Lactobacillus plantarum survival during the osmotic dehydration and storage of probiotic cut apple

Journal of Functional Foods 38 (2017) 519–528

Contents lists available at ScienceDirect

Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Lactobacillus plantarum survival during the osmotic dehydration and storage of probiotic cut apple Kassandra Emser, Joana Barbosa, Paula Teixeira, Alcina Maria Miranda Bernardo de Morais ⇑ Universidade Católica Portuguesa, CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital, 172, 4200-374 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 28 June 2017 Received in revised form 4 September 2017 Accepted 13 September 2017

Keywords: Probiotic Lactobacillus plantarum Osmotic dehydration Sorbitol Water activity Colour

a b s t r a c t The feasibility to incorporate Lactobacillus plantarum in apple cubes during the osmotic dehydration (OD) was investigated. The effects of 40 and 60 °Brix osmotic solutions of sucrose or sorbitol on the viability of L. plantarum during the OD at 37 °C and 1013 or 150 mbar was evaluated. The storage at 4 °C and a quick simulation (2 h) of the digestion of probiotic apple cubes through the gastro-intestinal tract were also performed and the viability of the probiotic evaluated. Lactobacillus plantarum got incorporated in the osmotically apple cubes (107–108 cfu/g) with preference for 40 °Brix solutions and it maintained the viability of 107 cfu/g during a 6 day-storage at 4 °C. L. plantarum also survived (107 cfu/g) during the simulation of the digestion. Colour changes of the probiotic apple cubes occurred after OD and storage. Therefore, osmotically dehydrated apple cubes incorporated with L. plantarum could be a new probiotic food. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Conventional foods containing probiotics often originate from dairy products as yoghurt and kefir. As the demand for probiotic functional products and the preference of consumers for dairyfree products are increasing, studies have been performed to incorporate probiotics also into several types of processing of fruits and vegetables. Non-dairy probiotic products, besides supplements, include fruit and vegetable drinks, fruit purees and pieces, jam and powders, chocolate and even flan (Barbosa, Borges, & Teixeira, 2015; Borges et al., 2016). Different fruits, e.g. banana, strawberry, mango and many others, have been investigated for their suitability as probiotics carriers. The apple fruit matrix was shown to be highly applicable for probiotics, may be because of its high porosity and, therefore, easy incorporation of probiotics (Espírito Santo et al., 2012). Another explanation may be associated to the cellulose in apple, which is not digested and could serve as a protective matrix for probiotics through the intestinal tract (Kourkoutas, Kanellaki, & Koutinas, 2006). Rêgo et al. (2013) demonstrated the compliance of apple as a fruit matrix for probiotic survival over time, as they studied hot air-dried apple cubes with incorporated L. plantarum during 65 days of storage and found only minimal viability losses of 1 log cfu/g. ⇑ Corresponding author. E-mail address: [email protected] (A.M.M. Bernardo de Morais). https://doi.org/10.1016/j.jff.2017.09.021 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

Among other probiotics, the Gram-positive and aerotolerant L. plantarum has been extensively studied and is nowadays one of the most commonly used probiotic in the production of functional food. Traditionally it is found in fermented plant products, such as sauerkraut, Korean kimchi and sourdough (Lee & Lee, 2010; Vries, Vaughan, Kleerebezem, & Vos, 2006; Vuyst et al., 2014). Lactobacillus plantarum 299 v is marketed for probiotic functional foods production as for example probiotic drinks and capsules (Shah, 2001; Tuzen, 2017; UltraFlora, 2017). Lactobacillus plantarum 299 v originates from the intestinal human mucosa and is sold as an especially beneficial probiotic because of its claimed adherence and following colonization on gastrointestinal cells, which is of main importance to be considered as a probiotic bacterium (Probi AB, Sweden, 2017; Vries et al., 2006). The market for fresh-cut fruits has been growing in recent years induced by consumer demand for food products that are fresh-like, healthy minimally processed, nutritious and convenient (FAO, 2010). To maintain the quality of these fresh-cut products techniques are required to preserve organoleptic characteristics and to guarantee a reasonable shelf life. Osmotic dehydrated apple containing probiotics would be suitable for the growing market of fresh-cut or minimally processed fruits. The application of freshcut fruits into other products brings new possibilities to the food industry, which have been barely exploited yet. In osmotic dehydration, pre-treated sliced fruits are immersed into an osmotic solution, for example, a sugar syrup (fruits) or a

