Thermal analysis of chemically and mechanically modified pectins

ARTICLE IN PRESS

FOOD

HYDROCOLLOIDS Food Hydrocolloids 21 (2007) 1101–1112 www.elsevier.com/locate/foodhyd

Thermal analysis of chemically and mechanically modified pectins Ulrike Einhorn-Stolla,, Herbert Kunzeka, Gerhard Dongowskib a

Technische Universitaet Berlin, Food Science and Food Chemistry, Food Quality and Material Science, Koenigin-Luise-Strasse 22, D-14195 Berlin, Germany b German Institute of Human Nutrition Potsdam-Rehbruecke, Research Group Food Chemistry and Preventive Nutrition, D-14558 Nuthetal, Germany Received 6 February 2006; accepted 4 August 2006

Abstract Highly methoxylated citrus pectins were modified chemically (demethoxylation and amidation) and mechanically (disaggregation) and examined with a combined simultaneous thermal analysis (differential scanning calorimetry (DSC), thermogravimetry (TG) and differential thermogravimetry (DTG)) in a dynamic inert nitrogen atmosphere in the temperature range from 20 to 450 1C. The pectin degradation was observed in one single DSC or DTG peak, respectively, in the temperature range between 210 and 270 1C. The parameters of the degradation peak varied systematically in dependence on the degree of modification. All chemically modified pectins were more sensible to thermal degradation than their unmodified reference materials. The extrapolated onset, peak and offset temperatures of the DSC and DTG signals as well as the weight losses decreased with increasing degree of modification. The maximum reaction enthalpy was higher for all chemically modified pectins than for their unmodified reference pectins. The maximum degradation velocity decreased after demethoxylation and increased after amidation. The mechanically degraded pectins showed a completely different behaviour. Their thermal degradation started earlier and ended later with decreasing molecular weight (MW). The maximum reaction enthalpy and velocity were reduced after modification. Not only experimental modifications but also the pectin origin had a considerable influence on the thermal degradation of the pectins. The extrapolated onset temperatures of the three reference pectins varied already by about 10%. In combination with other methods, the thermal analysis is suitable for a relatively quick and reproducible characterisation of structural changes and of state transitions, occurring during preparation and modification of pectins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Pectin; Demethoxylation; Amidation; Disaggregation; Thermal analysis; DSC; Thermogravimetry

1. Introduction According to Rahman and McCarthy (1999), ‘‘the state of a system or material can be defined by listing its properties’’. These properties are described by chemical as well as by physical and other material parameters. Thermoanalytical investigations are often used for a qualitative and/or quantitative examination of physicochemical parameters (Tomassetti, Campanella, & Aureli, 1989; Utschick & Schultze, 2000). For instance, investigations of thermal sample decompositions (e.g. pyrolysis) or state transitions (e.g. glass transition) are relatively easy, reveal accurately even small material differences and are widely applied for food components (e.g., John & Shastri, 1998; Roos & Karel, 1991), especially for carbohydrates. Corresponding author. Tel.: +49 30 31471798; fax: +49 30 31471799.

E-mail address: [email protected] (U. Einhorn-Stoll). 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.08.004

For decades, these investigations were focused on starch and cellulose as well as flour because of their importance for the human nutrition (e.g. Gloyna, Schreck, Schierbaum, & Westphal, 1991; Muenzing, 1992; Radlein, Piskorz, & Scott, 1991; Sahai & Jackson, 1996; Sievert & Wuersch, 1993; Vasanthan & Bhatty, 1996). In the past years, however, also other food polysaccharides were intensively investigated, such as cyclodextrins (Trotta, Zanetti, & Camino, 2000), gums (Zohuriaan & Shokrolahi, 2004) or several biomass-derived materials (Fisher, Hajaligol, Waymack, & Kellogg, 2002). Previous investigations of apple cell wall materials, using also thermal analysis (Gloyna & Kunzek, 1998; Godeck, Kunzek, & Kabbert, 2001), found differences in the thermal degradation behaviour in dependence on the degree of methoxylation (DM) of the pectin component of the apple materials. The thermal behaviour allowed conclusions on the state of the pectin component and on

