M224 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 4, 2005Published on Web 5/4/2005

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M: Food Microbiology & Safety

JFS M: Food Microbiology and Safety

Examination of Exopolysaccharide Producedby Lactobacillus delbrueckii subsp. bulgaricusUsing Confocal Laser Scanning and ScanningElectron Microscopy TechniquesKKKKKELELELELELVINVINVINVINVIN K.T K.T K.T K.T K.T. G. G. G. G. GOHOHOHOHOH, R. D, R. D, R. D, R. D, R. DEREKEREKEREKEREKEREK H H H H HAISMANAISMANAISMANAISMANAISMAN, , , , , ANDANDANDANDAND H H H H HARJINDERARJINDERARJINDERARJINDERARJINDER S S S S SINGHINGHINGHINGHINGH

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: E: E: E: E: Exxxxxopolysaccharopolysaccharopolysaccharopolysaccharopolysaccharide (EPS) pride (EPS) pride (EPS) pride (EPS) pride (EPS) produced boduced boduced boduced boduced by y y y y LLLLLactobacillus delbrueckii actobacillus delbrueckii actobacillus delbrueckii actobacillus delbrueckii actobacillus delbrueckii subspsubspsubspsubspsubsp. . . . . bulgaricusbulgaricusbulgaricusbulgaricusbulgaricus NCFB 2483 (2483) NCFB 2483 (2483) NCFB 2483 (2483) NCFB 2483 (2483) NCFB 2483 (2483)was examined using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) tech-was examined using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) tech-was examined using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) tech-was examined using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) tech-was examined using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) tech-niques. These microscopy techniques were used to probe the location and distribution of EPS in milk permeate–niques. These microscopy techniques were used to probe the location and distribution of EPS in milk permeate–niques. These microscopy techniques were used to probe the location and distribution of EPS in milk permeate–niques. These microscopy techniques were used to probe the location and distribution of EPS in milk permeate–niques. These microscopy techniques were used to probe the location and distribution of EPS in milk permeate–based media. In CLSM, lectin (SBA) (from based media. In CLSM, lectin (SBA) (from based media. In CLSM, lectin (SBA) (from based media. In CLSM, lectin (SBA) (from based media. In CLSM, lectin (SBA) (from Glycine maxGlycine maxGlycine maxGlycine maxGlycine max) Alexa Fluor 488 conjugate was used to stain the EPS. The EPS) Alexa Fluor 488 conjugate was used to stain the EPS. The EPS) Alexa Fluor 488 conjugate was used to stain the EPS. The EPS) Alexa Fluor 488 conjugate was used to stain the EPS. The EPS) Alexa Fluor 488 conjugate was used to stain the EPS. The EPSappeared randomly distributed as aggregates in the culture media. The CLSM technique was simple to perform withappeared randomly distributed as aggregates in the culture media. The CLSM technique was simple to perform withappeared randomly distributed as aggregates in the culture media. The CLSM technique was simple to perform withappeared randomly distributed as aggregates in the culture media. The CLSM technique was simple to perform withappeared randomly distributed as aggregates in the culture media. The CLSM technique was simple to perform withminimal sample preparation, was nonintrusive, and allowed in-situ examination of EPS. At high magnificationsminimal sample preparation, was nonintrusive, and allowed in-situ examination of EPS. At high magnificationsminimal sample preparation, was nonintrusive, and allowed in-situ examination of EPS. At high magnificationsminimal sample preparation, was nonintrusive, and allowed in-situ examination of EPS. At high magnificationsminimal sample preparation, was nonintrusive, and allowed in-situ examination of