520

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

salt solution (vegetables) for a certain period of time at a temperature above room temperature and until 60 °C (Chavan & Amarowicz, 2012). The difference of the osmotic pressure between the food and the solution results in the diffusion of water from the food into the solution, and diffusion of the solute from the solution to the food (An et al., 2013). The osmotic dehydrated products have a moisture content around 10–40%, an aw between 0.6 and 0.9 and an extended shelf life (Barbosa-Cánovas, Fontana, Schmidt, & Labuza, 2007). For example, intermediate moisture products with an aw from 0.75 to 0.85 are shelf stable for at least 90 days at refrigeration conditions, with an optimum temperature of 3–5 °C (Nayyar et al., 2002). Osmotic dehydration (OD) offers many advantages compared to other techniques of fruit preservation. With the applied low temperature, organoleptic characteristics of the product, including flavour and colour, are retained. The activity of PPO and, therefore, the enzymatic browning are inhibited because the products are immersed in the solution, not exposed to O2. In addition, the incorporated sugar limits PPO activity. After OD, a sweeter, lower acid product, with the same shape and less volume, is obtained. A sweeter product may be favourable for consumers and a decreased volume reduces further costs of processing, storage and transport. The entire process is simple and more economic than the conventional hot air drying (Chavan & Amarowicz, 2012). Huerta-Vera et al. (2017) used OD with 40, 50 and 60 °Brix sucrose solutions at 35 °C to enrich banana slices with Lactobacillus rhamnosus encapsulated in a double emulsion. These authors used a vacuum pulse of 50 mbar at the beginning of the osmotic process and L. rhamnosus got incorporated and survived at levels above 107 cfu/g in the osmodehydrated bananas. Flores-Andrade et al. (2017) also studied the effect of vacuum at the same temperature on the impregnation of these L. rhamnosus microcapsules in apple slices using OD with the same sucrose solutions, and they found a higher impregnation with an initial vacuum pulse of 20 min. In addition, the survival of probiotics decreased with increasing osmotic pressure of the solution. The number of viable cells in the osmodehydrated apple was in the range 106–108 cfu/g d.b. Genevois, de Escalada Pla, and Flores (2017) studied the effects of simultaneous fortification of iron and Lactobacillus casei on pumpkin tissues. In this study, the probiotic concentration remained above 107 cfu/g for 14 days at 8 °C, but the viability was affected by the mineral incorporation. In an osmotic dehydration process with fruits, normally a sucrose solution is used as the osmotic agent. Another option can be a sorbitol solution, which has been studied recently by Assis, Morais, and Morais (2017). In the performed study, the initial osmotic dehydration rate of apple samples with a sorbitol solution was higher compared to a sucrose solution with the same soluble solids content (Assis et al., 2017). Sorbitol has 60% of the sweetness of sucrose and 2.4 kcal/g compared with sucrose, which contains 4 kcal/g (SPI Polyols, 2017). Sorbitol is a low-cariogenic substance compared to sucrose, because, when consumed in low amounts, it does not decrease the pH of the plaque enamel, which may lead to demineralisation (Burt, 2006). In addition, sorbitol is considered a low-digestible carbohydrate and low-glycemic, because it is more slowly absorbed in the small intestine and, consequently, not fully digested. With the resulting lower caloric value, sorbitol could be a helpful component in diets for consumers suffering from diabetes or trying to reduce and stabilize weight (Livesey, 2003). Sorbitol may also play a role as prebiotic. Prebiotics are indigestible polyand oligosaccharides, which are the main substrate for beneficial microorganisms and selectively promote their growth, composition and activity in the gastrointestinal tract (Roberfroid, 2007). As prebiotic, sorbitol has a positive impact on the general bowel function by stimulating the growth of probiotics and preventing

