Innovative Food Science and Emerging Technologies 11 (2010) 203–209

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Innovative Food Science and Emerging Technologies

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Evaluation of fresh-cut apple slices enriched with probiotic bacteria

Christian Rößle a,⁎, Mark A.E. Auty b, Nigel Brunton a, Ronan T. Gormley a, Francis Butler c

a Ashtown Food Research Centre (Teagasc), Ashtown, Dublin 15, Irelandb Moorepark Food Research Centre (Teagasc), Fermoy, Co. Cork, Irelandc Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin 4, Ireland

⁎ Corresponding author. Tel.: +353 1 8059529; fax: +E-mail address: christian.roessle@teagasc.ie (C. Rößl

1466-8564/$ – see front matter © 2009 Elsevier Ltd. Aldoi:10.1016/j.ifset.2009.08.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 May 2009Accepted 28 August 2009

Editor Proof Receive Date 10 September 2009

Keywords:ProbioticLactobacillusLGGFresh-cutAppleNon-dairy

The aim of this study was to apply a probiotic microorganism (Lactobacillus rhamnosus GG; LGG) to fresh-cutapple wedges (cultivar Braeburn) and measure entrapment and stability of the microorganism. Instrumentaleating quality parameters (Colour Lab, texture, soluble solids, titratable acidity and pH) and sensoryacceptability were also monitored to investigate if application of the probiotic significantly influenced eatingquality. Apple samples were cut into skin-on wedges and were dipped in an edible buffer solution containingapproximately 1010cfu/ml of LGG. LGG were enumerated on each test day (0, 2, 4, 6, 8 and 10) on wholewedges, on wedges flushed with a buffer solution (2% tri-sodium citrate), and on the flush-off liquid itself. Allthree samples sets contained ca. 108cfu/g over the test period, which is sufficient for a probiotic effect, and iscomparable to counts of probiotic bacteria in commercially available dairy products. This included the sampleset of wedges which had been flushed with buffer solution indicating good adherence of the bacteria over thetest period. Physicochemical properties of the apple wedges containing LGG compared to the controlremained stable over the 10 day period. Cryo scanning electron microscopy and confocal scanning lasermicroscopy demonstrated good adherence of LGG to the surface of apple wedges.Industrial relevance: Probiotic dairy foods, e.g. yoghurts, are well recognised by most consumers andcommand a significant market share. However, many people are allergic or intolerant to dairy products andan alternative option is desirable. Minimally processed freshly prepared fruits are a popular item and areperceived as healthy by consumers. They are therefore an ideal vehicle for incorporation of other functionalcomponents such as probiotics. Therefore, a probiotic bacterium was applied to fresh-cut apple wedges. Thiswill provide an alternative probiotic food choice for consumers and could be particularly appealing tochildren. The process for making this product is relatively simple and the product would retail from theconventional chill counters of supermarket stores. It is likely that its price would be competitive with existingprobiotic dairy products.

353 1 8059550.e).

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© 2009 Elsevier Ltd. All rights reserved.

1. Introduction

In the early years bacteria were generally regarded as undesirableand the cause of many diseases. More recently scientific research hasdone much to reduce their negative image. In particular, muchresearch has been aimed at searching for healthy bacteria andcomponents which can beneficially affect health conferring bacteria.Probiotics belong to the former category and are classically defined as‘live microbial feed supplements which beneficially affect the hostanimal by improving its intestinal microbial balance’ (Fuller, 1989).Several scientific studies have shown that microbial cells have abeneficial effect on the health and well-being of the human host(Salminen et al., 1998a,b) if directed in the right amounts (Dave andShah,1997; Kailasapathy & Rybka,1997; Brown& Valiere, 2004; Prado,

Parada, Pandey & Soccol, 2008). Some studies have indicated thatregular consumption of viable probiotics can confer a number ofhealth benefits such as a reduction of cholesterol (Anderson &Gilliland, 1999; Nguyen, Kang & Lee, 2007), control of gastrointestinalinfections (McFarland et al., 1995; Saavedra, Bauman, Oung, Perman &Yolken, 1994), improvement of lactose tolerance (Hove, Norgaard &Mortensen, 1999; Kim & Gilliland, 1983; Shah, 2007), improvement ininflammatory bowel disease (Lammers et al., 2003) , inhibition ofsome cancers (Aso et al., 1995; Cross, 2002), anti-diabetic properties(Matsuzaki, Yamazaki, Hashimoto & Yokokura, 1997; Yadav, Jain &Sinha, 2007), anti-diarrhoeal effects (Nomoto, 2005), and immunesystem stimulation (De Moreno de LeBlanc et al., 2008; Cross, 2002).