EPS. At high magnificationsusing SEM, the EPS aggregates appeared as web-like structures distributed through the interstices of the proteinusing SEM, the EPS aggregates appeared as web-like structures distributed through the interstices of the proteinusing SEM, the EPS aggregates appeared as web-like structures distributed through the interstices of the proteinusing SEM, the EPS aggregates appeared as web-like structures distributed through the interstices of the proteinusing SEM, the EPS aggregates appeared as web-like structures distributed through the interstices of the proteinmatrix. The web-like structures were apparent, especially in the sample treated with Flavourzyme, which hydrolyzedmatrix. The web-like structures were apparent, especially in the sample treated with Flavourzyme, which hydrolyzedmatrix. The web-like structures were apparent, especially in the sample treated with Flavourzyme, which hydrolyzedmatrix. The web-like structures were apparent, especially in the sample treated with Flavourzyme, which hydrolyzedmatrix. The web-like structures were apparent, especially in the sample treated with Flavourzyme, which hydrolyzedthe milk proteins. The formation of the intricate web-like structures could be attributed to the dehydration of the EPSthe milk proteins. The formation of the intricate web-like structures could be attributed to the dehydration of the EPSthe milk proteins. The formation of the intricate web-like structures could be attributed to the dehydration of the EPSthe milk proteins. The formation of the intricate web-like structures could be attributed to the dehydration of the EPSthe milk proteins. The formation of the intricate web-like structures could be attributed to the dehydration of the EPSduring the critical point drying process of the SEM specimens. The present investigation also found that the 2483during the critical point drying process of the SEM specimens. The present investigation also found that the 2483during the critical point drying process of the SEM specimens. The present investigation also found that the 2483during the critical point drying process of the SEM specimens. The present investigation also found that the 2483during the critical point drying process of the SEM specimens. The present investigation also found that the 2483EPS networEPS networEPS networEPS networEPS network rk rk rk rk remained intact at neutremained intact at neutremained intact at neutremained intact at neutremained intact at neutral or loal or loal or loal or loal or low pH (apprw pH (apprw pH (apprw pH (apprw pH (approooooximately 3.9). Hximately 3.9). Hximately 3.9). Hximately 3.9). Hximately 3.9). Hooooowwwwwevevevevevererererer, the 2483 EPS was highly suscep-, the 2483 EPS was highly suscep-, the 2483 EPS was highly suscep-, the 2483 EPS was highly suscep-, the 2483 EPS was highly suscep-tible under alkaline pH conditions. Increasing the pH from 8 to 10 appeared to destroy EPS structure as indicatedtible under alkaline pH conditions. Increasing the pH from 8 to 10 appeared to destroy EPS structure as indicatedtible under alkaline pH conditions. Increasing the pH from 8 to 10 appeared to destroy EPS structure as indicatedtible under alkaline pH conditions. Increasing the pH from 8 to 10 appeared to destroy EPS structure as indicatedtible under alkaline pH conditions. Increasing the pH from 8 to 10 appeared to destroy EPS structure as indicatedbbbbby a loss of its y a loss of its y a loss of its y a loss of its y a loss of its “““““rrrrropopopopopy” chary” chary” chary” chary” characteracteracteracteracteristics as wistics as wistics as wistics as wistics as well as the EPS levell as the EPS levell as the EPS levell as the EPS levell as the EPS levelselselselsels.....

