Calcium Binding Restores Gel Formation of Succinylated Gelatin and

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Calcium-binding restores gel formation of succinylated gelatin and reduces brittleness with preservation of the elastically stored energy. Harmen H.J. de Jongh, and Diana Baigts Allende J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01962 • Publication Date (Web): 08 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Agricultural and Food Chemistry

Calcium-binding restores gel formation of succinylated gelatin and reduces brittleness with preservation of the elastically stored energy.

Diana Baigts Allende1, and Harmen H.J. de Jongh1,2, #

1

TI Food and Nutrition, P.O. Box 557, 6700 AN, Wageningen, the Netherlands

2

ProtIn consultancy, Rozenstraat 19E, 3702VL Zeist, the Netherlands

#

Corresponding author: Harmen H.J. de Jongh, TI Food and Nutrition, P.O. Box 557, 6700

AN, Wageningen, the Netherlands; Tel.: +31 317 486160; e-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

2

To better tailor gelatins for textural characteristics in (food) gels their interactions are

3

destabilized by introduction of electrostatic repulsions and creation of affinity sites for

4

calcium to ‘lock’ intermolecular interactions. For that purpose gelatins with varying degrees

5

of succinylation are obtained. Extensive succinylation hampers helix formation and gel

6

strength is slightly reduced. At high degrees of succinylation the helix propensity,

7

gelling/melting temperatures, the concomitant transition enthalpy and gel strength become

8

calcium-sensitive and relatively low calcium-concentrations restore these properties largely.

9

Although succinylation has a major impact on the brittleness of the gels formed and the

10

addition of calcium makes the material less brittle compared to non-modified gelatin, the

11

modification has no impact on the energy balance in the gel where all energy applied is

12

elastically stored in the material. This is explained by the unaffected stress relaxation by the

13

network and high water holding capacity related to the small mesh sizes in the gels.

14 15 16 17 18 19 20 21

Keywords:

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Gelatin, protein gel, succinylation, calcium-binding, rheology

23


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24

INTRODUCTION

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Collagen is an abundant protein in tissues organized with a highly ordered molecular axis in

26

fibrous networks contributing to the spatial cellular structure 1. Gelatin is the product of

27

structural and chemical degradation of collagen, that still resembles most of the functional

28

properties of the original molecule. Gelatin is widely used in a variety of applications in

29

different sectors of the food industry, as bakery (to promote emulsification, or gelling and

30

stabilization properties), dairy (stabilization and texture), processed meat (water-binding),

31

confectionery (texture), as well as in specific products like jam, jelly and low-fat spreads

32

(promoting creaminess, fat reduction and mouth feel)

33

functional properties in production of foods makes it a valuable tool for developing new and

34

more attractive or tailored products for consumers. One of the most important quality

35

parameters in processed foods is its texture. The importance of understanding microstructure

36

formation of gelatin networks and their intermolecular interactions lies in the established

37

relationship between sensorial perception (mouth-feel) of food-based protein gels and their

38

structural building blocks. Gel structure has an important influence on the mechanical

39

responses, which are controlled by the mechanism of network-formation and mechanical

40

breakdown of the network 4.

41

A primordial attribute of gelatin-gels is the ability to elastically store energy that can be

42

recovered when an applied deformation relaxes. Viscoelastic solid foods can be studied by

43

rheological measurements, within the linear regime (small deformation) or in the typically

44

non-linear regime including fracture events (large deformation). When deformation energy is

45

applied to a gel, there are different ways to store or dissipate the applied energy 5. The stored

46

energy has been measured by the recoverable energy in uniaxial compression experiments. It

47

has been hypothesized that the microstructural properties of the network set the magnitude of

48

dissipation and thereby determine the recoverable energy. The profound stiffness of gelatin

2,3

. The versatility of gelatin in



49

gels has been related to the presence of long triple helices (strands) connected by flexible

50

regions 6. The ability of gelatin molecules to assemble into triple helices relates to the net

51

charge of the molecules that affects the number of junctions along a strand and their

52

interaction efficiency. Gelatin molecules connected at random points produce shorter strands

53

giving rise to more cross-linked or branched structures and yield consequently weaker

54

networks 6. Typically, for fibrous networks, like that of gelatin, a number of structural motifs

55

are known to dictate the macroscopic functionality. These are: (i) the mesh size of the

56

network as set by the average distance between junction zones, which is protein concentration

57

dependent, (ii) the length of the dominant structural motif, like the triple helix in gelatins

58

(inherent to the type of gelatin), and (iii) the thickness, and thereby flexibility of the structural

59

motifs (fixed in gelatins). The role of the interaction energy between strands is less clear.