Over the last years the minimally processed foods market has beenextended especially due to an increase of the fresh-cut fruits market(Buckley, Cowan & McCarthy, 2007; Gorny, 2003). Development ofhealth promoting foods is one of the key drivers for the food industrydue to an increasing demand for foods enriched with physiologicallyactive components such as probiotics (Mark-Herbert, 2004). Probiotic

204 C. Rößle et al. / Innovative Food Science and Emerging Technologies 11 (2010) 203–209

dairy foods are a well established product and are recognised by mostconsumers as a healthy product. However, many people are allergic orintolerant to dairy products and an alternative option such as fruitswould be desirable (Rivera-Espinoza & Gallardo-Navarro, 2008).Therefore there is a need for non-dairy products enhanced withprobiotic bacteria (Betoret et al., 2003; Heenan, Adams, Hosken &Fleet, 2004; Yoon,Woodams & Hang, 2006). The aim of this studywas,therefore, to apply a probiotic microorganism (Lactobacillus rhamno-sus GG; LGG) to fresh-cut apple wedges thereby producing a doublyfunctional food product, i.e. the inherent functionality of the applewedges plus the added functionality of LGG.

2. Materials and methods

2.1. Culture

The commercial strain L. rhamnosus GG (LGG) was obtained fromMoorepark Food Research Centre (Teagasc; Fermoy, Co. Cork, Ireland).

2.2. Preparation of probiotic solution

Lyophilized cultures were grown in MRS (Oxoid Ltd., Hampshire,UK) broth overnight at 37 ºC for approximately 15 h. The cells werethen washed with citric acid-sodium: citrate buffer (pH 3.8) bycentrifugation (5810R; Eppendorf AG, Hamburg, Germany) at7000 rpm for 15 min. This was repeated until the supernatant wasclear (approximately 3–4 times). Washed cells were then re-suspended in citric acid: sodium citrate buffer [pH 3.8; 1:10 (w/v)].

2.3. Sample preparation

Apples (cultivar Braeburn) were purchased in a local supermarket,washed inwater, cored (20 mmdiameter stainless-steel cork borer) andcut with a stainless-steel knife into wedges (each ca.10 g). Five skin-onwedges from each of five apples (chosen randomly) were used forinfusion. The wedges were then dipped for 10 min in probiotic solutionat a 1:1 solution/wedge ratio containing approximately 1010cfu/ml ofLGG. Thewedges were then drained for 2 min and dipped for 2 min in a6% (w/v) solution at a 3:1 solution/wedge ratio of browning inhibitorNatureseal®AS1 (AgriCoat Ltd., Great Shefford,UK). Natureseal®AS1 is acommercial available anti-browning agent and is widely used forinhibition of browning in the fresh-cut fruit industry. The wedges wereagain drained for 2 min packed in clear trays (15 cm×10.5 cm×3 cm;Versatile Packaging, Silverstream, Ireland), heat sealedwith a breathingfilm (O2 transmission of <2 ml /24 h/23 °C; water vapour transmissionof <6 g/24 h/38 °C) using a modified atmosphere packaging machine(Ilpra Foodpack Basic V/G, Ilpra, Vigenovo, Italy) and stored at 2–4 ºCfor 10 days. Breaburn apple wedges were dipped in Natureseal® AS1browning inhibitor as described above and were used as a controltreatment. Apple wedges with probiotics and control wedges wereprepared in3 replicates and testswere carried outon the samples onday0, 2, 4, 6, 8 and 10.

