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International Dairy Journal 34 (2014) 125e134

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairyj

Isolation and characterisation of exopolysaccharide-producingWeissella and Lactobacillus and their application as adjunct culturesin Cheddar cheese

Kieran M. Lynch a, Paul L.H. McSweeney b, Elke K. Arendt b, Thérèse Uniacke-Lowe b,Sandra Galle b, Aidan Coffey a,*

aDepartment of Biological Sciences, Cork Institute of Technology, Rossa Avenue, Bishopstown, Cork, Irelandb School of Food and Nutritional Sciences, University College Cork, Ireland

a r t i c l e i n f o

Article history:Received 20 November 2012Received in revised form14 June 2013Accepted 17 July 2013

* Corresponding author. Tel.: þ353 21 433 5486.E-mail address: aidan.coffey@cit.ie (A. Coffey).

0958-6946/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.idairyj.2013.07.013

a b s t r a c t

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potentialfor use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures inCheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 andLactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 105 to107 Da. Both strains were used in the production of miniature Cheddar cheeses that employed a con-ventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that usedpurified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient)had higher moisture levels compared with the control cheese made using starter alone. Inclusion of theadjunct strains had no detectable negative effects on cheeses in terms of proteolysis.

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1. Introduction

In cheese production, adjunct cultures are defined as “selectedstrains of cheese-related microorganisms that are added to thecheese milk to improve development of cheese sensory quality” (ElSoda, Madkor, & Tong, 2000). Generally, they are non-starter lacticacid bacteria (NSLAB) strains usually isolated from cheese, chosenfor specific properties or quality enhancements that they confer onthe manufactured cheese (Settanni & Moschetti, 2010). Morerecently, non-dairy strains have been investigated also (Di Cagno,Quinto, Corsetti, Minervini, & Gobbetti, 2006b).

NSLAB and adjuncts primarily affect cheese maturation andresultant quality and flavour through their proteolytic activities. Incombinationwith starter proteinases, the action of NSLAB peptidasesresults in the formation of free amino acids (FAAs) that are the pre-cursors of many flavour and aroma compounds, with the combinedbreakdown of large “bitter” peptides (Sousa, Ardo, & McSweeney,2001). Milesi, McSweeney, and Hynes (2008a) found that a Lactoba-cillus plantarum adjunct increased the levels of secondary proteolysis

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(i.e., FAAs), which could impact on cheese quality. In addition, the useof adjunct strains isolated from non-dairy ecosystems could providenovel enzymatic activities in cheese, but their effects are strain-dependant (Di Cagno et al., 2006b).

NSLAB and adjunct strains are now also being chosen for reasonsbeyond adding to the sensorial aspects of cheese. Bacteriocin-producing Lactobacillus adjuncts have also been used for the bio-preservation of cheeses against Listeria (Mills et al., 2011). The useof strains mediating probiotic properties also give additional healthattributes to the cheese which is acting as a probiotic deliveryvehicle (Gardiner, Ross, Collins, Fitzgerald, & Stanton,1998; Settanni& Moschetti, 2010). Such applications have the potential to increasethe value of cheese.

Exopolysaccharides (EPS) produced by members of the cheesemicrobiota or by selectively added adjunct strains have been in-creasingly investigated in recent years for both their technologicaleffects in cheese and for their potential health benefits. The currentdefinition of lactic acid bacteria (LAB) EPS defines them as being“located in the extracellular medium without covalent bonds with[the] bacterial membrane” (Badel, Bernardi, & Michaud, 2011). EPSare divided into two categories: heteropolysaccharides and homo-polysaccharides. Heteropolysaccharides are composed of more thanone sugar monomer type and are synthesised within the cell from

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134126

sugar nucleotides and then excreted. Homopolysaccharides arecomposed of a single sugarmonomer (either D-glucose or D-fructose)and are synthesised in the extracellular environment from sucrose byan enzyme (glycansucrase) secreted by the bacterium (Badel et al.,2011; De Vuyst & Degeest,1999;Monsan et al., 2001;Welman, 2003).

EPS are technologically important in the dairy fermentation in-dustry for their rheological and textural effects on products. Forexample, they have been used in reduced-fat and half-fat cheeses toimprove the textural and rheological properties of these cheeses.Removal of fat results in a more dense protein network, thusaffecting the cheese texture (Mozzi et al., 2006). Studies have shownthat the use of EPS-producing starters in these circumstances resultsin higher moisture levels, with the microstructure of reduced-fatcheese made with EPS-producing starter being similar to full-fatcheeses, as evidenced by electron microscopy (Hassan & Awad,2005). Costa et al. (2010) manufactured Cheddar cheese with anEPS-producing Lactococcus lactis starter and its isogenic, non-EPS-producing variant. The presence of EPS significantly increased themoisture levels; this was postulated to be the result of trapping ofwater by the EPS through hydrogen bonding.

Another important aspect of LAB EPS is their potential healthbenefits to the consumer. Several health-promoting activities ofLAB EPS have been studied and these include immunomodulatoryactivity (Wu et al., 2010), antioxidant potential (Liu et al., 2011),antimicrobial activity (Wang, Ganzle, & Schwab, 2010) and prebi-otic potential (Salazar, Gueimonde, Hernandez-Barranco, Ruas-Madiedo, & de los Reyes-Gavilan, 2008).

The objective of this study was to assess the suitability of anumber non-dairy, homopolysaccharide-producing LAB for use asadjuncts in cheese. The type of EPS produced by the strains in thisstudy are glucans consisting of a-(1,6) linkages, i.e., dextrans.Dextrans have previously been shown to have prebiotic potential(Olano-Martin, Mountzouris, Gibson, & Rastall, 2000) and havebeen approved by the European Commission for use as an additivein bakery products (Anonymous, 2000). A miniature Cheddar typecheese model was used and the effects of the adjuncts on thecomposition, biochemical properties and proteolysis in cheesewere assessed.

