Isolation and characterization of hydrocolloids fr

(Cissampelos pareira )leaves

B.Vardhanabhuti a,*,S.Ikeda b

a

Department of Food Science and Technology,Chiangrai Rajabhat University,Chiangrai 57100,Thailand

b

Department of Food and Human Health Sciences,Osaka City University,3-3-138Sugimoto,Sumiyoshi-ku,Osaka 558-8585,Japan

Abstract

Hydrocolloids from monoi leaves were extracted using cold water and precipitated with alcohol.The hydrocolloid fraction contained 49.7%anhydrouronic acid,and 4.24%methoxyl groups.Degree of esterification of 48.45%was calculated assuming that the methoxyl groups are attached to anhydrouronic acid only.Atomic force microscopy revealed that the monoi hydrocolloid consisted of linear chains with occasional long branches.The large variability in end-to-end lengths along individual chains suggested a wide molecular weight distribution.The monoi hydrocolloid fractions exhibited high viscosity with shear thinning characteristic.Increasing temperature decreased the viscosity and pseudoplasticity.However,the effect of temperature seemed to be reversible.Addition of NaCl (0–100mM)or glucono-d -lactone (0–1.2%)increased the viscosity and pseudoplasticity of the hydrocolloid.q 2005Elsevier Ltd.All rights reserved.

Keywords:Hydrocolloid;Characterization;Atomic force microscopy;Viscosity

1.Introduction

Hydrocolloids are broadly used in food systems for various purposes,for example as thickeners,gelling agents,texture modifiers,and stabilizers.Large,linear,and flexible polysaccharides increase viscosity even at low concen-trations.This property allows hydrocolloids to be the major ingredient in liquid and semisolid type foods.Recently there has been an increase in the demand of hydrocolloids (Williams and Phillips,2000).The volume share of the ingredients depends on the security of their supply,quality and price.Hydrocolloids from plants have the advantage over those from animals because of their friendly image towards consumers.Starch,pectin,galactomannans,carra-geenans,alginates,as well as cellulose and its derivatives are the main plant hydrocolloids.A lot of work has been done to study the chemical composition and functionality of these polysaccharides.However,there is still place in the hydrocolloid market for new sources of plant hydrocolloids to meet the demand for ingredients with more specific functionality in foods.

Leaves are generally not a resource of hydrocolloids.Nevertheless,previous work on leaf gums has shown some interesting results.For example,polysaccharides from hsian-tsao leaves can form strong gels through the interaction with starch.This gelling behavior was strongly influenced by several factors such as extraction conditions,part of the plant from which they were extracted,starch types,and concentrations (Yang,Chen,and Lii,1982;Yang and Huang,1990).Stephen,Linder,and Churms (1992)found that treating the water extracted polysacchar-ides from leaves of Aloe ferox with aqueous oxalate,citrate or phosphate yielded viscous solutions.The extracted polysaccharides formed a gel on the restoration of Ca 2C .

Monoi (Cissampelos pareira )is a medicinal plant commonly found in the north and the northeast of Thailand.It is a woody vine climbing a considerable height over trees.Its silky leaves have been used for treating unhealthy sores as well as food.Once the leaves are squeezed in water and left overnight a gel will form.This research reports the characteristics of hydrocolloid from monoi leaves.The objective of this research was to extract and characterize monoi leaf polysaccharides as well as to study its flow properties.The effects of concentration,temperature,sodium chloride,and glucono-d -lactone (GDL)on flow behavior of polysaccharide were

investigated.

Food Hydrocolloids 20(2006)885–1

www.elsevier.com/locate/foodhyd

0268-005X/$-see front matter q 2005Elsevier Ltd.All rights reserved.doi:10.1016/j.foodhyd.2005.09.002

*Corresponding author.Tel.:C 19195132247;fax:C 19195157124.E-mail address:jeab906@yahoo.com (B.Vardhanabhuti).

2.1.Materials

The fresh monoi leaves were purchased from local farmers, washed with tap water,rinsed with deionized(DI)water,and then air-dried at ambient temperature(308C).To inactivate the enzymes naturally present,the leaves were heated in a hot air oven at958C for90min.The hydrocolloids extracted from heated leaves were used for characterization and viscosity study.For the study of hydrocolloid extracts of frozen leaves, the leaves were kept in a freezer before extraction.The moisture content of monoi leaves was determined prior to hydrocolloid extraction using the method in A.O.A.C.(1984). Commercial gums used in this study as references were xanthan gum and guar gum,which were the gifts from Lanna Foods and Supplies(Chiangmai,Thailand).Other chemicals were of reagent grade quality.