KKKKKeyworeyworeyworeyworeywords: exds: exds: exds: exds: exopolysaccharopolysaccharopolysaccharopolysaccharopolysaccharideideideideide, lactic acid bacter, lactic acid bacter, lactic acid bacter, lactic acid bacter, lactic acid bacteria, lectin, micria, lectin, micria, lectin, micria, lectin, micria, lectin, microscoposcoposcoposcoposcopyyyyy, alkali tr, alkali tr, alkali tr, alkali tr, alkali treatmenteatmenteatmenteatmenteatment

Introduction

Many bacterial strains secrete extracellular polymeric substanc-es that are viscoelastic in nature. The majority of these extra-

cellular polymers are polysaccharides. In some strains of bacteria,these polysaccharides can occur on the surface of cells. This layeris called “glycocalyx,” “capsule,” or “slime layer,” depending on thedegree of adhesion to the surface of the cell wall. In most cases, thepolysaccharides are secreted into the environment in the form ofslime (de Vuyst and others 2001). When this occurs, the polysaccha-rides are then known as exopolysaccharides (EPS). The productionof EPS is said to protect against adverse conditions such as osmoticstress, desiccation, phagocytosis, toxic compounds, and bacte-riophage infection (Cerning 1995b; de Vuyst and others 2001; Loo-ijesteijn and others 2001). However, others have suggested that thephage protection conferred by EPS may be very limited (Deveauand others 2002) and that the obvious advantage of EPS to growthor survival of bacterial cells remains unclear (Broadbent and others2003). The exact functions of EPS for the bacterial cells have notbeen completely elucidated (Ruas-Madiedo and others 2002). Inthe past decade, exopolysaccharides (EPSs) produced by lactic acidbacteria (LAB) have drawn the attention of food scientists, not onlyfor their physicochemical properties but also because of their Gen-erally Recognized as Safe (GRAS) status (Cerning 1990; Racine and

others 1991; Abbad-Andaloussi and others 1995; Cerning 1995b).Hence, EPS produced by LAB can be regarded as a natural thicken-er. The EPS produced by LAB has been used directly in situ toachieve functional benefits, for example, it is used to reduce syner-esis in yoghurt. The EPS may potentially be isolated, purified, andused as a functional ingredient in various food systems. However,the low production of EPS by most lactic acid bacteria is the mainbottleneck for its use as a functional ingredient. Understanding thedistribution of EPS in the fermented system is useful in the EPS iso-lation and purification regime. Several studies have used electronmicroscopy techniques to examine the EPS in yoghurt products(Schellhaass and Morris 1985; Toba and others 1990; Skriver andothers 1995). However, little research has been carried out usingconfocal laser scanning microscopy (CLSM) to observe the distribu-tion of EPS in milk permeate–based medium and to study the ef-fects of high pH on the EPS structure.

The objective of this study was to examine the location and dis-tribution of 2483 EPS in a milk permeate–based medium usingCLSM. The knowledge of EPS distribution in the fermented milksystem may allow the development of an effective EPS quantifica-tion and isolation regime.

Lectin (carbohydrate-binding proteins) conjugated with a fluo-rescent dye have been reported as an effective stain for carbohy-drate molecules (Yiu 1993; Hassan and others 2002) and was eval-uated in the present study. The images obtained using CLSM wereevaluated together with those obtained through scanning electronmicroscopy (SEM). The effects of pH on the physical structure ofthe 2483 EPS were also examined using both SEM and CLSM.

MS 20040770 Submitted 11/24/04, Revised 1/12/05, Accepted 2/14/05. Au-thor Goh is with Inst. of Food, Nutrition and Human Health, Massey Univ.,Private Bag 11 222, Palmerston North, New Zealand. Authors Haismanand Singh are with Riddet Centre, Massey Univ., Palmerston North, NewZealand. Direct inquiries to author Goh (E-mail: K.T.Goh@massey.ac.nz).

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Examination of EPS by CLSM and SEM . . .

Materials and Methods

Culture media preparation for microscopyCulture media preparation for microscopyCulture media preparation for microscopyCulture media preparation for microscopyCulture media preparation for microscopyThe bacterial strain used for the investigation, Lactobacillus del-

brueckii subsp. bulgaricus NCFB 2483 (NCIMB 702483), was ob-tained from the National Collection of Industrial and Marine Bac-teria (Aberdeen, Scotland) and denoted as 2483. The seed cultureof 2483 was grown in a 10% (w/v) reconstituted skim milk (RSM)and then inoculated (1% v/v) in different growth media. Fermen-tation was carried out at 37 °C for 24 h. The culture media and treat-ments applied were as follows:

Milk permeate (Fonterra Co-operative Group Ltd, Longburn,New Zealand) + 0.5% (w/v) yeast extract (Gibco-BBL). Note that themilk permeate was a by-product of the ultrafiltration of skim milk.

Milk permeate + 0.5% (w/v) yeast extract + 5% (w/v) skim milkpowder (SMP) (Fonterra Co-operative Group Ltd)

Milk permeate + 0.5% (w/v) yeast extract + 5% (w/v) SMP withFlavourzyme (Novozymes Ltd, Bagsvaerd, Denmark) treatment

Milk permeate + 0.5% (w/v) yeast extract + 5% (w/v) SMP with Fla-vourzyme treatment.