2.4. Enumeration of LGG in apple wedges

On each test day one wedge containing LGG was removed fromeach tray (n=3) and the surface was washed off with 2% tri-sodiumcitrate solution [1:1 (w/v)]. This wash off was serially diluted inmaximum recovery diluent (Oxoid Ltd., Hampshire, UK). Dilutionswere then plated on lactobacillus selective Rogosa Agar (Oxoid Ltd.,Hampshire, UK). The previously washed off wedges were thenmacerated in 2% tri-sodium citrate solution [1:10 (w/v)], seriallydiluted in MRD and plated on Rogosa Agar. A second wedge wasremoved from each tray (n=3) macerated in 2% tri-sodium citratesolution [1:10 (w/v)], withoutwashing off, serially diluted inMRD andplated on Rogosa Agar. All plates were incubated aerobically for 72–

96 h at 37 ºC followed by enumeration. This procedure gave the LGGcontent of: (a) thewash off liquid, (b) washedwedges, (c) the wash ofliquid+washed wedges and (d) wedges that were not washed withbuffer. All samples were serially diluted in duplicates and plated intriplicates.

2.5. Physicochemical evaluation

Physical and chemical properties were measured on day 0, 2, 4, 6, 8and 10 using five wedges per replicate of treated and control samples.Colour and firmness were measured first as described below. Sampleswere then homogenized for subsequent measurements.

2.5.1. Measurement of colourThe colour of the apple wedges was measured using a HunterLab

D25A DP-9000 colour meter (HunterLab, Reston, VA, USA). The colourfor 5 wedges per replicate was measured and expressed as a threedimensional Lab colour solid.

2.5.2. Measurement of firmnessThe firmness of apple wedges was measured on 5 wedges for each

treatment using a Texture Analyzer TA-XT2i (Stable Micro Systems,Godalming, UK) fitted with a 25 kg load cell and equipped with aWarner–Bratzler Blade. The wedge was fractured by a downwardmotion (10 mm/min) of a steel blade with a thickness of 3 mm. Themaximum force (highest value in N) applied to break the wedge wasused to quantify the firmness.

2.5.3. Measurement of soluble solids contentsSoluble solids contents (SSC) were measured using an Abbe

refractometer (2WAJ; Guru Nanak Instruments, New Delhi, India). Thescale was set to zero using the refractive index of water. Apple pulpwas squeezed throughmuslin. A dropwas placed on the refractometerglass prism and the percentage soluble solids content obtained.

2.5.4. Measurement of total titratable acidity and pHFor total titratable acidity (TTA) approximately 5 g of apple pulp

were diluted in 100 ml of distilled water and 3–4 drops of indicatorphenolphthalein was added. The solution was then titrated with a0.1 N NaOH solution beyond pH 8.1 (AOAC, 1995). The TTA calculatedas a percentage of malic acid [(ml NaOH×0.1 N/weight of sampletitrated)×0.067×100)].

pH was measured on homogenous apple pulp with an Orion pHmeter (420A; Thermo Fisher Scientific Inc., Waltham, MA, USA) whichwas calibrated prior to each measurement with phosphate buffers atpH 4.005 and 7.

2.6. Sensory evaluation

A blind study was used to evaluate the overall acceptability ofapple wedges containing LGG and control wedges by an untrained 25member taste panel between the ages of 23 and 67 years. Eachpanellist was given a plate containing 2 wedges (1 probiotic, 1control). Panellists were asked to quantify the using a scale from 0(unacceptable) to 6 (very acceptable). A score equal to three was usedas the threshold to produce acceptability. Tasting was performed in asensorial testing roomwith individual booths and controlled lighting.The evaluation was performed only on day 0.

