Hydrocolloid from leaves of Corchorus olitorius and its synergistic effect on κ-carrageenan gel strength

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FOOD

HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 819–825 www.elsevier.com/locate/foodhyd

Hydrocolloid from leaves of Corchorus olitorius and its synergistic effect on k-carrageenan gel strength Eiji Yamazakia,, Osamu Kuritaa, Yasuki Matsumurab a

Industrial Research Division, Mie Prefectural Science and Technology Promotion Center, Takachaya 5-5-45, Tsu, Mie 514-0819, Japan Laboratory of Quality Analysis and Assessment, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

b

Received 4 September 2006; accepted 26 March 2007

Abstract Hydrocolloid from leaves of Corchorus olitorius was fractionated with ammonium sulfate (50%, w/v) and extracted with distilled water. The yield of the hydrocolloid was 6.0% (w/w) based on dry material. The contents of total carbohydrate, anhydrouronic acid, ash, protein, and moisture of the hydrocolloids were 62.2%, 35.3%, 13.3%, 5.5%, and 16.1% (w/w), respectively. Degree of esterification of 19.4% was calculated assuming that the methoxyl groups were attached to only anhydrouronic acid. When the molecular weight distribution was checked by size-exclusion chromatography on Sepharose CL-4B, the hydrocolloid gave a single peak with a molecular weight of 1860 kDa. The rheological behavior of mixed gels made from k-carrageenan and the hydrocolloid at a total hydrocolloids concentration of 1% (w/w) was investigated by gel strength measurement. Then it was found that the hydrocolloid exhibited synergy with k-carrageenan in relation to gel strength. r 2007 Elsevier Ltd. All rights reserved. Keywords: Corchorus olitorius; Hydrocolloid; Ammonium sulfate fractionation; Synergy; k-carrageenan; Rheology

1. Introduction Many kinds of hydrocolloids obtained from natural resources are widely used in food systems for a variety of purposes, such as thickeners, stabilizers, gelling agents and texture modifiers. As a food hydrocolloid, it is usually mixed with other hydrocolloid(s) to apply to higher requests for practical use in food industries. For example, mixed gels obtained by addition of locust bean gum (LBG) to k-carrageenan are often utilized for expecting its synergistic effects (Rees, 1972). k-Carrageenan is a sulphated galactan which forms a gel on cooling, while locust bean gum (LBG) is a galactomannan which does not form a gel by itself. The mixture of these hydrocolloids forms a gel under conditions in which k-carrageenan does not, and the rheological behavior, such as gel strength, may be improved (Stading & Hermansson, 1993). Because introduction of new ingredient to real food systems may Corresponding author. Tel.: +81 59 234 8461; fax: +81 59 234 3982.

E-mail address: [email protected] (E. Yamazaki). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.03.009

change these structures and perceived textures, many explorations of new hydrocolloids from various sources for use in the food systems have been concentrated (Carvajal-Millan, Guilbert, Doublier, & Micard, 2006; Singthong, Ningsanond, Cui, & Goff, 2005; Vardhanabhuti & Ikeda, 2006). There are some interesting research reports about utilization of hydrocolloids obtained from plant leaves (Lai & Liao, 2002; Vardhanabhuti & Ikeda, 2006), although plant leaves are generally not source of hydrocolloids. Corchorus olitorius is native to Egypt and the Middle and Near East. Stem of the plant is an important source of fiver known as Jute in India and countries of the circumference, and leaves are so rich in vitamin B and beta-carotene that these have been eaten traditionally in Egypt and the Middle East as a medical vegetable. Recently Azuma et al. (1999) have reported the strong antioxidant activity of the leaves of the plant and revealed that the activity is attributed to some antioxidative phenolics. On the other hand, Ohtani, Okai, Yamashita, Yuasa, and Misaki (1995) have revealed that a large amount of water soluble polysaccharide is

