Effects of sugars on the gelation kinetics and texture of duck feet gelatin

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Effects of sugars on the gelation kinetics and texture of duck feet gelatin

Yau-Hoong Kuan

^b

, Abdorreza Mohammadi Nafchi

^a^,^b

, Nurul Huda

^b

, Fazilah Arif ﬁ n

^b

, Alias A. Karim

^b^,^*

aFood Biopolymer Research Group, Food Science and Technology Department, Damghan Branch, Islamic Azad University, Damghan, Semanan, Iran

bFood Biopolymer Research Group, Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

a r t i c l e i n f o

Article history:

Received 17 December 2015 Received in revised form 22 February 2016 Accepted 24 February 2016 Available online 26 February 2016

Keywords:

Duck feet gelatin Gelation kinetics Rheological properties Gel strength gelling temperature Melting temperature

a b s t r a c t

Gelatin extracted from avian sources, such as duck feet is a potential alternative to mammalian-derived gelatin. The effects of sugars (sucrose and lactose) at different concentrations (0, 5, 10, 20 and 40%) on the gelation kinetics, and thermal and rheological properties of duck feet gelatin (DFG) (6.67% w/w) were investigated using a mechanical rheometer. The secondary structure of the gelatin was investigated using Fourier transform infrared (FTIR) spectroscopy. The results showed that the addition of sugars affected the physicochemical and structural properties of the gelatin. The gelation rate constant (kgel) and gel strength decreased with increasing amounts of sugars at low concentration (i.e., 5e20% for sucrose and 5 e10% for lactose). These data suggest that the addition of sugars at these concentrations prevented gelatin chains from approaching each other kinetically during gelation. However, thekgeland gel strength increased upon further addition of sugars, likely due to the formation of additional small junction zones that led to development of a more rigid gel. Additionally, gelling and melting temperatures increased as the concentration of added sugars increased. Sucrose had more pronounced effects than lactose, probably because of its greater solubility and number of e-OH groups. These results illustrate the potential for use of DFG in confectionery products.

1. Introduction

Gelatin is a class of water soluble, high-molecular weight polypeptides derived from collagen. Gelatin is obtained by destroying the tertiary, secondary, and, in most cases, some aspects of the primary structure of the parent protein (i.e., collagen) (Ziegler & Foegeding, 1990). Gelatin can be obtained from the collagen-containing tissue of various animals. For food applications, it is generally derived from the skins, hides, and bones of either porcine or bovine sources (Mariod&Adam, 2013). Gelatin obtained from an acid-pretreated raw material is known as Type A (isoelectric point at pH 6.5e9.0), whereas that derived from alkaline- pretreated material is known as Type B (isoelectric point at pH 4.8e5.2) (Gomez-Guillen, Gimenez, Lopez-Caballero, &Montero, 2011). Currently, about 98.5% of the world's gelatin production is extracted from cattle hides, beef bones and pork skin (Karim &

Bhat, 2009). However, in some countries the use of pork is restricted for religious reasons, and in other countries cows have been afﬂicted with diseases that can be passed on to human con- sumers (Karim&Bhat, 2009). Thus, an alternative source of gelatin, such as from avian sources, is needed.

The food industry is a major user of gelatins for numerous applications due to its gelling ability when aqueous solutions are cooled. Gelatin forms gels similar to those polysaccharides by forming a micro-structural network (Mariod&Adam, 2013). The gelation of gelatin solution usually occurs at concentration greater than 0.5% by weight between 30 and 40 C, resulting from the partial return of the disordered gelatin molecules to the collagen triple-helix structure which act as the gel junction zones (Mariod&

Adam, 2013; Ziegler&Foegeding, 1990). However, when temperature increases, the gelation gel converts to a solution, thus gelatin gels tend to melt in the mouth (Morimura et al., 2002). These unique properties make gelatin a useful component of foods such as clear dessert jellies, mousses, fruit gums and marshmallows (Johnston-Banks, 1990). The gelation of gelatin are inﬂuenced by temperature, pH, ash content, methods of extraction, amino acid

*Corresponding author.

E-mail address:[email protected](A.A. Karim).

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Food Hydrocolloids

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composition, thermal history, concentration, and interaction with other food components such as sugars (Ledward, 1986).