Flavourzyme treatment was carried out by adding 100 �L of 10%(w/v) Flavourzyme solution (prepared in Milli-Q water) to a 10 gsample (adjusted to pH 7 using 1 M NaOH; Sigma-Aldrich, St. Lou-is, Mo., U.S.A.) and incubating in a water bath shaker at 50 °C for 4h. Control samples were prepared, which included a milk permeatemedium containing 5% (w/v) SMP plus 0.5% (w/v) yeast extract(without 2483), a 5% (w/v) lactose (Sigma-Aldrich) solution, and a3% (w/v) galactose (Sigma-Aldrich) solution.

Sample preparation for confocalSample preparation for confocalSample preparation for confocalSample preparation for confocalSample preparation for confocallaser scanning microscopylaser scanning microscopylaser scanning microscopylaser scanning microscopylaser scanning microscopy

The fermented sample (50 �L) was gently transferred to a con-cave glass slide. A solution of lectin (SBA) from Glycine max conju-gated with Fluor 488 (Molecular Probes, Eugene, Oreg., U.S.A.) at0.1% (w/v) was prepared, and 50 �L was added to the sample. Acover slip was then placed on the sample and the glass slide was leftfor about 30 min in the dark at approximately 20 °C. The sample wasthen viewed using a CLSM (Leica TCS 4D MMRBE, Leica Lasertech-nik, Heidelberg, Germany) equipped with an argon/krypton lasersource and a 100× oil immersion objective lens to give a 1000× mag-nification. An excitation wavelength of 488 nm was used. In addi-tion to the 2483 strain, 2 “non-ropy” strains of Streptococcus thermo-philus (ST1) and Lactobacillus helveticus (LH 30) were grown in 10%(w/v) skim milk media. The culture media were stained with thesame lectin conjugate and examined using the CLSM.

Sample preparation for scanning electron microscopySample preparation for scanning electron microscopySample preparation for scanning electron microscopySample preparation for scanning electron microscopySample preparation for scanning electron microscopyThe preparation of samples for SEM was adapted from the pro-

cedures described by Schellhaass and Morris (Schellhaass andMorris 1985). A template of cylindrical studs (4-mm dia and 10-mmlength) was made and used to create cavities in the agar for sampleencapsulation. A 3% (w/v) agar solution was poured almost to thebrim of the Petri dish, and the cylindrical studs were immersed inthe agar solution. The template was removed when the agar hadsolidified, leaving behind cylindrical cavities. Approximately 20 �Lof sample was pipetted into each of the cylindrical cavities at roomtemperature. This was subsequently overlaid with the agar andallowed to solidify. After solidification, small cubes encapsulatingthe sample were cut from the agar. The agar cubes were fixed over-night in 3% glutaraldehyde prepared in 0.1 M cacodylate buffer (pH7.2) at 4 °C. They were rinsed 3 times for 5 min each time in dilutedcacodylate buffer (1 part 0.1 M cacodylate to 1 part water). Second-ary fixation was carried out using 1% osmium tetroxide (OsO4) (Proc-

ite Tech., Thuringowa, Gld, Australia) in 0.1 M cacodylate buffer for1 h and followed by 3 rinses of the diluted cacodylate buffer at roomtemperature. The fixed samples were subjected to dehydrationusing a series of increasing concentration of acetone (25%, 40%,60%, 70%, 90%, and 2 changes of 100% for 20 min each). The finaldehydration step involved a Polaron E300 Critical Point Dryer (Bio-Rad/EBS, Agawam, Mass., U.S.A.) with liquid CO2. Each sample wasfractured (to expose the EPS), mounted on a double-sided tapeglued to an aluminum supporter and sputter coated with gold in asputter coater (Bal-Tec SCD 050, Liechtenstein, Switzerland). Thespecimen was then viewed using the 250 MK III Scanning ElectronMicroscope (Stereoscan, Cambridge, U.K.).