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present in leaves of C. olitorius. Further we have reported that dietary fivers obtained from the leaves have a high water-holding capacity (Yamazaki, Murakami, & Kurita, 2005). Thus leave of C. olitorius is an interested source of hydrocolloids. In this study, we report the characteristics of the hydrocolloid which was obtained by the ammonium sulfate fractionation from leaves of C. olitorius and its synergistic effect on the mixed gel made from k-carrageenan. 2. Materials and methods 2.1. Materials and chemicals Dried leaves of C. olitorius which was collected at Iga City in Japan was purchased from Agurinet Mie (Mie, Japan). Commercial k-carrageenan and (LBG) were kind gifts from Chuo Foods Material (KC-200S, Hyogo, Japan) and Sansho (Meypro-LBG, Osaka, Japan), respectively. Unless otherwise indicated, all chemical reagents were of the guaranteed reagent grade. 2.2. Fractionation and extraction of hydrocolloid from leaves of C. olitorius Dried leaves of C. olitorius was suspended with 50% (w/v) ammonium sulfate solution for 1 h at room temperature. The fractionation volume was adjusted to 30 g of ammonium sulfate solution /g of the leaves. The suspension was centrifuged at 7 k  g for 20 min and the precipitate was collected. Then the precipitate was suspended in distilled water and the extraction was performed at 10 1C overnight. After extraction, the suspension was centrifuged at 10 k  g for 20 min and the polysaccharide in the supernatant was subjected to be precipitated with final concentration of 55% (v/v) acetone. The polysaccharide of interest in the precipitate was dissolved in distilled water and lyophilized after through dialysis against deionized water and weighed. It was named the hydrocolloid from leaves of C. olitorius: HLC. 2.3. Viscosity measurement Viscosity measurement was performed with a Brookfield viscometer (Toki Sangyo, Tokyo, Japan) at 23 1C. Five g of dried leaves of C. olitorius was suspended in 100 mL of distilled water or 150 g of 50% (w/v) ammonium sulfate solution. 2.4. Determination of ash, protein and moisture content Ash content was determined by incinerating 1 g of sample in a furnace at 600 1C for 4 h. The subsequent ash was cooled and stored in a desiccator with P2O5 until weighing. Nitrogen (N) was determined by the Kjeldahl procedure and protein content was estimated as N x 5.7.

The moisture content was calculated as the weight loss after drying at 130 1C for 3 h. 2.5. Determination of neutral sugars Polysaccharide (200 mg) was hydrolyzed in 1.0 mL of 2.0 M trifluoroacetic acid for 2 h at 105 1C. The hydrolysate was dried-up with a rotary evaporator and reduced to their corresponding alditols by the addition of 500 mL of 1.0 M NH3 containing 10 mg of NaBH4 for 1 h at room temperature. After destroying the excess of NaBH4 by the addition of glacial acetic acid in an ice bath, acetylation was performed with 2.0 mL of acetic anhydride in the presence of 250 mL of 1-methylimidazole as a catalyst for 20 min at 40 1C. The acetylation was stopped by the addition of with 5.0 mL of distilled water and the alditol acetates were extracted with chloroform. The extracts were then analyzed by GC–MS (5971A, Agilent Technologies, Inc., Palo Alto, CA, USA) with a DB-5 capillary column (0.25 mm  28.5 m). 2.6. Determination of total carbohydrate and anhydrouronic acid content The content of total carbohydrate (galactose equivalent) was determined by the phenol–sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) after correction for interference from uronic acid. Total anhydrouronic acid (AUA) content was determined by the xylenol method (Walter, Fleming, & MacFeeters, 1993). Polysaccharide solution (500 mL) in 2% (w/v) NaCl was mixed with 4.0 mL of sulfuric acid in an ice bath, and heated in a boiling water bath for 10 min. Then the solution was mixed with 200 mL of glacial acetic acid containing 0.1% (w/v) xylenol, and rested for 10 min at room temperature. Then the reaction mixture was measured at absorbance 450–400 nm, while galacturonic acid solutions (0–100 mg/mL) were used to construct the standard curve for the determination of AUA content. Glucuronic acid and galacturonic acid contents were estimated by using a HPLC (Alliance 2960, Waters, Milford, MA, USA) with a refractive index detector (RID-10A, Shimazu, Kyoto, Japan) in a column of Sugar-D (4.6 mm ID  250 mm, Nakalai tesque, Kyoto, Japan). Elution was performed with CH3CN/25 mM phosphate buffer (pH 7.0) (70/30) at the flow rate of 1 mL/min at 40 1C. 2.7. Determination of degree of methylation Degree of methylation (DM) in polysaccharide was determined by the alcohol oxidase test (Klavons & Benetter, 1986). Polysaccharide (10 mg) was suspended in 10 mL of 0.5 M KOH for 1 h at room temperature. Adjusting the pH to 7.5 by the addition of 0.5 M phosphoric acid, total volume was measured up 25.0 mL in a mass flask by the addition of 50 mM phosphate buffer