2.2.Extraction of polysaccharides

The extraction of hydrocolloids from monoi leaves was performed using a method modified from that by Komae and Misaki(19).The dried and heated leaves were stirred in DI water for30min at room temperature,and then squeezed in cheesecloth.An equal volume of ethanol was added to the resultant viscous solution to precipitate the crude polysacchar-ide extract.The precipitate was washed successively with50% ethanol until a clear polysaccharide was obtained.It was then dewatered by washing successively with80%ethanol and acetone.The polysaccharide extract was dried in a vacuum oven(Eyela,Tokyo Rikakikai Co.,Ltd,Tokyo)at408C.

2.3.Composition of hydrocolloid

Methoxyl and anhydrouronic acid(AUA)contents were determined by the standard method of Owens,McCready, Shepard,Schultz,Pippen and Swenson(1952).Extracted hydrocolloid from monoi leaves(0.1g)was moistened with 5mL ethanol in a250mL conicalflask.To sharpen the end point,1g sodium chloride was added.100mL of carbon dioxide free distilled water and6drops of phenol red indicator were added.The mixture was titrated slowly with0.1N NaOH until the color of the indicator changed and persisted for at least 30s.The neutralized solution was saved for methoxyl determination.

2.4.Methoxyl content

Methoxyl(MeO)contents were determined by adding 25mL of0.25N NaOH to the neutral solution,mixing thoroughly,and allowing to stand for30min at room temperature in a stopperedflask.Twentyfive mL of0.25N HCl was then added and titrated with0.1N NaOH to the same end point as before.Assuming that the molecules are esterified with methoxyl groups only,the methoxyl content can be calculated as follows.

MeO%Z

meq of sodium hydroxide!31!100

wt of sampleðmgÞ

(1) where31is the molecular weight of the methoxyl group.

2.5.Anhydrouronic acid(AUA)

If the equivalent weight and methoxyl content of a uronide containing hydrocolloid are known,its AUA can be calculated as follows,assuming that the methoxyl groups are only attached to the carboxylic group of the uronide.

where176is the molecular weight of AUA

2.6.Degree of esterification

The degree of esterification(DE)can be determined according to the formula given below

%DE Z

meq for methoxyl

meq for methoxyl C meq for free acid

(3) 2.7.Atomic force microscopy(AFM)

The polysaccharide extract was dissolved in DI water (0.3%w/v)and dialyzed.The solution was applied to a column of DOWEX SBR-P(Dow Chemical,MI,USA),previously activated with4%NaOH,washed and equilibrated with DI water.Sample was then applied to a column of DOWEX HCR-S(Dow Chemical,MI,USA),previously activated with6% HCl,washed and equilibrated with DI water.After the ion exchange steps,samples were neutralized using1and0.1N NaOH before vacuum drying.Dried polysaccharide powders were dispersed into distilled water(1g/L)and heated at908C for30min to achieve complete solubilization.After cooling to room temperature,the solution was diluted with distilled water to a concentration of1mg/L.Aliquots(2m L)of the diluted samples were immediately spread onto freshly cleaved mica surfaces,air-dried,and imaged using a dynamic force(ac) mode of the microscope(SPA-400/SPI3800N,Seiko Instru-ments Inc.,Chiba,Japan)in air at room temperature.Samples were scanned at a scanning frequency of1Hz using a beam-shaped Si cantilever with a quoted spring constant of12N/m and a resonant frequency around140kHz.

2.8.Preparation of hydrocolloid solution

Hydrocolloid solutions at different concentrations(0.50, 0.75,and1%)were prepared by adding the powdered or

%AUA Z ðmeq of alkai for free acid C meq of alkali for methoxylÞ!176!100

weight of sample in mg

(2)

B.Vardhanabhuti,S.Ikeda/Food Hydrocolloids20(2006)885–1

886extracted hydrocolloid to vigorously stirred DI water of 60–808C.The solutions were stirred for2–3h to ensure complete solubilization.Volume adjustment was made after the solutions were cooled to room temperature.

2.9.Viscosity measurements

Measurements were made using a Physica MCR300 rheometer(Parr Physica,Denmark)with the courtesy of Metrohm Siam Ltd(Bangkok,Thailand).The Physica concentric cylinder measuring system was used in all experiments.The measuring system consisted of afixed cup and a rotating bob attached to a torque bar.The samples were sheared from0.1to2001/s.Flow behavior was described by the power law model

s Z k_g n or h Z k_g n K1(5) where s Z shear stress(Pa),h Z apparent viscosity(Pa.s), _g Z shear rate(1/s),n Zflow behavior index,and k Z consistency index.