Quantification of EPSQuantification of EPSQuantification of EPSQuantification of EPSQuantification of EPSThe fermented culture medium was 1st adjusted to neutral pH

with 1 M NaOH. Approximately 100 �L of 10% Flavourzyme solutionwas added to 10 mL of sample and incubated in a shaker for 4 h at50 °C. After protein hydrolysis, 100 �L of the sample was trans-ferred to a clean centrifuge tube containing 9.9 mL chilled ethanol(70% v/v) and kept at 4 °C overnight. The sample was centrifugedat 27000 × g for 40 min at 4 °C (Sorvall Centrifuge RC5C, SS-34 rotor,Dupont Co., Wilmington, Del., U.S.A.). The supernatant was dis-carded and the pellet was re-dispersed in 3 mL water. Chilled abso-lute ethanol (7 mL) was added to the solution and left overnight at4 °C for a 2nd ethanol precipitation. The sample was centrifugedonce more, and the pellet was dissolved in 1 mL water. Total carbo-hydrate was determined by the phenol sulfuric acid method(Dubois and others 1956). These procedures were also performedwith uninoculated medium. All assays were carried out in triplicate.The EPS value was obtained by subtracting the total carbohydratevalue in uninoculated medium from the amount in the fermentedmedium. Statistical analysis of data was carried out by using theGeneral Linear Model and Turkey multiple comparison tests fromMinitab® Release 14 software.

Effect of pH on EPSEffect of pH on EPSEffect of pH on EPSEffect of pH on EPSEffect of pH on EPSThe fermented culture medium (milk permeate + 0.5% [w/v]

yeast extract + 5% [w/v] SMP) was first treated with Flavourzyme.The enzyme-hydrolyzed medium was then adjusted to pH 8, 9, or10 using 1 M NaOH (Sigma-Aldrich). The samples were examinedusing CLSM and SEM. In addition, quantification of EPS in eachsample was carried out in triplicate.

Results and Discussion

Examination of EPS by CLSMExamination of EPS by CLSMExamination of EPS by CLSMExamination of EPS by CLSMExamination of EPS by CLSMThe use of the CLSM technique in conjunction with Lectin SBA

Alexa Fluor 488 conjugate was found to be effective for imaging thelocation and distribution of 2483 EPS in a culture medium. In all theCLSM micrographs, only the EPS was stained (Figure 1a and 1b).As expected, the control samples (without bacterial culture), name-ly milk permeate containing 5% (w/v) SMP and 0.5% (w/v) yeastextract; 5% (w/v) lactose; and 3% (w/v) galactose, showed no EPS.Furthermore, mannans, known to be present in yeast extract (Cern-ing 1995a), were not stained in the control sample. Note that thelectin conjugates dispersed in water showed negligible backgroundfluorescent intensities. From the control samples used in this study,the lectin conjugates did not stain proteins, lactose, mannans, orbacterial cells present in the media. In the fermented systems, theEPS was found to be randomly distributed, and large aggregatedstructures were apparent (Figure 1a and 1b). In the present study,much more EPS aggregates could be seen in the medium containingSMP (Figure 1b) than the culture medium without SMP (Figure 1a).

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Examination of EPS by CLSM and SEM . . .

An additional experiment was carried out on 2 non-ropy strains ofLAB (S. thermophilus ST1 and L. helveticus LH 30 obtained fromFonterra Co-operative Group Ltd) grown in a similar milk medium.The EPS aggregates in the non-ropy fermented broths were presentin lesser quantity than those observed in the ropy 2483 fermentedmedium. This observation was consistent with the levels of EPS pro-duced (approximately 90 to 110 mg/L for the non-ropy cultures asopposed to about 400 mg/L for the ropy culture). It was also notedthat in all cases, the bacterial cells were not visible, indicating thatthe EPS was not accumulated on the bacterial cell surface as a slimelayer. This was confirmed by the Maneval staining method (Corstvetand others 1982) performed using a light microscope, which showedthat no capsular polysaccharide was present for the 2483 culture.

