lable at ScienceDirect LWT - Food Science and Technology 77 (2017) 193e199 Contents lists avai LWT - Food Science and Technology journal homepage: www.elsevier .com/locate/ lwt Development of a potential probiotic lozenge containing Enterococcus faecium CRL 183 Juliana Jabur Polete Witzler a, *, Roseli Aparecida Pinto a, Graciela Font de Valdez b, Ana D�oris de Castro c, Daniela Cardoso Umbelino Cavallini a a Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutriç~ao, UNESP - Univ Estadual Paulista, Araraquara. Rodovia Araraquara Jaú, Km 01, 14800-903, Araraquara, SP, Brazil b Centro de Referência para Lactobacilos (CERELA) e Tucum�an, Chacabucco 145, 4000, San Miguel de Tucum�an, Argentina c Faculdade de Ciências Farmacêuticas, Departamento de F�armacos e Medicamentos, UNESP - Univ Estadual Paulista, Araraquara. Rodovia Araraquara Jaú, Km 01, 14800-903, Araraquara, SP, Brazil a r t i c l e i n f o Article history: Received 11 February 2016 Received in revised form 11 June 2016 Accepted 6 November 2016 Available online 9 November 2016 Keywords: Enterococcus faecium Sensorial analysis Viability Probiotic Streptococcus mutans * Corresponding author. Raposo Tavares Street, Num code 17011-020, Bauru, S~ao Paulo, Brazil. E-mail address: jujabur@yahoo.com.br (J.J.P. Witzl http://dx.doi.org/10.1016/j.lwt.2016.11.011 0023-6438/© 2016 Elsevier Ltd. All rights reserved. a b s t r a c t In this work, a diet lozenge with the microencapsulated probiotic strain Enterococcus faecium CRL 183 was developed. The microbiological, physicochemical and sensorial characteristics were analyzed and the anticariogenic potential of the strain was also evaluated. The results showed that the complex coacervation microencapsulation technique enables higher viability during storage at room temperature. Lozenges were produced through three treatments: PCecontrol formulation; PPP1eprobiotic formula- tion; PPP2eprobiotic formulation with inulin. The probiotic strain had the viability decreased after lozenges production and during the storage from 5.53 to 4.83 log CFU/g at the beginning to 1.05e1.86 log CFU/g after 28 days, for both PPP1 and PPP2. Formulations were different concerning water activity, moisture and color (p < 0.05). All formulations remained microbiologically safe during the storage period. Association of probiotic bacteria and inulin improves the lozenges flavor acceptance and the purchase intent. The anticariogenic evaluation showed that the probiotic bacteria were able to survive in saliva and inhibit the growth of Streptococcus mutans ATCC 25175 in this study. The lozenge with E. faecium CRL183 and inulin represents an alternative to the diversification of the probiotic segment which aggregates anticariogenic potential. However, a process refinement and use of gas resistant packaging to preserve the probiotic viability, is necessary. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Probiotics and oral health have evoked new researches in the last years. Results indicate that probiotics provide important ben- efits to the oral health, preventing caries, helping periodontal and halitosis disease treatment and reducing the amount of bacteria related to oral pathology (Cagetti et al., 2013; Haukioja, 2010). The action mechanism of probiotics in the oral cavity involves the formation of biofilms, immune system modulation and competition with bacteria that cause caries and periodontal dis- eases, such as Streptococcus mutans (Çaglar, Kargul, & Tanboga, 2005). ber 9-72, Apartment 84, zip er). The probiotic strain Enterococcus faecium CRL 183, isolated from Tafí cheese (typical of Tucuman, Argentina), has several proven health effects, as it modulates the lipidic profile, intestinal micro- biota and immune system, reduces colon and breast cancer risks, and improves symptoms related to ulcerative colite (Cavallini et al., 2009, 2011; Celiberto, 2014; Kinouchi, 2006; Rossi, Vendramini, Carlos, Oliveira, & Valdez, 2003, Rossi et al., 2008; Sivieri et al., 2008). It is known that such strain is safe to human consumption and presents gastrointestinal resistance, although, its oral effects have not been investigated so far (Saavedra, Taranto, Sesma, & Valdez, 2003). The viability