2.10.Effect of concentration

Hydrocolloid solutions were prepared at0.5,0.75,and1% concentrations.The viscosity was measured at258C.

2.11.Effect of temperature

To study the effect of temperature,hydrocolloid solutions were prepared at1%concentration.Viscosity measurements were made at4,10,20,40,60,and808C.To study the stability after heating and freezing,the hydrocolloid solutions werefirst measured at258C.They were then heated to808C or cooled to K108C and held for1h.The temperature was then returned to 258C and the viscosity measured again.

2.12.Effect of NaCl

Hydrocolloid solutions were prepared at0.5%.Sodium chloride was added to givefinal concentrations of0,20,40,60, 80,and100mM.Viscosity measurements were performed at 258C.

2.1

3.Effect of GDL

Hydrocolloid solutions were prepared at0.5%.Glucono-d-lactone was added to givefinal concentrations of0,0.2,0.8, and1.2%.Solution viscosity was measured at258C.

3.Results and discussion

3.1.Extraction of hydrocolloid

Extraction of polysaccharide from plant sources can be done with various solvents.Diluted acid such as0.1N HCl is generally used in commercial pectin extraction;however some hydrolysis will occur depending on the conditions.Sodium bicarbonate or sodium carbonate was used to extract gum from hsian-tsao leaves(Lai,Tung,and Lin,2000).Cold water extraction of seeds of Ficus awkeotsang yielded an acidic polysaccharide(Komae and Misaki,19).In this study,we used cold water extraction in order to avoid hydrolysis.Stirring the leaves in DI water for30min yielded a green viscous solution.After squeezing through cheesecloth and centrifu-gation,the color of the solution changed to orange.By washing with ethanol and acetone,the color was removed and hydrocolloid precipitated.Yields for dried and frozen leaves of22.8G3.5%and32.2G1.9%were obtained,respectively. The yield of commercial pectin from apple pomace was reported to be17%(Kertesz,1951).Thus,the yield from monoi leaf extraction looks promising for commercial production. 3.2.Characterization of hydrocolloids

It was found that the monoi leaf hydrocolloid contained anhydrouronic acid and methoxyl group,a characteristic in common with pectin.In order to establish that the hydrocolloid has a pectic nature,10mg/100L of a pectinase enzyme (Lallzyme HC(Lallemand,Canada))was added to a0.5% hydrocolloid solution.The resulting viscosity was compared to the viscosity of the solution prior to enzyme addition. Enzymatic treatment resulted in a significant decrease in the viscosity of the hydrocolloid.For example,the viscosity at the shear rate of61s K1reduced from305to3.8mPa s.This result indicates that the hydrocolloid contains pectin.

Percent AUA and methoxyl were49.70G0.27and4.24G 0.00,respectively.The amount of methoxyl group was in the same order of magnitude as found for pectin from lime and apple(Kravtchenko,Voragen,&Pilnik1992).The degree of esterification was calculated to be48.45G0.25which indicates that the monoi leaf hydrocolloid may show similar function-alities as low methoxyl pectin.Low methoxyl pectin can form gels without sugar in the presence of divalent cations (Nussinovitch,1997).Thus,the monoi leaf hydrocolloid may be useful in making low sugar or calcium pectate gels. However,the low AUA content of the extract suggests that the extract is not pure.Further purification is needed in order to obtain higher%AUA and make it better suited for commercial use like pectin.On the other hand,other components in monoi hydrocolloid may be responsible for the unique properties of the polysaccharide.Gelation and stabilization properties of the hydrocolloid are being investigated.

3.3.Atomic force microscopy(AFM)

Fig.1shows an AFM image of the monoi leaf polysaccharide deposited onto mica.Uniform coverage of fibrous structures was obtained in the present preparation conditions.Somefibrous structures have long branches with the lengths ranging up to a few hundreds nanometers,while the heights are fairly uniformly around0.5–0.8nm,suggesting that these are individual polymer chains.The end-to-end lengths along individual chains vary,approximately from several tens

B.Vardhanabhuti,S.Ikeda/Food Hydrocolloids20(2006)885–1887

to 500nm or even longer,exhibiting a wide molecular weight distribution of this polysaccharide fraction.Reported AFM images of tomato pectin have revealed the existence of long branches presumably consisting of polygalacturonic acid attached to a main chain which are similar to the results presented here (Round et al.,1997,2001).The existence of long branches in the monoi leaf polysaccharide implies that its functionalities such as rheological characteristics and gelation abilities are significantly influenced by the distribution of branching points on the backbone and that of the lengths of branches.