One of the unique advantages of using CLSM is its ability to opti-cally section specimens (Blonk and Van Aalst 1993). As such, minimalsample preparation is required and the specimen does not involvefixing, dehydrating, embedding, and sectioning steps. This not onlyreduces the time spent, but more importantly, physiological process-es and physical structures can be examined undisturbed. In addi-tion, artifacts caused by the harsh chemicals used in sample prepa-ration for electron microscopy are absent (Vodovotz and others1996). In the present study, the selection criteria of a suitable lectin(conjugated to a fluorescent dye, lectin SBA Alexa Fluor 488) wasbased on the affinity for �-galactopyranosyl residues reported to bepresent in many EPS structures of lactic acid bacteria (Gruter andothers 1993). The composition consisted of the EPS by the 2483strain consisted of galactose, glucose, rhamnose, and mannose in theratio of approximately 5:1:0.6:0.5, with traces of glucosamine (Gohand others 2005). Commercially, several lectins with affinity for dif-ferent sugar residues are available from various sources. Some exam-ples of lectins include Concanavaline A with specificity for terminal�-D-mannosyl and �-D-glucosyl residues, Anguilla anguilla withspecificity for �-L-fucosyl residues, and Bandeiraea simplicifolia withspecificity for terminal �-D-galactosyl and N-acetyl-�-D-galac-tosaminyl residues (Kennedy and others 1995).

Examination of EPS by SEMExamination of EPS by SEMExamination of EPS by SEMExamination of EPS by SEMExamination of EPS by SEMThe micrographs of the 2483 culture obtained using SEM were

comparable to typical ropy cultures observed by other researchers(Schellhaass and Morris 1985; Toba and others 1990; Skriver andothers 1995). In the milk permeate medium, bacterial cells could beseen entrapped in a web-like matrix (Figure 2a). The web-like ma-trix was much thicker in the culture medium containing SMP andobscured the bacterial cells (Figure 2b). Among the matrix wereaggregates that appeared to be present in larger quantities in themedium containing SMP. When the medium containing SMP wastreated with Flavourzyme to hydrolyze the milk proteins, the ag-gregates were mostly reduced but the web-like matrix remained(Figure 2c). This suggests that the aggregates were most likely to beproteins, whereas the web-like matrix was likely to be the EPS.

The images seen in the SEM micrographs may be explained bythe illustration given in Figure 3. When the pH value decreases toabout 3.9 after 24 h of fermentation at 37 °C, casein micelles are de-stabilized and form a gel with a series of channels and poresthrough which the EPS in the continuous phase can flow (Figure3a). In the culture medium, the bacterial cells are entrapped withinand around the protein matrix. The bacterial cells are believed to bepartially responsible for the void spaces in the protein matrix (Kalaband others 1983; Toba and others 1990). The EPS produced by thebacteria was secreted into the continuous phase and filled the in-terstices and channels of the protein matrix and reinforced the pro-tein matrix structure possibly through EPS-protein interactions.The presence of EPS increased the viscosity of the continuous

This was consistent with the different levels of EPS determined bychemical analysis (about 150 mg/L as opposed to about 400 mg/L).These levels are within the typical range of EPS (110 to 1400 mg/L)produced by other LAB strains (Bouzar and others 1996; Mozzi andothers 1996; Degeest and others 2001). The different area and in-tensity of brightness (fluorescence) observed in the micrograph(Figure 1b) was due to different EPS concentrations and probablydue to partial obscuration of the fluorescence by the coagulatedprotein matrix. When the protein matrix was removed by enzymatichydrolysis, the EPS appeared to form larger aggregates (Figure 1c).The exact mechanism for the formation of EPS aggregates is still un-clear. A likely reason could be the poor solvent quality of the hydro-lyzed culture medium (approaching that of a theta solvent) (Flory1953) for the EPS molecules. Under such conditions, there is astronger attraction of macromolecule segments to each other thanwith the solvent molecules, forming the aggregated EPS structures.

Figure 1—Micrographs from confocal laser scanning mi-croscopy (CLSM). (a) 2483 culture grown in a milk perme-ate medium containing 0.5% (w/v) yeast extract; (b) 2483culture grown in a milk permeate containing 0.5% (w/v)yeast extract and 5% (w/v) skim milk powder; (c) 2483culture with Flavourzyme treatment at pH 7.