60

In a previous study this strand-strand interaction was impaired by the introduction of steric

61

moieties along the strands (via Maillardation with glucose), with limited impact on helix

62

propensity, slightly reducing gel firmness and slowing down the kinetics of network

63

formation, but without impact on the recoverable energy 7. The aim of this work is to

64

destabilize strand-strand interactions by electrostatic repulsion and creating affinity sites for

65

divalent cations to ‘lock’ the junctions. It is hypothesized that in this way the ability to

66

dissipate energy will be affected. For that purpose gelatins are chemically modified on the

67

available lysine-residues using succinic anhydride with varying degrees of substitution,

68

converting positive charges into negative ones. Using calcium ions the strands, bearing now a

69

higher negative charge density, could become stabilized at their inter-strand junction zones.

70

The materials are chemically and physically characterized, tested for helix propensity and

71

thermal behavior and the impact of the modification on the mechanical behavior is studied

72

using small and large deformation rheology.

73


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MATERIALS and METHODS

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Gelatin modification by succinylation

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Gelatin type A (PGS from pork skin, Bloom 150 and an average molecular weight of ~ 124

77

kDa) was kindly provided from Rousselot® (Gent, Belgium). The material was succinylated

78

according to a protocol described elsewhere 8. To this end, 1 % w/v gelatin was dispersed in

79

0.02M phosphate buffer (pH 8), stirred for 1 hour at 60°C and cooled to 40 °C to dissolve all

80

material. Succinic anhydride (239690-250G, Sigma-Aldrich) was gradually added in small

81

aliquots (~10 mg portions) to the gelatin solutions at continuous stirring at 40 °C up to the

82

fixed amounts between 5 and 35 mg per g of gelatin (see Table I), in order to obtain batches

83

with different degrees of modification. The pH was continuously adjusted to 8.0 using a pH-

84

stat by titration with 1M NaOH. After addition of the succinic anhydride the solutions were

85

stirred for another 30 min. The solutions were extensively dialyzed (Thermo Scientific 10

86

kDa cutoff, 22 mm diameter) against deionized water (at room temperature; pH 6.8) in four

87

repetitions with equilibration times of 6 hours each, and subsequently lyophilized. A reference

88

sample was subjected to the same procedure without addition of succinic anhydride.

89 90

Chromogenic OPA-assay

91

To quantify the degree of succinylation (DS) the availability of lysine-residues was

92

determined using the specific reaction between ortho-phthaldialdehyde (OPA) and free

93

primary amino groups in the proteins in the presence of 2-(dimethyl amino) ethanethiol

94

hydrochloride (DMA) as described previously 9. The measured absorbance was corrected with

95

that of a sample containing non-reacted reagent. A calibration curve was obtained by diluting

96

the OPA reagent with a series of L-leucine (1 mM stock). All assays were done in triplicate.

97

The DS is expressed as 100% minus the percentage of non-reacted primary amines relative to

98

the reference material.



99 100

Apparent isoelectric point

101

To determine the apparent isoelectric point, freeze dried gelatin (reference and modified), was

102

dissolved in miliQ water at a concentration of 0.5 % (w/v). Zeta potential (ζ) values were

103

determined as a function of pH at 20°C using a Zetasizer Nano ZS (Malvern Instruments,

104

UK). Automatic titration was performed with 0.1M HNO3 and 0.1M NaOH solutions under

105

continuous stirring. The isoelectric point was evaluated as the pH where the detected zeta

106

potential value of the material (mV) was zero. For each sample 5 runs were performed. All

107

experiments were performed at least in duplicate.

108 109

Calcium-binding measurements

110

A 0.1 M CaCl2 stock-solution was prepared by dissolving calcium chloride dihydrate (Sigma-

111

Aldrich, Germany) in the appropriate acetate buffer (pH 6.5). The concentration of free Ca+2

112

ions was measured using a calcium ion selective electrode device ( Ca ISE, connected to an

113

Orion Star A214 ISE meter). The potentiometric readings (mV) of the electrode was

114

calibrated using standard CaCl2 solutions (0.5 to 30 mM) with addition of ISA solution to

115

match the ionic strength in all solutions. Gelatin samples (0.9 % w/v; pH 6.5) were titrated

116

with CaCl2 and the corresponding amount of mol calcium bound per mol protein (assuming

117

an average molecular weight of 124 kDa) was established using a Scatchard analysis (see

118

elsewhere for details 10). This higher protein concentration was chosen to increase the

119

sensitivity in the experiment by providing a higher absolute number of calcium-binding sites,

120

but to refrain from too viscous solutions. The contribution of the potassium-acetate buffer

121

with gelatin was neglected in the determination of calcium activity and all samples were

122

adjusted to a similar ionic strength as described previously 11. All experiments were

123

performed in triplicate at 20°C.