pathogenic bacteria. It can also be associated with the cancer prevention, the decrease of LDL-cholesterol, the improvement of the immune system and the production of bacteriocins (Bielecka, Biedrzycka, & Majkowska, 2002; Mandal, Sen, & Mandal, 2009; Schley & Field, 2002). However, as sorbitol is only partly used by the microorganisms in the gastro-intestinal tract, an elevated intake of sorbitol may cause laxation by increasing water content of the stool. Moreover, gastro-intestinal symptoms as diarrhea, abdominal pain, cramps and flatulence were reported after an overload consumption of sorbitol (Livesey, 2001). An acceptable daily intake for sorbitol has not been estimated, because the substance has been approved and found to be non-toxic with a laxation threshold of 50 g/day (Zumbe, Lee, & Storey, 2001). Nevertheless, consuming more than 20 g/day of this polyol is not recommended and an intake of only 7–14 g/day can already exert adverse effects in some individuals (Burt, 2006; Rowe, Sheskey, & Quinn, 2009). The objective of this study was to investigate if probiotics can be incorporated during the OD of fruits, focussing on L. plantarum and apple as the fruit for OD. In addition, the impact of sucrose and sorbitol as osmotic agents on the incorporation was also studied. The third purpose was the examination of the viability of L. plantarum in the apple cubes during storage and to determine quality changes in terms of colour during the OD and storage. The viability of the probiotic was also evaluated after a quick simulation of the digestion of probiotic apple cubes through the gastro-intestinal tract. 2. Materials and methods 2.1. Probiotic strains and growth conditions The probiotic culture L. plantarum 299 v were purchased from Probis Probiotika (Lund, Sweden). The bacteria strains were grown aerobically on de Man, Rogosa and Sharpe (MRS) agar (Lab M, Bury, UK) at 37 °C for 24 h and stored at
80 °C in MRS broth (Pronadisa, Madrid, Spain) containing 30% v/v glycerol. Before use, each strain was sub-cultured twice in 10 mL MRS broth (Lab M). 2.2. Sample preparation 2.2.1. Apple sample Apples, variety ‘Royal Gala’, were graciously provided from Campotec, Portugal and stored at 4 °C. The fruits (3–5 aleatory apples) were washed for 5 min in an aqueous 7500 ppm active chlorine solution and then cut in cubes (12  12  12 mm) with a vegetable cutter (Secret de Gourmet, France). The apple cubes were immersed in sterile Ringer’s solution (Merck, Darmstadt, Germany) for 3 min to prevent enzymatic browning and residual sodium solution was then removed from the surface of the samples by drying the cubes carefully with tissue paper. In order to determine the soluble solids content of the apples, around 100 g fresh apple was mixed with a handmixer and the resulting juice was measured with a hand refractometer (Atago, Guangzhou, China) before carrying out the OD. The soluble solids content of the apple was 15.3 ± 1.3 °Brix. 2.2.2. Preparation of inoculum From MRS agar incubated at 37 °C for 24 h, one colony of each probiotic was transferred to 10 mL of MRS broth and incubated in the same conditions. For the final inoculum, 0.1 mL of the last culture was transferred to 10 mL of MRS broth (1:100) and incubated at 37 °C for 24 h to reach stationary phase. The probiotic culture was centrifuged for 10 min at 7000 rpm and 20 °C (Hettich Zentrifugen Rotina 35R, Tuttlingen, Germany). The supernatant

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

was discarded and the cell pellets were washed twice in sterile Ringer’s solution (Merck, Darmstadt, Germany) and centrifuged under the same conditions. The pelleted cells were re-suspended in 10 mL Ringer solution. 2.3. Osmotic dehydration 2.3.1. Osmotic solutions Osmotic solutions of 20, 40 and 60 °Brix were prepared with commercial sucrose and sorbitol (Fagron Iberica, Barcelona, Spain) in deionized water. Each osmotic solution was autoclaved (JSM, Model JSM 75L PL, Portugal) at 121 °C during 15 min in glass flasks before being used. 2.3.2. Osmotic dehydration with L. Plantarum in the osmotic solution The inoculum of Lactobacillus plantarum 299v was prepared as described above, but instead of re-suspending the pelleted cells in Ringer solution, all the pellet was re-suspended in the same volume of sterile osmotic solution to obtain the maximum concentration of inoculum in the solution. Apple cubes, prepared as described above, were immersed into solutions of 40 and 60 °Brix of sucrose and sorbitol (with resuspended probiotic) with a mass ratio of apple to the solution of 1:4. These conditions were based on a previous study, which had identified the mass ratio of sample to solution of 1:4 as an alternative to 1:10 in the OD at the atmospheric pressure, as lower quantities of osmotic solution and, therefore, solute were required to carry out the OD process to the same level of dehydration (Assis et al., 2017). Each sample (approx. 25 and 100 g of apple cubes, for experiments at atmospheric and vacuum pressures, respectively) was placed in the osmotic solution with L. plantarum in a hermetic container, which were kept in a water-bath at 37 °C and 50 rpm, during 24 h. The osmotic dehydration was carried out at atmospheric pressure and under vacuum (150 mbar). The impregnation of the probiotic took place during OD. All experiments were performed in duplicate. 2.4. Probiotic enumeration Enumeration was performed in the apple samples and osmotic solutions, in order to know if L. plantarum were incorporated in apple samples or, if not, if they were viable in the osmotic solution after OD. To enumerate probiotics in the apple samples, one apple cube (approx. 1 g) was washed in sterile deionized water and blotted gently with tissue paper, and added to 9 mL of sterile Ringer’s solution in a stomacher bag. After trituration of the apple sample in a stomacher (BagMixerÒ 400 P, Interscience, France) for 4 min, serial decimal dilutions were performed in sterile Ringer’s solution. From osmotic solutions, 1 mL was added to 9 mL of sterile Ringer and serial decimal dilutions performed. Samples of the apple cubes and the osmotic solution were taken at t = 0, 6 and 24 h (for vacuum, only at t = 0 and 24 h). All samples of apple and solution were treated as described previously. Each sample and respective dilutions were plated on MRS agar, in duplicate, by the drop count technique (Miles, Misra, & Irwin, 1938). Colony counting was performed after incubation at 37 °C for 24 h. The experiments were performed in duplicate.