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Examination of EPS by CLSM and SEM . . .

phase, which explains why yoghurt made with a ropy culture usu-ally reduces syneresis. Figure 3b shows the distribution of EPS with-in the protein matrix. The bacterial cells have been deliberatelyexcluded to simplify the illustration. The web-like appearance isprobably due to dehydration of EPS resulting from sample prepa-ration techniques (Kalab 1993). The proteins appear as depositsand aggregates among the web-like structures (Figure 3c). In theprotein hydrolyzed samples, the SEM micrographs reveal a moredistinct EPS web-like structure, which remains intact after the Fla-vourzyme treatment (Figure 3d).

Effect of pH on the EPS structureEffect of pH on the EPS structureEffect of pH on the EPS structureEffect of pH on the EPS structureEffect of pH on the EPS structureTreatment under alkali conditions have been applied to isolate

polysaccharides (Nielsen and Jahn 1999), to remove EPS from thesurface of LAB cells (Forde and Fitzgerald 1999), or to remove acetylgroups from some polysaccharides (Sutherland 1997). The effects ofincreasing pH on the EPS structure were examined using the CLSMand SEM. The culture medium used was a milk permeate–based

medium containing 0.5% (w/v) yeast extract and 5% (w/v) SMP. In-creasing the pH of culture medium from 3.9 to 10 with 1 M NaOH ledto a progressive reduction of EPS clusters as observed by CLSM (Fig-ure 4a to 4c). At pH 10, no EPS could be seen (Figure 4c).

Under the condition of increasing pH, it was noted that the vis-cosity and ropy characteristics were reduced markedly. The reduc-tion in viscosity was not only due to the probable modification ofthe EPS structure but also to the dissociation of the casein aggre-gates. At pH 10, the extent of protein aggregation was markedlyreduced as seen in the micrographs obtained using SEM (Figure 5aand 5b). The original ropy characteristic of the culture mediumcould hardly be detected. The EPS web-like structure was alsolargely destroyed as seen in the Flavourzyme treated sample at pH10 (Figure 5c). This suggests that the native EPS chemical structureis modified and disrupted under the alkaline pH. However, at thispH, the bacterial cells remain intact (Figure 5b). Analysis of the EPSlevels showed a drastic decrease at pH 8 and above (Figure 6). Thissuggests that the native EPS structures are modified under the al-

Figure 2—Micrographs from scanning electron microscopy(SEM). (a) 2483 culture grown in a milk permeate mediumcontaining 0.5% (w/v) yeast extract; (b) 2483 culture in amilk permeate containing 0.5% (w/v) yeast extract and 5%(w/v) skim milk powder; (c) 2483 culture with Flavourzymetreatment at pH 7. Arrows indicate protein aggregates.

Figure 3—Pictorial representation of the fermented milk withthe presence of exopolysaccharide (EPS). (a) Protein aggre-gates forming a matrix under acidified pH in the fermentedculture medium; (b) EPS filled the interstices of the proteinmatrix; (c) EPS network formed within the protein matrixafter scanning electron microscopy (SEM) processing; (d)EPS network after Flavourzyme treatment under SEM.

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Examination of EPS by CLSM and SEM . . .

kaline pH. The detrimental effect of alkali treatment on polysaccha-ride produced by Pseudomonas aeruginosa has been reported pre-viously (Pier and others 1978) but none on EPS from LAB.

Further studies of polysaccharidesFurther studies of polysaccharidesFurther studies of polysaccharidesFurther studies of polysaccharidesFurther studies of polysaccharidesusing lectin conjugatesusing lectin conjugatesusing lectin conjugatesusing lectin conjugatesusing lectin conjugates

In some polysaccharides such as gellan, pectin, gum arabic (Bairdand Smith 1989), and EPS produced by different strains of LAB (vanden Berg and others 1995; van Casteren and others 1998; Urashimaand others 1999; Ricciardi and others 2002), rhamnose is a commonsugar found among the repeat units of the polymer molecules. Be-cause rhamnose is not a common sugar used in growth media, itspresence can be used as an effective detection site for most EPS. Theuse of rhamnose as a detection site is effective because it is easier todetermine the composition of polysaccharides (for example, by highperformance liquid chromatography) than to unravel the terminalends of the sugar residues (for example, by methylation analysis) of

Figure 4—Micrographs from confocal laser scanning mi-croscopy (CLSM) with 2483 in a milk permeate containing0.5% (w/v) yeast extract and 5% (w/v) skim milk powder(SMP). The culture medium was treated with Flavourzyme(50 °C, 4 h) and adjusted to respective pH using 1 M NaOH.(a) pH 8; (b) pH 9, and (c) pH 10.