The use of gelatin in food for nutritional and functional purposes dates back many centuries, but our understanding of the relationship between structure and function has developed only during the last 30e40 years (Owusu-Apenten, 2004). How gelatin acts in a complex food system and how other ingredients alter its structure, function, and intrinsic properties need to be studied to obtain reproducible information for development of potential applications of gelatin. Gelatin can be studied in either simple systems or in real food systems (Luyten, Vereijken, & Buecking, 2004; Owusu- Apenten, 2004). These methods are entirely different and provide different information. In structure/function studies using a simple system, care is taken to avoid interactions with other food components during the experiment (Owusu-Apenten, 2004). In real food systems, the interaction of gelatin with other components is studied. For example, sugar is commonly applied in gelatin food systems, as sugar is fundamental to many food products based on gelatin. Table jellies, desserts and confections all include mixtures of sugars and gelatin in varying proportions (Johnston-Banks, 1990). Therefore, it is of considerable practical importance to un- derstand how sugars affect the gelation properties of gelatin in order to predict the rheological properties of gelatin-sugar composites.

Sugars and polyols are known to stabilize the gels prepared from globular proteins and ﬁbrous proteins by enhancing the overall hydrogen bonding structure of water and thus strengthening the hydrophobic interaction of the gels (Gekko, Li,&Makino, 1992). The rheological properties of gelatinesugar mixtures have been studied previously (Gekko et al., 1992; Gekko &Koga, 1983; Naftalin &

Symons, 1974; Shimizu&Matubayasi, 2014). The extent of stabilization depends on the effect of the sugar on the solvent properties of water (i.e., the sugar's stereochemical structure) (Oakenfull&

Scott, 1986).Naftalin and Symons (1974)suggested that the stabilization of sugar on the gel structure occurs through hydrogen bonds involving water and form ternary complexes that stabilize the gel. Thermodynamic studies of many solute/sugaresolvent interactions in gelatins (Gekko et al., 1992; Shimizu&Matubayasi, 2014) have revealed that the proteins are stabilized by sugars predominantly through a strengthening of hydrophobic interactions or a preferential hydration mechanism, originating from the water-structure-making characteristic of polyhydric compounds (Gekko et al., 1992). Because these compounds can stabilize globular and ﬁbrous proteins, comparative studies of solvent perturbation of gelation kinetics could lead to a better understanding of their role in the stabilization of gelatin gels.

The effects of various electrolytes and sugars on the properties of gelatins have been studied extensively (Gekko et al., 1992; Gekko

&Koga, 1983; Naftalin &Symons, 1974; Shimizu &Matubayasi,

2014), but the effects of lactose monohydrates have not been investigated. Lactose monohydrate is a disaccharide similar to sucrose, but its molecular weight differs slightly from that of sucrose due to differences between their monomers. Lactose, with a molecular weight of 360.32, consists of monosaccharides, glucose, and galactose joined via glycosidic bonds. In contrast, sucrose consists of glucose and fructose and has a molecular weight of 342.30 (Tester&Karkalas, 2003). The melting point for lactose falls within the range of 201e202 C, while that for sucrose is 160e186 C (Tester & Karkalas, 2003). In addition, their solubilities differ greatly. At 25C, the solubility (by weight) of sucrose is 67.89%, whereas that of lactose is only 21.6% (Tester&Karkalas, 2003). This characteristic plays an important role in the effects of sugar interactions on the gelation properties of gelatin.

By-products of the poultry processing industry are a readily available potential source of gelatin. In this paper, the gelation

behavior of duck feet gelatin (DFG) and gels made of DFG containing sucrose and lactose in different concentrations was investigated. Experiments were conducted to characterize changes in physicochemical properties, kinetics of gelation, gel strength, and gelling and melting temperatures caused by the addition of sugars.

Understanding the underlying mechanisms by which sugars affect the physicochemistry of DFG will provide the basis for further research into potential applications of this type of gelatin in food products.

2. Materials and methods

2.1. Materials

DFG was extracted from duck feet of the commercial strain of the Cherry Valley type of Peking ducks (Anas domesticus) procured from a halal certiﬁed breeding farm located in the northern region of Penang, Malaysia. The extraction of DFG followed a previously described method (Almeida&Lannes, 2013). Commercial bovine gelatin (BG) extracted from bovine skin (G9391, Type B), sucrose, and lactose monohydrate were acquired from the SigmaeAldrich Company Ltd. (Petaling Jaya, Selangor, Malaysia). All reagents used were of analytical grade and were used without further puriﬁcation.