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state transitions during the preparation of the apple materials. In other previous works it was found, that the DM or the degree of amidation (DA) of pectins had also considerable effects on their stabilising ability in whey protein emulsions, on their thermodynamic compatibility with milk proteins or on their ability to form conjugates with these proteins by heating in a maillard-type reaction (Einhorn-Stoll, Glasenapp, & Kunzek, 1996; EinhornStoll, Salazar, Jaafar, & Kunzek, 2001; Einhorn-Stoll, Ulbrich, Sever, & Kunzek, 2005). So, an examination of possible effects of these different modifications of isolated pectins on their thermal behaviour became a subject of further interest. There are several studies dealing with the thermal analysis of pectins. Appelquist, Cooke, Gidley, and Lane (1993) carried out investigations with differential scanning calorimetry (DSC) in a temperature range from 20 to 120 1C and found an endothermic melting process for several polysaccharides, including pectin, in a temperature range between 50 and 80 1C. Iijima, Nakamura, Hatakeyama, and Hatakeyama (2000) examined phase transitions of pectins with sorbed water using DSC in a temperature range from 150 to 180 1C. They detected an endothermic

peak in dry highly methoxylated pectin (HMP) at approximately 150 1C and explained it with a phase transition from a crystalline to an amorphous structure. This peak, however, was not detected in any acid demethyoxlated and or amidated pectins, and the authors assume that the crystalline region is destroyed during the

Table 2 Characterisation of the reference highly methoxylated pectins (HMP) HMP 1

HMP 2

HMP 3

DM (%) [Z] (cm3/g)

75.8 634.0

74.8 562.7

78.9 436.2

Ash (%) Protein (%) Galacturonan (%)

1.63 1.83 83.1

2.11 2.01 77.4

3.65 2.05 62.6

Sugar composition Rhamnose (%) Arabinose (%) Xylose (%) Galactose (%) Glucose (%)

1.15 3.71 0.35 4.25 0.9

1.3 2.51 0.25 4.45 0.9

0.95 2.71 0.35 4.35 0.9

Table 1 Parameters of the chemical characterisation and thermal analysis of the pectins [Z] (cm3/g)

Ton DSC (1C)

TP DSC (1C)

Toff DSC (1C)

Ton DTG (1C)

TP DTG (1C)

Toff DTG (1C)

DTDTG (K)

vmax (%/min)

DmDTG (%)

Dm (%)

Alkaline demethoxylated pectins HMP 1 75.8 83.1 P 30 min 55.9 82.1 P 60 min 40.1 80.7 P 90 min 36.9 77.0 P 120 min 27.9 72.8

634.0 367.8 350.0 321.5 317.8

250.3 234.5 228.5 228.4 226.3

263.8 247.9 242.1 242.7 241.2

276.4 259.2 253.6 254.3 253.4

240.6 224.6 219.1 217.6 215.1

257.4 243.1 237.2 237.3 235.4

271.7 253.8 249.5 249.8 248.7

31.1 29.2 30.4 32.2 33.6

19.1 18.2 16.9 16.9 16.2

37.3 32.5 33.1 32.8 32.8

68.0 66.3 64.4 67.1 66.0

Amidated pectins HMP 2 0 3M 1h 11.3 3M 2h 15.8 6 M 0.5 h 16.0 6M 1h 22.5 3M 4h 25.5 6M 4h 34.1 3 Ms 1 h 3.6 3 Ms 2 h 9.9 3 Ms 4 h 12.7 6 Ms 1 h 18.4 6 Ms 2 h 25.7 6 Ms 4 h 26.7

77.4 85.5 80.5 79.1 80.8 91.5 89.2 80.2 83.4 79.4 85.4 81.1 75.3

562.7 472.2 429.7 406.6 381.2 414.9 291.1 417.2 419.9 421.9 423.4 374.3 417.6

249.4 244.2 242.9 243.4 242.9 242.5 242.3 246.7 244.8 243.6 243.6 242.0 242.4

262.0 252.7 250.9 251.1 250.3 249.2 249.8 256.0 253.7 251.9 251.1 249.0 249.1

272.8 260.3 258.1 258.0 257.0 255.4 256.8 264.2 261.6 259.0 258.0 256.0 255.7

241.1 240.7 239.9 240.6 240.1 239.8 239.5 241.2 240.7 240.0 240.6 239.8 239.2

255.9 249.9 248.3 248.6 247.8 246.9 247.5 252.3 250.4 249.0 248.5 247.2 246.8

268.0 257.0 254.6 254.6 253.0 252.0 253.7 260.3 257.9 255.8 254.6 252.7 252.5

26.9 16.3 14.7 14.0 12.9 12.2 14.2 19.1 17.2 15.8 14.0 12.9 13.3

18.8 27.1 34.6 32.6 33.5 37.0 29.1 25.2 28.0 30.5 31.9 35.4 35.3

34.5 30.6 29.5 29.3 28.3 28.7 28.0 31.3 31.7 30.5 30.0 29.1 28.7

70.1 66.7 65.1 63.9 64.1 64.4 63.4 67.0 64.2 66.0 63.2 62.0 61.0

Mechanolytically degraded pectins HMP 3 78.9 62.4 P 2h 75.8 63.4 P 5h 75.9 63.7 P 10 h 74.9 62.5 P 25 h 75.5 62.7 P 50 h 75.6 62.8 P 75 h 74.4 61.4