Figure 6—The levels of exopolysaccharide (EPS) inFlavourzyme treated culture media containing 0.5% (w/v)yeast extract and 5% skim milk powder in milk permeateat pH 3.9, 5, 6, 7, 8, 9, and 10. The pH was adjusted using0.5 M NaOH solution. Determination of EPS was carriedout in triplicate. Error bar indicates mean ± SD. Differentletters represents EPS levels that were significantly differ-ent at P � 0.05.

Figure 5—Micrographs from scanning electron microscopy(SEM) with 2483 in a milk permeate containing 0.5% (w/v)yeast extract and 5% (w/v) skim milk powder (SMP). (a andb) culture medium adjusted to pH 10 using 1 M NaOH. (c)culture medium treated with Flavourzyme (50 °C, 4 h) andadjusted to pH 10 using 1 M NaOH.

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different EPS molecular structures. The use of a lectin specific onlyto rhamnose may improve the accuracy for targeting EPS. Althoughlectins specific to rhamnose have been isolated from the eggs of cat-fish (Hosono and others 1999), they are not commercially available.An area that is worth further investigation is to relate the intensity offluorescence to the EPS level. This may require an additional step tohydrolyze the proteins to minimize obscuration of fluorescence. Inaddition, apart from fluorochrome, lectin conjugated with other com-pounds such as electron-dense gold and enzyme-linked peroxidasecan be used as labels for electron microscopy and colorimetric meth-ods for detecting EPS respectively. These types of lectin conjugatesare already available commercially but have not yet been exploredfor this purpose. However, if no lectins or stains are specific for anEPS, the fluorescent-antibody technique (Corstvet and others 1982)for making highly specific labels could be considered.

Conclusions

The use of CLSM, together with fluorescent lectin conjugates,was proven effective for imaging EPS in milk permeate–based

media. The CLSM micrographs reveal that EPS occur as aggregatesin the culture medium. Unlike SEM, the sample preparation proce-dures for CLSM did not require rigorous chemical processing stepsthat could introduce artifacts and complicate image interpretation.The success of CLSM relied primarily on the selection of an appro-priate fluorescent stain, SBA lectin Alexa Fluor conjugate. This stainhad no effect on proteins, lactose, mannans or bacterial cellspresent in the media. Instead, the fluorescent lectin conjugateshad strong affinity for the EPS due to the presence of galactopyra-nosyl residues in the EPS chemical structure. The SEM micrographshowed EPS as a web-like structure distributed through the inter-stices of the protein matrix. The present study provided greater un-derstanding of the distribution of EPS in the fermented milk systemand may be useful in the development of an effective EPS isola-tion regime as well as providing a better understanding of the rheo-logical properties of a ropy ferment.

From this study, it was also found that at neutral and low pH(about pH 3.9), the EPS aggregates remained intact. However, asthe pH was increased (pH 8 to 10), the EPS aggregates appeared tobe disrupted. Furthermore, the observed ropy characteristics of theculture medium were much reduced. Caution will need to be exer-cised if alkali is to be used for the isolation of the 2483 EPS. Theeffect of alkali treatment on EPS structure would require furtherstudies because polysaccharides are generally known to be stableunder mild alkaline conditions.

AcknowledgmentsThis study was funded by Fonterra Co-operative Ltd and the NewZealand Foundation for Research, Science and Technology throughthe Technology for Business Growth program. The authors would liketo acknowledge the help given by Elizabeth Nickless for using theCLSM, and Dough Hopcroft and Raymond Bennett for processing andviewing the samples for SEM. The authors would like to thank Profes-sor Richard Archer and Professor Ian Maddox for helpful discussions.

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