521

placing approximately 3.5 g of apple sample in the measuring container. The aw of the initial apple cubes was 0.993 ± 0.003. The moisture content and the dry matter of the apple samples were determined by placing them in an oven (Binder, Germany) at 105 °C for 24 h (until constant weight). The moisture content of the initial apple was 87.3 ± 1.0%. Two measurements were performed for each duplicate. 2.6. Storage After 24 h-OD, around 6 g apple samples were taken from each solution and stored in individual petri dishes sealed with parafilm (Bemis Company Inc., Neenah, Wisconsin, USA) at 4 °C for 6 days to study the L. plantarum survival in the samples and the colour changes. Enumeration of L. plantarum was performed on days 4 and 6 as described above (Section 2.4). Water activity and moisture content were determined at the end of storage. 2.7. Colour determination The colour of fresh and 24 h-osmotically dehydrated apple samples was evaluated with a colorimeter (Minolta. Chromameter CR300, Osaka, Japan). The colour of the latter samples was also evaluated on the 4th and 6th days of storage. Lightness (L⁄), Redness (a⁄) and Yellowness (b⁄) were measured. The total colour difference (DE) was calculated by the following equation:

DE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2   2 ðL0
L Þ þ ða0
a Þ2 þ ðb0
b Þ

ð1Þ

the index ‘‘0” indicating the sample before OD (t = 0). Tenfold measurements were performed. 2.8. Gastro-intestinal tract simulation 2.8.1. Inoculum After a 24 h-OD at 37 °C and atmospheric pressure and a 4 daysstorage at 4 °C, apple cubes incorporating L. plantarum were used as inoculum in the experiment of a simulation of the gastrointestinal tract. 2.8.2. Simulated gastro-intestinal conditions A simulation of the gastro-intestinal tract was performed following the method described by Barbosa et al., 2015, with some modifications. One apple cube (approx. 1 g) from each sample osmotically dehydrated with 40 or 60 °Brix sucrose or sorbitol solutions at atmospheric pressure was triturated in a stomacher for 120 s and placed in a glass flask with 49.0 mL of Buffered Peptone Water (BPW, Lab M) adjusted to pH 3.0 with Hydrochloric Acid (1 M HCl, Pronalab) and with 1000 units/mL of a filter sterilized solution of pepsin (Sigma) to simulate the stomach conditions. Samples were taken at time 0 (time of inoculation) and after 30 min and 60 min (quick gastric transit simulation). To simulate the conditions of the small intestine, a sterile solution of bile salts was added (final concentration of 0.3% (w/v), Pronadisa), after increasing the pH from 3.0 to 7.0 with a sterile solution of Sodium Hydroxide (1 M NaOH, Pronalab). Again, samples were taken at time 0 (time of bile salts addition) and every 30 min for a total of 60 min (quick digestion simulation). Enumeration of survivors was done as described in Section 2.4. Two replicates were performed.

2.5. Water activity and moisture content determination 2.9. Statistical analysis The water activity (aw) was determined at a constant temperature of 21 ± 1 °C, before and after OD, with a hygrometer (Aqualab Series 3, Decagon Devices Inc., Pullmam, Washington, USA), by

The statistical analysis was performed using IBM SPSSÒ Statistics 20.0 for WindowsÒ (2012, SPSS Inc., Chicago, USA).