2. Materials and methods

2.1. Bacterial strains and growth media

A Weissella strain and three Lactobacillus strains (two Lactoba-cillus reuteri strains and one Lactobacillus casei strain) were selectedas potential adjuncts for this study from a bank of EPS-producingLAB (Table 1). The starter culture used was L. lactis R604 DVS(direct vat set; Chr. Hansen, Cork, Ireland). Strains were stocked in40% glycerol and stored at �80 �C. Prior to experiments, Weissellaand Lactobacillus strains were cultured on MRS agar (Lab M Ltd.,Lancashire, UK) and incubated anaerobically overnight at 30 �C and

Table 1List of strains examined as potential adjuncts, their rRNA typing matches and properties

Species Originalsource

EPS (linkage)

Weissella cibaria MG1 (98% identity with W. cibariaLMG 17699T 16S rRNA gene sequence)

Sourdough Glucan homoglucooligosac

Lactobacillus casei C12 (98% identity with L. caseiATCC393 16S rRNA gene sequence)

Cheese Heteropolysa

Lactobacillus reuteri ff2hh2 (97% identity with L. reuteriJCM1112 16S rRNA gene sequence)

Porcine gut Glucan homofructooligosa

Lactobacillus reuteri cc2 (97% identity with L. reuteriJCM1112 16S rRNA gene sequence)

Porcine gut Glucan homoand fructooli

a Typical yield in EPS-broth (g L�1).

37 �C, respectively. Anaerobic conditions were generated by usingan anaerobic jar and an AnaeroGen AN0025A gas pack (OxoidLimited, Hampshire, UK). Strains were then inoculated at 1% (v/v)into MRS broth (Sigma, St Louis, MO, USA) and grown overnight.The starter was cultured on M17 agar (Lab M Ltd.) and incubatedanaerobically overnight at 30 �C followed by overnight cultivationin M17 broth (Sigma). Two of the four potential strains wereselected as adjuncts for inclusion in cheese-making trials based ontheir ability to grow in milk (10% reconstituted skim milk, RSM;Becton Dickinson, Franklin Lakes, NJ, USA) and the novel charac-teristics of the EPS produced.

2.2. Characterisation of strains by 16S typing

DNA was extracted from overnight bacterial cultures (1%, v/v,inoculum) using a High Pure PCR Template Preparation Kit (Roche,West Sussex, UK). The universal primers described by Weisburg,Barns, Pelletier, and Lane (1991), namely fD1 (AGA GTT TGA TCCTGG CTC AG) and rP2 (ACG GCT ACC TTG TTA CGA CTT) were used toamplify the 16S rRNA gene sequence. PCRwas performed on a T3000thermocycler (Biometra, Goettingen, Germany). The reactionmixture (50 mL) consisted of BioMix Red (MyBio, Kilkenny, Ireland;25 mL), 10 pmol each primer (1 mL each), sterile water (21 mL) andbacterial DNA (2 mL). The amplification program consisted of aninitial “hot start” of 94 �C for 4min, followed by 30 cycles of 94 �C for30 s, 61 �C for 30 s, 72 �C for 1 min, and final elongation at 72 �C for7 min. Amplified target sequences were purified with a High PurePCR Product Purification Kit (Roche), and sequenced (Eurofins MWGOperon, Ebersberg, Germany) prior to analysis using the BLASTdatabase.

2.3. EPS screening, isolation and purification

The synthesis of EPS was determined by growing the strains onMRS agar supplement with various concentrations of substrate car-bohydrates. These were one of 10% (w/v) sucrose, 8% (w/v) sucrose,2.9% (w/v) maltose, 2.9% (w/v) raffinose, 2.5% (w/v) glucose or acombination of carbohydrates. After incubation at 30 �C or 37 �C for 5days, the synthesis of EPS was observed by visual appearance ofmucoid colonies. EPS was isolated and purified by the method of DiCagno et al. (2006a) with modifications. Briefly, cells were incubatedfor 48 h in “EPS-broth” consisting of (in g L�1): tryptone, 10; yeastextract, 5; meat extract, 5; dipotassium hydrogen phosphate trihy-drate, 2.6; potassium di-hydrogen phosphate, 4; cysteine hydro-chloride, 0.5; ammonium chloride, 3; sucrose, 100 (or other substratecarbohydrates, as appropriate); Tween 80, 1 mL; magnesium sul-phate, 0.1; manganese sulphate, 0.05; and vitamin mix, 1 mL (allreagents from Sigma). The vitamin mix consisted (0.2 g L�1 each) ofcobalamin, folic acid, nicotinic acid amide, panthothenic acid, pyri-doxal phosphate and thiamine (all from Sigma). Cells were removedby centrifugation at 11,700� g for 20 min at 4 �C and the EPS was

of produced exopolysaccharides (EPS).

and oligosaccharide produced EPS molecularmass (Da)

Typical EPS yielda inEPS-broth (g L�1)

polysaccharide (a-1,6) andcharide

5 � 106e4 � 107 36

ccharide 5 � 103e105 0.7

polysaccharide (a-1,6 and a-1,4) andccharide

105e4 � 107 3.6

polysaccharide (a-1,6 and a-1,4)gosaccharide

105 2

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134 127

precipitated with 3 volumes of chilled 96% (v/v) ethanol. Afterstanding overnight at 4 �C, the resultant precipitate was collected bycentrifugation at 20,900� g for 20min at 4 �C. The EPS was dissolvedin distilledwater, dialysed against distilledwater at 4 �C for 2e3 days,and lyophilised. The lyophilised EPS was weighed and the yieldcalculated per litre of culture.

