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FOODHYDROCOLLOIDS

0268-005X/$ - s

doi:10.1016/j.fo

Abbreviations

EPS, exopolysa

ELISA, enzyme

saline; HRP, ho�Correspond

fax: +458625 1

E-mail addr

Food Hydrocolloids 21 (2007) 1062–1071

www.elsevier.com/locate/foodhyd

Screening of probes for specific localisation of polysaccharides

D. Arltofta,b,�, F. Madsena, R. Ipsenb

aPhysical Food Science, Danisco A/S, Edwin Rahrsvej 38, 8220 Brabrand, DenmarkbDepartment of Food Science, KVL, Rolighedsvej 30, 1958 Frederiksberg C, Denmark

Received 2 March 2006; accepted 20 July 2006

Abstract

Polysaccharides can be used as gelling or stabilising agents in foods. In order to investigate how polysaccharides affects the rheology

and sensory characteristics of foods, an in situ method for direct localisation is required. The aim of this study was to investigate the

specificity and stability of the monoclonal pectin antibody JIM7, anti-carrageenan polyclonal antibody and the lectins wheat germ

agglutinin (WGA) and concanavalin A (ConA). An enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of these

probes for compounds frequently found in dairy products. The compounds chosen were pectins, carrageenans, galactomannans,

starches, exopolysaccharides (EPS), alginate, xanthan gum, gelatine and skimmed milk powder. The impact of exposure to low pH,

0–2% salt and 0–40% sugar on the antigen-binding activity of the probes was tested. The results showed that WGA bound specifically to

chitosan and hetero-EPS at neutral pH, and while ConA bound strongly to hetero-EPS, it also exhibited various degrees of affinity for all

the other hydrocolloids tested. However, the ability of the investigated lectins to bind to hetero-EPS almost disappeared under conditions

similar to those in yogurt. JIM7 was very stable and highly specific for pectins, also under conditions similar to those present in yogurt.

The polyclonal antibody was very stable and displayed potential as probe for all the polysaccharides tested. Due to the broad specificity,

however, the identity of the polysaccharides present must be known. Confocal laser scanning microscopy (CLSM) images using the

probes for specific localisation of pectin and carrageenan in dairy products are shown.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Direct immunostaining; Hydrocolloid; ELISA; Dairy products; Stability; Specificity; JIM7; WGA; Concanavalin A; Antibody; Microstructure

1. Introduction

Polysaccharides are widely used to gel and stabilise foodproducts and various attempts have been made to assessrelations between the microstructure of polysaccharidesand their gelling and stabilising characteristics (Beaulieu,Turgeon, & Doublier, 2001; Hemar, Tamehana, Munro, &Singh, 2001; Turgeon & Beaulieu, 2001; Walkenstrom,Kidman, Hermansson, Rasmussen, & Hoegh, 2003). Linksbetween the protein-microstructure and rheology havebeen indicated (Auty, Fenelon, Guinee, Mullins, & Mulvi-hill, 1999; Bourriot, Garnier, & Doublier, 1999) and it has

ee front matter r 2006 Elsevier Ltd. All rights reserved.

odhyd.2006.07.020

: WGA, wheat germ agglutinin; ConA, concanavalin A;

ccharides; CLSM, confocal laser scanning microscopy;

-linked immunosorbent assay; PBS, phosphate-buffered

rse radish peroxidase

ing author. Tel.: +458943 5333/+453528 3276;

077.

ess: ditte.arltoft@danisco.com (D. Arltoft).

been possible to establish relations between the polysac-charide-microstructure and the texture/rheology of pro-ducts employing polysaccharides as a stabiliser (Arltoft,Madsen, & Ipsen, 2006). In order to establish such linksspecific probes are needed. Covalent labelling has been thesole method employed for specific localisation of poly-saccharides in foods (Tromp, van de Velde, van Riel, &Paques, 2001; van de Velde, Weinbreck, Edelman, van derLinden, & Tromp, 2003). However, within other researchareas, antibodies have been used to identify specificstructures in situ (Celi et al., 2003; Iwamoto, Burrows,Born, Piepkorn, & Bothwell, 2000; Keller, Winde, Terpe,Foerster, & Domschke, 2002), suggesting the possibility touse antibodies to localise polysaccharides in situ in foods.In order to fully explore the possibilities of in situ

staining, microscopical methods without the need forslicing, freezing or dehydration of the specimen areadvantageous due to the imaging of the least perturbedmicrostructures. Confocal laser scanning microscopy

ARTICLE IN PRESSD. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–1071 1063

(CLSM) offers the possibility of collecting signal fromvarious probes emitting light at different wavelengths andhence, possess the ability to evaluate the localisation of oneingredient relative to others, in the hydrated state. Othermicroscopical methods, such as environmental scanningelectron microscopy and atomic force microscopy, with theability to look at hydrated specimens only offer morphol-ogy to distinguish between different components.