522

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

Kolmogorov-Smirnov test was used to verify the normality of the data of water activity, moisture content, colour and enumeration of probiotics at the different conditions. The Levene’s test was used to verify the homoscedasticity, equality of variances. When the normality and the homoscedasticity was verified, ANOVA was used to detect significant differences between the conditions. Tukey’s multiple range test was used to determine statistically significant differences. A significance level of 5% was assumed. All results are presented as the average ± standard deviation (SD).

3. Results and discussion 3.1. Viability of Lactobacillus plantarum after OD The 60 °Brix solutions were used in the OD in order to have a higher process rate (Assis et al., 2017). Keeping in mind any adverse effect of an excess intake of sorbitol, it was aimed to avoid osmotically dehydrated apple cubes with too high sorbitol concentration and, therefore, 40 °Brix solutions were also tested. The ratio of 1:4 had proved to be an efficient ratio for OD of apple cubes (Assis et al., 2017). At the time of incubation (t = 0), counts of L. plantarum in the 40 and 60 °Brix solutions were 9.9 ± 0.1 log cfu/mL. This initial value was considered N0. The probiotic showed viability in both the solutions and in the apple cubes where it was incorporated during the OD. In Fig. 1 the log-unit percentage differences (%Dlog) between the osmotic solution and the apple cubes are shown, for OD at 37 °C at normal pressure (Fig. 1a) and in vacuum (Fig. 1b). At atmospheric pressure, Dlog of 15.7 ± 3.9% were found for apple cubes at t = 6 h, for both osmotic agents and both solution concentrations used (Fig. 1a), meaning that the counts of L. plantarum were around 8 log cfu/mL. There does not seem to be a clear tendency for the viability after the 6 h-OD. The differences between the solutions (at t = 0) and the apple cubes after the 24 h-OD ranged from 13.9 ± 1.1% Dlog, for 40 °Brix sorbitol, to 22.6 ± 5.1% Dlog, for 60 °Brix sucrose. After this OD time, L. plantarum could survive better in a lower concentrated solution, i.e. 40 °Brix. Krasaekoopt and Suthanwong (2008) showed that lower incorporation of L. casei into guava and papaya pieces was obtained with 30 °Brix solutions compared to 15 °Brix solutions and explained the inhibition of bacteria by the high sugar content. In fact, the degree of protection afforded by a given solute during drying processes is species- and strain-dependent (Carvalho et al. 2004). The duration of 6 h of the OD could be sufficient for the incorporation of L. plantarum into apple cubes, because the longer time of 24 h decreased the viability of L. plantarum in apple cubes for more concentrated solutions. In relation to the osmotic agent, there was no significant difference in the viability of L. plantarum in the apple cubes osmotically dehydrated in sucrose or sorbitol solutions. These findings go along with Assis et al.’s, who did not find significant differences in the sugar gain (around 16%), therefore, in the total soluble solids of apple cubes osmotically dehydrated in the two types of solution with 60 °Brix and at 40 °C, using the same mass ratio of sample to solution, i.e., 1:4 (Assis et al., 2017). The incorporation of the probiotic could have followed a pattern similar to the solute gain during OD. The water loss in that study was not significantly different after OD in both solutions either (Assis et al., 2017). In vacuum, counts of L. plantarum in apple cubes after the 24 hOD in 40 and 60 °Brix sucrose solutions were not significantly different (p > 0.5) than their osmotic solutions at t = 0 and, so, % Dlog relative to these conditions were low (Fig. 1b). However, the same behaviour does not occur in 40 and 60 °Brix sorbitol solutions. Differences between survivals in the solution (at t = 0) and the apple