2.2. Methods 2.2.1. Gel formation

A series of sucrose and lactose stock solutions were prepared at concentrations of 0%, 5%, 10%, 20% and 40%, respectively, prior to gel formation. DFG, at ambient temperature (~25C), were weighed and added into the prepared solutions at concentration of 6.67% (w/

v), respectively. The dispersions were then allowed to hydrate for 1 h at room temperature (~25C) prior to dissolve in the 65 C water bath for 15 min, with occasional swirling until all gelatins were properly dissolved in the stock solutions. A small amount of sample was used for viscoelasticity measurements. Bovine gelatin (BG) (model) was also prepared in the same manner.

2.2.2. Fourier transform infrared (FTIR) spectroscopy analysis The prepared gelatin solutions were allowed to stand for 30 min at room temperature before being gelled in the refrigerator at 10C for 16 h. Subsequently, the gelatin gels were freeze-dried for FTIR analysis. The freeze-dried gelatins were milled into powder and ground with potassium bromide (KBr) powder (Merck KGaA, Darmstadt, Germany) at a ratio of 1 mg of gelatin to 100 mg of KBr. The KBr powder was stored and dried at 120C to eliminate moisture prior to use. A pellet was prepared using a press and was immediately placed in the sample holder. The FTIR spectra were recorded in the region of 4000e400 cm¹for 32 scans. The back- ground spectrum was collected before each scan. The spectra from a given sample were smoothed, baseline corrected, normalized, and averaged for qualitative interpretation of the spectra. Corrected band heights were used for FTIR analysis and were obtained using the software Omnic version 6.2 (Thermo Scientiﬁc, Waltham, MA, USA).

2.2.3. Gelation kinetics

Gelation kinetics of DFG containing sucrose and lactose were determined by measuring the viscoelastic properties of each sample using a stress-controlled rotational rheometer (AR-1000N, TA Instrument Ltd., New Castle, DE, USA) (Fonkwe, Narsimhan,&Cha, 2003). The rheometer with a cone-plate geometry (40 mm, 2angle with 51mm truncation gap) was attached to the instrument with the aid of silicon oil (Sigma cat. No. 14615-2), and the sample

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holding region was sealed with an insulated cover to prevent evaporation during heating. The sample was placed in the sample holding region and cooled from 24C to 4C at a predetermined rate of 1C/min. The temperature of the rheometer was controlled by the Peltier plate attachment while the rheometer oscillated sinusoidally. Subsequently, the temperature was maintained at 4C for 3 h. Values of the storage/elastic modulus (G⁰) and loss modulus (G⁰⁰) over theﬁxed time period at a frequency of 1 Hz and constant strain of 5% were recorded. The strain used was found previously to be within the linear viscoelastic region (LVR) for the gelatin gels (data not shown).

The evolution of G⁰ over time during the holding phase of gelation wasﬁtted to a logarithmic equation of the following form:

Gt ¼k_gelln t_gel

þC (1)

whereG_tis the value ofG⁰at timet,Cis a constant,t_gelis gelation time, andkgelis the gelation rate constant. BG gel was used as the model to obtain the target storage modulus (G⁰_ref) after 3 h of cooling and gelling (holding) at 4C. k_gel, values for all systems investigated were obtained byﬁtting Equation(1)to the experimental data. Consequently, the time (tmodel) required for a gelling system to reachG⁰_ref was calculated using the following equation:

t_model¼eð^G⁰refCÞ=^k^gel (2)

2.2.4. Determination of gel strength

After the holding phase of 3 h,G⁰andG⁰⁰were determined over a frequency range of 0.01e10 Hz at 4C. Gel strength was determined from the frequency response of the gels. The storage complianceJ⁰ was calculated as follows:

J⁰¼ G⁰

G⁰²þG⁰⁰² (3)

The gel strength,G⁰_N, is related to the value of storage compli- ance,J⁰_N, at the frequency at whichG⁰⁰exhibits a shallow dip (Ferry, 1980) as follows:

G⁰_N¼ 1

J⁰_N (4)

2.2.5. Gelling and melting temperatures

The gelation and melting temperatures of the gelatin samples were determined using the dynamic temperature sweep rheological test. The chosen strain of 5% and the chosen frequency of 1 Hz for measurement were within the LVR. The samples were initially maintained at a temperature of 5C for 10 min to allow for equil- ibration. Gelatin samples were then heated on a Peltier plate from 5C to 50C and cooled back to 5C, both at a scanning rate of 1C/

min. The gelation temperature (Tg) was taken to be the temperature at which the elastic modulus began to dramatically increase in value and the viscous modulus began to decrease in value (i.e.,G⁰ andG⁰⁰ crossover occurred) during the cooling scan. The melting temperature (Tm) was taken to be the temperature at which theG⁰ andG⁰⁰crossover occurred during the heating scan.