3.4.Effect of concentrations on flow behavior

Flow behavior of hydrocolloid from monoi leaves appeared to be pseudoplastic or shear thinning (Fig.2).The viscosity lost on shearing was regained on standing (data not shown).Hydrocolloids such as xanthan gum,galactomannans or pectin exhibit high viscosity at low concentration and strong shear thinning (Towle and Christensen,1973).Linear and stiff molecules have a large hydrodynamic size,which contributes to high viscosity and pseudoplasticity.Shear thinning is the result of an

orientation effect.As shear rate is increased,the polymer molecules,which are long,randomly positioned chains,become increasingly aligned in the direction of flow resulting in less interaction between adjacent polymer chains.The pseudoplastic property of gums allows liquid food to be

pumped easily and imparts a pleasant body and mouth feel to the food.The flow behavior index (n )and consistency index (k )values obtained by fitting the viscosity versus shear rate data to the power law model (Eq (5))are given in Table 1.Increasing the monoi hydrocolloid concentration increased the pseudo-plasticity and viscosity as shown by an increase in n and k values,respectively.When compared with the linearly branched xanthan gum and guar gum,the monoi hydrocolloid exhibited lower viscosity and less pseudoplasticity at the same concentration.

3.5.Effect of temperature on flow behavior

Effects of temperature on hydrocolloid viscosity vary depending on hydrocolloid species.Xanthan gum can maintain viscosity at high temperature (Sworn,2000;Rocks,1971)while an increase in temperature from 20to 808C results in a drop of the viscosity of galactomannans by 50%(Wielinga,2000).Also temperature dependency of viscosity of monoi hydrocolloid was examined (Fig.3).As temperature increased,the viscosity decreased (lower k ,Table 2).The higher flow behavior index indicated that at higher temperature the solutions were less pseudoplastic.This effect was more pronounced at low shear rate.The effect of heating

Table 1

Comparing flow behavior index (n )and consistency index (k )of Monoi gum,Xanthan gum,and Guar gum Gum type Concentration (%)n a

k a

Monoi gum

0.500.84G 0.010.19G 0.000.750.75G 0.020.96G 0.2010.67G 0.02 2.77G 0.48Xanthan gum

0.500.25G 0.02 1.36G 0.070.750.19G 0.00 2.46G 0.0810.18G 0.01 3.77G 0.15Guar gum

0.500.61G 0.010.66G 0.020.750.58G 0.02 2.13G 0.081

0.49G 0.02

5.65G 0.37

a

Mean G standard deviation.

Fig. 2.Flow behaviors of hydrocolloid from monoi leaves at different concentrations:(C )0.5%hydrocolloid;(:)0.75%hydrocolloid;(&)1%hydrocolloid.

Fig.1.(a)AFM image of monoi leaf polysaccharide.Image size is 2m m !2m m.(b)The height profile of the cross-section highlighted in the image (a).

B.Vardhanabhuti,S.Ikeda /Food Hydrocolloids 20(2006)885–1

888

and freezing on the viscosity of hydrocolloid is shown in Fig.4.The monoi hydrocolloid showed the same flow behavior after heating or freezing.Several hydrocolloids,such as xanthan gum and guar gum,have shown the excellent stability during freeze-thaw cycling,the property known as freeze-thaw stability (Downey,2002).Xanthan gum also shows stability over high temperature.Our result suggests that monoi leaf hydrocolloid may also be utilized in products that require stability after heating or freezing.Future work needs to be done to look at the stability of monoi gum after extended or repeated exposure times to heating and freezing (Table 3).3.6.Effect of NaCl on flow behavior

The effects of salts on the viscosity of various gums have been established.Xanthan gum,galactomannans,and methyl as well as hydroxypropyl methyl cellulose maintain their viscosity with the addition of salts (Williams and Phillips,2000).Carboxymethyl cellulose (CMC)loses its viscosity when salts are added.The viscosity of dilute pectin solutions (up to 0.5%)decreased with the addition of monovalent cations because of the suppression of charges on carboxyl groups,thus weakening repulsion between adjacent chains and allowing closer association (Nussinovitch,1997;Towle and Christen-sen,1973).In this study,the effect of sodium chloride on flow behavior of monoi hydrocolloid (0.5%)was investigated.As shown in Fig.5,sodium chloride greatly affected the flow