of probiotic bacteria is important in order to pro- vide health benefits to the consumer. Therefore, the microorganism should survive adverse conditions during processing and storage (pH and temperature variations and oxygen exposure), besides resisting the gastrointestinal transit (Folign, �e, Daniel, & Pot, 2013). Probiotic microorganisms used to maintain the oral health must as mailto:jujabur@yahoo.com.br http://crossmark.crossref.org/dialog/?doi=10.1016/j.lwt.2016.11.011&domain=pdf www.sciencedirect.com/science/journal/00236438 http://www.elsevier.com/locate/lwt http://dx.doi.org/10.1016/j.lwt.2016.11.011 http://dx.doi.org/10.1016/j.lwt.2016.11.011 http://dx.doi.org/10.1016/j.lwt.2016.11.011 J.J.P. Witzler et al. / LWT - Food Science and Technology 77 (2017) 193e199194 well stay longer in the mouth and resist the oral environment. Lozenges are candies manufactured in a simple way, which can deliver a variety of compounds as they disintegrate slowly in the mouth (Edwards, 2001). Considering the time in which lozenges are kept in the mouth, they could start a local effect such as reduction of caries and other oral pathologies risks, representing a potential vehicle for probiotic microorganisms. Microencapsulation, a process that surrounds microorganisms in a polymeric membrane, confers greater protection to probiotic cells, allowing their inclusion in products stored at room temper- ature and their release under specific conditions (Tripathi & Giri, 2014). Thus, this study aimed at developing a probiotic diet lozenge with the addition of microencapsulated and lyophilized E. faecium CRL 183, evaluating its anticariogenic potential, in vitro. 2. Material and methods 2.1. Material Diet lozenges with probiotic strain E. faecium CRL 183 addition (CERELA - Centro de Referência para Lactobacilos, Argentina). 2.2. Methods 2.2.1. Probiotic bacteria 2.2.1.1. Probiotic culture preparation. An aliquot (1.0 mL) from a probiotic stock culture (E.faecium CRL 183) maintained at �80.0 �C in sterilized milk with yeast extract, glucose and glycerol addition, was inoculated into 10.0 mL of Man Rogosa and Sharp Media (MRS) (Acumedia®) and incubated at 37.0 �C, for 16 h. After this, 0.5 mL was again inoculated into 5.0 mL of MRS and incubated at 37.0 �C, for 16 h. 2.2.1.2. Probiotic culture microencapsulation and freeze drying. Two methods were used to microencapsulate E. faecium CRL 183 cells. In method 1 (M1), the extrusion technique was carried out ac- cording to Lee, Cha, and Park (2004), with modifications. The pro- biotic suspension (200.0 g/L, 8.99 log CFU/g) was added to a sterile solution composed by sodium alginate (Synth®, Brazil, 20.0 g/L), glycerol (Synth®, Brazil, 50.0 g/L), MRS broth (Acumedia®, USA, 5.5 g/L), xanthan gum (Synth®, Brazil, 2.6 g/L), tween 20 (Synth®, Brazil, 1.0 g/L) and inulin GR (Beneo®Orafti, Belgium,10.0 g/L). After complete homogenization, this solution was dripped in a sterile calcium chloride (Vetec®, Brazil, 74.0 g/L) solution. After 15min, the beads were rinsed with sterile distilled water. Method 2 (M2) used the complex coacervation technique, car- ried out as described by Baracat, Nakagawa, Freitas, and De Freitas (2004) and Oliveira (2006), with changes. In order to promote coacervation, pectin (Vetec®, Brazil, 20.0 g/L) and casein (Vetec®, Brazil, 20.0 g/L) were dispersed (8% of total solids) in distilled water with constant mechanical stirring. The pH was adjusted to 8.0 (±0.1) with NaOH solution (4.0 M), and then, this dispersion was sterilized. The E. faecium CRL 183 suspension (200.0 g/L, 8.99 log CFU/g) was added to the cooled dispersion, under aseptic condi- tions, and the pH fall monitored (pH ¼ 4.8). The microcapsules obtained through M1 and M2 were frozen at�20.0 �C. Then, the samples were freeze-dried (Modulyod Freeze Dryer, Thermo Electron Corporation), and stored in sterile glass flasks at room temperature (25.0 ± 2.0 �C). 2.2.1.3. Viable cells count. The E. faecium count was performed in free cells and freeze-dried materials (M1 and M2). The analyses were performed immediately after obtaining the freeze-drying microcapsules (T ¼ 0) and weekly, during the storage or by the time the count reached values below 6 log colony forming unity (CFU)/g. Cell viability was determined by the pour plating technique in M17 agar (Difco®, USA), in aerobic conditions at 37.0 �C, for 48 h. The results were expressed as log CFU/g (Rossi et al., 2008). 