436.2 250.4 188.5 116.6 50.7 35.6 32.6

223.2 218.9 217.4 216.1 215.8 215.0 215.2

237.2 238.1 236.7 236.8 236.2 237.0 237.1

246.3 250.8 250.0 250.6 250.9 252.9 252.6

217.6 214.0 213.4 212.3 210.9 210.4 210.0

233.8 233.5 232.5 232.1 231.9 232.5 232.4

243.4 247.4 247.2 247.5 247.0 248.5 248.5

25.8 33.4 33.8 35.2 36.1 38.1 38.5

20.1 15.8 15.3 14.4 14.2 13.2 13.4

32.5 33.5 33.3 32.7 32.4 32.7 32.6

64.6 66.4 65.2 65.4 63.6 63.1 65.0

Sample

DM (%)

74.8 62.3 56.6 56.5 50.2 45.2 34.2 67.2 60.8 55.2 52.8 45.3 39.5

GC (%)

DA, degree of amidation; DM, degree of methoxylation; GC, galacturonan content; [Z], intrinsic viscosity; Ton/Tp/Toff, extrapolated onset, peak and offset temperature of the DSC/DTG signal; DTDTG, peak width of the DTG-sigma; vmax, maximum degradation velocity; Dm, weight loss during the DTG signal/whole reaction; Amidated pectins Ms were prepared in a small scale.

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experiments with plant cell walls, containing pectin, at 15–90 1C and found an influence of the DM on the glass transition of calcium-treated cell wall materials. Aguilera, Cuadros, and del Valle (1998) used the DSC to detect thermal phase transitions in low moisture apple products, containing pectin, during heating from 20 to 200 1C. They found an endothermic peak between 130 and 160 1C, caused by water evaporation for the apple material as well as for commercial apple pectin. At approximately 200 1C, there seems to begin an exothermic peak in the pectin sample, but this effect was not further examined and discussed. Furthermore, pectin gels were investigated using thermal analysis. For instance, Iijima Hatakeyama, Nakamura, and

0 0.2 –2 DSC [mW/mg]

exo

DTG

DSC

0

100 90

–4

80

–6

70

–8

60

-0.2

DTG [%/min] TG [%]

chemical reaction. These two studies are restricted to a temperature range below pectin degradation. Fisher et al. (2002) examined, among other biomass materials, also a HMP and found a softening or melting as well as a bubble formation of pectin particles and a weight loss of about 70% below 300 1C, resulting from a primary decomposition by heating the material up to 400 1C. Gloyna (2004) investigated a highly methoxylated citrus pectin with combined DSC and thermogravimetry (TG) and found two peaks, an endothermic peak for water evaporation around 100 1C and an exothermic pectin degradation peak in the range of 180–270 1C. Phase and state transitions in cell wall materials are often examined by DSC. Lin, Yuen, and Varner (1991) made

1103

–10 50

-0.4 TG

–12 40 -0.6 –14 100

50

150

200

250 T [°C] [ C]

300

350

400

30

450

Fig. 1. Typical diagram of a thermal degradation of citrus pectin with DSC, TG and DTG curves.

0.2

Texon 214.6°C exo

DSC [mW/mg]

0

Texoff 253.3°C -0.2

-0.4

-0.6

Tp 237.3°C 160

180

200

220 T [°C] [ C]

240

260

280

300

Fig. 2. Calculation of the degradation temperatures in DSC and DTG curves: Texon and Texoff are the extrapolated onset and offset temperatures and Tp is the peak temperature of the signal.

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Hatakeyama (2001, 2002) examined pectin gels thermomechanically. Other authors investigated the thermal transition of fruit systems containing pectins at different water contents (Barra, Di Matteo, Vittoria, Sesti Osseo, & Cesaro, 2000), and the water uptake by pectin and other different hydrocolloids by DSC (Bhaskar, Ford, & Hollingsbee, 1998). In several studies, an influence of the sample structure and structure modifications on the thermal behaviour for starch (Gloyna et al., 1991), cyclodextrins (Trotta et al., 2000), biomass materials (Fisher et al., 2002) or cellulose (Zohuriaan & Shokrolahi, 2004) was postulated. No systematic investigation was made up to now, however, concerning the effect of chemical and physical modifications on the thermal behaviour of dry isolated pectins in a temperature range including pectin degradation, though this knowledge is crucial for the further understanding of the physical state and state transitions, resulting from structural changes during preparation and processing of pectin materials. The physical state is, beside the chemical structure, essential for the application of pectins. It has an effect, e.g. on the water solubility and, thus, on the production of jam and jelly with modified pectins as gelling agents. State and structure are important also for the compatibility and interactions of pectins with other food components (proteins, water) during food storage and processing and, moreover, might have an influence on the action of pectin as a soluble dietary fibre component in the human intestine. The aim of this paper was, therefore, to investigate systematically the thermal behaviour of various modified pectins. The results should allow conclusions on the influence of structural changes on the physical parameters and, as a consequence, contribute to a better understanding of state and process-dependent state transitions of pectins.