behavior of monoi hydrocolloids.Unlike pectin solutions,addition of NaCl from 0to 100mM increased the viscosity (k )and pseudoplascity (n )of hydrocolloid solutions.The k value increased from 0.29G 0.02to 8.53G 0.31while the n value decreased from 0.81G 0.00to 0.30G 0.00.Adding NaCl at the concentration of 0–100mM decreased the pH of the system from 3.74to 3.05.NaCl is the most popular salt used in food as flavoring and preserving agents.Its primary effect on polyelectrolyte is considered to shield electrostatic repulsions among charged groups on the polymer chain.This can lead to a reduction in viscosity due to less expansion of the molecules or an increase in viscosity due to intermolecular association.The decline in pH and the increase in viscosity with NaCl addition as observed in this study indicate that soluble intermolecular complexes are formed due to reduced charge on the molecules.The results suggest that monoi gum may be used to thicken foods containing low concentrations of NaCl.3.7.Effect of GDL on flow behavior

The viscosity of some hydrocolloids,such as xanthan gum and methyl cellulose,remains stable at low pH (Sworn,2000;Murray,2000).Some hydrocolloids such as galactomannans,CMC,and gum karaya,lose their viscosity at low pH (Glicksman,1982).Effect of GDL on flow behavior of monoi leaf hydrocolloid is shown in Fig.6.Addition of 0–1.2%GDL resulted in an increase in viscosity and pseudoplasticity of 0.5%hydrocolloid solution.The pH of

Fig.3.Effect of temperature on flow behavior of 0.5%monoi hydrocolloid:(%)48C;(&)108C,(:)208C;(C )408C;(!)608C;(B )808C.

Table 2

Comparing flow behavior index (n )and consistency index (k )of 0.5%Monoi hydrocolloid at different temperature Temperature (8C)N a

k a

40.66G 0.00 1.20G 0.06100.68G 0.000.91G 0.04200.75G 0.000.52G 0.04400.83G 0.000.23G 0.02600.88G 0.030.11G 0.0080

0.94G 0.04

0.05G 0.01

a

Mean G standard deviation.Fig. 4.Effect of freezing and heating on flow behavior of 0.5%monoi hydrocolloid.

Table 3

Comparing flow behavior index (n )and consistency index (k )of 0.5%Monoi hydrocolloid at different NaCl concentration NaCl (mM)n a

k a

00.81G 0.000.29G 0.02200.77G 0.020.43G 0.01400.56G 0.03 1.62G 0.29600.46G 0.03 3.07G 0.61800.34G 0.00 6.22G 0.12100

0.30G 0.00

8.53G 0.31

a

Mean G standard deviation.

B.Vardhanabhuti,S.Ikeda /Food Hydrocolloids 20(2006)885–18

the solutions decreased from 3.90to 3.24.Similar to the NaCl effect,reduced charge with GDL addition led to the formation of soluble intermolecular complex which led to higher viscosity of the solutions.It may suggest that a smaller amount of the monoi hydrocolloid can be used to thicken acidified foods such as drinks and juices.4.Conclusion

Cold water extraction of hydrocolloid from dried and frozen monoi leaves yielded 22.8and 32.8%hydrocolloid,respectively.The extracted hydrocolloid contained 49.7%AUA, 4.24%MEO in weight %,and 48.45DE.The macromolecules appeared to be occasionally branched linear chains with a wide molecular weight distribution.Flow behavior of monoi hydrocolloids exhibited shear-thinning characteristic.Increasing the solution concen-trations from 0.5to 1%increased the viscosity and pseudoplasticity.The viscosity of monoi hydrocolloids was

also found to be influenced by temperature.Increasing temperature from 4to 808C resulted in a decrease in viscosity and pseudoplasticity.Flow behavior remained stable after heating or freezing.Addition of NaCl from 0to 100mM or GDL from 0to 1.2%resulted in an increase in viscosity and shear thinning behavior.It can be seen that monoi hydrocolloids may be utilized in foods to give viscosity,body,and mouth feel with the ability to remain stable over the range of temperature.Examples of food applications are bakery fillings,frozen thickened products,pourable dressing,sauces and gravies,dairy products as well as drinks.Acknowledgements

This research was funded by Rajabhat Institute Council currently part of Commission on Higher Education,Thailand.The authors are grateful to Professor Katsuyoshi Nishinari (Osaka City University)for his support and helpful suggestion on hydrocolloid chemistry.Sincere appreciation went to Sriwon Chaisuk,Penpan Kanpinyo,and Surin Boonsai (Chiangrai Rajabhat University)for their helpful information regarding monoi plant.We would like to thank Lanna Foods and Supplies (Chiangmai,Thailand)and Metrohm Siam Ltd (Bangkok,Thailand)for donating the commercial gums and the use of their equipments.

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