2.2.2. Production of diet lozenge The diet lozenges were prepared with xylitol (Labonathus®, Brazil), gelatin (Gelita®, Brazil), inulin GR (Beneo® Orafti, Belgium), mint flavor (Firmenich®, Brazil), microencapsulated and freeze- dried Enterococcus faecium CRL 183, and water. Three formulations were defined: control formulation (PC) with xylitol (89.60 g/100 g), water (8.40 g/100 g), gelatin (1.50 g/100 g) and mint flavor (0.50 g/100 g); potentially probiotic formulation 1 (PPP1) with xylitol (88.41 g/100 g), water (8.25 g/100 g), gelatin (1.47 g/100 g), microencapsulated and freeze-dried E. faecium CRL 183 (1.38 g/100 g) and mint flavor (0.50 g/100 g); and potentially probiotic formulation 2 (PPP2) with xylitol (85.66 g/100 g), water (8.25 g/100 g), gelatin (1.47 g/100 g), microencapsulated and freeze-dried E. faecium CRL 183 (1.38 g/100 g), inulin (2.75 g/100 g) and mint flavor (0.50 g/100 g). In order to reduce the amount of E. faecium added to the pro- biotic formulations, the microorganism suspension was previously concentrated by centrifugation. The lozenges were produced in three batches, through the agglutination of ingredients by the gelatin solution, followed by molding and drying at 35.0 �C for 20 h (Edwards, 2001), stored in plastic bags (low density polyethylene) and kept in a temperature- controlled chamber (Fanem®, Biochemical oxygen demand), at 23.0 ± 0.5 �C. 2.2.3. Characterization of the diet lozenge 2.2.3.1. Microbiologic evaluation. Samples containing 25.0 g of each formulation were collected and diluted in 225.0 mL of peptone water. Serial dilutions were subsequently prepared with the same diluent and used for the viability and safety analyses of the lozenges. Countings of E. faecium in the product were weekly enumerated by the pour plating technique, using 1.0 mL of each dilution in M17 agar followed by aerobic incubation at 37.0 �C, for 48 h. The results were expressed in log CFU/g (Rossi et al., 2008). Microbiologic safety assays were determined in triplicate, through yeasts and molds, thermotolerant coliforms and total mesophiles counts. The thermotolerant coliforms analysis were carried out in Petrifilm® (3 M®, USA), while the total mesophiles one was performed in PCA (Plate Count Agar, Himedia , India) and incubated at 37.0 �C, for 48 h. The yeasts and molds analysis was performed in PDA (Potato Dextrose Agar, Himedia , India) and incubated at 30.0 �C, for 120 h. 2.2.3.2. Physicochemical assessments. All formulations were analyzed for their physicochemical characteristics. The pH, water activity, moisture and color were determined fortnightly, in triplicate. The pH was determined using a digital potentiometer (Qualx- tron®, Model 8010), through the dissolution of a 3.0 g-sample in 20.0 mL of distilled water. The water activity (Aw) was measured in a 3.0 g-sample, using a specific device (Aqualab® CX2). The mois- ture content was measured in an infrared analyzer (Sartorius®, MA35). A colorimeter (Hunterlab® Color Quest XE) was used for the assessment of color parameters. 2.2.3.3. Sensory evaluation. The sensory panel was composed of 112 untrained individuals. The acceptance test of attributes Table 1 Viability of the E. faecium CRL 183 during the storage period. Time (days) M1 M2 Inoculum 0 5.74 ± 0.03 a A 8.73 ± 0.06 a B 8.84 ± 0.04 a B 14 3.35 ± 0.05 b A 8.59 ± 0.02 b B 6.97 ± 0.05 b C 28 e 8.65 ± 0.03 abc A 5.06 ± 0.04 c B 42 e 8.59 ± 0.06 bcd A 2.50 ± 0.07 d B 56 e 8.13 ± 0.05 e e 70 e 8.05 ± 0.03 ef e 84 e 8.38 ± 0.02 g e 98 e 8.49 ± 0.05 bdgh e 112 e 8.39 ± 0.02 ghi e 126 e 8.31 ± 0.02 gij e 140 e 8.31 ± 0.02 gijk e 154 e 8.02 ± 0.01 efl e 168 e 8.30 ± 0.01 gijkm e 182 e 8.03 ± 0.05 efl e 196 e 8.31 ± 0.02 gijkm e 210 e 7.43 ± 0.03 n e 224 e 7.57 ± 0.06� e 238 e 7.13 ± 0.05 p e 252 e 7.16 ± 0.02 p e 266 e 6.93 ± 0.01 q e 280 e 5.93 ± 0.02 r e Results are presented as means ± standard deviation, in log CFU/g. Means in the same column followed by different lowercase letters are significantly different by the