cubes after the 24 h-OD in sucrose or sorbitol solutions were around 1 log-unit and 2 log-units, respectively. Therefore, survival of L. plantarum in apple cubes osmotically dried in sucrose solutions was improved in comparison with sorbitol solutions and should be a better choice for an OD in vacuum. There were no significant differences in the L. plantarum survivals between 40 and 60 °Brix solutions for both osmotic agents used. Other authors have investigated the impact of carbohydrates, including sucrose and sorbitol, on the survival of lactobacilli. Ferreira et al. (2005) observed a 60% higher survival of L. sakei during spray drying when it had previously been grown in the presence of sucrose. However, Linders, De Jong, Meerdink, and Van’t Riet (1997) claimed that, although sorbitol and sucrose were protective for L. plantarum during fluidized bed drying, sucrose was less successful. Also, in Perdana et al.’ study (2014), lowmolecular weight carbohydrates like sorbitol provided better stabilization of L. plantarum during spray drying. Corcoran, Stanton, Fitzgerald, and Ross (2005) demonstrated an enhanced survival of L. rhamnosus in gastric acid juice with metabolisable sugars, such as sucrose. In a more acid apple environment, sucrose could support the survival of lactobacillus. In the OD process, not only the carbohydrate solution, but also the apple matrix may benefit the survival of L. plantarum. Vacuum impregnation of probiotics showed to be an efficient used method for the fortification of fruit matrices, possibly because of a fast introduction of external liquids into the fruits (Betoret et al., 2003; Zhao & Xie, 2004). Krasaekoopt and Suthanwong (2008) applied a pressure of 50 mbar for 5–15 min in the vacuum impregnation of L. casei in papaya and guava pieces and noted an irreversible destruction of the porous fruit matrix and a decreased incorporation of L. casei, after only 10 min in vacuum. Interestingly, both the counts in the osmotically dehydrated apple and the osmotic solutions after the 24 h-OD were not significantly different (p > 0.05). Since no measurement was performed at 6 h in vacuum, it may be speculated that an equilibrium was reached of L. plantarum in the solution. Small differences were also shown between OD performed with the two different solutes, but the use of sucrose did ameliorate survival compared to sorbitol. Especially noteworthy is the OD in vacuum, as even after a 24 h-OD, no significant differences (p > 0.05) were detected in the viability of probiotic from sucrose solutions between the osmotic solutions at t = 0 and apple cubes after 24 h-OD, as already mentioned. The reason could be the higher resulting water content in relation to the correspondent experiments with sorbitol (Fig. 2). The probiotic L. plantarum most probably suffered from osmotic stress and, therefore, did not survive as well in an apple cubes with reduced water content and aw. Ribeiro et al. (2014) demonstrated a 1-log-unit reduction of L. plantarum incorporated in strawberry and banana, as well as a 3-log unit reduction of the probiotic in kiwi after an air-drying process, and suggested that the bacteria counts decreased probably due to the reduced water content. Even though lactobacilli can adapt easily to changes in their environments, the solute concentration in the environment should be relatively constant (Poolman & Glaasker, 1998). An increased osmolarity in the environment, which is the case of the sorbitol and sucrose solutions, leads to osmotic stress, in consequence of a water flow from the inside of the cell to the environment, and, therefore, a reduction of the cell turgor, a shrinkage of cells and an intracellular increase of fruit acids (De Angelis & Gobetti, 2004; Ribeiro et al., 2014). To some extent, L. plantarum can balance intra- and extracellular concentrations of sucrose and lactose, but osmotic stress can also be detrimental (Jordan, Hutchings, & Mascher, 2008; Van de Guchte et al., 2002). Santivarangkna, Kulozik, and Foerst (2006) found that sorbitol has a positive effect during vacuum drying of Lactobacillus helveticus, but only with the

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

523

Fig. 1. Viability of L. plantarum in apple cubes during/after OD with 40 or 60 °Brix solutions of sucrose or sorbitol at 37 °C at atmospheric pressure (a) and in vacuum of 150 mbar (b) in relation to the viability in the initial osmotic solution, expressed in Dlog (Dlog (%) = (log(N0,solution)
log(N))/log(N0,solution) * 100).

addition of 1%; with 100% sorbitol, the bacteria survived to a lesser extent than without any addition and probably suffered osmotic stress. The viability of the probiotic during OD may also depend on the structure of the fruit. Ribeiro et al. (2014) found that strawberry had the highest cell count after immersion in L. plantarum suspension and attributed this fact to the high porosity of this fruit, which probably promoted the adherence of the probiotic to the fruit. Apple is also a high porous fruit. Considering the OD time, it can be considered that longer OD times cause higher viability losses of L. plantarum inside the apple cubes. After the 24 h-OD in normal atmosphere and in vacuum, the counts of bacteria in 60 °Brix sorbitol solutions decreased significantly (p < 0.05), while for sucrose solutions no changes were found. 3.2. Relation between the moisture content and water activity and the viability of Lactobacillus plantarum in osmotically dehydrated apple cubes Fig. 2 shows the viability of L. plantarum in apple cubes in relation to the moisture content and the aw after 24 h-OD. No significant differences (p > 0.05) were found among % Dlog corresponding to the different values of moisture content and aw of samples osmotically dehydrated with 40 and 60 °Brix sucrose or sorbitol solutions either at atmospheric pressure or in vacuum. However, a tendency can be derived from both the moisture contents as well as the aw values. For higher moisture contents and