2.7. Cryo scanning electron microscopy (Cryo-SEM)

Apple wedges (control and probiotic-treated, day 0) wereexamined using cryo scanning electron microscopy. Both the treatedapple surfaces and fracture surfaces were analysed to investigatewhether probiotic bacteria had penetrated the apple tissue. Forfracture surfaces, thin sections approximately 5×5×2 mm were cut

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with a razor blade and mounted into a slotted aluminium sampleholder using OCT embedding compound. The sample was thenplunge-frozen into liquid nitrogen slush (−196 °C) and transferredunder vacuum to the cold stage using the Alto 2500 cryo-transferdevice (Gatan Ltd., Oxford, UK). After sublimation at−95 °C for 2 min,the sample was cooled to −125 °C, sputter coated with platinum andtransferred to the cold stage for imaging at −125 °C. Secondaryelectron images were obtained using a Zeiss Supra 40VP field emissionscanning electron microscope (Carl Zeiss SMT Ltd., Cambridge, UK)operating at 1 kV and a working distance of 5–6 mm.

2.8. Confocal scanning laser microscopy (CSLM)

Confocal scanning laser microscopy (CSLM) in conjunction with insitu viability staining was used, based on a method by Auty et al.(2001), to visualise the distribution and viability of probioticorganisms on the surface of the wedges at day 0 and day 10. Briefly,one drop of Live/Dead BacLight® viability stain (Biosciences Ltd, DunLaoghaire, Ireland) was added to probiotic-treated surface of the applewedge, cut parallel to the treated surface to ensure a horizontalsurface when mounted on a microscope slide with a coverslip on top.After 5 min incubation at room temperature, images were acquiredusing a 488 nm argon gas laser in conjunction with a 514 nmexcitation filter and emission peaks set at 560 nm and 650 nm usinga Leica SP5 confocal microscope (Leica Microsystems GmBH, Man-nheim, Germany). When excited by the laser, viable cells fluorescegreen whilst non-viable cells fluoresce red. Duplicate samples wereexamined and a minimum of 5 images acquired for each sample.

2.9. Statistical design

The statistical design was 2 dips (control, probiotic)×6 test days(0, 2, 4, 6, 8 ,10)×3 replicates with 35 degrees of freedom (df)followed by ANOVA [(Genstat 5 Version 3.2); Lawes Agricultural Trust,Rothamsted, Harpenden, UK].

3. Results and discussion

3.1. Survival of LGG in apple wedges

LGG was tested for its ability to survive on apple wedges at 2–4 °C.To indicate the location of LGG on the apple wedges various sampleswere taken. The number of LGG was measured in (a) dipped wedgessubsequently washed with tri-sodium citrate buffer [2% (w/v)],(b) the wash off liquid, (c) counts of washed wedges+wash offliquid, and (d) dipped unwashed wedges. The wedges washed offwith 2% tri-sodium citrate solution for 2 min had a cfu/g count of 108

on day 0 (results presented in Fig. 1). This count for the post-washed(PW) wedges remained stable throughout the 10 day storage periodat 2–4 °C. Tri-sodium citrate buffer [2% (w/v)] was used to wash off(WO) the surface of the apple wedges and LGG counts in this WO wasmeasured to indicate the adherence of LGG to the apple wedgesurface. In fact it was expected to have more LGG in the wash off thanin the remaining wedge. However the results show that theconcentration of the WO was 107cfu/g therefore significantly lower(P<0.001) than the counts (108cfu/g) from the remaining wedge(Fig. 1). This was not expected therefore Cryo-SEM and CSLM wasconducted to get a better understanding of nature of the adherence ofLGG to the apple wedges. As a third sample a wedge was taken andenumerated without washing it off prior with solution. Therefore theresults of the whole wedge should show similar concentrations of theWO and PW wedge together. There was a slight difference, howeverthis was not significant (P>0.05). Overall all measurements haveshown that LGG did survive the 10 days storage and was adsorbedin some manner on the apple wedges (Fig. 1). There is no clearagreement on the minimum concentration of probiotic intake to

achieve a beneficial effect on the host. While some researcherssuggest concentrations higher than 106cfu/ml (Kurman & Rasic, 1991;Dave & Shah, 1997) are required, others suggest a concentration of atleast 107 and 108cfu/ml (Kailasapathy & Rybka, 1997; Lourens–Hattingh & Viljeon, 2001). Some researchers even stipulate thatconcentrations of 109cfu/ml (Prado et al., 2008) are to be needed. Inany case, all apple wedges had a concentration of 108cfu/g over the10 days storage period. LGG was chosen for this trial as it is acommercially available bacteria and it has also been reported to beresistant to gastrointestinal conditions (Succi et al., 2005). Thepossibility of encapsulating the bacteria to make it even moreresistant to gastrointestinal conditions would be also an option(Pimentel-Gonzalez, Campos-Montiel, Lobato-Calleros, Pedroza-Islas& Vernon-Carter, 2009).