3. Results and discussion

3.1. FTIR spectroscopy analysis

The FTIR spectroscopy analysis was conducted out to investigate

the effect of added sucrose and lactose on the properties of DFG.

Figs. 1 and 2show a qualitative comparison of the FTIR spectra for sucrose and lactose, respectively. Six regions of the spectra (amides A, B, I, II, and III and afingerprint region) were investigated.Tables 1 and 2list the locations and assignments of each peak for sucrose and lactose, respectively. Identification and interpretation of the peaks obtained were conducted following Shurvell (2002). The major amide bands (A, B, I, II, and III) were observed in all spectra, but variations in wavenumber and peak height were observed. In all cases, amide A tended to join with amide B, which showed that the CH₂stretch bands were associated with the dimeric intermolecular association of the carboxylic group. The spectra for DFG containing sucrose and lactose could be differentiated from the DFG control based on differences in thefingerprint region, particularly in the peak within 1400 to 900 cm¹. These bands are associated with stretching of the CeOeC or CeOH bonds or bending of the CeOeH group for carbohydrates.

To evaluate the effects of the sugars on DFG, the spectra for samples with and without added sucrose was compared (Fig. 1, Table 1). The average location of the amide A band for all samples was close to the free NH stretching frequency at 3520e3320 cm¹, but it tended to shift to a lower frequency with increasing concentration of sucrose from 5% to 40%. A peak near 3563 cm¹was observed only for sucrose at high concentrations (20% and 40%), indicating the presence of OeH stretching vibration, particularly for polysaccharides group. The amide B band was found at 2936e2928 cm¹ and represented CH antisymmetric and symmetric stretching foreCH3andeCH2in aliphatic compounds. The amide I band was found to be associated with the secondary structure of protein; it was located at 1663e1649 cm¹, and indi- cated the characteristic triple helix and random coil structure of gelatin (Muyonga, Cole,&Duodu, 2002). The position of the amide II band is also indicative of changes in the secondary structure of gelatin, although this band is usually considered to be more sen- sitive to hydration than to structural changes in protein (Benjakul, Oungbho, Visessanguan, Thiansilakul,&Roytrakul, 2009).Table 1 illustrates the tendency for all three peaks (amides B, I, and II) to shift to a higher wavenumber with increasing sucrose concentration. In contrast, no difference was found for the amide III band located at around 1240e1237 cm¹, which is responsible for the random coil or disordered structure (Cai&Singh, 1999). To compare the absorption intensity among spectra, the peak height of all amide bands were quantitatively measured and compared. The amount of gelatin in the KBr pellet was carefully controlled to ensure the same amount of gelatin was used for all FTIR analyses.

An increased intensity in allﬁve amide bands (A, B, I, II, and III) with the increasing sucrose concentration was associated with the increase in molecular order in gelatin which may be correlated with the increased gel textural properties, particularly gel strength as compared to the control (Sow&Yang, 2015).

DFG containing lactose was evaluated in the same way, and spectra with similar characteristics were observed (Fig. 2,Table 2).

The amide A band was located within the range of 3420 to 3341 cm¹, and was indicative of free NH group stretching. The amide B band was found at 2936e2928 cm¹, illustrating CH antisymmetric and symmetric stretching for eCH₃ and eCH₂ in aliphatic compounds. The amide B band tended to shift to a higher wavenumber with increasing lactose concentration. Peaks observed only for 20% and 40% lactose were related to theeNH3þ

stretching vibration for amino acids. The amide I band was located at 1663e1649 cm¹and represented the characteristic triple helix structure of gelatin (Muyonga et al., 2002). The amide II peak located at 1335 cm¹ disappeared with the addition of lactose, which illustrated the tendency of this peak to shift to a lower wavenumber to join with amide III band. This tendency suggests

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Fig. 1.FTIR spectra of duck feet gelatin (DFG) containing 5%, 10%, 20%, and 40% sucrose.