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124 125

Calorimetric analysis

126

The enthalpic transition of succinylated solution samples 3% (w/v) in 20 mM acetate-buffer

127

(pH 6.5) in the absence or presence of calcium (0- 20mM) during heating-cooling ramps (60-

128

5°C and vice versa) was measured by differential scanning calorimetry (TA Instrument

129

Q1000 TzeroTM DSC) under ambient conditions. Gelatin solutions (20 µL) were placed in

130

aluminum pans and sealed followed of heating-cooling ramps The heat flow was recorded at

131

2°C/min speed rate in duplicated for each modified and reference sample relative to a protein-

132

free sample. Temperature and enthalpy transition values were obtained by integration of

133

melting and cooling thermograms using the Universal Analysis V1.7F software (TA

134

instruments).

135 136

Optical rotation measurements

137

The triple helix propensity in gelatin in the absence or presence of calcium was monitoring by

138

optical rotation at 436 nm using a Perkin Elmer 341 Polarimeter equipped with a

139

thermostatted water bath (HAAKE Phoexix II C25P model). Solutions were prepared by pre-

140

equilibration at 60°C for 1 hour and subsequent cooling from 50-5°C at 0.5°C/min. Samples

141

(0.5 % w/v; 20 mM acetate buffer pH 6.5) were incubated in a cell with path length of 10 mm.

142

The specific rotation, (∝)λ=∝/cl , where α is the optical rotation angle in deg at λ= 436 nm, c

143

is gelatin concentration (g/mL) and l the length of optical path (dm) was continuously

144

measured. The helix content (h) at a given concentration is determined by means of Eq (1).

145

h=((α)λ-(αcoil)λ)/(( αcollagen)λ-(αcoil)λ)

146

Representative values for (α coil)λ and (α collagen)λ at 436nm of -256 and -800 deg are used

147

respectively 12.

148


( Eq. 1)


149

Mechanical deformation

150

The evolution of the storage and viscous modulus (G’ and G’’ respectively) is monitored

151

during temperature ramps for (modified) gelatin solutions in 20 mM acetate-buffer (pH 6.5)

152

(3% w/v) in the presence of 0-20 mM calcium. Strain-controlled dynamic oscillatory

153

measurements were carried out in an Anton Paar MCR 502 rheometer (Anton Paar, Graz,

154

Austria) using a concentric cylinder geometry. A scan rate of 0.5°C/min was used during

155

cooling and heating ramps from 5-50°C and vice versa.

156

For large deformation studies, preformed 3 % w/v gelatin gels were used, prepared as

157

described above ,cooled to room temperature (20 ±1°C) and equilibrated for 24 hours.

158

Uniaxial compression to fracture experiments were performed using an Instron universal

159

testing machine (model 5543, Instron International LDT, Edegem, Belgium) equipped with a

160

plate-plate geometry. A uniaxial (de)compression speed of 1 mm/s was used. Cylindrical

161

gelatin gels were prepared in 50 mL (diameter 21.7 mm) syringes pre-lubricated with

162

paraffin-oil and cut with a wire in self- supported specimens of 20 mm height. To determine

163

the fracture properties the gels were compressed for 90% of the original height and the stress

164

response was monitored as a function of applied strain.

165

Determination of the recoverable energy (RE) was established by applying a compression-

166

decompression cycle up to a strain of 20% at a deformation speeds of 1 mm/s. The work

167

necessary to compress the samples up to this strain (Wc) was calculated from the area below

168

the stress-strain curve. After reaching the maximal strain, the samples were decompressed

169

immediately at the same speed and the work (Ws) released by the gel specimen was

170

determined accordingly. The results were expressed as RE (%) = 100% * Ws/Wc. All gels

171

were analyzed at least in triplicate.

172

In a similar set-up as described above stress relaxation was evaluated for specimens prepared

173

in the same way. The gels were compressed up to a set strain of 20% at a compression speed


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174

of 1 mm/sec and held at the maximal loading strain for five minutes. During this time interval

175

the stress was recorded with a step-resolution of 60 ms. Every sample was analyzed at least in

176

duplicate. Analysis of the time intervals was performed using a two exponential decay non-

177

linear regression curve-fitting procedure (normalized stress = A1*exp(-τ1/t)+A2*exp(-

178

τ2/t)+A0), as described in detail elsewhere 13. This provided two relaxation times τ1 and τ2

179

with corresponding proportions A1 and A2. A0 reflects in this analysis the stress-contribution

180

that does not dissipate in the experimental time regime of 5 minutes. Typically in all cases R2

181

–values >0.995 were obtained. A higher order of exponents did not gave fits with higher R2-

182

values. For this study, with an experimental time scale for the RE measurements of ~8 sec,

183

only the fast relaxing contribution is reported.