We identified four commercially available probes ashaving potential for specific localisation of polysacchar-ides. The probes comprised antibodies raised againstspecific polysaccharides and lectins known to have anaffinity for certain sugar molecules, specifically the lectinsconcanavalin A (ConA) and wheat germ agglutinin(WGA), and the monoclonal pectin antibodies JIM5 andJIM7. We also produced a polyclonal antibody fromrabbits injected with hybrid-carrageenan. ConA and WGAconjugated with a fluorophore have been used for in situ

localisation of exopolysaccharides (EPS) (Folkenberg,Dejmek, Skriver, & Ipsen, 2005; Hassan, Frank, & Qvist,2002; Hassan, Ipsen, Janzen, & Qvist, 2003). The culturesupernatants of the monoclonal rat antibodies JIM5 andJIM7 have been used for specific localisation of pectin inplants (Jauneau, Roy, Reis, & Vian, 1998). JIM5 has beentested previously, and was for this reason not included inthe present study (Arltoft et al., 2006). Conjugated with afluorophore, JIM5 has been successfully used for in situ

localisation of pectin in yogurt and model milk gels(Arltoft et al., 2006).

The binding of antibodies and lectins to their respectiveepitopes is facilitated by various interactions such ashydrogen bonding, electrostatic-, Van der Waals- andhydrophobic forces (Padlan, 1994; Weis & Drickamer,1996). These interactions are affected by changes in ionicstrength and pH. Such changes away from physiologicalconditions will be referred to as stress or stressingconditions in this paper. Dairy product environments arehighly variable in terms of pH (3.5–7.5), sugar concentra-tion and ionic strength. Hence, it is uncertain whether anantibody or lectin with documented specificity andfunctionality under physiological conditions of pH andionic strength will retain this functionality in a dairyproduct environment. The term stability will in this paperrefer to the retention of antigen-binding activity (function-ality) under stress. In addition, it is important to knowwhether a given probe will bind to other molecules presentin the sample—implying that the affinity of probes forother compounds must be assessed. Knowledge of thesefactors is a prerequisite when using antibodies or lectins forspecific localisation in foods.

The aim of this study was to screen ConA, WGA, JIM7and the polyclonal anti-carrageenan antibody as probes forspecific localisation of polysaccharides in foods. For thispurpose, their affinity for a range of compounds frequentlyfound in foods and their ability to bind at variable pH, saltand sugar concentrations were tested prior to applicationin model dairy systems (yogurt and dairy dessert).

2. Materials and methods

2.1. Commercial probes

The commercial probes employed were the monoclonalrat IgA JIM7 (PlantProbes, Leeds, UK); ConA–AlexaFluor 488 conjugate, and WGA–Alexa Fluor 488 con-jugate (both from Molecular Probes, Inc., Eugene, USA).

2.2. Production and purification of polyclonal anti-

carrageenan antibodies

Three rabbits each had four subcutaneous injections of250 ml 1% hybrid-carrageenan solution (Table 1) mixedwith alhydrogel and Freunds complete adjuvant. Therabbits were subsequently boosted with five injections of250 mL every 2nd week, for a total of 12 weeks, using asolution as described above, but with Freunds incompleteadjuvant. The blood was collected after 12 weeks andserum separated and stored at �18 1C.To separate the antibodies from other serum compo-

nents, we used Protein A sepharose (Amersham Bios-ciences AB, Uppsala, Sweden). The binding buffer usedwas phosphate-buffered saline (PBS) (0.14M NaCl,2.7mM KCl, 7.8mM Na2HPO4 � 2H2O, 1.5mM KH2PO4,pH 7.2) with 0.5M NaCl. The elution buffer was 0.1Mglycine-HCl buffer, pH 2.5. Concentration and bufferexchange were performed using Vivaspin concentrators(molecular weight cut off value ¼ 50,000, Vivascience AG,Hannover, Germany). The purified antibody was added2mM NaN3 for preservation and stored at 4 1C.