the pH was kept in this range for 30, 60, 90 and 120 min, respectively, by further adding of potassium carbonate. The volume of the pectin solution was recorded and, after the demethoxylation time, the same volume 95% ethanol was added to the pectin-carbonate solution and the pectin was precipitated within 15 min. Afterwards, the supernatant was removed by squeezing and the pectin was suspended in a 10% (w/v) solution of HCl, kept for 30 min and squeezed again. This procedure was repeated, in order to remove the potassium carbonate completely. The HCl was then removed by washing with 70% (w/v) ethanol several times until no precipitate was formed on mixing the filtrate with AgNO3. The pectin was dehydrated by a water–ethanol exchange, made by washing twice with 95% (w/v) ethanol, and dried in a drying oven at 50 1C. Amidation: The method is a modified procedure according to Anger and Dongowski (1988). The dry pectin HMP 2 was suspended in a cold solution containing 60% ethanol and 3 or 6 M ammonia and stirred at 5 1C for 30 min to 4 h.

Ton DSC

Ton DTG Toff DSC

Tp DTG

Tp DSC Toff DTG

280

260

T [°°C]

1104

240

220

2. Materials and methods 200 25

2.1. Preparation of materials

35

45

(a)

65

75

65

75

50 ΔT DTG [°C]/ Δm DTG [%]

All chemically modified pectins were prepared from different non-standardised charges of highly methoxylated citrus pectin (HMP) from Herbstreith and Fox (Neuenbu¨rg, Germany). The mechanically disaggregated pectins were made from HMP from Copenhagen Pectin A/S (Lille Skensved, Denmark). The preparation parameters and the composition of the samples are shown in Tables 1 and 2. The HMP 1 and 2 were washed with an ethanolic solution of hydrochloric acid as described previously (Einhorn-Stoll et al., 2005), in order to remove ions, residual low-molecular sugars and other impurities. Alkaline demethoxylation: The HMP 1 was dissolved in distilled water overnight. The sample was kept at 5 1C during the following demethoxylation step, in order to inhibit b-elimination as far as possible. Then, a 10% (w/v) solution of potassium carbonate was added until the pH of the pectin solution was between 10.5 and 11.0. Afterwards,

55 DM [%]

peak width weight loss

40

30

20

10 25 (b)

35

45

55 DM [%]

Fig. 3. Thermal analysis of demethoxylated pectins: (a) degradation temperatures; and (b) peak width and weight losses in dependence on the DM.

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dated pectins with HCl containing solution, however, reduced also residual low-molecular sugars and other impurities. In order to keep the comparability, the according HMP had to be purified, too.

The ammonia solution was then removed by filtration and the pectin was washed three times with 60% ethanol. During the second and third washing step, the pH of the suspension was adjusted to 1.5 with HCl and kept at this value for 30 min. The HCl was then removed by washing with 70% (w/v) ethanol several times until no precipitate was formed on mixing the filtrate with AgNO3. The pectin was dehydrated by a water–ethanol exchange, as described above, and dried in a drying oven at 50 1C. For mechanical disaggregation the dry pectin was treated in a laboratory vibration mill ‘‘Vibratom’’ (Siebtechnik Mu¨lheim, Gemany) with ceramic spheres (diameter 10–15 mm) at room temperature for 2–75 h according to Bock, Anger, Kohn, Malovikova, Dongowski, and Friebe (1977) and Dongowski and Bock (1989). The HMP 3 was not washed as described above, because this would reduce the comparability with the disaggregated pectins. The treatment of the demethoxylated and ami-

2.2. Characterisation of pectins The DM, the galacturonan content and, in case of the amidated pectins, the DA were determined according to a combined titrimetric method used by Herbstreith and Fox (1993). The intrinsic viscosity [Z] was determined using a capillary viscosimeter method as described by EinhornStoll et al. (2001). The protein content of the HMP was analysed according to Kjeldahl (N  6.25), the ash was determined in a muffle furnace at 525 1C. The polysaccharide composition of the HMP was determined by GC analysis of the alditol acetates of the sugars after combined enzymatic and acidic