Fig. 2.FTIR spectra of duck feet gelatin (DFG) containing 5%, 10%, 20%, and 40% lactose.

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that structural changes occur in proteins due to hydration (Benjakul et al., 2009). The overall intensities of amides A, B, I, II and III increased with increasing amounts of lactose, suggesting the relationship with increased molecular order and increased gel strength

(Sow&Yang, 2015).

3.2. Gelation of gelatins

Fig. 3shows the evolution of storage and loss moduli (G⁰andG⁰⁰, respectively) with time for BG (model) and DFG (control) cooled at a rate of 1C/min and held at 4C for 3 h. Upon cooling, the rate of G⁰for both gelatin dispersions increased rapidly for about 1 h; the rate continued to increase at longer times but at a diminishing rate.

However, the rate never reached a constant value (Stainsby, 1977).

In both cases,G⁰was much higher thanG⁰⁰ after about 10 min of gelation, indicating the presence of a gel upon cooling (Choi, Lim,&

Yoo, 2004). The time interval in which the rate of increase ofG⁰was large represents the period during which the gel structure was being formed. Once the gel network was established, the rate of increase ofG⁰with time decreased. The gelation of gelatin involves inter-chain associations that lead to the formation of cross-links or junction zones separated byﬂexible strands that are stabilized by

non-covalent interactions (Ziegler&Foegeding, 1990). The gelation process of gelatin gels involves the structural transformation from coil to helix during cooling (i.e., formation of junction zones from the renaturation process involving nucleation, growth, and annealing), which depends greatly on concentration, pH, and temperature (Harrington & Rao, 1970). In our study, G⁰ also increased with increasing time held at aﬁxed temperature, which is indicative of increased of gel strength of the gelatin gels. Sub- sequently, theG⁰continued to increase gradually at a diminishing rate, indicating that the existing linkages were reorganizing continually as the gels annealed.

The kinetics of rigidity development in gelatin gels support the idea that gelation reﬂects a transition from a primary to a secondary crystallization process, and that secondary crystallization starts earlier or primary crystallization ends earlier if the ageing temperature is higher (te Nijenhuis, 1981). When gelatin gels mature at high temperatures, only a few collagen-like junctions form and the large remainder of each chain is disordered, resulting in weak gels (Ledward, 1986).Fonkwe et al. (2003)reported that a more elastic gel was formed at 4C compared to 8C. Therefore, 4C was used as the gelation temperature throughout the study described herein. Fonkwe et al. (2003) also reported that the Table 1

Location and assignment of the peaks identiﬁed in FTIR spectra for gels prepared with duck feet gelatin (DFG) containing sucrose.

Region Peak wavenumber (cm¹) Assignment and remarks^a

Control 5% Sucrose 10% Sucrose 20% Sucrose 40% Sucrose

Amide A e e e 3563 3563 OH stretch coupled with H-bond (polysaccharides)

3420 3402 3404 3390 3389 NeH stretch coupled with H-bond

Amide B 2928 2929 2928 2935 2936 CH antisymmetric and symmetric stretching

Amide I 1649 1663 1654 1661 1657 C]O stretch/hydrogen bond coupled with COO^-

Amide II 1544 1557 1557 1557 1550 NH bend coupled with CN stretch

1449 1448 1414 1434 1432 CH2bending (scissors) vibration

1335 1250 1258 1279 1280 CH2wag of proline and glycine

Amide III 1239 1237 1237 1240 1239 NH bend stretch coupled CN stretch

Fingerprint 1080 1054 1052 1063 1064 CeO skeletal stretch

e 990 991 992 1000 CeO skeletal stretch

e 926 923 911 991 CH2out-of-plane wag

e 862 867 858 859 CeH deformation vibration (carbohydrate)

654 e e 684 685 CeC Skeletal stretch

e 538 546 544 548 CeC Skeletal stretch

e e e 469 471 CeC Skeletal stretch

aPeaks were identiﬁed by referring toShurvell (2002).

Table 2

Location and assignment of the peaks identiﬁed in FTIR spectra for gels prepared with duck feet gelatin (DFG) containing lactose.