2.3. Testing specificity—enzyme-linked immunosorbent

assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) was usedto evaluate the affinity of the probes for each of thecompounds listed in Table 1. Microtiter plates (Multisorp,Nunc A/S, Roskilde, Denmark) were coated with 100 mL of0.05% hydrocolloid in PBS (if necessary the hydrocolloidswere heated to 80 1C for up to 30min in order to solubilise)in each well and left overnight at 4 1C. Prior to the assay200 mL blocking buffer was applied for 1 h at roomtemperature in order to block the unoccupied binding sites(Table 2). Wells were washed 3 times with 200 mL washingbuffer (Table 2). The probes were diluted in PBS, asappropriate (Table 3). To each well 100 mL of the dilutedprobe was added and left for 1 h at room temperature. Thewells were washed 5 times with 200 mL washing buffer. Inorder to identify the lectins, an additional step for WGAand ConA was included, using an anti-Alexa 488 poly-clonal antibody derived from rabbit (Molecular Probes), asno anti-lectin horse radish peroxidase (HRP) conjugatedantibody was available. This additional step included100 mL anti-Alexa 488 antibody diluted 1:750 in antibodybuffer (Table 2) for 1 h at room temperature, followed by5 washes. Finally, 100 mL HRP-conjugated antibody

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Table 2

Buffers used for ELISA and their composition

Buffer name Composition

Washing buffer PBS, 0.05% Tween 20

Blocking buffer PBS, 0.1%BSA

Probe buffer PBS

Antibody buffer PBS, 0.05% Tween 20, 0.1% BSA

Table 3

The initial concentration of the probes, if known; their dilution used in

ELISA assays and the antibody used for assessing the amount of probe

binding to the antigen

Probe Dilution Assessing

antibody

Culture supernatant JIM7 1:400 HRP-Anti-rat

WGA 1mg/ml in PBS 1:50,000 Anti-Alexa 488

ConA 1mg/ml in PBS 1:400 Anti-Alexa 488

Protein A purified polyclonal

antibodies 2mg/ml in PBS

1:100 HRP-Anti-rabbit

Anti-Alexa 488 antibody 1mg/ml

in PBS

1:750 HRP-Anti-rabbit

Table 1

Compounds used for evaluating the specificity of JIM7, polyclonal antibody, WGA and ConA

Hydrocolloid Identity Supplier

High-ester block structure pectin Non-standardised (%DE ¼ 70) Danisco A/S

Low-ester pectina Experimental sample P41 (%DE ¼ 41)* Danisco A/S

Low-ester amidated pectin GRINDSTEDs Pectin LA410 Danisco A/S

Hybrid-carrageenanb Non-standardised (kappa:iota ratio 2:3) Danisco A/S

Chitosanc 48% deacetylated Advanced Biopolymers AS

Locust bean gum (LBG) GRINDSTEDs LBG 147 Danisco A/S

Alginate GRINDSTEDs Alginate FD155 Danisco A/S

Guar gumd Edicol 40-70 Lucid Colloids Ltd.

Xanthan gum FN Jungbunzlauer

Modified starch, maize C*PolarTex 06748 Cerestar

Hetero-EPS Lactococcus lactis subsp. cremoris Danisco A/S

Homo-EPS Lactobacillus sakei Danisco A/S

Iota-carrageenan C3799 Sigma

Kappa-carrageenan C1263 Sigma

Lambda-carrageenan C3889 Sigma

Amylose, potato 10130 Fluka

Amylopectin, maize 10120 Fluka

Gelatin 48723 Fluka

Skimmed milk powder Milex 240 Arla foods

aPositive control polysaccharide for JIM7. *Willats et al. (2000).b,c,dPositive control polysaccharides for polyclonal antibody, WGA and ConA, respectively.

D. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–10711064

(Table 3) diluted 1:750 in antibody buffer was added toeach well. The plate was left for 1 h at room temperatureand then washed 5 times with 200 mL washing buffer ineach well. Hundred microlitres substrate solution (12mLdeionised water with 8mg o-phenylendiamine dihydroclor-ide (DakoCytomation A/S, Glostrup, Denmark) and 5 mLH2O2 (30%)) was added to each well and left forapproximately 5min. The reaction was stopped by adding50 mL 0.5M H2SO4. The absorbance at 492 nm was

measured using a Power Wave 200 microplate reader(Biotek instruments, Winooski, USA). All plates included apositive control employing the specific antigen of the probeas well as negative controls with and without antigen. Aminimum of four replications was used.