higher aw, % Dlog presented a tendency to be lower for the OD with 40 °Brix sucrose at atmospheric pressure and in vacuum, this meaning that L. plantarum survived better. Following this pattern, apple cubes osmotically dehydrated in 60 °Brix sorbitol solutions, with the lowest water content and aw, tended to contain smaller amounts of L. plantarum. Exceptions were apple cubes osmotically dried in the 60 °Brix sucrose solution at 37 °C and at normal atmosphere, which showed similar viability, in spite of the moisture content and the aw being higher than in apples from OD in the 60 °Brix sorbitol solution. Also, L. plantarum in apple samples dehydrated under vacuum presented a tendency to survive less in the 40 °Brix sorbitol solution than in the 60 °Brix sucrose solution, although the aw and moisture content of osmotically dried apple from 40 °Brix sorbitol was significantly higher (p < 0.05). As discussed before, an explanation could be a better viability of the probiotic with sucrose than with sorbitol. From this point of view, it cannot be claimed that either the sucrose or sorbitol, or 40 or 60 °Brix osmotic solutions are more recommended for the incorporation of L. plantarum into apple cubes, but the osmotic stress could play an important role in the survival of L. plantarum, while OD and the resulting water content in the apple matrix may also be crucial. 3.3. Storage of apple cubes incorporated with Lactobacillus plantarum after OD Factors as levels of oxygen, pH, storage temperature, the presence of competing microorganisms, among others, determine the

524

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

Fig. 2. Moisture content and water activity of apple cubes incorporated with L. plantarum after OD (24 h) with 40 or 60 °Brix solutions of sucrose or sorbitol at 37 °C and atmospheric pressure (a) and in vacuum of 150 mbar (b) (Dlog (%) = (log (N0,solution)
log(N))/log(N0,solution) * 100).

shelf life of a probiotic product, i.e., the viability of probiotics in the food matrices (Coman et al., 2012). The storage temperature of 4 °C has been proven to be the best temperature to maintain the viability of probiotic in a fruit matrix over time in several studies (Barbosa et al., 2015; Borges et al., 2016; Ribeiro et al., 2014). In the present work, the viability of L. plantarum during storage at 4 °C was high (Fig. 3a and b). After a 6 day-storage at 4 °C the log reductions were higher for L. plantarum in apple samples from 40 °Brix sucrose solutions with 0.9 log-unit reduction, compared with the 4 day-storage. Lactobacillus plantarum presented tendency to increase in apple cubes from 60 °Brix solutions (Fig. 3a). In Fig. 3b the behaviour during storage of L. plantarum in apple cubes from the OD at 37 °C in vacuum is depicted. Overall, after 4 days, the reductions observed in the viability of the probiotic were all inferior to 1 log-unit. A reduction of 0.8 log-units was found for the dehydrated apple samples from the 40 °Brix solutions and a reduction of 0.7 log-units was also observed in apple cubes from 60 °Brix solutions. No reduction of L. plantarum occurred in apple samples osmotically dehydrated with sorbitol. After 6 days, log cfu/mL of L. plantarum increased in sucrose solutions, but not in sorbitol solutions compared to storage after 4 days. No consistent tendency can be drawn from analysing the storage of apple cubes from the different treatments. Differences in the viability of the probiotic among samples were irrelevant. Therefore, L. plantarum survived without any significant log reductions (p > 0.05) in all samples during a 6 day-storage at 4 °C. Other authors claimed a positive effect of carbohydrates during the storage of L. plantarum. Carvalho et al. (2002) found that