2.4. Characterisation of EPS monosaccharide composition andstructure

The composition and structure of the EPSs produced by Weis-sella cibaria MG1, L. reuteri ff2hh2, L. reuteri cc2 and L. casei C12were determined as described by Schwab, Mastrangelo, Corsetti,and Gänzle (2008). Oligosaccharides were analysed as describedby Galle, Schwab, Arendt, and Gänzle (2011).

2.5. Growth of potential adjunct strains in milk

Ten percent RSM was sterilised by autoclaving at 110 �C for10 min. Fresh cultures from �80 �C stocks were cultivated in MRSprior to washing in phosphate buffered saline (PBS). The PBS cellsuspension was inoculated into fresh 10% RSM to a final concen-tration of 105 cfu mL�1. Milks were incubated under conditionsoutlined previously for the growth of individual strains, for 18 h.Plate counts were performed in triplicate at 2 h intervals. Growth ofthe strains in MRS broth was examined in parallel.

2.6. Miniature Cheddar cheese manufacture

Raw milk was obtained from a local dairy farm and pasteurisedat 63 �C for 30 min. Miniature Cheddar cheeses were manufacturedunder aseptic conditions from 200 mL batch-pasteurised milk usingthe procedure of Milesi, McSweeney, and Hynes (2008b). Sixminiature (20 g) Cheddar cheeses were prepared as outlined inTable 2, with six replicates of each. The six replicates for a particularcheese treatment weremade on six different days, with six differenttreatments being made in any one particular cheese-making day.Three different batches of milk were used over the course of thetrial. Manufacture was performed under a laminar air-flow hoodusing sterile utensils on each cheese-making day. Cheeses weresalted by immersion in sterile brine (20%, w/v, sodium chloride,0.05%, w/v, calcium chloride, 30 min at room temperature) and thenwiped dry, vacuum packed and ripened at 8 �C for 90 days.

Substrate carbohydrates were added to cheeseemilk at a con-centration of 5% (v/v) and were prepared as 40% (w/v) solutions insterile deionised water and filter-sterilised through a 0.45 mm filter.Purified EPS was added at a level of 0.4 g to 200 mL cheeseemilk.Fresh adjunct cultures were grown for 18 h, washed once in PBS andadded to achieve a final concentration of 105 cfu mL�1 in the

Table 2Cheeses manufactured in this study.a

Cheese Component

A Control (no adjunct or EPS ingredient)B W. cibaria MG1C W. cibaria MG1 þ 5% (v/v)b sucroseD L. reuteri cc2E L. reuteri cc2 þ 4% (v/v) sucrose þ 1% (v/v) glucoseF EPS ingredient, 0.2% (w/v) in cheeseemilk

a All cheeses contained L. lactis R604 starter culture. Adjuncts were added at afinal titre of 105 cfu mL�1.

b A total of 5% (v/v) substrate carbohydrates was used in contrast to 10% (w/v)used during initial EPS screening to minimise the levels of carbohydrates added tothe cheeses.

cheeseemilk. Starter culturewas added to cheeseemilk at a level of0.03% (w/v) in DVS form.

2.7. Differentiation of bacterial species isolated from cheese byspecies-specific PCR

During the microbiological analysis at 7, 14, 30, 60 and 90 days,10 colonies were randomly picked from a countable MRS enumer-ationplate and stocked in a cryopreservative both composed of a 1:1mixture of MRS broth and 80% glycerol. Isolates were storedat�20 �C until DNA extraction. Genomic DNAwas extracted using aphenolechloroform method of Coakley and Ross (1996) withmodifications. A ribolyser (MagNA Lyser, Roche) was used for 45 s atspeed setting 4500 to lyse the cells.

Genus/species-specific PCR was carried out using genomic DNAextracted from the isolated bacterial colonies. For the Weissellaadjunct, genus-specific primersWeissgrp (50-GAT GGT TCT GCTACCACT AAG-30) and reverse primer (50-GGN TAC CTT GTT ACG ACT TC-30) were used (Schillinger et al., 2008). For the Lactobacillus adjunct,species-specific primers Lreu-1 (50-CAG ACA ATC TTT GAT TGT TTAG-30) and Lreu-4 (50-GCT TGT TGG TTT GGG CTC TTC-30) were used(Agnes, Lettner, Krammer, Mayer, & Kneifel, 2005). The PCRmixturewas composed of 25 mL Bioline BioMix Red, the primers (0.5 mMeach) and 1 mL DNA (100 ng). ForWeissella the PCR conditions were:an initial denaturation step at 94 �C for 2 min followed by 33 cyclesof denaturation at 94 �C for 1 min, annealing at 55 �C for 1 min,elongation at 72 �C for 90 s, followed by a final extension at 72 �Cfor 7 min. For Lactobacillus the PCR conditions were: an initialdenaturation step of 95 �C for 5 min followed by 35 cycles ofdenaturation at 95 �C for 1 min, annealing at 60 �C for 1 min,elongation at 72 �C for 1 min, and final extension at 72 �C for 7 min.