2.4. Testing stability—ELISA

When performing in situ staining of polysaccharides infoods, the probes must be able to bind under realistic foodconditions, which will normally include a certain concen-tration of both salt and sugar. Hence, we tested the abilityof the antibody probes to bind to their antigen under theconditions created by 12 combinations of three levels ofNaCl: 0, 1 and 2w/v% and four levels of sucrose: 0, 10, 20and 40w/v% either with the PBS buffer (i.e. 0% added saltis 0.15M NaCl) or 25mM succinic acid buffer pH 3.5 (i.e.0% added salt is 0% salt) as base. Additionally, thestability of JIM7, WGA and ConA was tested underconditions similar to yogurt, as pectins and EPS arecommon stabilisers in yogurt. In order to simulate yogurtconditions acidic whey-based stress buffers were designed.The whey was produced by chemically acidifying recon-stituted milk (17% skimmed milk powder) with 3.3%glucono-d-lactone (GDL) to a pH of 4.0 and centrifugingfor 30min at 9000g. The supernatant (acidic whey) wasfiltered through a 0.45 mm filter (Sartorius AG, Goettingen,Germany). The pH of the acidic whey buffer was 3.9. Thelectins were tested in acidic whey and acidic whey with 10%sugar added, whereas JIM7 was challenged with all thecombinations of salt and sugar concentrations listed abovefor the PBS base. The stability of the polyclonal antibodywas not tested in acidic whey since carrageenan is rarely

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used in low pH products. PBS served as the positive controlbuffer. The ELISA procedure was almost identical to thespecificity tests, the only difference being that the probeswere diluted in stress-buffers (at the concentration listed inTable 3), and that the positive control polysaccharide foreach probe was at the bottom of the wells (Table 1). Allconditions were tested in at least three wells on twoseparate occasions. All plates included a positive controlwith the probe diluted in PBS and the preferred antigenand negative controls with and without antigen.

2.5. Using the probes for specific localisation of

hydrocolloids in situ

In order to use the probes for specific localisation ofhydrocolloids in situ, we separated the antibodies fromother culture supernatant/serum components. For this weused KappalockTM Sepharose 4Bs (Zymed LaboratoriesInc., USA) and protein A Sepharose (Amersham Bios-ciences, Uppsala, Sweden) respectively, and subsequentlyconjugated a fluorophore directly to the antibodies usingcommercially available mono- and polyclonal conjugationkits (Molecular probes). The methods for purification andconjugation have been described in detail (Arltoft et al.,2006). The conjugation of Alexa Fluor 647 to JIM7resulted in more than 2mole of dye per mole of antibody.For conjugation of Alexa Fluor 488 to the polyclonalantibody pH 7.0 was used as a compromise betweenretaining the activity of the antibodies and conjugating asufficient amount of Alexa Fluor 488 (0.6mole dye/moleantibody). ELISA was used to verify the activity of theantibody–fluorophore conjugate.

We used JIM7-Alexa Fluor 647 conjugate to localisepectin in a whole milk yogurt fermented with YOMIXs

410 (Danisco A/S) and using 0.12% low-ester pectin asstabiliser (GRINDSTEDs Pectin SY640, Danisco A/S).The polyclonal antibody was employed to localise carra-geenan in a gelled dairy dessert comprising 87.2% skimmedmilk, 9% sucrose, 2.4% cream, 1% native maize starch,0.3% GRINDSTEDs Carrageenan CL314 MX and 0.1%vanilla flavour NI T03134 (Danisco A/S). Production wascarried out in a pilot plant as follows. Milk was heated to40 1C, the premixed dry ingredients were added to the milkwhile stirring and subsequently left 30min for completehydration. Then the mixture was heated to 90 1C, kept for10min and hot-filled into plastic beakers of 155mL andtransferred to cold storage.