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(pH 7.5). Then 0.5 mL of the solution was reacted with 0.5 mL of alcohol oxidase (1.0 U/mL) at 25 1C for 20 min. Two mL of coloring reagent (0.02 M acetylacetone/2.0 M ammonium acetate/0.05 M acetic acid) was added to the reaction mixture and incubated at 60 1C for 15 min. Color of the reaction mixture developed at absorbance 412 nm was recorded, while methanol solutions (0–10 mg/mL) were used to construct the standard curve for the determination of the methyl ester linkages in HLC. The DM was calculated as molar ratio of methanol to uronic acid. 2.8. Pectin gel preparation Pectin gel preparation was performed at two conditions, such as for low methoxyl pectin (LMP) and for high methoxyl pectin (HMP). At LMP condition (Ralet, Axelos, & Thibault, 1994), hydrocolloid (10 mg) was dissolved in 1 mL of distilled water and heated in a boiling bath for 30 min. Cationic calcium was added to a final concentration of 3% (w/w). Then it was left overnight at 4 1C. At HMP condition, hydrocolloid (10 mg) was dissolved in 500 mg of distilled water and 500 mg of sucrose was added to the solution. The mixture was heated in a boiling bath for 30 min and citric acid (1 M) was added slowly to reach 0.05 M in final concentration. Then it was also left overnight at 4 1C. 2.9. Amino acid analysis Sample was dissolved in 6 N HCl containing 1% (w/v) phenol and heated at 110 1C under N2 atmosphere for 21 h. The amino acid composition was determined by the method of Bidlingmeyer, Cohen, and Tarvin (1984) with a Pico Tag System (Waters Corp., Milford, MA). 2.10. Gel diffusion reaction with Yariv reagent Petri dishes containing 1% (w/v) of agar gel in 10 mM Tris buffer (pH 7.3) together with 0.9% (w/v) NaCl and 10 mM CaCl2 were used. One mg/mL of Yariv reagent (Biosupplies Australia Pty Ltd. Parkville, Australia) was delivered to a central well (diameter 3 mm) and sample as well as gum arabic (which was used as a positive control) was put in a well 1 cm from the center of the plate. Gels were left at 4 1C overnight.

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injected onto the column and then 1.0 mL fractions were collected. 2.12. Mixed gel preparation Polysaccharides powder were suspended separately in distilled water and stirred for 3 h at room temperature. The hydrocolloids were mixed to give a total hydrocolloids concentration of 1.0% (w/w) and the composition will be denoted k-carrageenan/HLC or LBG, for example 80/20 means a mixture of 80% k-carrageenan and 20% HLC or LBG. The mixed solutions were stored at 4 1C overnight and then heated to 90 1C under continuous stirring and kept at this temperature for 10 min. Each of the mixed solutions was poured into a cylindrical container (height 22 mm, diameter 11 mm) and stored at 4 1C overnight. The mixed gels obtained were analyzed after maintaining at 23 1C beforehand. k-Carrageenan–calcium gel was prepared as follows. Calcium lactate and k-carrageenan were dissolved separately in distilled water. Then the k-carrageenan solution was heated to 90 1C and the calcium solution was added to the hot k-carrageenan solution and kept this temperature for 10 min. Final k-carrageenan concentration was a constant (0.9%, w/w) and cationic calcium concentration was varied from 0.0055% to 0.022% (w/w). The mixed solution was poured into a cylindrical container (height 22 mm, diameter 11 mm) and stored at 4 1C overnight. The gel obtained was analyzed after maintaining at 23 1C beforehand. 2.13. Gel strength measurement Gel strength measurement was performed using a Rheoner II (Yamaden, Tokyo, Japan) with a cylindrical plunger 6 mm in diameter at a compression rate of 1 mm/s at 23 1C. A sample of the mixed gel in a cylindrical container (height 22 mm, diameter 11 mm) was used for the gel-breaking strength measurement without putting it out from the container. 2.14. X-ray fluorescence analysis Each metal contents in HLC was determined by X-ray fluorescence analysis based on the fundamental parameter method under irradiation at diameter 30 mm using a XRF1700 (Shimazu, Kyoto, Japan).