0.2 exo 0 DSC [mW/mg]

1 -0.2 3 -0.4 2 -0.6 4 5

-0.8 200

220

240

260

280

300

280

300

T [°C] [ C]

(a) 0

DTG [%/min]

-5

-10

2

5

1

4 3 -15

-20 200 (b)

220

240

260 [ C] T [°C]

Fig. 4. Thermograms of the demethoxylated pectins: (a) DSC curves and (b) DTG curves; decreasing DE with increasing number: 1 ¼ HMP 1; 2 ¼ P 30 min; 3 ¼ P 60 min; 4 ¼ P 90 min; and 5 ¼ P 120 min; 30–120 min is the demethoxylation time.

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hydrolysis of the pectins according to Blakeney, Harris, and Stone (1983), modified by Gloyna, Kabbert, and Kunzek (1997). Details of the preparation and modification parameters and of the chemical analysis of the pectins are given in Tables 1 and 2. 2.3. Thermoanalysis Before the investigations, all samples were dried at room temperature and at about 6 mbar in a desiccator, containing P4O10 as dryı´ ng agent, for at least 2 weeks. The simultaneous thermal analysis (DSC and TG; see Fig. 1) was carried out using a STA 409 C-device (Netzsch, Selb, Germany) according to Gloyna and Kunzek (1998), using the following conditions: linear heating rate 10 K/min from 20 to 450 1C, dynamic inert nitrogen atmosphere (75 mL/ min), 85 mL Pt crucible without lid, empty crucible as reference, sample weight approximately 20 mg. All runs were performed at least in duplicate, mostly three times. The instrument was calibrated by a standard procedure following the recommendations of the Gesellschaft fuer thermische Analyse (Society of Thermal Analysis—GEFTA) (Cammenga, Eysel, Gmelin, Hemminger, & Ho¨hne, 1992; Ho¨hne & Cammenga, 1990). Extrapolated onset, peak and offset temperatures were calculated with the Netzsch software as shown in Fig. 2.

showed two main peaks during the thermal analysis of the pectins. The first endothermic peak between 50 and 150 1C with a maximum at approximately 100 1C is ascribed to water evaporation and was found and identified for example also by Gloyna (2004) for citrus pectin, Aguilera et al. (1998) and Godeck et al. (2001) for dry apple products containing pectin and by Appelquist et al. (1993) for several other polysaccharides. The second exothermic pectin degradation peak between 210 an 270 1C was found also by Godeck et al. (2001) and Gloyna (2004). In contrast to Appelquist et al. (1993) or Iijima et al. (2000), however, no melting peak was observed in any of the pectin materials of this study, so this phase transition can be excluded. The weight loss resulting from water evaporation in the TG curves of the investigated pectins was marginal because of the pre-drying treatment of the samples. The main weight loss was found for the pectin degradation step. Therefore, the influence of the pectin modifications on the thermal behaviour will be discussed only for the temperature range of the pectin degradation peak from 200 to 300 1C. Ton DSC Tp DTG

Tp DSC Toff DTG

280

260 T [°°C]

3. Results and discussion

240

220

200 0

10

(a)

20 DA [%]

30

40

50 ΔT DTG [°C]/ Δm DTG [%]

The results of the pectin analysis and of the themoanalytical investigations are shown in Tables 1 and 2. A typical diagram of the thermal degradation (see Fig. 1) consists of two original curves: The TG curve records the weight loss Dm during heating and the DSC curve describes changes in the reaction enthalpy during the degradation. The differential thermogravimetry (DTG) curve is the first derivation of the TG curve and reports the degradation velocity. For the determination of the beginning or ending of a degradation process, extrapolated temperatures are used (Fig. 2): Texon is the extrapolated onset temperature at the start and Texoff is the extrapolated offset temperature at the end of a reaction. The maximum peak temperature Tp DTG of the DTG signal characterises the maximum reaction velocity vmax. The signal width DTDTG ¼ Texoff DTGTexon DTG characterises the duration of the reaction. DmDTG is the weight loss between Texon DTG and Texoff DTG and Dm the total weight loss between 20 and 450 1C. The DTG and DSC signals are used for the discussion of the thermal degradation in the present study. Generally, a small peak width DTDTG seems to reflect the degradation of a relatively uniform sample matrix and low Texon in the DTG and DSC signals can be considered as an indicator for a low thermal stability of a sample (Godeck et al., 2001). The residue of any thermal degradation of the pectins was a black char powder. The typical DSC curves (Fig. 1)

Ton DTG Toff DSC

peak width weight loss

40

30

20

10 0 (b)

10

20 DA [%]

30

40

Fig. 5. Thermal analysis of amidated pectins of decreasing molecular weight (DMW): (a) degradation temperatures; and (b) peak width and weight losses in dependence on the DA.