The microstructure of pectin and carrageenan wasvisualised using a Leica TSP2 confocal laser-scanningmicroscope (Leica, Mannheim, Germany). For staining thepectin JIM7-Alexa Fluor 647 was diluted 1:10 in whey,centrifuged for 3min at 7300 g and 10 mL of the super-natant (�1 mg JIM7-Alexa Fluor 647) was added andspread out to approximately 2 cm2 on the cover glass with aspatula. Approximately 1 g of specimen was gently addedon top and left in a hydrated chamber for at least 30min at15 1C before microscopy. FITC was used to localise the

protein. For localising carrageenan in the dairy dessert, theconjugate of polyclonal antibody with Alexa Fluor 488were used at �8 mg per staining due to the lower affinityand fewer conjugated dye molecules. The �8 mg of AlexaFluor 488-conjugated antibody present in 10 mL of PBSwas added and spread out to approximately 2 cm2 on thecover glass with a spatula. A specimen of the dairy dessertapproximately sized 20mm� 12mm� 4mm was gentlyremoved from the original container with a sharpenedspatula, carefully added on top of the antibody solutionand left in a hydrated chamber for at least 30min at 15 1Cbefore microscopy. Protein was visualised using reflectionmode at 488 nm.

3. Results

3.1. Specificity of probes

Fig. 1 shows the specificity of JIM7 and the polyclonalantibody. JIM7 had high affinities (475%) for all thepectins tested and also exhibited a high affinity for guargum and a medium affinity (25–75%) for locust bean gum(LBG). The polyclonal antibody raised against hybridcarrageenan had a broad specificity (Fig. 1). It showedequal affinity for all the polysaccharides investigated, but,most importantly, no affinity for skimmed milk protein orgelatine was present. In other words, when only onepolysaccharide is present in a dairy system, localisationshould be possible using the polyclonal antibody.Fig. 2A shows that WGA exhibited high affinity for

chitosan and hetero-EPS and low affinity (10–25%) forxanthan gum. The affinity for all other compounds testedwas negligible (o10%). ConA showed broad, but variableaffinity. Very high affinity for hetero-EPS, high affinity forguar gum, xanthan gum and LBG, and medium affinitytowards most of the compounds tested, including gelatinewere determined. Even some binding to skimmed milkpowder was assessed (Fig. 2B).

3.2. Stability

As seen from Fig. 3 JIM7 maintained a high antigen(pectin) binding activity at most of the conditions tested.Sugar was an inhibiting factor for the antigen bindingactivity of JIM7, independent of pH and salt. But 40% ofsugar was necessary to decrease the affinity from high tomedium. The pH was not an inhibiting factor by it self asseen from the antigen binding activity in Fig. 3B comparedto Fig. 3A. Furthermore, JIM7 was stable towards highsalt concentrations, only very low salt levels decreased theaffinity, as seen from the medium affinity at pH 3.5 with0% added salt (Fig. 3B). The acidic whey exerted aninhibiting effect on the antigen binding activity of JIM7(Fig. 3C) that cannot be explained by pH, or by the highionic strength present, as the affinity at pH 3.5 and 2% saltwas higher than at the same conditions in whey. Thecombination of acidic whey and 40% sugar strongly

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JIM7

Fig. 1. The affinity of JIM7 and the polyclonal antibody for a range of compounds shown as a percentage of the affinity to low-ester pectin P41 and

hybrid carrageenan, respectively. The columns show the average results from one trial with four replications. Bars indicate standard deviation.

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ConA, pH 7.2

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Fig. 2. The affinity of lectins for a range of compounds shown as a percentage of the affinity to the positive control polysaccharide. (A) WGA in PBS;

(B) ConA in PBS. The columns show the average results of one trial with 4 replications. Bars indicate standard deviation. ND is not determined numbers.

D. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–10711066

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JIM7 Polyclonal antibodies (A)

(B)

(C)

Fig. 3. The relative affinity of JIM7 and the polyclonal antibodies for their antigen at different stressing conditions as assessed by ELISA. The stressing

conditions are all combinations of three levels of salt: 0%, 1% and 2%; four levels of sugar: 0%, 10%, 20% and 40% at (A) in PBS pH 7.2, (B) in 25mM

succinic acid buffer pH 3.5 and finally, (C) in acidic whey. The positive control was in all cases PBS pH 7.2. The columns show the average results obtained

in two independent trials with three replications each. Bars indicate the average standard deviation of the two trials.