3.2. Physicochemical evaluation

No significant difference in instrumental colour values (Hunter-Lab) between control apple wedges and infused apple wedges wasnoted (P>0.05). However, during chill storage (2–4 °C) Hunter Lvalues increased up to day 2 (Table 1), indicating a whitening effect,while L values between day 2 and 10 showed a slight decrease for thecontrol (from 69.3 to 66.7) and probiotic apple wedges (from 69.1 to66.7). Even though L values were significantly different (P<0.001)over the 10 day storage period these differences were very small inpractical terms. In terms of browning levels samples from bothtreatments were visually still acceptable after 10 days of storage at 2–4 °C indicating that the browning inhibitor Natureseal® AS1 had asimilar effect on the control and the probiotic wedges. This outcomewas also confirmed by Hunter a (redness) values which decreasedbetween day 0 and day 2 for the control and day 0 and 4 for probioticapple wedges (Table 1). As the storage progressed a values increasedfor the control (from −3.32 to −2.52) and the probiotic apple slices(from−2.81 to−1.61) but remained negative throughout the 10 days(Table 1) indicating no development of redness an indicator forbrowning. There was no significant difference for Hunter b values forcontrol apples and probiotic apple wedges and also the storage periodhad very little effect on the yellowness of the apple wedges (P<0.05).Natureseal® products have been studied extensively and have shownto be effective in prevention of colour deterioration (Rupasinghe,Murr, DeEll & Odumeru, 2005; Toivonen, 2008; Rößle, Gormley &Butler, 2009). There is also a possibility that the probiotic solutionwhich is mainly citric acid sodium citrate buffer could have a positiveeffect on retaining the colour in apple wedges as citric acid has beenreported to have an anti-browning effect (Pizzocaro, Torreggini &Gilardi, 1993; Rojas-Graü, Soliva-Fortuny &Martìn-Belloso, 2008) or itcould be due to low pH-induced inhibition of the enzyme responsiblefor browning development PPO (Garcia & Barrett, 2002).

Shear values for control apple wedges and probiotic apple wedgesshowed no significant difference (P>0.05, Table 1). However, shearvalues changed significantly (P<0.001) over the 10 day storage period.Between day 0 and 2 shear values increased which could be due to theAS1browning inhibitor. AsNatureseal®AS1has calciumcontent of 90 mg100 g−1 (Rößle et al., 2009) the firming effect could be due to cross-linking of both cell wall and middle lamella pectin by calcium ions (Ricoet al., 2007). Control (from 38.6 N to 36.1) and probiotic apple (from 36.3to 31.8) lost their firmness between day 2 and 10 but the shear values atday 10 were similar to the firmness measured on day 0 (Table 1).Natureseal® AS1 had less firming effect on the probiotic wedges whichcould be due to the fact that the wedges were previously dipped for10 min in probiotic solution which could induce tissue softening.

Soluble solid contents (SSC)werenot influencedbydipping applewedgesin probiotic solution as indicated inTable 1 (P>0.05). SSC ranged from11.8 to12.2% for both control and apple wedges containing LGG.

Values for total titratable acidity (TTA) for control apple wedgesand infused apple wedges showed no difference as shown in Table 1

Fig. 1. Enumeration of L. rhamnosus GG after incubation on Rogosa agar for 72–96 h of post-washed apple wedges (a), wash off solution (b), wash-off solution+post-washed applewedges (c) and unwashed apple wedges (d) in log10 cfu/g over 10 days storage at 2–4 °C (Fischer test day: P<0.001; LSD 0.031; Fischer test sample: P<0.001; LSD 0.029; dataaveraged from 3 replicates).