2.8. Microbiological analysis of cheeses

Cheese samples (2 g) were homogenised in 18 mL of 2% (w/v)tri-sodium citrate solution for microbiological analysis and serialdilutions were prepared in sterile quarter-strength Ringer’s solu-tion. Counts were performed in duplicate on all trial cheeses after 7,14, 30, 60 and 90 days of ripening. Starter bacterial titres weredetermined using M17 agar after incubation at 30 �C for 3 days. Theadjunct cultures and NSLAB, in experimental and control cheesesrespectively, were determined on MRS agar (Lab M Ltd.), with pHadjusted to 5.5 (Briggiler-Marcó et al., 2007) (MRS-5.5) to inhibitstarter growth, after incubation at either 30 �C (Weissella) or 37 �C(Lactobacillus) for 5 days under anaerobic conditions.

2.9. Gross composition of cheeses

The pH values were measured as a single measurement of eachtrial cheese at 7, 14, 30, 60 and 90 days by insertion of a combina-tion electrode connected to a pH meter (Orion, Boston, MA, USA)into cheese slurry 1:10. Samples of 30-day-old cheeses were ana-lysed in duplicate for moisture by oven drying at 103 � 1 �C (IDF,1982) and salt (Fox,1963). Nitrogen content was determined on 90-day-old cheeses by themacro-Kjeldhal method (IDF,1993) as singlemeasurement on each trial cheese.

2.10. Assessment of proteolysis in cheeses

Peptide profiles of pH 4.6-soluble fractions (Kuchroo & Fox, 1982)from 90-day-old cheeses were obtained by reverse-phase high per-formance liquid chromatography (RP-HPLC) using a system whichconsisted of a Waters Acquity Ultra-Performance Liquid Chroma-tography (UPLC) H-Class Core System with an Acquity UPLC TUVDetector (dual wavelength) and Acquity Column Heater 30-A. The

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134128

system was interfaced with Empower 3 software (WatersCorp.,Milford, MA., USA). The core system included an Acquity UPLC H-Class quaternary solvent manager, an H-Class Sample Manager-FTNand a CH-A column heater. The column used was an Acquity UPLCBEH C18 1.7 mm, 2.1 � 50 mm column. Elution was monitored at214 nm and a mobile phase of two solvents, A, 0.1% (v/v) trifluoro-acetic acid (TFA, sequential grade, Sigma, USA) in deionised HPLCgrade water (Milli-Q system, WatersCorp., USA) and B, 0.1% (v/v) TFAin HPLC grade acetonitrile (Lab-scan Ltd., Dublin, Ireland) was used.

Freeze-dried soluble fractions of the pH 4.6-soluble extractsfrom cheese were dissolved in solvent A (10 mg mL�1). Thesamples were centrifuged at 15,000� g for 10min in an Eppendorfcentrifuge. The samples were then filtered through a 0.45 mmcellulose acetate filter (Sartorius GmbH, Gottingen, Germany) and6.9 mL of the filtrate was injected on the column at an eluent flowrate of 0.46 mL min�1. Separation was achieved using thefollowing gradient: 100% A for 0.37 min, 50% B (v/v) for 6.91 min,95% B (v/v) for 1.14 min. The column was washed with 100% B (v/v) for 0.45 min, followed by equilibrationwith 100% A for 1.13 minbefore the next injection. Data acquisition was from 0 to 7.28 min.Time between injections was 1 min at 100% solvent A. Somesamples were run twice to measure the reproducibility of thepatterns in the above conditions.

Casein degradation was assessed by electrophoresis in poly-acrylamide gels (urea-PAGE) (12.5% T, 4% C, pH 8.9) on 90-day-oldcheeses using a Protean II vertical slab-gel unit (Bio-Rad Labora-tories Ltd., Herts, UK) according to the method of Andrews (1983)with modifications. Gels were stained directly with CoomassieBrilliant Blue G250 and destained in distilled water until thebackground was clear, as described by Blakesley and Boezi (1977).

Proteolysis was also quantified on duplicate samples of eachtrail cheese by the trinitrobenzene-sulphonic acid (TNBS) method(Adler-Nissen, 1979). A calibration curve was prepared usingleucine (Sigma) as standard (range 0.0e1.0 mmol L�1 Leu), and theresults were expressed as mg Leu per cheese.

2.11. Monosaccharide analysis of cheeses

A0.2 g quantity of cheesewasused. Thiswashomogenised in1mLof 2M sulphuric acid (H2SO4) and incubating for 2h at 80 �C for proteinprecipitation and carbohydrate hydrolysis. Cheese particles wereremoved by centrifugation (10 min, 9000 � g) and supernatant wasdiluted 1:5 in distilledwater for further analysis. Glucose and fructoseconcentrations were determined by HPLC (Agilent 1200 series) usingan Aminex 87H column (300 mm � 7.8 mm, Bio-Rad, Mississauga,Canada) coupled to a refractive index detector (Agilent 1200). Themonosaccharides were eluted from the column with 5 mmol L�1

H2SO4 at a flow rate of 4 mL min�1 and a temperature of 70 �C.

2.12. Statistical analyses

Statistical analysis of compositional data was carried out usingMinitab version 16 (Minitab Inc., State College, PA, USA). Data for pHand TNBS were compared between samples at day 90 of ripeningand data for salt and moisture contents were compared betweensamples at day 30 were compared at day 30. Data for all parametersmeasured were examined for normality using the AndersoneDarling normality test at a significance level of 0.1, i.e., when calcu-lated p values were found to be <0.1, it was assumed that the datawere not normally distributed. For normally distributed data (pHand moisture values), one-way analysis of variance (ANOVA) wascarried out using a significance level of 0.05. For sample data thatwere not normally distributed (salt and TNBS values), the KruskaleWallis non-parametric test was used to make inferences about theequality of medians between the samples. If the p values from the

KruskaleWallis testwere found to be greater than a pre-determinedvalue of 0.05, it was concluded that none of the treatment effectswere significant and treatment medians were equal.