184



185

RESULTS and DISCUSSION

186

Characterization of the material

187

The conceptual idea behind this study is to enable a fortification of the strand-strand

188

interaction points by affinity-binding with calcium. For that purpose different degrees of

189

succinylation of the protein was performed. Table I presents the different materials obtained

190

when gelatin is subjected to different degrees of succinylation (DS) as described in the

191

method section. The DS shows a linear relation with the amount of succinic anhydride added,

192

indicating that the number of lysine groups available for chemical modification is not limiting

193

and/or hampered by steric or electrostatic constraints. It can also be observed that, where the

194

reference material has an isoelectric point (IEP) of around 7.5, the lowest DS already yields

195

an apparent IEP just below 5. The IEP gradually decreases further with increasing DS to 4.4

196

when ~82% of all lysine residues are modified with negative succinate groups. From its

197

amino acid composition this gelatin contains about 27 lysines per 1000 residues and with an

198

average molecular weight of 124 kDa it implies that for the highest DS about 26 succinate

199

groups have been introduced per molecule. All experiments described in this work have been

200

carried out at a pH of 6.5, above the isoelectric point of all succinylated variants, but below

201

that of the reference material.

202

Figure 1 shows the triple helix content as obtained from optical rotation measurements during

203

a cooling ramp down to 5 °C for aqueous solutions of reference and succinylated gelatins with

204

different DS. It can be observed that even a high DS does not affect the onset temperature for

205

helix formation. Moreover, a DS of 43% does not impair the extent of helix induction, while

206

this is marginally affected at a DS of 65%. Only for a DS of 82% a significant reduction of

207

the helix content is found of about 20-25%. Recent work showed that the ability of gelatin to

208

form helices decreased when the availability of lysine decreased 14. Prolonged incubation at


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209

low temperature did not increase the helicity of this latter sample, illustrating that the

210

observed reduction is not the result of a slower kinetic process.

211

Overall, it can be concluded that a series of succinylated gelatins can be prepared that, when

212

studied above their isoelectric point, all exhibit the ability to adopt a triple helical

213

configuration essential for developing a spatial network.

214 215

Gelling behavior in the absence of calcium

216

The gelling properties of the various materials have been studied using small deformation

217

rheology at concentrations well above the critical gel concentration (3 % w/v). The

218

subsequent cooling and heating traces are shown in Figure 2. It can be observed that up to a

219

DS of 65% the introduced succinate groups have only very limited impact on both

220

gelling/melting temperatures as well as on the gel strength developed. Only at a DS of 82% a

221

significant loss in gel strength (factor of 5-6) was observed and the gelling temperature shifted

222

from 17 to 11 °C. Also the helix melting temperature was lowered. Lowering the cooling rate

223

from 0.5 to 0.2 °C/min had a marginal effect on the transition temperature and no significant

224

impact on G’ (results not shown).

225

The gelling temperatures observed with small de formation rheology are confirmed by those

226

obtained from thermal calorimetric analysis (Table II). The corresponding transition enthalpy

227

(related to cooperative thermal processes) decreases with increasing DS and shows a strong

228

reduction for the highest DS of 82%. A lower transition enthalpy was suggested to result from

229

a less cooperative assembly of the triple helices as found by Dardelle and co-workers 15 using

230

different cross linkers that distorted the gelatin strand formation locally. Alternatively, the

231

energy content related to the melting process (Table II last column) shows much higher values

232

compared to those found for the gelling and a smaller dependence on the DS. This suggests



233

that during the gel assembly the kinetics of strand-strand interactions is strongly hindered by

234

the electrostatic repulsions, making the process of network formation less cooperative.

235 236

Calcium binding

237

Figure 3 shows the calcium-binding curves for reference and succinylated gelatin at

238

concentrations below the critical gel concentration (0.5 % w/v). It can be observed that

239

reference gelatin (net negatively charged under the conditions) shows a moderate calcium-

240

binding and with increasing succinate content (all positively charged) the number of calcium-

241

ions that are bound to the protein steadily increases. The fact that no plateau value is reached

242

for any of the samples indicates that the binding affinity to molecular gelatin is relatively

243

weak and there is a continuous equilibrium between bound and non-bound calcium that is

244

shifted towards the bound state by increasing the bulk calcium concentration.

245

The macroscopic impact of the presence of calcium on gel formation is evaluated by

246

assessment of the physical state of protein solutions (pre-equilibrated at 60 °C), after cooling

247

to 20 °C, qualitatively in terms of whether the sample appears liquid (free flow), viscous (a

248

lumpy cohesive flow) or gelled (immobilized) at room temperature. In all cases visually

249

transparent samples (liquids/gels) were obtained. It can be observed in Figure 4A that in the

250

absence of calcium for reference gelatin a concentration higher than 0.8 % (w/v) is needed to

251

obtain a viscous solution and higher than 1.0 % to obtain a gel. In the absence of added

252

calcium it can also be observed that with increasing DS the protein concentration required to

253

obtain a viscous or a gel state shifts to higher protein concentrations. This can be explained by

254

the increased inter-molecular electrostatic repulsions with increasing DS. Figure 4B shows the

255

same samples in the same concentration range but when cooled to room temperature in the

256

presence of 5 mM calcium. Where for reference gelatin no effect of added calcium is present,

257

for all succinylated samples a fortification of the protein network can be observed and most


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258

effectively at higher DS. For example, gelatin with a DS of 26% behaves in the presence of 5

259

mM calcium physically like reference gelatin. 1.2% (w/v) Gelatin with a DS of 82% behaves

260

like a liquid in the absence of calcium, but is gelled in the presence of 5 mM calcium.