2.11. Size-exclusion chromatography 3. Results and discussion Sepharose CL-4B (GE Healthcare, Piscataway, NJ, USA) was used for size-exclusion chromatography. The column (1.1 cm ID  47.5 cm) was eluted by 50 mM phosphate buffer (pH 6.8) containing 0.1 M NaCl at the flow rate of 0.15 mL/min at 23 1C. The column was calibrated with Dextran 2000, 500, 70, and 40 (GE Healthcare, Piscataway, NJ, USA). Hydrocolloids that were dissolved in the same buffer (up to 1.0 mg/mL) were

3.1. Extraction of polysaccharide from leaves of C. olitorius Extraction of polysaccharides from plant sources is commonly performed with complex process such as fractionation with suitable solvent(s), separation and/or purification by column chromatography, and so on. An increase of viscosity of polysaccharide solution is often

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observed during extraction process. It is important for extraction of polysaccharides to simplify the process without increasing of the viscosity from at least two reasons. First, a complex-process may not only decrease the yield, but degrade of the polysaccharide. Second, an increasing viscosity will decrease an efficiency of the work. When leaves of C. olitorius are suspended in distilled water, a drastic increase of viscosity of the suspension is observed and, therefore, it is difficult to extract polysaccharide without the addition of a lot of water to decrease the viscosity of the suspension. In this study, leaves of the C. olitorius were firstly treated with ammonium sulfate solution, though ammonium sulfate treatment is commonly performed at the last stage for the purification of polysaccharides from sources where impurities such as proteins and/or nucleic acid exist (Cui & Chisti, 2003). As shown in Table 1, HLC was fractionated as a precipitate by treating with ammonium sulfate without increasing of the viscosity. After extracting with distilled water and precipitation with acetone, yield of 6.0% based on dry material was obtained.

Content (%, w/w) Ash Protein (N  5.7) Moisture

13.3 5.5 16.1

Total carbohydratea Rhamnose Arabinose Galactose Glucose Mannose

62.2 4.4 8.8 12.4 0.8 0.5

Uronic acidb Galacturonic acid Glucuronic acid

35.3 25.2 10.1

a

The content of total carbohydrate (galactose equivalent) was determined after correction for interference from uronic acid. b Uronic acid was determined as anhydrogalacturonic acid.

Table 3 Amino acid composition (mol%) of the hydrocolloid from leaves of Corchorus olitorius (HLC)

3.2. Characterization of HLC The chemical composition of HLC is shown in Table 2. Ash, protein (derived from nitrogen) and moisture are 13.3, 5.5 and 16.1 (%, w/w), respectively. In the neutral sugar compositional analysis, galactose (12.4%), arabinose (8.8%), rhamnose (4.4%), glucose (0.8%), and mannose (0.5%) were detected as the major neutral sugars in HLC. It was also revealed that HLC is rich in uronic acid (35.3%), a characteristic in common with pectins. Pectins are chain like polymers, consisting of galacturonic acid linked through the 1 and 4 positions, with a portion of carboxyl groups esterified with methanol (Be Miller, 1986). Then the DM and the compositional analysis of uronic acid were measured about HLC. DM, which is of importance for the use of pectins in food industry as a gelling agent (Renard & Thibault, 1993), was estimated to be 19.4%; pectins with DM lower than 50% is classified as LMP and have an ability to form gel without sugar in the presence of divalent cations (Iglesias & Lozano, 2004). Therefore the ability to form gels of HLC at the condition of LMP or HMP was tested and it was revealed that HLC did not gel at both conditions. On the other hand, the content of glucuronic acid was calculated to be approxiTable 1 Viscosities of the dried leaves powder of Corchorus olitorius in distilled water and 50% (w/v) ammonium sulfate solution Viscosity (Pa s)a Distilled water Ammonium sulfate 50% (w/w)

Table 2 Chemical composition of the hydrocolloid from leaves of Corchorus olitorius

50 0.78

a Five grams of sample was suspended in each solvent (100 g) and the viscosities were measured by using a Brookfield viscometer at 23 1C.

Amino acid

Mol%

Alanine Valine Leucine Glycine Aspartic acid Threonine Glutamic acid Serine Isoleucine Lysine Phenylalanine Proline Methionine Tyrosine Arginine Hydroxy-proline Histidine

13.6 10.5 10.2 9.0 8.8 7.2 7.1 6.1 6.0 4.4 3.5 2.9 2.8 2.4 2.4 1.5 1.4

mately 45 mol% among uronic acids in HLC by HPLC analysis while pectins are almost composed of galacturonic acid. From the gelling property and the uronic acid analysis, the content of pectin of HLC, if any, would be rather low. The amino acid composition of the protein present in HLC is shown in Table 3 and comparable to that of the pectic arabinogalactan-proteins (AGPs) from Vernonia kotshchyana Sch. Bip. Ex Walp (Nergard et al., 2005). It is well known that the protein moiety of AGPs is found to contain the amino acid hydroxyproline (Tan, Qiu, Lamport, & Kieliszewski, 2004) which is also present in HLC (Table 3). On the other hand, the sugar analysis showed relatively high content of arabinose, galactose, rhamnose and glucuronic acid (Table 2). These results suggest a