3. Results and discussion

3.1. Characterisation of Lactobacillus and Weissella strains andtheir exopolysaccharides

The strains used in this study (Table 1) were primarily chosenbased on their EPS-producing ability and the type and amounts ofEPS produced. W. cibaria MG1 was isolated from sourdough andproduces large amounts (up to 36 g L�1) of high molecular massglucan with a-1,6 linkages (i.e., dextran) in the presence of 10%sucrose (Fig. 1a). Glucooligosaccharides (GOS) were formed inaddition to EPS when the acceptor maltose was present (8% sucroseplus 2.9% maltose). GOS formationwas indicated by the presence ofpanose or higher oligosaccharides of the glucosylated panose series(Schwab et al., 2008). Strain MG1 synthesised small amounts ofpanose, but mostly glucosylated panose with a degree of poly-merisation of up to 14 (Fig. 1b).

TheWeissella genus arose from the reclassification of Leuconostocparamesenteroides and some related ‘atypical’ heterofermentativelactobacilli (Collins, Samelisl, Metaxopoulos, & Wallbanks, 1993).W. cibaria was described in 2002 and is closely related to Weissellaconfusa (Björkroth et al., 2002). Weissella species have variablehabitats but are mainly associated with fermented foods (Huys,Leisner, & Björkroth, 2012). W. cibaria and W. confusa are particu-larly associated with, and are members of the natural microbiota ofsourdoughs (Galle, Schwab, Arendt, & Gänzle, 2010), but have alsobeen found in human faeces and the human gut, respectively (Huyset al., 2012).

Both L. reuteri strains cc2 and ff2hh2 were isolated from theporcine gut and produce glucan (dextran) with a-1,6 and a-1,4linkages in the presence of 8% (w/v) sucrose plus 2.5% (w/v)glucose, and 8% (w/v) sucrose plus 2.9% (w/v) maltose, respectively(Fig. 1d). These L. reuteri strains formed fructo-oligosaccharides(FOS) in addition to EPS when the acceptor raffinose was present(8%, w/v, sucrose plus 2.9%, w/v, raffinose; Fig. 1c). L. reuteri is foundwidely in the intestines of animals and humans and is believed tobe one of a few truly autochthonous Lactobacillus species in thehuman intestine (Reuter, 2001). It is a well-known biofilm former, acapacity that is thought to enable it to persist in the intestinal tract(Ganzle & Schwab, 2009). Additionally, strains of L. reuteri are usedas probiotics (Valeur, Engel, Carbajal, Connolly, & Ladefoged, 2004).L. casei C12 was isolated from cheese and produces a hetero-polysaccharide composed of glucose, galactose and rhamnose inthe presence of 10% (w/v) sucrose (Table 1).

Di Cagno et al. (2006b) previously used strains from sourdoughas adjuncts in the manufacture of miniature Caciotta cheese, whileDe Angelis, Curtin, McSweeney, Faccia, and Gobbetti (2002) usedL. reuteri as adjunct in cheese-making. This study takes a novelapproach in selecting strains for use as adjuncts based on theirability to produce homopolysaccharide, and also to evaluate puri-fied EPS as an added ingredient in cheese-making.

3.2. Growth of potential adjunct strains in milk

The ability of each adjunct strain to grow in 10% RSMwas testedand used as a criterion for selection as adjunct for use in cheese-making trials. All strains grew in milk (Fig. 2). Both W. cibariaMG1 and L. casei C12 grew well reaching levels of 107 cfu mL�1

within 10 h. In the case of the L. reuteri strains, both grew tomaximum numbers of between 104 and 105 cfu mL�1, but subse-quently decreased within 10 h.

Fig. 1. (a) Exopolysaccharide hyper-production byW. cibaria MG1 on MRS agar in the presence of 10% sucrose after incubation at 30 �C for 48 h; this EPS is a dextran consisting of a-(1,6) linkages. (b) High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showing formation of glucooligosaccharides by W. cibariaMG1 in the presence of 8% sucrose with 2.9% maltose during fermentation in MRS at 30 �C for 16 h; pan: panose standard; pan-(glu)n: glucosylated panose; n: degree of poly-merization. (c) HPAEC-PAD showing formation of fructo-oligosaccharides by L. reuteri ff2hh2 (top) and L. reuteri cc2 (middle) in the presence of 8% (w/v) sucrose with 2.9% (w/v)raffinose during fermentation in MRS at 30 �C for 16 h; raffinose control (bottom). (d) Size exclusion chromatography of EPS produced by L. reuteri ff2hh2 and L. reuteri cc2(including dextran control).

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134 129

The slow growth of these L. reuteri strains may be due to the factthat the carbohydrates in milk are not good substrates for this in-testinal Lactobacillus. In addition, as with NSLAB in general, thesestrains lack proteinase activity to allow them to use casein as agrowth substrate. Indeed, NSLAB tend to grow poorly in milk(Stanley, 1998), but proliferate well in ripening cheese, indicatingthat poor growth in milk is not necessarily an indicator of poorperformance in cheese.

The relatively strong growth of W. cibaria MG1 in milk isinteresting as the original description of W. cibaria indicated thatthis species is unable to ferment lactose and galactose (Björkrothet al., 2002). However, more recent study of dextran-producingWeissella strains found that out of eight strains only oneW. cibaria strain was able to weakly ferment lactose while theothers could not. Most strains were able to ferment galactose(Bounaix et al., 2010). Di Cagno et al. (2006b) found that of thecheeses made with adjuncts originating from sourdoughs, cheesemade with W. confusa 14A had the highest FAA level, highaminopeptidase activity and high free fatty acid (FFA) levels. Insome cases, values were higher than with adjunct NSLAB strainsthat were cheese isolates. These observations may explain thegrowth of W. cibaria MG1 in milk, this genus having relatively highproteolytic and lipolytic capabilities; however, enzymatic activitiesare strain specific.