261

To elaborate on this effect, the impact of calcium on the gel properties has been studied for all

262

materials. To illustrate this the gel properties of reference gelatin and that with a DS of 82% is

263

presented in more detail below.

264 265

Calcium-sensitivity in gel formation

266

Figure 5 shows the small deformation rheology of 3% (w/v) gelatin solutions (reference and

267

DS 82%) when applying a cooling ramp to pre-equilibrated samples at 50 °C to 4 °C and

268

subsequently a heating ramp up to 50 °C in the presence of different calcium-concentrations.

269

For reference gelatin (Figure 5A) there is no detectable impact of the presence of added

270

calcium up to a concentration of 20 mM, nor in gelling or melting temperature, nor in the

271

maximal gel strength developed. For the succinylated gelatin it was already shown in Figure 2

272

that in the absence of calcium both the gelling temperature and the gel strength were

273

significantly reduced. Figure 5B shows, that addition of calcium ‘restores’ both the gelling

274

temperature and gel strength to those observed for reference gelatin. For DS 65% it has been

275

found that where in the absence of calcium (see Figure 2) a slightly reduced G’ was observed

276

at 5 °C, in the presence of 4 mM of calcium no differences with the reference gelatin was

277

observed anymore (results not shown).

278

This behavior is confirmed when these systems were analyzed calorimetrically, as illustrated

279

in Figure 6. Where for reference gelatin no impact is observed of the presence of calcium both

280

on the gelling temperature and on the concomitant enthalpy change, for the 82% succinylated

281

sample the gelling temperature steadily increases from 11 to 17 °C. Moreover, the

282

concomitant enthalpy change increases from almost negligible in the absence of calcium to



283

about 10 J/g in the presence of 10 mM calcium. This is still considerably lower (~ 30-40%)

284

compared to that observed for reference gelatin. For the 65% DS sample the transition

285

temperature and enthalpy were fully restored by the addition of 10 mM calcium.

286

In order to evaluate whether the calcium acts at the level of strand-strand interactions or at the

287

level of helix assembly, optical rotation of these samples is followed. Figure 7 shows the

288

optical rotation as monitored during cooling for reference (panel 7A) and 82% succinylated

289

gelatin (panel 7B) at concentrations below the critical gelling concentration in the presence of

290

different calcium concentrations. In line with the above results calcium does not have an

291

impact on the helix formation of reference gelatin. As was already shown in Figure 1, in the

292

absence of calcium highly succinylated gelatin has a lower helix propensity. This can, as

293

shown in Figure 7B, be partly (for about half of the loss of helicity in the absence of calcium)

294

restored by the presence of 20 mM calcium. The increased helicity as induced by the presence

295

of calcium does, however, not fully explain the enhancement of the gel mechanical properties

296

as shown in Figure 5, suggesting that the effect of calcium is not limited to the ability to adopt

297

a triple helix only.

298

To study this in more detail, the effect of 20 mM calcium on the helix content and maximal

299

gel strength are determined and presented in Table III for the different succinylated materials.

300

It can be observed that the helicity steadily declines with increasing DS in the presence of 20

301

mM calcium. The storage modulus on the other hand shows a maximum at a DS of 43%,

302

where an enhancement of the gel strength of ~ 20% is found, and the helicity is reduced

303

compared to the reference. At higher DS G’ decreases again rather drastically, despite the fact

304

that under these conditions the helicity is not concomitant reduced.

305 306

Responses to large mechanical deformation


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307

The above-presented results show that extensive succinylation of gelatin does not prohibit gel

308

formation. At high DS helix formation is hampered, but still profound, and the gel strength is

309

only reduced slightly. The presence of calcium restores the material properties to those of

310

unmodified gelatin to some extent. There are two opposing aspects introduced by

311

succinylation of the protein. Increasing DS gradually reduces the helix propensity (Figure 1),

312

the cooperativity in network assembly (Table II), and the storage modulus of the gel (Figure

313

2) due to increased electrostatic repulsions. Alternatively, succinylation also increases

314

calcium-binding with moderate affinity (Figure 3), that leads to at a DS of 43% even to a

315

significant fortification of the gel elasticity (Table III). Obviously, calcium-binding does not

316

only affect the molecular interactions, but also acts as fortifier of strand-strand interactions.