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3.1. Demethoxylated pectins The thermal degradation of the demethoxylated pectins differed considerably from that of the reference material HMP 1, all degradation temperatures of the DSC and DTG signals were reduced (Table 1 and Figs. 3a and 4). The degradation started and ended earlier and the peaks were shifted to the left within the diagram with decreasing DM (Fig. 4a and b). The difference in the DTG signals was high (about 16 K) between the HMP 1 and the least demethoxylated pectin (P 30 min) and became smaller with increasing demethoxylation time. The maximum degradation velocity vmax was decreasing with the DM (Fig. 4b). The duration of the degradation (signal width DTDTG; Fig. 3b) was slightly decreasing from HMP 1 to P 30 min and increasing with further demethoxylation. At a heating rate of 10 K/min a total difference of 4 K is marginal and the degradation time can be considered to be nearly constant.

DSC [mW/mg]

0

1107

The weight loss DmDTG of the demethoxylated pectins was up to 5% smaller than that of the HMP 1 (Fig. 3b). The exothermic DSC peak (maximum reaction enthalpy) of the demethoxylated pectins is much higher than for the HMP 1 and is increasing with decreasing DM (Fig. 4a). The results of the thermal analysis of the demethoxylated pectins (degradation temperature shift, weight loss) are in agreement with those found by Godeck et al. (2001) for the apple materials containing HMP and low-methoxylated LMP. 3.2. Amidated pectins The amidated pectins were divided into two sample groups: amidated pectins of decreasing molecular weight (DMW, including also reference pectin HMP 2) and those of a nearly constant molecular weight (CMW). The latter were prepared by an amidation procedure in a very small

exo

1

-0.5 2 -1.0

6 7

-1.5

3 5 4

-2.0 200

220

240

260

280

300

T [°C] [ C]

(a) 0 -5

DTG [%/min]

-10 -15

1

-20 2

-25 7 -30

3

6

5

-35 -40 200 (b)

4

220

240

260

280

300

C] T [[°C]

Fig. 6. Thermograms of the amidated pectins of decreasing molecular weight (DMW): (a) DSC curves and (b) DTG curves; increasing DA and decreasing molecular weight with increasing number: 1 ¼ HMP 2; 2 ¼ 3M 1 h; 3 ¼ 3 M 2 h; 4 ¼ 6 M 0.5 h; 5 ¼ 6 M 1 h; 6 ¼ 3 M 4 h; 7 ¼ 6 M 4 h; 3 and 6 M is the molar concentration of ammonia of the amidation solution, 0.5–4 h is the amidation time.

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scale, so an increasing depolymerisation by b-elimination at higher DA could nearly be prevented. The results (Table 1 and Figs. 5–8) showed the same tendencies for both sample groups, but the differences were more distinct

Ton DSC

Ton DTG Toff DSC

Tp DTG

Tp DSC Toff DTG

280

3.3. Comparison of demethoxylated and amidated pectins

T [°°C]

260

240

220

200 0

10

20

30

20

30

DA [%]

(a)

50 ΔT DTG [°C]/ Δm DTG [%]

in the group DMW. The onset temperatures of the DTG signals were nearly constant, those of the DSC signals were slightly reduced (Figs. 6 and 8). The peak temperatures and the offset temperatures of both signals were decreasing continuously in both groups with increasing amidation. The weight losses DmDTG were smaller at higher DA, the peak width was considerably decreasing, too (Figs. 5 and 7). The maximum degradation velocity and the maximum reaction enthalpy were increasing with the DA (Figs. 6 and 8).

peak width weight loss

40

30

20

10 0 (b)

10 DA [%]

Fig. 7. Thermal analysis of amidated pectins of constant molecular weight (CMW): (a) degradation temperatures; and (b) peak width and weight losses in dependence on the DA.