Region Peak wavenumber (cm¹) Assignment and remarks^a

Control 5% lactose 10% lactose 20% lactose 40% lactose

Amide A 3420 3398 3379 3341 3344 NeH stretch coupled with H-bond

Amide B 2928 2929 2897 2896 2895 CH antisymmetric and symmetric stretching

e e e 2072 2072 eNH3þstretching vibration (amino acids)

Amide I 1649 1663 1659 1658 1658 C]O stretch/hydrogen bond coupled with COO^-

Amide II 1544 1554 1553 e e NH bend coupled with CN stretch

1449 1412 1431 1431 1430 CH2bending (scissors) vibration

1335 e e e e CH2wag of proline and glycine

Amide III 1239 1262 1260 1261 1261 NH bend stretch coupled CN stretch

Fingerprint 1080 1073 1030 1030 1030 CeO skeletal stretch

e e e 906 906 CeH deformation vibration (carbohydrate)

e 891 884 884 885 CeH deformation vibration (carbohydrate)

e e 771 771 771 CeH deformation vibration (carbohydrate)

e e 606 607 605 CeC Skeletal stretch

654 539 553 554 552 CeC Skeletal stretch

e e e 466 466 CeC Skeletal stretch

aPeaks were identiﬁed by referring toShurvell (2002).

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cooling rate affects the rigidity of the gels formed. The total amount of helix formation in the system depends greatly on the rate of cooling, as slower rates permit the formation of a greater degree of ordered helical structure than fast cooling rates (Ledward, 1986).

Therefore, the cooling rate chosen in this experiment was 1C/min to allow longer times for stabilization of the junction zones.

3.3. Effect of solutes on gelation rates

Fig. 3shows typicalG⁰/G⁰⁰against time proﬁles for the BG and DFG, whereas the proﬁles for gelatin/sucrose and gelatin/lactose gels are shown in Fig. 4. Gelation rates were calculated using Equation(1), and time taken for each dispersion to reach a target value was obtained from gelling of BG under the same conditions.

The time taken to reach the target value was then calculated using Equation(2). The experimental data obtained using a mechanical rheometer wereﬁtted into a logarithmic model (Equation(1)) to determine the gelation rates of the gelling systems with or without added sugars. The data for each system are summarized inTable 3 for sucrose andTable 4for lactose. Thek_gelvalues for each gelling system decreased upon addition of sugars and then increased upon further addition of sugars at high concentration. The same trend was observed for the time needed for the gelling systems to reach the target value obtained from gelling BG under identical conditions. Thekgelfor DFG decreased by about 43% upon addition of 20%

sucrose and by about 12% upon addition of 10% lactose. This reduction of gelation rate and increase of gelling time in the presence of sugars occurred because the sugars prevented gelatin chains from approaching each other kinetically during gelation.

Bryant and McClements (2000)andFonkwe et al. (2003)reported similar observations.

The kinetics of gelation may be inhibited by the rearrangement of gelatin chains in the presence of sugars, as the addition of sugars increases the viscosity of bulk water around the sugar molecules at the continuous phase. As a result, the thermal motion of the stabilized water decreases in gelatin-water-sugar systems and thus suppresses the motion of gelatin chains (Bryant &McClements, 2000; Kasapis, Al-Marhoobi, & Glannouli, 1999). Katsuka, Nishimura, and Miura (1992) reported similar observation for starch-water-sugar systems. Protein stabilization by sugars likely is the result of their effects on the structure of water and is related to the equatorial hydroxyl (e-OH) groups of sugars (Uedaira, Ishimura, Fig. 3.Evolution of storage and loss moduli during cooling and gelling of gelatin

dispersions of (A) bovine gelatin (BG) and (B) duck feet gelatin (DFG). Fig. 4.Evolution of storage and loss moduli during cooling and gelling of gelatin dispersions containing (A) 20% sucrose and (B) 10% lactose.

Table 3

Parameters for the logarithmic model used toﬁt the gelation proﬁles of gelatin gelling systems containing sucrose cooled at a rate of 1C/min and gelled at 4C for 3 h.