Tukey's test (p < 0.05) e M2 method and inoculums. Means in the same column followed by different lowercase letters are significantly different by the T-student test (p < 0.05) e M1 method. Means in the same line followed by different capital letters are significantly different by the T-student test (p < 0.05). M1 - E.faecium microencapsulated by the extrusion technique followed by freeze- drying. M2 - E.faeciummicroencapsulated by the complex coacervation technique followed by freeze-drying. Inoculum e free cells of the E.faecium. J.J.P. Witzler et al. / LWT - Food Science and Technology 77 (2017) 193e199 195 (appearance, color, flavor, taste, texture and overall acceptance) using a 9-point hedonic scale (1¼ disliked very much and 9¼ liked very much) and purchase intent test using a 5-point scale (1 ¼ would not certainly buy and 5 ¼ would certainly buy) (Meilgard, Civille, & Carr, 1988; Stone & Sidel, 1993) were per- formed immediately after lozenges processing. The consumers evaluated 3 formulations in a session. Each sample was coded by using a 3-digit random number and presented in a monadic form (approved by the Research Ethics Committee of the UNESP School of Pharmaceutical Sciences, Araraquara-Brazil, protocol number 661487). 2.2.4. Anticariogenic potential evaluation The probiotic strain survival capacity in saliva was evaluated in PPP1 and PPP2 samples and in free cells. Such analyses was carried out, as described by Haukioja (2009), with modifications. The saliva donated by a healthy volunteer was sterilized at 121.0 �C for 15 min and 1.0 g-samples of PPP1 and PPP2 formulations and 1.0 mL of free cells were inoculated into 10.0mL of saliva and incubated at 37.0 �C, for 24 h. E. faecium counts were enumerated in triplicate, at the beginning (T0 ¼ before incubation) and after 24 h of incubation, by pour plating technique, using 1.0 mL of each dilution in M17 agar, followed by aerobic incubation at 37.0 �C, for 48 h. The results were expressed in log CFU/mL (Rossi et al., 2008). The E. faecium CRL 183 ability to inhibit the multiplication of S. mutanswas measured with an agar well diffusion assay (Lewus& Montville, 1991). A 1.0 mL aliquot of Streptococcus mutans ATCC 25175 suspension cells (8.26 log CFU/mL) was inoculated into 10.0 mL of BHI broth (Brain heart infusion, Difco®, USA), and 1.0 mL of E. faecium cells (8.13 log CFU/mL) was inoculated into 10.0 mL of MRS. Both were incubated at 37.0 �C, for 16 h, followed by centri- fugation at 7000 rpm, for 10 min. The supernatant was discarded and the biomass re-suspended in 10.0 mL of peptone sterile water. To proceed with the well diffusion test, a layer of LAPTg medium (10.0 g yeast, 15.0 g peptone, 10.0 g glucose, 10.0 g tripton, 1.0 mL tween 80, 1.5 g agar and 70.0 g sucrose) was placed in triplicates of petri plates. After its complete solidification, 10.0 mL of a softer overlay of LAPTg medium (0.75 g agar) containing 10.0 mL of S. mutans was deposited in the plates. Following its solidification, 30.0 mL of E. faeciumwas deposited in a 5mm diameter well and the plates were incubated in aerobiose at 37.0 �C, for 24 h, the inhibi- tion halo being measured in millimeters. 2.2.5. Statistical analysis The results were expressed as mean ± standard deviation and statistical analyses of the averages were calculated by Analysis of Variance, Tukey and T-student test (p < 0.05), using the Biostat software. 3. Results and discussion 3.1. Viable cells counts It was established that the probiotic microorganism viability would be observed until reaching values below 6.0 log CFU/g and also, that the amount of probiotic cells added to the final product could be inferior to the range (8.0e9.0 log CFU in the daily portion) preconized in international surveys and local guides (Anvisa, 2008). The following aspects were considered: 1) the amount of inoculum added to the final product should be minimum so as to avoid un- pleasant sensory characteristics, and 2) the probiotic effect would be local, without the exposure to the conditions found in the gastrointestinal tract, therefore, an amount lower than the one suggested would be enough to observe the effects. The viability of E. faecium free cells, microencapsulated