317

For technological impact it is most relevant to study the gel responses to large deformation

318

from a product storage, handling and oral processing perspective. When evaluating the strain

319

required to create sufficient stress in the gel to result in fracture events (Table IV) it can be

320

observed that for reference gelatin the addition of calcium has no effect, but that the stress

321

evoked by the applied strain leading to fracture diminishes with 10-15% with increasing

322

calcium concentration. This situation is different for the gelatin with a DS of 82% (Table IV)

323

where in the absence of calcium almost half the strain is sufficient to initiate fracture

324

compared to the reference material. Calcium restores this property, and even to strains beyond

325

that observed for the reference material. Interestingly the fracture stress remains, independent

326

of the calcium concentration, approximately 30% lower than that of the reference material.

327

The picture that emerges is that increased electrostatics increases the structural heterogeneity

328

in the gel, thereby lowering the fracture strain. The addition of calcium “glues” the strands

329

together, smoothens the heterogeneities and thereby reduces brittleness of the material. As the

330

fracture stress is the result of unzipping of the triple helices and dissociation of the strands,

331

this is less affected by electrostatics or the presence of calcium.



332

Gelatins are renowned for their high recoverable energy and elastic nature 13. As these

333

rheological properties have been related to texture perception of food products 16, 17, it is

334

essential to evaluate how these varied electrostatics in the network assembly and calcium-

335

induced network stabilization affect these mechanical responses. Table IV presents the

336

recoverable energies (from a compression-decompression cycle up to a 20% compressive

337

strain, so below the observed fracture strains) for reference gelatin and that with a DS of 82%

338

as the two extreme cases. It can be observed that rheological response is not affected at all by

339

increased electrostatic repulsions between the individual network strands. Moreover, also the

340

additions of calcium has no impact. For the gelatins with the other DS-values comparable RE-

341

values between 93 and 95% were obtained and none of the samples showed a sensitivity

342

towards calcium (results not shown). These results underline that the RE is not related to the

343

elastic modulus or gel stiffness or a network characteristic, but set by the energy dissipation

344

modes as suggested in literature 13.

345

As gelatin networks have a fine coarseness with typical mesh sizes in the order 30 to 100 nm

346

18

347

enclosed liquid phase in the gel deforms along with the protein network, not giving rise to

348

significant energy dissipation by friction caused by serum flow through the protein network

349

13

350

stress is only marginally affected by succinylation or by the addition of calcium. As stress

351

relaxation, related to spontaneous rearrangements of the microstructure upon deformation of

352

the material, was shown to be a dominant energy dissipation mode for these gels 13, the

353

relevant stress relaxation times and their corresponding fraction of the total applied stress

354

have been determined. The results are presented in the last two columns of Table IV. It can be

355

observed that both the relaxation times and the corresponding fractions are not affected by

356

either succinylation nor by the addition of calcium. A relaxation time around 8 sec for 6% of

, their water holding capacity is consequently close to 100% 19. This indicates that the

. Also the occurrence of (micro-)fractures during deformation is not likely as the fracture


Page 16 of 34

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357

the total stress (the average numbers from Table IV), implies that this dissipation mode,

358

acting from the very start of the deformation applied (total cycle for compression-

359

decompression takes 8 seconds), accounts for about 2% of the energy loss during the RE-

360

measurement. Apparently, stress relaxation is set by the inherent molecule and maybe to a

361

limited extent by the ability to adopt triple helices, rather than strand-strand interactions of the

362

network. This explains why the RE is not affected by the succinylation or the restored storage

363

modulus by the addition of calcium.

364 365

Summarizing, in this work it is shown that extensive succinylation of gelatin does not prohibit

366

gel formation. Properties like helix propensity, gelling/melting temperatures and concomitant

367

transition enthalpy changes and gel strength are shown to become calcium-sensitive by high

368

degrees of succinylation. The presence of relatively low concentrations of calcium restores

369

these material properties majorly to those of non-modified gelatin. However, although

370

succinylation has a major impact on the brittleness of the gels formed and the addition of

371

calcium makes the material even less brittle compared to the non-modified gelatin, the

372

modification has no impact on the energy balance in the gel, where all the energy applied is

373

stored in the material. This insight allows food industries to tailor gelatins for texture

374

attributes.

375 376 377 378

ACKNOWLEDGMENTS

379

We gratefully acknowledge CONACYT for providing funding for Dr Baigts Allende.

380

Moreover we thank Eefjan Timmerman for technical and Peng Cho for experimental support.

381

Inge Gazi is acknowledged for performing initial mechanical deformation experiments.

382 ACS Paragon Plus Environment


383

REFERENCES

384

1.

Tzaphlidou, M.; Chapman, J.A.; Meek, K.M. A study of positive staining for electron

385

microscopy using collagen as a model system. I. Staining by phosphotungstate and

386

tungstate ions. Micron 1982, 13, 119-131.

387

2.

233-289

388 389

3.

Schreiber, R.; Gareis, H. In: Gelatin Handbook-Theory and industrial Practice, ed. by R. Schreiber and H. Gareis (Willey-VCH, 2007), pp. 63-71.