A comparison of the results of the degradations of the demethoxylated and of the amidated pectins (Table 3) shows some similarities but also several differences. Both pectins became more sensible to thermal degradation than their according reference HMP, their reaction enthalpy became higher and the weight losses during pectin degradation decreased with increasing degree of modification. Both modified pectins have functional groups (–COOH in the demethoxylated and additionally –CONH2 in the amidated pectins) which allow the formation of inter- and intra-molecular hydrogen bonds. It can be assumed that these hydrogen bonds favour the thermal degradation of the modified pectins. It is well-known that the hydrogen bonds between the amidated galacturonic acid residues play an important role for the physical state and also for the functional properties of the amidated samples, and, thus, also for the thermoanalytical behaviour and for the gelling properties (Voragen, Pilnik, Thibault, Axelos, & Renard, 1995). The maximum degradation velocity, however, was decreasing with increasing demethoxylation but increasing at higher DA, though the vmax values for the initial HMP 1 and HMP 2 were nearly the same. Moreover, the degradation time was much shorter for the amidated than for the methoxylated pectins. The pectin matrix seems to be more homogenous after amidation than after demethoxylation. This can be seen as an indicator for variations in the molecular structure and the state. They could result, on the one hand, from differences in the hydrogen bonds, formed between the randomly distributed carboxyl or the

Table 3 Comparison of the influence of different pectin modifications on the thermal degradation parameters

Texon DSC/DTG Tp DSC/DTG Texoff DSC/DTG DTDTG vmax Max. reaction enthalpy Dm DTG/total

Demethoxylation

Amidation

Mechanolysis

k k k — k m k

k k k k k m k

k — m m k k —

Texon/p/off, extrapolated onset, peak and offset temperature; DT, peak width; vmax, maximum reaction velocity; Dm, weight loss; DSC, differential scanning calorimetry signal; DTG, differential thermogravimetry signal; m, increase; k, decrease; —, no influence.

ARTICLE IN PRESS U. Einhorn-Stoll et al. / Food Hydrocolloids 21 (2007) 1101–1112

preferably blockwise distributed amino groups (Axelos & Thibault, 1991). On the other hand, the preparation procedures could have an influence, too. The demethoxylation was made in an aqueous pectin solution, the amidation in an alcoholic pectin suspension. Solved molecules are more de-folded and flexible and can, perhaps, undergo more or other structural and state changes than molecules of precipitated samples that are suspended in an alcoholic medium. An influence of substituents, which are able to form hydrogen bonds, was found also by Trotta et al. (2000) for modified cyclodextrins. The authors generally postulate an active participation of substituents in the thermal degradation for cellulose and similar materials. They also found a decreasing degradation temperature with an increasing steric hindrance in the polysaccharides. An additional influence of the type of the substituent on the thermal behaviour was found by Gloyna et al. (1991). They examined the thermal degradation of model systems

DSC [mW/mg]

0

1109

containing protein and polysaccharide components and found an action of the nitrogen from the protein components on the degradation of the carbohydrate component. Though the amino group of the amino acids is a better nucleophilic agent than the amido group of the amidated pectins, a similar influence on the thermal degradation reaction cannot be excluded. Altogether, the degradation mechanisms of the two chemically modified pectins are similar but not equal. 3.4. Mechanolytically depolymerised pectins All pectin modifications, despite of the amidation in group CMW, were accompanied by a more or less intensive depolymerisation (decreasing intrinsic viscosity, see Table 1). This could also have a more or less stronger influence on the thermal analysis of the modified pectins. Though the group CMW has already shown a singular effect of the amidation without molecular weight (MW)

exo

-0.5 1 -1.0

2 3

-1.5

4 5 6

-2.0 200

220

240

260

280

300

280

300

T [°C] [ C]

(a) 0 -5

DTG [%/min]

-10 -15 -20 1 -25

2 3 4

-30

200 (b)

5

6

-35 220

240

260 T [°C] [ C]

Fig. 8. Thermograms of the amidated pectins of constant molecular weight (CMW): (a) DSC curves and (b) DTG curves; increasing DA with increasing number: 1 ¼ 3 Ms 1 h; 2 ¼ 3 Ms 2 h; 3 ¼ 3 Ms 4 h; 4 ¼ 6 Ms 1 h; 5 ¼ 6 Ms 2 h; and 6 ¼ 6 Ms 4 h.

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1110

changes, additionally another HMP 3 was depolymerised mechanically in a vibration mill, in order to investigate the influence of the molecular weight as a single factor, too. All samples had a nearly constant DM and galacturonan content but a strongly decreasing intrinsic viscosity [Z] (Table 1). The thermoanalytical behaviour of these mechanically depolymerised pectins (Figs. 9 and 10) was quite different from that of the chemically modified samples. Their degradation started at lower and ended at higher temperatures than that of the corresponding HMP 3 (Fig. 9a), the peak width was clearly increasing with longer pectin disaggregation time (Fig. 9b). The peak temperatures of the DTG as well as of the DSC signals, however, were nearly constant. The maximum degradation velocity and maximum reaction enthalpy (DSC signal) were considerably reduced with decreasing MW (Fig. 10), the weight loss (Fig. 9b) was nearly unchanged.