Based on these results, strains W. cibaria MG1 and L. reuteri cc2were chosen as adjuncts for L. reuteri cc2 was particularly chosen,due to the novel EPS produced and because it grew best of the twoL. reuteri strains tested.

3.3. Microbiological analysis of cheeses

The two adjunct strains used in this study were the W. cibariaMG1 isolate from sourdough and L. reuteri cc2 isolate from theporcine gut. These were added to cheeseemilk at a level of105 cfu mL�1. Adjunct levels increased in all experimental cheesesexcept for cheese made with L. reuteri cc2 with added sucrosesubstrate. Of the two adjuncts selected for the study, W. cibariaMG1 grew better in cheese than did L. reuteri cc2. Levels ofW. cibariaMG1 remained constant at 105 cfu g�1 up to day 30 beforerising to 107 cfu g�1 in cheeses made with MG1 without substratecarbohydrate (data not shown). When substrate carbohydrate wasincluded, strain MG1 levels increased earlier, possibly through theutilisation of the sucrose as an additional carbon source, but thecounts at day 90 were slightly lower than those in cheese madewithout added carbohydrate.

It is likely that the Weissella strain was better able to adapt andpersist in the harsh cheese environment and utilise the availablenutrients, as suggested by its ability to grow inmilk. Bove et al. (2012)

Fig. 2. Growth of adjunct strains in 10% RSM (-) and MRS (A) over 18 h: a, Weissella cibaria MG1: b, Lactobacillus casei C12; c, Lactobacillus reuteri ff2hh2; d, Lactobacillus reutericc2. All points represent the mean of data from two independent experiments that were sampled in triplicate. Error bars show standard error.

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134130

compared the growth of Lactobacillus rhamnosus in MRS broth togrowth in a medium that mimicked the cheese environment (cheesebroth, CB). It was observed that during growth in CB, enzymesresponsible for citrate catabolism, acetate production, proteolyticactivity, and amino acid catabolism were increased, while enzymesresponsible for sugar transport, pentose phosphate pathway and cellwall biosynthesis were decreased when compared with growth inMRS broth. This reflects the adaption of the bacterium to its sur-rounding environment. Bove et al. (2012) suggested that L. rhamnosusmay use nucleotides as carbon sources during growth on CB. It ispossible that W. cibaria in our study adapts in a similar manner. DiCagno et al. (2006b) found that cheeses made with adjunctW. confusa had the highest FAA level, high aminopeptidase activityand high FFA levels when compared with other sourdough isolatesused as cheese adjuncts. Additionally, Ricciardi, Parente, and Zotta(2009) demonstrated the robustness of W. cibaria, modelling itsgrowth and showing that it could grow well over a wide range of pH(4e8), temperature (9e47 �C) and up to 12% salt.

Growth of L. reuteri cc2 was not as strong for most of theripening period (data not shown). This poor performance may beexplained by the strain being in the carbohydrate poor cheeseenvironment, as distinct from its natural carbohydrate-rich niche.De Angelis et al. (2002) included L. reuteri DSM20016 as an adjunctin cheese-making and stated that all adjuncts remained at levels ofabout 5 � 108 cfu g�1 throughout ripening. However, in that studythe initial inoculation level was 108 cfu g�1 and the ripening periodwas 40 days.

The presence of indigenous NSLAB was determined in the con-trol cheese. Here, the NSLAB counts increased later than adjunctlevels but reached similar levels within 30e60 days ripening. These

levels were comparable with typical NSLAB levels for Cheddarcheese (Fitzsimons, Cogan, Condon, & Beresford, 2001). To dis-tinguish the NSLAB from the adjuncts, a genus/species-specific PCRwas applied to colonies on the MRS enumeration plates (Agneset al., 2005; Schillinger et al., 2008). Ten random colonies wereisolated from a countable MRS plate at each enumeration time-point in all relevant cheeses. Based on the PCR result (inset,Fig. 3), the percentage of colonies confirmed as adjunct wasestimated.

Fig. 3 shows the calculated percentage colonies confirmed asWeissella adjunct over the 90-day ripening period. The represen-tative graph shows thatW. cibariaMG1 dominated during the earlystages of ripening, accounting for 70% of the isolated colonies atday 60. By day 90 the dominance of strain MG1 had decreased to38%. This shows that NSLAB became the prominent microbiota asexpected, but primarily confirms the survival of W. cibaria incheese. In addition, while average counts were 107 cfu g�1, PCRconfirmed growth of strain MG1 to levels of 108 cfu g�1. These re-sults indicate that during ripening, levels of W. cibaria MG1increased 1.5e1.8 log cycles. Similar levels have been observedwithLactobacillus strains used as adjuncts (Briggiler-Marcó et al., 2007;Hynes, Ogier, & Delacroix-Buchet, 2001; Milesi et al., 2008a).Growth of strain MG1 was similar in cheese made with addedsubstrate carbohydrate, indicating that in-situ EPS production didnot affect its growth.

Control cheeses were also tested by PCR for the presence ofadjunct (data not shown) and very low levels (6% on day 60 and 5%on day 90) were detected. The trend of starter culture growth wassimilar in all cheeses (data not shown), suggesting that addition ofadjuncts did not affect the starter performance.