390 391

Johnston-Banks, F.A. In: Food gels, Ed. by P. Harris (Elsevier, New York, 1990), pp.

4.

van Vliet, T. On the relation between texture perception and fundamental mechanical

392

parameters for liquids and time dependent solids. Food Qual and Pref. 2002, 13, 227-

393

236.

394

5

Properties. (Royal Society of Chemistry, Cambridge, 1993), pp 175-190.

395 396

van Vliet, T.; Walstra, P. In: Food Colloids and Polymer, Stability and Mechanical

6.

Gornall, J.L.; Terentjev, E.M. Helix-coil transition of gelatin: helical morphology and stability. Soft Matter 2008, 4, 544-549.

397 398

7.

Baigts Allende, D.; de Jongh, H.H.J. Food Hydrocoll.s 2015, 49, 82-88

399

8.

Kosters, H.A.; Broersen, K.; de Groot, J.; Simons, J.W.F.A.; Wierenga, P.A.; de

400

Jongh, H.H.J. Chemical modification as a tool to generate ovalbumin variants with

401

controlled stability. Biotechnol. Bioengineer. 2001, 84, 61-70.

402

9.

Church F.C.; Swaisgood, H.E.; Porter, D.H.; Catignani G.L. Spectrophotometric assay

403

using ο-phthaldialdehyde for determination of proteolysis in milk and isolated milk

404

proteins. J. Dairy Sci. 1983, 66, 1219-1227.

405

10.

Simons, J.W.F.A.; Kosters, H.A.; Visschers, R.W.; de Jongh, H.H.J. Role of calcium

406

as trigger in thermal β-lactoglobulin aggregation. Arch. of Biochem. and Biophys.

407

2002, 406, 143-152 .


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408


11.

Mekmene, O.; Gaucheron, F. Determination of calcium-binding constants of caseins,

409

phosphoserine, ctrate and pyrophosphate: a modelling approach using free calcium

410

measurement. Food Chem. 2011, 127, 676-682.

411

12.

Structural investigation. J. Phys. France 1988, 49, 319-332.

412 413

Djabourov, M.; Leblond, J.; Papon, P. Gelation of aqueous gelatin solutions: I.

13.

de Jong, S.; van Vliet. T.; de Jongh, H.H.J. The contribution of time-dependent stress

414

relaxation in protein gels to the recoverable energy that is used as tool to describe food

415

texture. Mech. of Time-Dependent Mater. 2015, in press

416

14.

Rbii, K.; Violleau, F.; Brambati, N.; Buchert, A.; Surel, O. Decrease of available

417

lysine in thermally treated gelatin followed by LC–UV: Influence on molar mass and

418

ability to helixes’ formation. Food Hydrocoll. 2011, 25, 1409-1412.

419

15.

Dardelle, G.; Subramaniam, A.; Normand, V. Determination of covalent cross-linker

420

efficacy of gelatin strands using calorimetric analyses of the gel state. Soft Matter

421

2011, 7, 3315-3322.

422

16.

of viscoelastic solid food materials. J. of Text. Stud. 1976, 7, 243–255.

423 424

Peleg, M. Considerations of a general rheological model for the mechanical behavior

17.

van den Berg, L.; Carolas, A.L.; van Vliet, T.; van der Linden, E.; van Boekel,

425

M.A.J.S.; van de Velde, F. Energy storage controls crumbly perception in whey

426

proteins/polysaccharide mixed gels. Food Hydrocoll. 2008, 22, 1404-1417.

427

18.

structure. Biopol. 2006, 84, 181-191.

428 429 430

Okuyama, K.; Xu, X.; Iguchi, M.; Noguchi, K. Revision of collagen molecular

19.

Urbonaite, V; de Jongh, H.H.J.; van der Linden, E.; Pouvreau, L. Origin of Water Loss from Soy Protein Gels. J. Agric. Food Chem. 2014, 7550-7558.

431



432

FIGURE CAPTIONS

433 434

Figure 1. Helicity as established from optical rotation measurements as described in the text

435

for aqueous solutions of reference and succinylated gelatin with different DS. The materials

436

with a DS of 82 (closed square) and 65% (closed circles) are indicated separately from the

437

other DS and reference (open cross, tringle, square and circle).

438 439

Figure 2. Storage modulus (G’) as a function of cooling/heating temperatures at 0.5°C/min

440

scan rate for reference and gelatin solutions with different DS (3% w/v). Only the traces with

441

a DS of 82 and 65% are indicated separately. The small arrows in the figure illustrate the

442

applied temperature ramp of cooling and subsequent heating of the sample.

443 444

Figure 3. Calcium binding curves for 0.5% w/v solutions of reference succinylated gelatin

445

with different DS as indicated in the graph. An ensemble averaged molecular weight of 124

446

kDa is used to calculate B.