Ton DSC

Ton DTG Toff DSC

Tp DTG

Tp DSC Toff DTG

280

During the mechanical treatment, the pectin chain is depolymerised and possibly also side chains are removed. Many of the interactions between and within the pectin molecules are destroyed. The material structure is less homogenous than the raw material, what delays the thermal degradation (longer degradation time and lower velocity). Because of the high input of mechanical energy, the system becomes less thermodynamically stable. This decreased the onset temperature of the thermal degradation and favoured, as an additional factor besides the reduced MW, the pectin solubility in water. The thermal degradation of the mechanically modified pectins proved to be quite different from those of the chemically modified pectins above (Table 3). This allows the conclusion, that, though the molecular weight has a considerable influence on the thermal degradation and the MW was mostly reduced during chemical modifications, other factors dominate the thermal degradation of demethoxylated and amidated pectins. All results concerning the thermal degradation of modified pectins were confirmed by Gloyna and Dongowski, investigating further series of demethoxylated, amidated and mechanolytically depolymerised pectins for another study. 3.5. Pectin origin

T [°C]

260

240

220

200 0

100

200 IV [cm

(a)

300

400

500

3/g]

ΔT DTG [°C]/ Δm DTG [%]

50

40

30

20

peak width weight loss

Not only pectin modifications, made in laboratory scale for scientific investigations, can be followed by thermal analysis. This is possible also in case of the composition as well as the structure, state and state transitions of pectin preparation, caused by the raw material or industrial processing. In this study, the properties of the original high methoxylated pectins proved to have a considerable influence on the thermal degradation stability, too. The two samples HMP 1 and HMP 2 of the alkaline demethoxylation and the amidation series are charges of citrus pectin, both made by Herbstreith (Germany). For the mechanical disaggregation, another citrus pectin HMP 3, made by Copenhagen Pectin (Denmark), was applied. Differences in the composition were found (Table 2) and the physical states of the pectins were most probably varying, too. The solubility of the HMP 3 (Copenhagen) was much better than that of the HMP 1 and HMP 2 (Herbstreith), what could be seen as an indicator of structural and state differences. The different pectin properties were reflected also by thermal analysis. The thermal degradation of the Herbstreith pectins HMP 1 and HMP 2 started about 25 1C later than that of the Copenhagen Pectin HMP 3 (Table 1), they were obviously more stable against thermal degradation.

10 0 (b)

100

200

300

400

500

4. Conclusions

IV [cm3/g]

Fig. 9. Thermal analysis of mechanically disaggregated pectins: (a) degradation temperatures and (b) peak width and weight losses in dependence on the molecular weight MW (MW as intrinsic viscosity [Z]).

The aim of this paper was a systematic investigation of the thermal behaviour of various modified pectins in order to get some information about the changes of structure as

ARTICLE IN PRESS U. Einhorn-Stoll et al. / Food Hydrocolloids 21 (2007) 1101–1112

1111

0.2 0

exo

DSC [mW/mg]

-0.2 -0.4

7 6

-0.6

4 3

-0.8

5

2 1

-1.0 -1.2 180

200

220

240

260

280

260

280

T [°C]

(a) 0

DTG [%/min]

-5

7 6

-10

5 4

3

2

-15 1 -20 180

200

220

(b)

240 T [°C]

Fig. 10. Thermograms of the mechanically disaggregated pectins: (a) DSC curves and (b) DTG curves; decreasing MW with increasing number: 1 ¼ HMP 3; 2 ¼ P 2 h; 3 ¼ P 5 h; 4 ¼ P 10 h; 5 ¼ P 25 h; 6 ¼ P 50 h; 7 ¼ P 75 h; 2–75 h is the milling time.

well as state transitions during the modifications. The results show convincingly, that the physical state, resulting from different raw materials, as well as from modifications of the molecular structure (different substituents or decreasing molecular weight), has a considerable influence on the thermal behaviour of pectins. The thermal degradability of the materials, their degradation time and velocity and also the weight loss during the pectin degradation have changed systematically. The results of the present study should be the starting point of further examinations about the connection between the physical state of pectin preparations and the parameters of their thermal degradation. The kinetic and the mechanism of the degradation reactions, as well as the structure of the volatile degradation products, should be studied in detail. Additionally, the consideration of otherwise modified pectins, such as enzymatic demethoxylated or acetylated, might be helpful for a deeper insight

into the complex interactions of structure, state and thermal degradation of pectins. The thermal analysis, in combination with other chemical and physical analytical means, is a suitable method for the investigation of the properties of pectins. This determination of the material properties allows conclusions on the state and possible state transitions of the pectin materials during preparation and processing.

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