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presence of AGPs in HLC. AGPs are typically consisted of short side chains containing arabinose and often rhamnose and glucuronic acid attached to a galactan back bone core (Gane et al., 1995) and some are linked to polysaccharides such as pectin (Oosterveld, Voragen, & Schols, 2002). To identify AGPs, the reactivity of HLC with Yariv reagent that is an indicator of AGPs was tested. It was found that HLC reacted positively with the Yariv reagent, indicating that AGPs was present in HLC. AGPs are almost found in higher-plant (Fincher & Stone, 1983) and play important roles of plant reproductive development, pattern formation and somatic embryogenesis (Nothnagel, 1997). Redgwell, Schmitt, Beaulieu, and Curti (2005) showed a potential of AGPs from coffee beans for an alternative source of the class of surface-active polymers, though few attempt have been done to characterize the rheological properties of AGPs. 3.3. Size-exclusion chromatography Fig. 1 showed the elution profile of HLC on Sepharose CL-4B eluted with 50 mM phosphate buffer (pH 6.8) containing 0.1 M NaCl. HLC obtained by the ammonium sulfate fractionation gave a single peak within the Kav ranging from 0.2 to 0.5 in reference to standard dextrans and the molecular weight of the peak was calculated to be 1860 kDa. Ohtani et al. (1995) reported previously that an acidic polysaccharide was isolated from leaves of C. olitorius via the cetylpyridinium chloride-complex formation and subsequent column chromatography. The acidic polysaccharide gave a single peak by Sepharose CL-6B column chromatography, calculating to be a molecular weight of 500 kDa, which is approximately 4 times lower than HLC obtained by the ammonium sulfate fractionation in this study. More, the sugar analysis of the acidic polysaccharide

Fig. 1. Size-exclusion chromatography on Sepharose CL-4B (1.1 cm ID  47.5 cm) of the hydrocolloid from Corchorus olitorius (HLC). Sample (1.0 mg) was injected on the column and eluted by 50 mM phosphate buffer (pH 6.8) containing 0.15 M NaCl. Fractions (1.0 mL) were assayed by the phenol sulfuric acid method.

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showed that uronic acid and rhamnose were estimated as main components to be 65% and 25%, respectively, while arabinose was not detected. These results are indicated that the acidic polysaccharide was different from HLC. The difference may be caused by the different fractionation used, since physicochemical properties of hydrocolloid generally influenced by several factors such as extraction and purification conditions.

3.4. Rheological results Fig. 2 showed the rheological behavior of mixed gels made from k-carrageenan and HLC or LBG by measuring the gel strength at total hydrocolloids concentration of 1.0% (w/w). Results were expressed as relative gel strength (%) ratio of the gel strength of 1.0% (w/w) k-carrageenan. As shown in Fig. 2, synergistic effects were observed for mixed gels made from k-carrageenan and HLC in the composition range I (from 97.5/2.5 to 50/50). In this range, addition of HLC to k-carrageenan resulted in gels with higher gel strength than that of the gel without HLC, and the highest gel strength that was found in the composition around 90/10 was approximately 2 times as high as that of the gel without HLC. In the composition range II (from 50/ 50 to 30/70) in Fig. 2, k-carrageenan/HLC mixtures were formed gels though those gels were weaker than that of the gel without HLC. In the composition range III (30/70) in Fig. 2, k-carrageenan/HLC mixtures did not gel. In addition, k-carrageenan/HLC mixtures in the composition range from 97.5/2.5 to 40/60 formed weak gels during storing at 4 1C without heat process, while k-carrageenan without HLC did not gel during storing at that condition. Similar phenomena were reported between k-carrageenan

Fig. 2. The rheological behavior of mixed gels made from k-carrageenan and the hydrocolloid from leaves of Corchorus olitorius (HLC, -&-) or locust bean gum (LBG, -K-) in relation to gel strength at total hydrocolloids concentration of 1.0% (w/w) at 23 1C. The effect of calcium on kappa-carrageenan gel (0.9%, w/w) was also plotted (n). Results were expressed as relative gel strength (%) ratio of the gel strength of 1.0% (w/w) k-carrageenan and reported values are the mean7SD (n ¼ 5).

