
KDGTPGQFGP(OH)PGAPGKGN(OH)H
+1.4
2036.88 Da
NEGTPGTGPAGPP(OH)GFHTPK(OH)W
+0.4
aP
(OH)
is hydroxproline, K(OH) is hydroxylysine, and N(OH) is 3-hydroxyasparagine.
531
bEstimated
532
terminal (7.8), and C-terminal (3.5).
using the pKa values of amino acid residues K (10.2), H (7.0), E (4.6), D (4.6), N-
533
28
ACCEPTED MANUSCRIPT 535
Figure Legends
536
Figure 1:
537
Sephadex G-50 column. Hydrolysis was done at an alcalase-to-gelatin ratio of 1:15 (w/w) at
538
pH 9.0, 45 C for different hydrolysis times (10, 15, 20, 25, and 30 min).
539
gelatin hydrolysates on ice crystal growth in an ice cream mix after 7 thermal cycles between
540
-14 and -12 C at the rate of 3 oC/min: (a) ice cream mix alone (control); (bf) ice cream mix
541
+ 4 wt% gelatin hydrolysate obtained at 10, 15, 20, 25, and 30 min hydrolysis time,
542
respectively.
543
hydrolysis time.
544
Figure 2:
545
hydrolysis time.
546
Figure 3:
547
hydrolysate obtained under optimum hydrolysis conditions, i.e., 20% (w/w) fish gelatin in
548
deionized water at pH 9.0 and 45 oC treated with alcalase at an enzyme-to-substrate ratio of
549
1:15 (which was equivalent to 0.176 Anson units/g gelatin) for 25 min.
550
correspond to fractions between the vertical dotted lines that were pooled together.
551
Effects of fraction 1, 2 and 3 (from A) on ice crystal growth in an ice cream mix after 7
552
thermal cycles at -14 to -12 C. (a) Ice cream mix (control); (bd), ice cream mix + 4 wt% of
553
fraction 1, fraction 2, and fraction 3, respectively.
554
Figure 4:
555
Figure 5:
556
exchanger: The column was eluted with 20 mM phosphate buffer (pH 7.0) for the first 100
(A) Elution profiles of alcalase-hydrolyzed fish gelatin hydrolysates on a
(B) Effect of
(C) The size distribution of ice crystals formed in B, plotted as a function of
MALDI-TOF mass spectrum of gelatin hydrolysate obtained at 25 min
(A) Sephadex G-50 size exclusion chromatographic profile of fish gelatin
Fractions 1, 2, and 3 (B)
MALDI-TOF mass spectrum of fraction 2. (A) Elution profile of fraction 2 on Sulfopropyl-Sephadex C-25 cation
29
ACCEPTED MANUSCRIPT 557
mL, followed by elution with 0 – 0.5 M NaCl gradient in 20 mM phosphate buffer (pH 7.0).
558
SP1 and SP2 refer to anionic and cationic peptide sub-fractions, which were pooled
559
separately and lyophilized after exhaustive dialysis against water using a 500 Da cut-off
560
membrane.
561
cream mix after 7 thermal cycles at -14 to -12 C: (a) Ice cream mix (control); (b) ice cream
562
mix + 4 wt% SP1 fraction; (c) ice cream mix + 4 wt% SP2 fraction.
563
Figure 6. MALDI-TOF mass spectrum of the SP2 fraction.
(B) Effect of SP1 and SP2 peptide fractions on ice crystal growth in an ice
564
Figure 7:
Effect of the SP2 fraction on ice crystal growth in an ice cream mix after 7 and 25
565
thermal cycles at -14 to -12 C. (A) and (C) control ice creams after 7 and 25 cycles
566
respectively; (B) and (D) ice cream + 4 wt% SP2 after 7 and 25 cycles, respectively.
567
Figure 8:
568
solution after 7 thermal cycles at -14 to -12C.
569
sucrose solution + 4 wt% fraction 2; (c) 23% sucrose solution + 4 wt% SP2 fraction.
Effects of fraction 2 and the SP2 fraction on ice crystal growth in 23% sucrose
570 571 572 573 574 575
30
(a) 23% sucrose solution (control); (b) 23%
ACCEPTED MANUSCRIPT 577
Figure 1A:
578
Figure 1 B:
579 580 581 582 583 584 585 586
(A)
(B)
(D)
(E)
(C)
587 588 589 590 591 592 593 594 595 596
31
(F)
ACCEPTED MANUSCRIPT 598
Figure 1C:
599 600
25
602 603 604
Ice crystal size (µm)
601
20
15
10
5
0 0
10
20
Hydrolysis Time (min)
32
30
ACCEPTED MANUSCRIPT 606
Figure 2:
607
608 609
33
ACCEPTED MANUSCRIPT Figure 3:
Fraction 1
613
30
614
A
Absorbance at 225nm
25
615 616 617
Fraction 2
612
Fraction 3
611
20 15 10
618
5
619
0 150
200
250
620
300
350
400
450
500
550
Elution volume (mL)
621 622 623 624
B
625 626 627
(a)
(b)
(c)
34
(d)
ACCEPTED MANUSCRIPT 629
Figure 4: MALDI of Fraction 2
630
631 632 633 634 635 636 637
35
ACCEPTED MANUSCRIPT 639
Figure 5A:
640
6
0.50 0.40 0.35
4
0.30 3
0.25 0.20
2
0.15 0.10
1
0.05 0
0.00 0
50
100
150
200
Elution Volume (mL)
641
Figure 5B
642
(A)
(B) 36
(C)
NaCl Concentration (M)
Absorbance at 225nm
0.45 5
ACCEPTED MANUSCRIPT 643
Figure 6:
644
645 646 647 648 649 650 651 652
37
ACCEPTED MANUSCRIPT 654
Figure 7: A,B 7cyles; C, D, 25 cycles; SP2
655 656 657
Figure 8:
658 659 660 661 662 663 664
(a)
(b)
665 666 667
38
(c)