Gelling systems Gelation rate

G⁰_ref kgel(Pa) C R² T6731-Model (h)

BG (model) 6731 1633.50 4892.9 0.91 3

DFG (Control) 6731 912.73 2904.6 0.92 66

DFGþ5% sucrose 6731 888.45 2835.7 0.91 80 DFGþ10% sucrose 6731 853.50 2701.3 0.92 112 DFGþ20% sucrose 6731 517.80 1470.0 0.94 25,855 DFGþ40% sucrose 6731 963.76 3107.0 0.92 43 Logarithmic model equation: G⁰_ref¼kgellnðtÞ þC where G⁰_ref ¼ target storage modulus (Pa) obtained from bovine gelatin (BG) dispersion,kgel¼gelation rate constant (Pa),t¼gelation time (h) andC¼constant. DFG: duck feet gelatin.

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Tsuda,&Udaira, 1990).Uedaira and Ishimura (1989)suggested that sugar molecules containing a large number of e-OH groups have stronger stabilizing effects on the structure of water surrounding the sugar molecules, which is very much related their solubility in water. The solubilities of sucrose and lactose in water are 2 g/ml and 0.2 g/ml, respectively, which indicates that the average number of e-OH groups in lactose is less than that in sucrose (Tester &

Karkalas, 2003). Because sucrose had a greater inﬂuence on the kgeland gelling time than lactose (Tables 3 and 4), thus indicating that the structure development of gelatin was retarded upon addition of sugars was in the following order: sucrose>lactose.

However, thekgelwas increased upon further addition of sugars (i.e., 40% sucrose and 20% lactose). The mean number of e-OH group increases and the amount of water available for gelation of gelatin decreases with increased sugar concentration. Thus, the annealing and crystallization of sugar molecules become more pronounced relative to the gelation of gelatin upon cooling. Additionally, because the solubility of sucrose in water is higher than that of lactose, a more pronounced increase ofkgelwas observed for the high concentration sucrose gels compared with the lactose gels upon cooling.

3.4. Effect of solutes on gel strength

Knowing the gel strength of a gelatin gel is crucial for predicting its physical characteristic in actual application in food products.

Ledward (1986)reported that the strength of gels prepared from gelatin is due to an inherent structural feature of gelatin known as the rigidity factor. Upon cooling, cross-linking occurs when pyrrolidine-rich regions of the gelatin molecules serve as nucleation sites for potential junction zones through their tendency to adopt the poly-^L-proline II conﬁguration. The rigidity factor depends greatly on this ability, which is ascribed to the heterogeneity of gelatin size and shape and the distribution of imino acids within the polypeptide chain (Ledward, 1986).Fig. 5shows a typical frequency spectrum for DFG gel containing 5% sucrose, which was used to evaluate the gel strength using Equations(3) and (4). Gel strength analysis (Tables 5 and 6) of the DFG samples with added sugars showed a trend similar to that of gelation rate. Gel strength decreased upon addition of 20% sucrose and 5% lactose but was higher for increased concentrations of the sugars. This suggests that the intermolecular interaction of the gelatin-sugar composite was weaker than that of the control.Yoshimura et al. (2000)reported that the reduction ofG⁰values in the presence of sugar could result from the increased distance between entangled points of the molecular chain segment, which would decrease the number of entangled points in the gelatin networks. In other words, the protein would aggregate slowly because of the increased viscosity of

the continuous phase upon addition of sugars, thus preventing the gelatin chains from approaching each other kinetically (Bryant&

McClements, 2000).

The increase of gel strength upon further addition of sugars may be due to a stabilization effect. Evidence derived from both spectroscopy and thermodynamics shows that sugars interact with water to an extent that depends on their molecular structure (James&Rintoul, 1982; Oakenfull&Scott, 1986). The mechanism of stabilization involves mutual stabilization of the collagen structure of gelatin and the three-dimensional hydrogen-bonded structure of the neighboring water molecules. As a result, the junction zones become smaller but more numerous and produce a more extended gel network that increases the rigidity of the gel (Oakenfull&Scott, 1986).

3.5. Effect of solutes on gelling and melting temperatures

Typical viscoelastic proﬁles (Fig. 6) were obtained to determine theTgandTmof the gelatin systems (Tables 5 and 6). TheTgandTm

for both sucrose and lactose in the gelatin systems increased with increasing concentration of sugars. Naftalin and Symons (1974) suggested that there must be a strong sugaregel interaction (i.e., via hydrogen bonding) such that water is involved in a way that differs from its normal involvement with sugars or gels individu- ally. They explained that the phenomenon is derived from the formation of hydrogen bonding via water molecules (SeHOHeGel) and the formation of small“pockets”of water molecules in which their orientation correlation time is greatly enhanced. Thus the rate of exchange with free water becomes relatively slow. The addition of sugars also increases the rigidity of the gel, as additional small junction zones and a more extended gel network are created (Oakenfull&Scott, 1986). Thus, a higher temperature is required to overcome the intermolecular forces created by the addition of sugars.