by complex coacervation (M1) and extrusion technique (M2) followed by freeze-drying is shown in Table 1. At first (T ¼ 0), the population of free cells was 8.84 log CFU/g. After the microencapsulation by complex coacervation (M2), fol- lowed by freeze-drying, the count was 8.73 log CFU/g, showing that the technique did not affect the E. faecim survival (p < 0.05). Only after 210 days of storage, 1 log cycle reduction was observed in the probiotic cells population (M2). The results obtained show that the complex coacervation technique was able to protect the strain, for 273 days, since only at the 280th day of storage the viability reached values below 6.0 log CFU/g. This long storage period was superior to that of Oliveira (2006), who also used complex coacervation to microencapsulate probiotic strains. The viability of E. faecium microencapsulated by the extrusion technique (M1) followed by freeze-drying, suffered substantial reductions of 3.1 log at the beginning (T ¼ 0) and 5.49 log after 14 days of storage. Therefore, the extrusion technique is not suitable to protect E. faecium CRL 183 cells. The use of alginate as a wall material results in porous beads, which can allow the passage of internal material to the external environment (Mandal, Hati, Puniya, Khamrui, & Singh, 2014; Rokka & Rantamaki, 2010). Thus, we hypothesize that E. faecium passed to the external environment reducing the microcapsules' viability. This result differs from that of Haghshenas et al. (2015) who observed higher Lactobacillus plantarum viability in capsules ob- tained through the extrusion technique, using at least 1.5% of alginate as a wall material. The present study used 2.0% of alginate in the wall material composition, however, since the effect and performance of a bacterium are strain dependent, it is not possible to observe the same results for strains in the same species (Sha, 2007). J.J.P. Witzler et al. / LWT - Food Science and Technology 77 (2017) 193e199196 E. faecium free cells had their viability reduced below 6.0 log CFU/g after 28 days of storage at room temperature. Comparing such a result to the one achieved with complex coacervation, it is possible to say that the technique associated with freeze-drying raised the probiotic viability in 200 days. Thus, the coacervation technique (M2) was selected for the next step of the study. 3.2. Lozenges preparation The viable counts of the concentrated and microencapsulated E. faecium (M2) before being added to the probiotic lozenge for- mulations, were 10.62 log CFU/g. It was added in enough quantity to reach 7.0 log CFU/g of lozenges in formulations PPP1 and PPP2. Considering that the average weight of each lozenge is 1.5 g, the ingestion of one unit would not be enough to reach the viable cell counts of 108e109 CFU/g recommended in the daily portion of the final product. It is important to emphasize that probiotic lozenges focus would be a local effect in the oral cavity. In such cases, lower viable counts would be accepted if the benefits were proved. 3.3. Diet probiotic lozenges characterization 3.3.1. Microbiological evaluations 3.3.1.1. Viable counts. The survival of E. faecium in the lozenges formulations is shown in Table 2. The viable counts in both PPP1 and PPP2 formulations were reduced to values below 6.0 log CFU/g at the initial time (T ¼ 0). The viable counts in PPP1 formulation suffered a reduction superior to 2.0 log after 7 days of storage, whereas a reduction of 1.53 log was observed after 21 days in formulation PPP2, which might indicate a protective inulin effect. However, this trendwas notmaintained until the end of the storage period. After 28 days, the bacteria viability was severely reduced in both probiotic formulations, thus, the viability follow up ended. The rapid loss of probiotic viability was not expected, since the microencapsulated E. faecium presented high viability for nine months, at room temperature. Several factors can influence the survival of probiotic bacteria including the processing temperature, oxygen incorporation, pH, mechanic stress, raw material and in- gredients (Shah, 2000). Lozenges preparation stages do not involve high temperature, oxygen incorporation or mechanic