Fig. 3. Graph showing total bacterial titres in the adjunct cheeses combined with the average percentage colonies confirmed as Weissella by genus-specific PCR in the six cheesereplicates during the ripening period. Inset: genus-specific PCR for Weissella performed on the colonies e lanes 1 and 15, molecular weight standard; lanes 2e11, genus-specific PCRon isolates from cheeses made with Weissella strain MG1 as adjunct; lanes 16e23, genus-specific PCR on isolates from control cheeses (no adjunct); lane 12, PCR control (notemplate DNA); lane 13, positive control (DNA from pure culture of W. cibaria MG1).

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134 131

3.4. Cheese composition

Compositional data for all cheeses is shown in Table 3. Values ofexperimental cheeses (Table 3, cheeses BeF) were compared withthe control cheeses (Table 3, cheese A). Statistical analysis ofcompositional data showed no significant difference betweentreatments for pH, moisture and pH 4.6-soluble protein. Therewereno significant differences in salt levels at 30 days in cheeses thatjust contained adjuncts, purified EPS, and the control cheeses. Saltlevels between the control and cheeses with added substrate car-bohydrate were statistically different (p < 0.05); however, the dif-ference was minor and occurred only between the control andcheeses made with L. reuteri cc2 plus substrate carbohydrates.

While there were consistent differences in the moisture levelsbetween the control and experimental cheeses, these differencesindividually were not statistically significant. There were increasedmoisture levels in all cheeses containing EPS-producing adjunct oradded EPS ingredient when compared with the control. Moisture

Table 3Microbial counts and gross composition of miniature Cheddar cheesesa produced wiingredient.b

Cheese Starter count(log cfu g�1,day 90)

Adjunct count(log cfu g�1,day 7)

Adjunct count(log cfu g�1,day 90)

pH(day 90)

Saltday

A 11.05 Not added Not added 5.09 1.8B 11.80 5.78 7.57 4.93 1.8C 10.99 5.73 7.18 4.89 2.0D 12.10 5.01 7.26 4.93 1.6E 11.48 5.09 5.85 5.10 2.3F 12.02 No adjunct No adjunct 4.96 1.7

a Cheeses were: A, control cheese; BeF, experimental cheeses made with the additioncc2, (E) L. reuteri cc2 plus substrate carbohydrates, (F) addition of EPS as ingredient. NSL

b Abbreviations are: S/M, salt in moisture; FAAs, free amino acids. No statistically signifi(day 90) or pH 4.6-soluble protein (day 90); salt was significantly different (p < 0.05) bet(cheese E). Microbial counts are the means of six replicates and compositional data are

levels were, on average, between 1 and 3% higher in the experi-mental cheeses compared with control cheeses. Studies haveshown that cheese made with EPS-producing starters have highermoisture levels (Hassan & Awad, 2005). Costa et al. (2010) manu-factured Cheddar cheese with an EPS-producing L. lactis starter andits isogenic, non-EPS-producing variant. The presence of EPSsignificantly increased themoisture levels, which was postulated tobe the result of trapping of water by the EPS through hydrogenbonding. For this reason, EPS-producing cultures have particularlybeen used in reduced-fat and half-fat cheeses to improve thetextural and rheological properties of these cheeses.

Thehighestmoisture levelswere in cheesesmadewithW. cibariaMG1 as adjunct alone, cheeses made with W. cibaria MG1 adjunctwith substrate sucrose, and cheeses with the MG1 dextran EPSadded as an ingredient. To investigate the level of EPS present incheeses, carbohydrate analysis was performed on each cheese type.Following hydrolysis, glucose and fructose levels were assessed byHPLC. As expected, higher levels of glucose were identified in the

thout (control) or with EPS-producing adjuncts or with addition of purified EPS

(%, w/w,30)

Moisture(%, w/w,day 30)

S/M (%, w/w) FAA (day 90)(mg Leucheese�1)

pH 4.6-solubleprotein(day 90) (%)

5 � 0.29 30.3 � 5.0 6.13 � 0.71 7.63 � 2.27 0.65 � 0.051 � 0.14 33.0 � 6.3 5.68 � 1.31 8.22 � 2.31 0.70 � 0.055 � 0.19 32.7 � 5.4 6.47 � 1.49 7.79 � 2.09 0.67 � 0.087 � 0.21 31.1 � 4.0 5.44 � 0.89 7.45 � 1.85 0.70 � 0.074 � 0.38 31.5 � 5.2 7.54 � 1.28 7.57 � 2.09 0.64 � 0.056 � 0.27 31.9 � 4.3 5.63 � 1.38 7.89 � 3.32 0.68 � 0.08

of (B) W. cibaria MG1, (C) W. cibaria MG1 plus substrate carbohydrate, (D) L. reuteriAB counts on control cheeses at day 90 were 7.88 log cfu g�1.cant difference between samples (p> 0.05) was found for pH (day 90), moisture, FAAween the control and cheeses made with L. reuteri cc2 plus substrate carbohydratesthe means of three replicates. Values shown are means � standard deviation.

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134132

cheeses containing purified EPS, typically up to 17 mmol kg�1 ofcheese by comparison with 3 mmol kg�1 in the case of controlcheeses. Cheeses made using the adjunct culture alone exhibited aglucose level of 5mmol kg�1, while cheeses made using the adjunctculture with sucrose had a higher level (14 mmol kg�1), but this islikely to have resulted from hydrolysis of the sucrose substrate,which is supported by the fact that the moisture levels in bothadjunct cheeses were similar.

This is the first study to use EPS as an added ingredient incheese-making. In this study, the EPS ingredient was added at 2 gper litre of cheese milk, resulting in a slight but consistent increasein moisture. This suggests that if the level was increased, it has thepotential to increase moisture retention in the cheeses. The Euro-pean Union Commission (Anonymous, 2000) has approved the useof dextran EPS up to a level of 5% in bakery products, suggestingthat at this level there would be no risk to the consumer. It isworthy of mention that the presence of such levels of EPS couldpotentially have a beneficial effect on the consumer, as suggestedby the findings of Olano-Martin et al. (2000) who showed thatdextran has prebiotic potential.