Table 4

Affinity of JIM7 for pectins, LBG and guar gum when diluted in acidic

whey as a percentage of the activity determined at neutral pH with ELISA

JIM7’s affinity for the 13 other compounds tested was negligible. The

mean (standard deviation) of four replications is shown

Hydrocolloid JIM7 activity (%)

High-ester pectin 79.0 (5.1)

Low-ester pectin 66.0 (6.7)

Amidated pectin 69.4 (10.0)

Locust bean gum 0.7 (0.1)

Guar gum 4.1 (0.6)

D. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–1071 1067

decreased the affinity of JIM7. The affinity for LBG andguar gum, assessed at neutral pH, disappeared in acidicwhey (Table 4).

The polyclonal antibody were very stable and main-tained a high affinity for carrageenan at all the conditionstested except for the medium affinity assessed at pH 3.5,2% salt and 40% sugar. The salt concentration did notaffect the affinity of the polyclonal antibody within therange tested, nor did the pH. Increasing sugar decreasedthe antigen binding activity of the polyclonal antibody. Butonly 40% sugar combined with pH 3.5 and 2% saltdecreased the affinity to below 50% of the positive control.

When WGA was tested in acidic whey with and without10% sugar, the affinity for chitosan decreased to14.870.8% and 19.170.7% of the positive control,respectively, whereas the affinity for hetero-EPS disap-

peared (3.670.3% and 3.770.2%). WGA’s affinity for allthe other hydrocolloids tested was negligible (WGA datanot shown in figure). The affinity of ConA also decreased

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Fig. 4. The affinity of ConA in acidic whey and acidic whey with 10% sugar for a range of compounds shown as a percentage of the affinity to guar gum:

the positive control polysaccharide. The columns show the average results of one trial with four replications. Bars indicate standard deviation. ND is not

determined numbers.

Fig. 5. CLSM image of low-ester pectin in yogurt. Green is protein

stained with FITC and red is JIM7-Alexa Fluor 647 conjugate staining

pectin. Scale bar is 10 mm.

D. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–10711068

when tested in acidic whey with and without sugar (Fig. 4),although binding was still assessed to almost all thecompounds investigated. Notably, the high affinity forhetero-EPS at pH 7.2 disappeared when both WGA andConA was diluted in acidic whey.

3.3. Using the probes for specific localisation of

hydrocolloids in situ

Fig. 5 shows the use of the JIM7-Alexa Fluor 647conjugate for localising pectin in whole milk stirred yogurt

with 0.12% low-ester pectin. The pectin was located assmall entities in the vicinity of the protein aggregates.Fig. 6 shows the use of the polyclonal antibody

conjugated with Alexa Fluor 488 for localising carrageenanin a dairy dessert. In Fig. 6B the signal from the polyclonalantibody exhibited a gelled network connecting andstabilising the larger protein aggregates. In Fig. 6A, fromthe reflection mode, the same area with signal from thepolyclonal antibody also showed a slight signal. Stainingthe same dairy dessert with FITC (image not shown)showed a similar slight signal in the areas surrounding thedensely stained protein aggregates. Furthermore, FITCstaining revealed that most of the non-stained part of Fig. 6was constituted of swollen starch granules.

4. Discussion

A lack of probes for specific localisation of polysacchar-ides in foods has been reported (van de Velde et al., 2003).Thus, we tested the specificity and stability of fourpotential probes for in situ localisation of polysaccharidesin foods. Each probe had, as expected, its own combinationof strengths and weaknesses.Monoclonal antibodies are produced by only one clone

and, thus, have only one type of antigen-binding site(Goding, 1996). In contrast, polyclonal antibodies havemany different antigen-binding sites, because of theirpolyclonal nature. These differences are, at least in theory,reflected both in the specificity and in their stabilitytowards stress. This means that the polyclonal antibodiesrecognise the injected antigen in many different ways and,hence, not in a highly specific manner. The monoclonalantibodies are very specific because they only have one wayof recognising the antigen. The same applies to stability.Conditions inhibiting the forces used by the monoclonalantibody to bind to its antigen will inhibit all the

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Fig. 6. CLSM image of a dairy dessert gelled with 0.3% carrageenan and

1% native starch. (A) Protein channel only, envisioned by reflection and

(B) overlay image with the carrageenan seen in red as stained by the

polyclonal antibody conjugated to Alexa Fluor 488. Scale bar is 50mm.