stress and the pH was close to neutrality (6.23e6.28), thereby, some ingredients were investigated. Possibly, the flavoring used could have played a role in probiotic survival, since alcohol is a common ingredient in flavoring com- positions. Thus, lozenges formulations PPP1 and PPP2 were pro- cessed againwithout it. However, the viability loss was very similar to that observed in formulations processed with flavoring. Xylitol is a polyol which is widely used as a sweetener by the food industry, and like other polyols and disaccharides it plays a Table 2 Viability of the E. faecium CRL 183 added to lozenges during the storage period. Time (days) PPP1 PPP2 0 5.53 ± 0.05 a A 4.83 ± 0.01 a B 7 3.35 ± 0.02 b A 4.15 ± 0.01 b B 14 2.71 ± 0.07 c A 4.01 ± 0.01 c B 21 2.52 ± 0.07 d A 3.30 ± 0.07 d B 28 1.86 ± 0.07 e A 1.05 ± 0.08 e B Results are presented as means ± standard deviation, in log CFU/g. Means in the same column followed by different lowercase letters are significantly different by the Tukey's test (p < 0.05). Means in the same line followed by different capital letters are significantly different by the T-student test (p < 0.05). PPP1 - Potentially probiotic formulation 1, with the E.faecium CRL 183. PPP2 - Potentially probiotic formulation 2, with the E.faecium CRL 183 þ inulin. role in cells protection during the drying process and storage period (Carvalho et al., 2004). Studies relating the use of polyols to pro- biotic viability loss were not found. Further studies are necessary to evaluate the E. faecium viability in relation to each ingredient present in lozenge formulations, so as to identify the ingredient involved in viability loss. The material used for wrapping the lozenges is another impor- tant factor that may be related to the decrease of probiotic cells viability. Polyethylene presents an excellent barrier to water vapor, however, it is permeable to gases (Jorge, 2013). The passage of oxygen from the external environment to the interior of the pack- age may have caused E. faecium viability loss in the lozenges. This interaction of oxygen with the probiotic strain probably was minimized during the storage of the microcapsules of E. faecium obtained by coacervation, considering that glass has excellent barrier properties and is impermeable to vapors and gases (Jorge, 2013). The results accomplished in the present study were different from those of Toiviainen et al. (2014), who used xylitol and sorbitol tabletes as Lactobacillus rhamnosus and Bifidobacterium lactis vehicle, focusing on the oral microbiota of adults. The viability of such probiotic tablets was maintained at 8.0 log CFU/g, during the 4 weeks of treatment. Çaglar, Kuscu, Cildir, Kuvvetli, & Sandali, (2008) used isomalt lozenges as Lactobacillus reuteri vehicles, focusing on the oral microbiota of young women. According to the study, the lozenges viability was 8.0 log CFU/g during the 10 days of treatment. 3.3.1.2. Microbiological safety. Microbiological counts during the storage of lozenges remained below 5.0 log CFU/g for total meso- philes; 4.0 log CFU/g for yeasts and molds; and 3NMP/ml for total and thermotolerant coliforms. These results confirm the suitability of processing operations and conformity to the local microbiolog- ical patterns of the category (Anvisa, 2001). 3.3.2. Physicochemical evaluations The results of physicochemical analyses of diet lozenges are shown in Table 3. The water activity, moisture and color values presented differences (p < 0.05) among formulations PC, PPP1 and PPP2. The lowest levels of water activity and moisture observed in formulations PPP1 and PPP2 are probably related to a lower water concentration (8.25 g/100 g), owing to the addition of the probiotic culture and inulin, which can reduce the water availability. The water activity range suggested for lozenges is 0.40e0.75 (Bussiere & Serpelloni, 1985). It is known that water activity values around 0.80 present susceptibility to yeast and mold growth. Since food components are concentrated, values below 0.60 are recom- mended in order to avoidmicrobial development (Bobbio& Bobbio, 2001). The addition of the probiotic strain and inulin made the loz- enges darker. Color analysis results proved such a fact, since the control formulation (PC) exhibited higher averages for parameter L* and lower ones for parameters a* and b* (p < 0.05). 