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(P>0.05). TA ranged from 0.53 to 0.59% malic acid with the highestvalue for control apples. The stability of TTA of probiotic apple wedgeswas expected as the pH (3.8) of the citric acid sodium citrate bufferwas adjusted according to the inherent pH of the apple batch. Prior totreatment 5 apples were chosen at random to determine their pH. Thisis very important as large differences in the inherent pH can be a riskin terms of microbial growth for minimally processed foods(Ahvenainen, 1996; Soliva-Fortuny, Elez-Martìnez & Martìn-Belloso,2004) and therefore result in a shorter shelf-life. However, the TTAand pH values for control and probiotic apple wedges showed similarpatterns over the 10 days storage period but they did not correlatevery well (R2=0.66).

Table 1Physicochemical and sensory properties of apple wedges containing Lactobacillus rhamnosus

Day L a b SS

Control 0 68.5±0.8 −2.22±0.3 16.0±0.1 122 69.3±0.8 −3.32±0.1 17.7±1.0 114 68.7±1.4 −2.85±0.5 16.7±0.3 116 68.3±0.5 −3.25±0.3 18.0±1.6 118 67.2±0.5 −2.09±0.2 17.1±0.6 12

10 66.7±0.5 −2.52±0.1 17.0±0.5 12Probiotic 0 68.7±0.4 −2.34±0.3 16.2±0.2 12

2 69.1±0.3 −2.62±0.1 16.3±0.1 114 68.6±1.8 −2.81±0.2 17.7±1.0 126 68.3±1.0 −2.27±0.1 16.9±1.1 128 66.5±.7 −1.84±0.3 16.1±0.4 11

10 66.7±1.1 −1.61±0.2 16.2±0.5 12F-testb dip NSc P<0.05 NS NF-test day P<0.001 P<0.001 P<0.05 N

a Overall acceptability from 0 (unacceptable) to 6 (very acceptable).b Fischer test.c Not significant.

Overall physicochemical properties were not strongly influencedby dipping apple wedges into probiotic solution containing LGG(Table 1). However, any negative effect could have been also inhibitedby post dipping wedges into browning inhibitor AS1.

3.3. Sensory evaluation

Sensory evaluation was conducted on day 0 to indicate thedifferences between sample sets of control and probiotic applewedges (Table 1). Panellists did not express a preference for applewedges containing LGG over control apples. 13 tasters preferredcontrol wedges while 12 preferred probiotic apple wedges. Similarly,

GG (probiotic) and control apple wedges (dipped in AS1) stored for 10 days at 2–4 °C.

C (%) TTA (%) pH Shear (N) Sensorya

.3±0.1 0.58±0.02 3.77±0.02 33.2±3.8 4.25±0.92.9±0.1 0.59±0.02 3.74±0.02 38.6±5.1 –

.8±0.3 0.53±0.01 3.79±0.02 38.3±3.9 –

.9±0.2 0.56±0.01 3.75±0.02 36.3±3.3 –

.1±0.1 0.56±0.01 3.76±0.02 36.3±5.5 −

.1±0.1 0.55±0.02 3.76±0.03 36.1±4.9 –

.0±0.2 0.56±0.02 3.76±0.01 28.4±4.4 4.20±1.27.9±0.1 0.55±0.02 3.78±0.02 36.3±3.7 –

.1±0.1 0.55±0.02 3.76±0.03 34.9±6.3 –

.0±0.1 0.58±0.03 3.75±0.02 34.7±3.3 –

.9±0.1 0.55±0.02 3.76±0.02 32.1±3.6 –

.0±0.3 0.58±0.01 3.75±0.01 31.8±5.0 –

S NS NS NS NSS NS NS P<0.001 –

Fig. 2. Cryo scanning electron micrograph of probiotic-treated surface (day 1) of applewedge. Bar=10 μm.

Fig. 3.Magnified view of cryo scanning electronmicrograph of probiotic-treated surface(day 1), showing rod-shaped bacteria. Arrows indicate crystals possibly due to frozenbuffer solution. Bar=1 μm.