D. Arltoft et al. / Food Hydrocolloids 21 (2007) 1062–1071 1069

monoclonal antibodies. Whereas for the polyclonal anti-bodies a condition that inhibits the binding of one antigen-binding site may not necessarily inhibit the binding of adifferent site.

4.1. Specificity

We found that the monoclonal JIM7 was much morespecific than the polyclonal antibody. The affinity of JIM7

for different pectins corresponded to what has been foundearlier by Willats, Gilmartin, Mikkelsen, and Knox, (1999).However, JIM7 has not previously been tested for cross-reactivity against other types of polysaccharides. The cross-reactivity of JIM7 against guar gum and LBG assessed inthis study was also found for the pectin monoclonal ratIgG2a antibody JIM5 (Arltoft et al., 2006) but to a lesserextent: 2972.2% for guar gum and 9.270.7% for LBG.Guar gum and LBG are both galactomannans, LBGhaving half the galactose residues of guar gum. As theaffinity for the galactomannans decreased with the amountof galactose residues, this indicates that the cross-reactivityof JIM5 and JIM7 may be caused by the galactose residues.The polyclonal antibody exhibited a broad specificity

against polysaccharides, implying that they could be usedto localise almost any type of polysaccharide. However, theELISA results, indicating affinity of the polyclonal anti-body for starch, contrast with the image in Fig. 6 wherethere was no indication of binding to swollen starchgranules. This will be further discussed in Section 4.3.ConA exhibited broad specificity, indicating that it

should only be used for localisation of a polysaccharidewhen the identity of all the polysaccharides in the sample isknown and all possible controls are employed. WGAseemed much more specific, binding only to chitosan andhetero-EPS. WGA was tested at much higher dilution inorder to get absorbance below 2.0. Both lectins have beenemployed for localisation of EPS (Folkenberg et al., 2005;Hassan et al., 2002, Hassan et al., 2003; Strathmann,Wingender, & Flemming, 2002). The present study furtherrevealed the strong affinity of both lectins for hetero-EPS.However, the affinity decreased strongly under conditionssimilar to those of yogurt. This may be overcome byincreasing the concentration of the lectin, diluting it inwhey before use, centrifuging and then using the super-natant only. We have used this method successfully withJIM5 for localising pectin in yogurt and a similarprocedure was used for localising EPS with lectins (Hassanet al., 2002).

4.2. Stability

A probe for localising polysaccharides in dairy productsin general would be exposed to low pH in yogurt, highsugar concentrations in desserts, and high ionic strength incheeses. Thus, it is important to test the antigen-bindingactivity of potential probes under relevant conditions. Aspectins are commonly used in yogurt, the ability of JIM7 tobind to pectin at conditions similar to yogurt is important.The decreased binding assessed at pH 3.5 with very lowionic strength has little practical relevance for most foodproducts, as a physiological ionic strength will normally bepresent. JIM7 showed high tolerance to low pH and highionic strength when compared with the monoclonal ratantibody JIM5, which we have tested earlier (Arltoft et al.,2006). It appears that JIM7 is probably more suitable forlocalisation of pectin in yogurt than JIM5.

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No other studies investigating the stability of rat IgA orpolyclonal antibodies have been encountered in the litera-ture. However, studies on the stability of other mouse andhuman monoclonal antibodies have showed that theantibodies generally retain antigen-binding activity at pH5–9 (Castle et al., 2002; Jiskoot et al., 1991; Riggin,Clodfelter, Maloney, Rickard, & Massey, 1991; Usami,Ohtsu, Takahama, & Fujii, 1996), and their stability towardshigh and low ionic strength is variable (Haskell, Buchegger,Schreyer, Carrel, & Mach, 1983; Kammer, 1983).

The polyclonal antibody were even more stable towardsthe stressing conditions than JIM7. The broad affinitytowards all the polysaccharides tested implies that thecontent of polysaccharides in a sample must be knownbefore the microstructure is evaluated. However, thistechnique needs further clarification, as the polyclonalantibody appeared to exclusively bind to carrageenan inthe CLSM image where native maize starch was alsopresent. This will be thoroughly discussed in Section 4.3.

The specific activity of WGA against chitosan could beuseful in specifically localising chitosan in mixtures withmilk-protein and a range of other hydrocolloids. Thestrongly decreased activity against chitosan in acidic wheyindicates that WGA is not able to detect chitosan in yogurt.However, as mentioned above, increasing the concentra-tion of WGA may compensate for the decreased activity.The concentration used in the ELISA was very low(1:50,000).