3.3.3. Sensory evaluation The sensory assessment of lozenges was conducted at the initial time of storage. As an unexpected rapid loss of viability was observed in formulations PPP1 and PPP2, it was not possible to conduct another sensory assessment. The acceptability and pur- chase intent are shown in Table 4 and Fig. 1, respectively. All formulations had the attributes scores above 6.0, ranging from “liked slightly” and “liked moderately”, however, formulation PPP2 achieved 5.75 and 5.68 for the appearance and color. Con- sumers have declared a positive purchase intent (would certainly or probably buy the product), especially for PC and PPP2 formulations Table 3 Physicochemical characterization for the three lozenges formulations (PC, PPP1 and PPP2). PC PPP1 PPP2 Water activity 0.79 ± 0.01A 0.77 ± 0.02 B 0.75 ± 0.02 C Moisture (%) 2.31 ± 0.07A 2.12 ± 0.07 B 1.99 ± 0.06 C pH 6.28 ± 0.08A 6.25 ± 0.05 A 6.23 ± 0.07 A Color L* 92.77 ± 0.90 A a* �0.20 ± 0.07 A b* 1.30 ± 0.08 A L* 85.84 ± 0.92 B a* 0.38 ± 0.09 B b* 7.00 ± 0.29 B L* 88.64 ± 0.78 C a* 0.51 ± 0.09 C b* 8.13 ± 0.39 C Results are presented as means ± standard deviation. Means in the same line followed by different capital letters are significantly different by the Tukey's test (p < 0.05). PC e Control formulation, without the probiotic strain. PPP1 - Potentially probiotic formulation 1, with the E.faecium CRL 183. PPP2 - Potentially probiotic formulation 2, with the E.faecium CRL 183 þ inulin. L* ¼ luminosity, from black to white. a* ¼ from green to red. b* ¼ from blue to yellow. Table 4 Sensory evaluation of lozenges formulations PC, PPP1 and PPP2. PC PPP1 PPP2 Appearance 7.26 ± 1.43 A 6.11 ± 1.53 B 5.75 ± 1.51 B Color 7.59 ± 1.2 A 6.13 ± 1.49 B 5.68 ± 1.48 B Flavor 6.40 ± 1.25 A 6.32 ± 1.42 A 6.47 ± 1.56 A Taste 6.18 ± 1.71 A 6.43 ± 1.67 A 6.97 ± 1.40 B Texture 6.09 ± 1.79 A 6.23 ± 1.63 A 6.61 ± 1.44 A Overall Acceptance 6.58 ± 1.49 A 6.21 ± 1.39 A 6.58 ± 1.21 A Results are presented as means ± standard deviation. Means in the same line followed by different capital letters are significantly different by the Tukey's test (p < 0.05), n ¼ 112. PC e Control formulation, without the probiotic strain. PPP1 - Potentially probiotic formulation 1, with the E.faecium CRL 183. PPP2 - Potentially probiotic formulation 2, with the E.faecium CRL 183 þ inulin. J.J.P. Witzler et al. / LWT - Food Science and Technology 77 (2017) 193e199 197 (48.6% and 62.2%, respectively). Formulations PPP1 and PPP2 had inferior averages on appear- ance and color (p < 0.05) as compared to PC. Such result reflects the addition of microencapsulated and freeze-dried E. faecium which changes the lozenges color and could have been perceived as a defect. Flavor, texture and global impression had no differences Fig. 1. Lozenges Purchase intent. PC e Control formulation, without probiotic strain. PPP1 - Potentially probiotic formulation 1, with E. faecium CRL 183. PPP2 - Potentially probiotic formulation 2, with E. faecium CRL 183 þ inulin. among the formulations (p < 0.05). The addition of probiotic associated with inulin improved taste perception, indicating that the changes in appearance and color had no negative effect on this attribute. This was confirmed by the purchase intent, where formulation PPP2 had the lowest negative results, that is, 12.7% of consumers would not probably or certainly buy the product. Satisfactory results have been found in recent studies with different categories of food products and the addition of probiotic microorganisms (Lalicic-Petronijevic et al., 2015; Pimentel, Madrona, & Prudencio, 2015). However, no studies relating the effect of probiotic bacteria addition on the sensory characteristics of lozenges or similar products, were found. 