3.5. Proteolysis in cheeses

Breakdown of caseins and peptides by rennet and microbialenzymes, respectively, is important in cheese for the formation offlavour and aroma compounds that give a particular cheese type itscharacteristic attributes (Smit, Smit, & Engels, 2005). Proteolysiswas measured by TNBS, urea-PAGE of pH 4.6-insoluble N and RP-HPLC of pH 4.6-soluble N.

Assessment of primary proteolysis by urea-PAGE of the pH 4.6-insoluble extracts (Fig. 4) showed no differences between the

Fig. 4. Urea-polyacrylamide gel electrophoretograms of the pH 4.6-insoluble extracts of CheLanes 1 and 7, control cheeses; 2 and 8, cheeses made with W. cibaria MG1; 3 and 9, cheeseL. reuteri cc2; 5 and 11, cheese made with L. reuteri cc2 plus substrate carbohydrates; 6 and

control and experimental cheeses after 3 months of ripening. Thissuggests that the adjunct cultures did not contribute to primaryproteolysis and this is in agreement with previous studies whereno differences in primary proteolysis were observed in cheesesmade with and without adjunct Lactobacillus cultures (Di Cagnoet al., 2006b; McSweeney et al., 1994). These microorganisms areknown to be weakly proteolytic and thus do not contributeconsiderably to the hydrolysis of casein during cheese ripening (DiCagno et al., 2006b; Fox, McSweeney, & Lynch, 1998). Primaryproteolysis is mainly attributed to the action of residual coagulant,milk plasmin, and the proteolytic activity of starter culture strainproteinases (Costa et al., 2010; Dabour, Kheadr, Benhamou, Fliss, &LaPointe, 2006).

HPLC was used to examine secondary proteolysis and peptideformation. There were no qualitative differences apparent betweenthe control and experimental cheeses after 3 months ripening.Fig. 5 shows an overlay of the peptide profiles from control repli-cates and replicates containing W. cibaria MG1 as adjunct alone.From the position of the peaks it can be seen that very similarprofiles were obtained in both cheeses.

These results demonstrate that the adjuncts and treatmentsused in this study did not greatly alter the peptide profiles of thecheeses. This could be seen as a positive, but should be studied inmore detail in large-scale cheese trials. The primary reason forinclusion of EPS producers in cheese is not necessarily to impact onflavour compounds, and thus, inclusion of W. cibaria is not likely tohave a negative impact on flavour or quality.

The concentration of total FAA in the pH 4.6-soluble extracts ofcontrol and experimental cheeses were determined using the TNBSmethod after 90 days ripening (Table 3). All cheeses except thosecontaining L. reuteri cc2 as adjunct had higher concentrations of

ddar cheeses at 90 days. Shown are extracts from two different trials (individual days).s made with W. cibaria MG1 plus substrate carbohydrate; 4 and 10, cheese made with12, cheese made with purified EPS as ingredient. SC, sodium caseinate standard.

Fig. 5. Overlay of all ultra-performance liquid chromatography chromatograms obtained from control cheeses and experimental cheeses containing W. cibaria MG1 as adjunct atday 90 of ripening.

K.M. Lynch et al. / International Dairy Journal 34 (2014) 125e134 133

FAA. The highest were found in cheeses containing W. cibaria MG1as adjunct, indicating a contribution to secondary proteolysis andpotential contribution to cheese flavour. Again, this would beworthy of investigation in large-scale cheese trials where cheeseflavour could be assessed, given that the peptide profiles wereunaltered in the experimental cheeses. FAA levels have previouslybeen observed to be higher in cheeses containing adjunct lacto-bacilli (Broome, Krause, & Hickey, 1990; Hynes et al., 2001). DiCagno et al. (2006b) used a number of strains originating fromsourdough as adjuncts in a Caciottamodel cheese system and foundhighest concentrations of FAA in cheese made with a W. confusastrain as adjunct. Additionally, this strain was found to have highpeptidase activity when compared with the other adjuncts origi-nating from sourdough. Proteolytic activity of adjuncts is oftensignificant and sometimes desired, since FAA are precursors toflavour and aroma compounds in cheese (Briggiler-Marcó et al.,2007). The adjuncts used in this study appear not to affect prote-olysis; however, as stated, a large-scale trial would be necessary toassess quality and flavour changes in these cheeses.

4. Conclusion

In conclusion, this study confirms the growth and survival of asourdough W. cibaria strain in cheese-making. The findings indicatethat thisstrainmayhave thepotential tobeauseful adjunct, increasingthemoisture retentionof cheesewithout significantlyaffecting cheeseproteolysis and therefore the characteristic flavour and aroma prop-erties of Cheddar cheese. In addition, it has been shown that the useofpurified dextran EPS as ingredient has the potential to increasemoisture retention in cheese; however, use at a higher concentrationof possibly up to 5% would be likely to exert a greater effect. Use ofW. cibaria or its EPS as an ingredientmay contribute to cheese textureas well as having a potential health-promoting effect.

Acknowledgements

This work was supported by the Irish Department of Agricul-ture: Food Institutional Research Measure (F.I.R.M.), Project

Reference 08RDCIT600 and The Irish Research Council for Science,Engineering & Technology (IRCSET) Project Reference RS/2011/13.The authors would like to thank Felicia Ciocia for her assistanceduring the Cheddar cheese-making process.

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