447 448

Figure 4. Qualitative assessment of gelatin solutions (reference and with various DS) at

449

different protein concentrations in terms of liquid, viscous, or gel (definitions see text) in the

450

(A) absence or (B) presence of 5mM calcium. Protein solutions are pre-heated for 30 min at

451

60 °C, subsequently cooled to 20 °C and assessed after 1 hour equilibration at this

452

temperature.

453 454

Figure 5. Small deformation rheology on 3% (w/v) (A) reference and (B) succinylated

455

(DS=82%) gelatin during a cooling and subsequent heating ramp between 50 and 5 °C in the


Page 20 of 34

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456

presence of different calcium concentrations (0-20 mM) as indicated in the graphs. The

457

arrows indicate the cooling-heating cycle.

458 459

Figure 6. Calorimetric analysis of the cooling ramp from 50 to 5 °C of a 3% w/v reference

460

(left panels) and 82% succinylated (right panels) gelatin in the presence of different

461

concentrations of calcium. Upper panels present the onset of the gelling temperature and the

462

lower panels show the concomitant change in enthalpy.

463 464

Figure 7. Helix fraction as established from optical rotation measurements for reference (top

465

panel) and 82% succinylated (lower panel) gelatin in the presence of various calcium

466

concentrations (0-20 mM) as indicated in the panels during cooling from 50 to 5 °C.




Page 22 of 34

Page 23 of 34


TABLES

Table I Characterization of gelatin materials subjected to different degrees of succinylation (DS), as determined from the chromogenic OPA assay, and the apparent isoelectric point, as established from zeta-sizer measurements as described in the method section.

Material (code)

mg succinic anhydride

DS (%)

Apparent IEP

added per gr gelatin

Reference

0

0

7.5

A

5

12

4.9

B

10

26

4.7

C

20

43

4.5

D

30

65

4.5

E

35

82

4.4

Estimated error in determination of DS as determined by the OPA-assay is ± 3%



Page 24 of 34

Tabel II Calorimetric analysis of Reference and succinylated gelatin. Transition Transition enthalpy Material

temperature gelling

Transition enthalpy gelling (J/g)

(% DS)

(°C)

melting (J/g)

Reference

20.3 ± 0.9

15.0 ± 1.8

40.2 ± 1.6

12

17.8 ± 0.8

13.2 ± 0.9

39.2 ± 1.4

26

18.2 ± 0.7

9.2 ± 0.8

37.4 ± 1.6

43

17.9 ± 0.7

7.2 ± 0.5

35.9 ± 2.1

65

17.3 ± 0.6

6.3 ± 1.1

34.4 ± 1.2

82

11.8 ± 0.9

0.8 ± 0.4

28.5 ± 0.9


Page 25 of 34


Tabel III Helix propensity (optical rotation) and maximal gel strength (small deformation rheology of reference and various succinylated gelatins in the presence of 20 mM calcium. Material

Helicity (fraction)

(% DS)

at 5 °C

Reference

0.45 ± 0.02

760 ± 20

12

0.44 ± 0.03

780 ± 30

26

0.42 ± 0.02

830 ± 40

43

0.40 ± 0.02

930 ± 50

65

0.37 ± 0.03

550 ± 40

82

0.34 ± 0.03

280 ± 30

Maximal G’ (Pa)



Page 26 of 34

Tabel IV Responses to large deformation of 3% w/v reference and 82% succinylated gelatin gels for various calcium concentrations. The compression speed is 1 mm/sec and the experiments were performed at room temperature. The RE-data are obtained from a compression-decompression cycle (up to a 20% strain) and the stress relaxation data are obtained from a compression and strain-hold experiment.

Fracture stress

Fracture strain

RE

Stress

(kPa)

(-)

(%)

relaxation

[Calcium]

Reference

82 % DS

Time

a

Fraction

(sec)

(-)

0 mM

27.5 ± 1.6

0.76 ± 0.08

94.6 ± 0.10

8.6

0.06

4 mM

25.2 ± 1.4

0.77 ± 0.12

94.5 ± 0.11

8.4

0.05

10 mM

25.7 ± 1.5

0.79 ± 0.11

94.9 ± 0.03

8.5

0.06

20 mM

24.2 ± 1.6

0.81 ± 0.09

94.9 ± 0.06

8.5

0.06

0 mM

16.2 ± 1.8

0.42 ± 0.11

93.3 ± 0.07

7.4

0.10

4 mM

18.3 ± 1.2

0.77 ± 0.08

93.9 ± 0.11

7.7

0.11

10 mM

20.3 ± 1.5

0.86 ± 0.10

94.2 ± 0.06

8.2

0.10

20 mM

19.7 ± 1.4

0.94 ± 0.04

94.4 ± 0.07

8.2

0.07

a: estimated error ± 0.3 b: estimated error ± 0.02


b

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TOC Graphic 243x186mm (120 x 120 DPI)


Page 34 of 34

Calcium Binding Restores Gel Formation of Succinylated Gelatin and

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