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and LBG (Tako & Nakamura, 1986; Tako, Qi, Yoza, & Toyama, 1998). It has been well known that synergistic effects are observed for mixed gels made from k-carrageenan and LBG (Rees, 1972). When mixed gels were prepared with k-carrageenan and LBG at the same condition as HLC, the maximum synergistic effects were observed in the hydrocolloids composition range around 60/40–50/50 and the highest gel strength was approximately 4 times as high as that of the gel without LBG, which was approximately 2 times as high as that of the mixed gel made from k-carrageenan and HLC in the composition range 90/10 (Fig. 2). However, the synergistic effects of mixed gels made from k-carrageenan and LBG at the composition range around 90/10 were apparently lower than that of the k-carrageenan and HLC in the same composition range (Fig. 2). It meant that the addition of HLC was able to improve the k-carrageenan gel strength at the lower concentration than that of LBG. The gelation mechanism of k-carrageenan depends on the counter-ion present (Hermansson, Ericsson, & Jordansson, 1991) and the most common counter-ions in food applications are potassium, sodium and calcium. Then the metal contents of HLC were determined by X-ray fluorescence analysis and it was revealed that calcium (5.18%, w/w) was detected as the major counter-ion present while content of potassium was considerably low (0.19%, w/w) and sodium was not detected (Table 4). To confirm whether these synergistic effects between k-carrageenan and HLC were caused in the presence of calcium or not, the effect of calcium on the gel strength of k-carrageenan was measured under a constant concentration of k-carrageenan (0.9%, w/w) and the results were plotted in Fig. 2. The k-carrageenan gel strength increased with the addition of calcium. When the concentrations of cationic calcium was 0.011% (w/w) that was almost 2 times as high as that of present in the mixed gel made from k-carrageenan/HLC (90/10), the relative gel strength increased by 178% (Fig. 2). However, it was apparently lower than that of the mixed gel made from k-carrageenan/ HLC (90/10). These results indicated that the influence of cations present in HLC on the gel strength would be low.

Table 4 Metal contents of the hydrocolloid from leaves of Corchorus olitorius (HLC) Metal

% (w/w)

Ca K Mg Fe Zn Mn Na

5.18 0.19 0.16 0.06 0.01 0.01 n.d.a

a

Not determined.

The gelation process of k-carrageenan is highly complex and involves a coil-helix transition followed by aggregation and network formation (Rees, 1981; Smidsrød & Grasdalen, 1982). Addition of LBG to k-carrageenan is enhanced a couple network with specific junction (Turquois, Rochas, & Taravel, 1992) and as a result, improvement of the rheological properties of the mixed gel is achieved. In gelling milk desserts, k-carrageenan is often used for its excellent stabilizing effect attributing to a high specific interaction between k-carrageenan and k-casein (Stanley, 1990). To improve the gel strength of such milk desserts with addition of LBG, it is necessary to consider the following two problems. First, when LBG is added to a constant amount of k-carrageenan, the flavor-release of such milk desserts might be decreased because total hydrocolloids are increased. Second, when the total hydrocolloids are constant, the stabilizing effect of k-carrageenan must be lowered and may cause a separation of milk whey. On the other hand, comparing to LBG, HLC would be expected to give the synergy with k-carrageenan at rather small amounts. Hence the utilization of HLC to k-carrageenan for such milk desserts would be of advantage against LBG. In order to understand the mechanism of the synergistic effect between k-carrageenan and HLC, structural feature of HLC, interaction of HLC with k-carrageenan, and microstructure of the mixed gel will be further studied. These are now investigated and will be reported separately.

4. Conclusion The present paper reported the basic chemical and physicochemical properties of this new hydrocolloid from leaves of C. olitorius obtained by the ammonium sulfate fractionation. It has revealed that HLC exhibited unique rheological behavior such as a strong synergy with k-carrageenan in relation to gel strength. The synergistic effect on mixed gel made with k-carrageenan was different from mixed gels with LBG in the following respect; in case of addition of HLC, the maximum synergistic effect was observed at the sugar composition (k-carrageenan/HLC) of 90/10 while that of k-carrageenan/LBG were observed around 60/40–50/50. The difference implies that HLC could not only be an alternative to LBG in many applications, but may introduce new functions to k-carrageenan and other hydrocolloids. Thus C. olitorius is an interesting source of hydrocolloid though further investigation should be done in order to fully explore the potential of HLC.

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