The effects onTgandTmcaused by the addition of sucrose were even more pronounced than those of lactose. This indicates that the rigidity of the gels containing sucrose was higher, particularly due to its greater solubility in water and greater number of e-OH groups.

4. Conclusions

In conclusion, added sugars affect the gelation of gels prepared from DFG by lowering the k_gel at lower sugars concentrations, probably due to the prevention of gelatin chains from forming a gel network. However, kgel increased when sugars were added at Table 4

Parameters for the logarithmic model used toﬁt the gelation proﬁles of gelatin gelling systems containing lactose cooled at a rate of 1C/min and gelled at 4C for 3 h.

Gelling systems Gelation rate

G⁰_ref kgel(Pa) C R² T6731-Model (h)

BG (model) 6731 1633.50 4892.9 0.91 3

DFG (control) 6731 912.73 2904.6 0.92 66

DFGþ5% lactose 6731 863.67 2769.4 0.92 98 DFGþ10% lactose 6731 800.82 2624.4 0.92 169 DFGþ20% lactose 6731 890.41 2995.9 0.93 66 DFGþ40% lactose 6731 923.51 3052.9 0.92 54

Logarithmic model equation: G⁰_ref¼kgellnðtÞ þC where G⁰_ref ¼ target storage modulus (Pa) obtained from bovine gelatin (BG) dispersion,kgel¼gelation rate constant (Pa),t¼gelation time (h) andC¼constant. DFG: duck feet gelatin.

Fig. 5.Typical frequency spectrum for a duck feet gelatin (DFG) gel containing 5% of sucrose when cooled at 1C/min to 4C and gelled for 3 h.

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higher concentration, and sucrose had a more pronounced effect than lactose. Similar trends were observed for gel strength. Added sugars stabilize DFG gels by increasing the thermodynamic stability of the collagen fold, as illustrated by increasing gelling and melting temperatures with increasing sugar concentration. Again, sucrose had a more pronounced effect than lactose, probably due to its greater solubility in water and number of e-OH groups. Based on these results, we postulate that the added sugars act indirectly and inﬂuence the mutual interactions of the polypeptide and the solvent. The data obtained from this study suggest that the addition of

sugars greatly affects the properties of DFG and that DFG may have potential applications in confectionery and dairy based products.

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Table 5

Effects of sucrose concentration on gelation temperature, melting temperature, and storage modulus of duck feet gelatin (DFG) during cooling at a rate of 1C/min, holding for 3 h.

Gelling systems Gelation temperature,Tgel(C) Melting temperature,Tm(C) G⁰at end of holding period (Pa) Gel strength at end of holding period,Gn(Pa)

DFG (control) 21.9 29.2 3751 3751.31

DFGþ5% sucrose 22.3 29.8 3563 3563.36

DFGþ10% sucrose 27.3 30.2 2750 2750.46

DFGþ20% sucrose 33.3 39.5 2099 2099.20

DFGþ40% sucrose 35.1 44.6 4267 4267.54

Table 6

Effects of lactose concentration on gelation temperature, melting temperature, and storage modulus of duck feet gelatin (DFG) during cooling at a rate of 1C/min, holding for 3 h.

Gelling systems Gelation temperature,Tgel(C) Melting temperature,Tm(C) G⁰at end of holding period (Pa) Gel strength at end of holding period,Gn(Pa)

DFG (control) 21.9 29.2 3751 3751.31

DFGþ5% lactose 21.6 29.4 3570 3570.13

DFGþ10% lactose 22.6 29.8 3796 3796.11

DFGþ20% lactose 23.5 31.5 4074 4074.24

DFGþ40% lactose 24.9 32.1 4148 4148.17

Fig. 6. Typical proﬁles of viscoelastic properties (G⁰and G⁰⁰values) of duck feet gelatin (DFG) gels (6.67%) containing (A) 5% sucrose and (B) 20% lactose.

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