3.4. Anticariogenic potential evaluation The E. faecium CRL 183 survival in saliva is shown in Table 5. The probiotic strain was able to propagate in saliva in the three forms evaluated: free cells, PPP1 and PPP2 lozenges formulations. The viable counts observed after 24 h increased 0.82 log CFU/ml in the free cells, whereas in formulations PPP1 and PPP2 they increased 3.64 and 3.31 log CFU/ml, respectively, showing that E. faecium CRL 183 resists human saliva. Haukioja (2009) evaluated the survival of Table 5 Viability of E. faecium CRL 183 inoculated in saliva. t ¼ 0 t ¼ 24 h PPP1 5.45 ± 0.03 a A 9.09 ± 0.02 a B PPP2 5.66 ± 0.06 b A 8.97 ± 0.03 b B Inoculum 8.04 ± 0.06 c A 8.86 ± 0.03 c B Results are presented as means ± standard deviation, in log CFU/g. Means in the same column followed by different lowercase letters are significantly different by the Tukey's test (p < 0.05). Means in the same line followed by different capital letters are significantly different by the T-student test (p < 0.05). PPP1 - Potentially probiotic formulation 1, with the E.faecium CRL 183. PPP2 - Potentially probiotic formulation 2, with the E.faecium CRL 183 þ inulin. J.J.P. Witzler et al. / LWT - Food Science and Technology 77 (2017) 193e199198 various probiotic strains in saliva and all were viable after the inoculation, but none presented increases. No studies were found in the literature reporting that the presence of xylitol could stimulate the probiotic multiplication, although, it is known that such ingredient can reduce the cell multiplication rate and acid production, leading to a reduction in the S. mutans population (Marsh & Martin, 2005). The inhibition potential of E. faecium CRL 183 in relation to S. mutans was proved through the formation of a 1.7 ± 0.3 mm diameter halo (Fig. 2), showing that the probiotic strain inhibited S. mutans growth under these conditions, thus, reducing the dental caries risk. Local and systemic effects of probiotic microorganism are related to the inhibition of some pathogenic strains, by the competition for binding sites, adhesion reductions, co-aggregation, production of organic acids, hydrogen peroxide and/or bacteriocin- like compounds and immune-modulation (Cagetti et al., 2013; Hassanzadazar, Ehsani, & Mardani, 2014). Redondo (2008) showed that E. faecium CRL 183 and Lactobacillus helveticus 416 have the ability of auto-aggregation (80.0%), co-aggregation (25.4%), besides inhibiting the E. coli 0157:H7 adhesion to intesti- nal cells Caco 2. The same strains did not produce antimicrobial substances in relation to different pathogenic strains (E. coli 0157:H7, L. monocytogenes V2 and S. enteritidis). In the present study, it is not possible to state the exact mech- anism involved in the inhibition of S. mutans, since specific tests have not been conducted. The anticariogenic effect should be better investigated through analyses involving the oral microbiota and biofilm formation. Fig. 2. Agar well plates diffusion assay, with the inhibition halo. Indicator strain: (inoculated in the culture medium): Streptococcus mutans ATCC 25175. Probiotic strain (inoculated in the well): Enterococcus faecium CRL 183. 4. Conclusions The present study showed that the complex coacervation technique was efficient in preserving the E. faecium CRL 183 viability, at room temperature. However, the food matrix was not suitable for the probiotic veiculation, due to the severe loss of viability in formulations PPP1 and PPP2. The probiotic strain investigated was able to survive and multiply in human saliva and also inhibit the multiplication of Streptococcus mutans ATCC 25175, in the study conditions. 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Introduction 2. Material and methods 2.1. Material 2.2. Methods 2.2.1. Probiotic bacteria 2.2.1.1. Probiotic culture preparation 2.2.1.2. Probiotic culture microencapsulation and freeze drying 2.2.1.3. Viable cells count 2.2.2. Production of diet lozenge 2.2.3. Characterization of the diet lozenge 2.2.3.1. Microbiologic evaluation 2.2.3.2. Physicochemical assessments 2.2.3.3. Sensory evaluation 2.2.4. Anticariogenic potential evaluation 2.2.5. Statistical analysis 3. Results and discussion 3.1. Viable cells counts 3.2. Lozenges preparation 3.3. Diet probiotic lozenges characterization 3.3.1. Microbiological evaluations 3.3.1.1. Viable counts 3.3.1.2. Microbiological safety 3.3.2. Physicochemical evaluations 3.3.3. Sensory evaluation 3.4. Anticariogenic potential evaluation 4. Conclusions Acknowledgments References