The broad affinity of ConA indicates that ConA shouldonly be used when complete knowledge of the hydro-colloids present in the sample is available and that imagesof the microstructure observed with ConA should beinterpreted with great caution and all possible controlsshould be employed.

No reports of stability studies on lectins have beenfound.

4.3. In situ localisation

The in situ localisation of pectin and carrageenan inFigs. 5 and 6, respectively, showed that the directimmunostaining technique of JIM7 and the polyclonalantibodies could be employed with a good signal-to-noiseratio.

The localisation of pectin in the stirred yogurt as smallentities in the vicinity of the protein aggregates corre-sponded to the localisation of another low-ester pectin withJIM5 in stirred yogurt (Arltoft et al., 2006). A high localcalcium level caused by the increased solubility of calciumphosphate at the low pH combined with the affinity of thenegatively charged pectin for the positively chargedk-casein may have caused this very dense pectin-structurein the vicinity of the protein aggregates.

The polyclonal antibody showed a gelled networksurrounding and connecting the larger protein aggregates.In reflection mode a slight staining of small particles wascolocalised with the gelled network. We believe that these

particles were protein, and not fat nor starch. Milk fatglobules would be easily recognised due to their globularmorphology. To our best knowledge starch leaked from thegranules (amylose) does not reflect light, hence, it is notlikely that the particles in the gelled network are amylose.Furthermore, a similar slightly stained structure has beenobserved colocalised with the carrageenan network in dairygels composed of only skimmed milk and hybrid-carragee-nan, i.e. without starch (manuscript under preparation).Lastly, an accepted theory explaining the effectiveness ofcarrageenan as a milk protein stabiliser proposes thatcarrageenan forms a coupled network with the caseinmicelles (Langendorff et al., 2000), thus rendering itplausible that the observed particles are protein. Themicrostructure seen in the dairy dessert in Fig. 6corresponds to the microstructure seen in similar gels, asevaluated by Nunes, Raymundo, and Sousa (2006) wherethe areas constituting the carrageenan network areunspecifically visualised with rhodamine.We are puzzled by the fact that the polyclonal antibody

did not bind to the swollen starch granules, identified byFITC staining, contrary to the ELISA results, where theywere found to bind to amylose, amylopectin and modifiedstarch. Further investigations were made using the poly-clonal antibody together with the JIM5 antibody to localisepectin and carrageenan in model milk systems. This alsoindicated that the polyclonal antibody did not bind at thesame locations as JIM5 (results not shown). Thus, eventhough the polyclonal antibody is able to bind to otherpolysaccharides, they seem to prefer carrageenan when it ispresent. However, it should be evaluated whether thepolyclonal antibody only bind to carrageenan and not toother polysaccharides when in situ or whether the antibodyprefer carrageenan when it is present and whether, whencarrageenan is absent, the antibody can be used as probesfor other polysaccharides.

5. Conclusions

Major differences were found in the binding specificity ofthe probes and their stability towards stress. Even thoughthe same general interactions are involved in the specificbinding between the probes and the polysaccharides, thebalance between the interactions is unique to each probe.Hence, the specificity and stability towards stress differed.JIM7 is a strong potential candidate for pectin localisa-

tion under all the conditions tested, except for very lowionic strength. But caution must be taken if guar gum and/or LBG are present in the samples.The polyclonal antibody was very stable towards the

stressing conditions frequently encountered in foods andwas successfully used to localise carrageenan in a dairydessert. However, because of the broad affinity for otherpolysaccharides, caution must be taken when otherpolysaccharides are present in the specimen evaluated.The two lectins tested both bound strongly to EPS at

neutral pH. WGA is very specific and can probably be used

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successfully to localise chitosan. ConA showed affinity forseveral hydrocolloids but also bound to gelatine. Thebroad specificity implies that caution must be used wheninterpreting images with ConA if more than one poly-saccharide is present in the sample. Due to the stronglyreduced activity of the lectins in acidic whey specialpreparation procedures are probably needed in low pHfoods like yoghurt.

Acknowledgements

The financial support by the Ministry of Science,Technology and Innovation is highly appreciated. Sincerethanks go to Tove Christensen and Anne Sahlgren(Danisco A/S) for kindly providing the isolated homo-and hetero-EPS.

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