Fig. 4.Magnified view cryo scanning electronmicrograph of probiotic-treated (day 1) ofapple wedge. Arrow indicates rod-shaped bacteria on wedge surface. Bar=2 μm.

Fig. 5. Magnified view of cryo scanning electron micrograph of control fracture (day 1)of apple wedge. It focuses on the fracture not on the surface of the apple wedge.Bar=1 μm.

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overall acceptability of the samples was the samewith amean value of4.25 for control and 4.20 for probiotic apple wedges showing thatprobiotic apple wedges were accepted well by the panellist (P>0.05,Table 1). The softer texture of the probiotic apple wedges compared tothe control apple wedges was the main reason for choosing controlover probiotic wedges. The time between dipping apples in AS1 andthe actual taste panel was too short to allow AS1 to have a positiveeffect on the textural properties of the apple wedges hence thenegative comments on the texture. Also some panellist disliked theslight lactic acid odour of the wedges with LGG as they commentedthat it is an odour which they do not expect in context with applewedges. However, some comments indicated that LGG gave the applewedges a unique and pleasant odour. Taste panel tests were conductedon day 0 only for logistic reasons. This aspect of the work must,therefore, be considered preliminary and will be addressed in furthertrials relating to intellectual property aspects of the research findingsand know-how. These results are preliminary and future work willfocus on the sensory evaluation once the microbial safety status hasbeen confirmed. The effect of LGG on the natural micro flora of theapple wedges and the microbial safety of the product are currentlybeing investigated by and ISAFruit partner.

Fig. 6. Cryo scanning electron micrograph of control (day 1) apple wedge. Frozenfracture face showing parenchyma cells. Bar=10 μm.

Fig. 7. Confocal scanning laser micrograph of probiotic treated apple wedge surface(Day 0). Labelled with Live/Dead BacLight® viability stain. Viable bacteria are green;non-viable bacteria are red. Bar=50 μm.

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3.4. Cryo-SEM observation

Cryo-SEM revealed the presence of numerous rod-shaped bacteriaon the cut, treated surface of the apple wedges (Fig. 2). Highermagnification showed the presence of needle-like crystals associatedwith the bacteria (Fig. 3). The needle-like crystals were probably dueto the crystallisation of buffer salts. No bacteria were seen within theapple tissue either within cells or at intercellular junctions (Fig. 4). Nobacteria were seen on the surface (Fig. 5) or fracture (Fig. 6) of thecontrol sample.

Fig. 8. Confocal scanning laser micrograph of probiotic treated apple wedge surface(Day 10). Labelled with Live/Dead BacLight® viability stain. Viable bacteria are green;non-viable bacteria are red. Bar=50 μm.

3.5. Confocal scanning laser microscopy

CSLM of apple wedge surfaces revealed large numbers of unevenlydistributed viable bacteria on day 0 (Fig. 7). High numbers of viablebacteria were still present at day 10 but there appeared to be a relativedecrease in the number of non-viable bacteria (Fig. 8). This reflects theresults shown in Fig.1 as therewas a decrease in viable bacteria on day10 compared to day 0 for all apple wedges containing LGG.

4. Conclusion

Physicochemical and sensory evaluation indicated that dippingapple wedges in a solution containing probiotic bacteria resulted inwedges of acceptable quality with sufficient numbers of LGG adsorbedon to the surface for a probiotic effect. To make LGG even moreresistant, future work could be focused on the possibility ofmicroencapsulating the probiotic bacteria followed by infusion.Probiotic apple slices have shown to be a good alternative for allergicor intolerant people to dairy products and future studies should focusmore on these groups. To increase the possible beneficial effects ofprobiotic bacteria for the host, it could be combined with anappropriate concentration of probiotics.

Acknowledgments

This research (ISAFRUIT project) was part-funded by the EuropeanCommission [Thematic Priority 5 (Food Quality and Safety), 6thFramework Programme of RTD (Contract No. FP6-FOOD 016279)].

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