sorbitol produced more significant effects than inositol, fructose, trehalose, monosodium glutamate and propyl gallate toward maintaining viability of freeze-dried L. plantarum. The survival of L. plantarum in apple samples osmotically dehydrated with sorbitol solutions over 6 days-storage (Fig. 3a and b) could be explained by the protective effect of sorbitol during storage, as shown by Carvalho et al. (2002). In general, sugar can have a positive effect on the viability of L. plantarum during storage. Charalampopoulos and Pandiella (2010) studied the behaviour of L. plantarum during storage of fermented cereal extracts and observed a higher viability in malts extracts than in barley and wheat extracts and assumed it was due to their higher sugar content. This might explain the survival of this probiotic observed in the apple samples osmotically dehydrated in sucrose solutions (Fig. 3a and b). The short storage time of 6 days in this study would be sufficient for further handling in food industry as the osmotically dried apple cubes are intermediate moisture products meant to be further processed. In several other studies, longer storage periods at refrigeration temperature of L. plantarum incorporated in apple matrices were validated: Randazzo, Pitino, Licciardello, Muratore, and Caggia (2013) inoculated L. rhamnosus (109 cfu/g) into peach jam with 38–40 °Brix and showed an amount of 107 cfu/g in the jam after 78 days of storage at 5 °C. These authors also confirmed the high effectiveness of the peach fruit matrix for a survival of probiotics compared to a synthetic peach medium and noted additionally that lactobacilli changed pH and the composition of sugars in the jam during this period (Randazzo et al., 2013). Borges et al. (2016) studied the storage of hot air-dried apple powder with incorporated L. plantarum at 4 °C and observed reductions of less than 1 log cfu/g, over a 90-day period. Also, Rêgo et al. (2013) demonstrated a viability of L. plantarum in hot air-dried apple pieces over 65 days with a decrease of only 1 log cfu/g. However, in these two latter studies the products presented a much lower aw than the products of the present study.

3.4. Colour during OD and storage The colour changes (DE) of apple cubes during OD at 37 °C and at atmospheric pressure as well as in vacuum are depicted in Fig.4a and b. The colour tended to change in all samples from t = 0 to the end of the OD (t = 24 h), while over the storage period lower changes were observed. After 4 days of storage, no significant differences (p > 0.05) in DE were observed among samples osmotically dehydrated with 40 and 60 °Brix sucrose or sorbitol solutions either at atmospheric pressure or in vacuum, and DE was maintained relatively constant. The influence of the soluble solids may also have played an important role. Lower DE values were obtained for apple samples dehydrated in 60 °Brix solutions (Fig. 4a and b). This could be due to the protective effect of incorporated sugar, which prevents browning reactions. Krokida, Karathanos, and Maroulis (2000) studied the colour changes during the OD of apple samples and noticed that the infusion of sugar had positive effect on stability of L⁄, a⁄ and b⁄ values. L⁄ of fresh apple was 71.02 ± 3.30, a⁄ was
3.86 ± 0.96 and b⁄ 29.96 ± 2.40 The browning process led to a lower L⁄ (indicating a less light colour), higher a⁄ (a redder product) and lower b⁄ (a less yellow product) values over time. L⁄ values decreased less in apple samples from 60 °Brix solutions and b⁄ values were constant or even increased. (L⁄ 51.35 ± 3.19, b⁄ 30.36 ± 3.01). Increased b⁄values were observed for more yellowish apple samples from 60 °Brix solutions after OD and storage. A decrease in yellowness with a b⁄ = 24.00 ± 3.52 was observed for apple cubes from 40 °Brix solutions, after a 6 days storage. L⁄ was 45.08 ± 2.97 after storage of apple samples from 40 °Brix solutions.

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

525

Fig. 3. Survival of L. plantarum in apple cubes during storage at 4 °C after the OD (24 h) with 40 or 60 °Brix solutions of sucrose or sorbitol at 37 °C and atmospheric pressure (a) and in vacuum of 150 mbar (b).

The influence of the probiotic culture on the colour of the apple cubes was not studied. Randazzo et al. (2013) had not find any relation between added L. rhamnosus to peach jam and the colour parameters. 3.5. Gastro-intestinal tract simulation A functional product can only be claimed as probiotic, if it contains about 106 – 107 colony forming units of viable probiotic cells in 1 gram of product upon ingestion. During digestion, this number of viable microorganisms needs to be constant and, therefore, the survival of probiotics through gastro-intestinal tract is indispensable (FAO/WHO, 2002). In this work, a quick digestion in a simulated gastro-intestinal tract was performed of the dehydrated apple cubes containing L. plantaru, according to the method proposed by Barbosa et al. (2015). No significant changes (p > 0.05) in the viability of L. plantarum were found in apple cubes dehydrated in either 40 or 60 °Brix sucrose solutions, as well as 40 or 60 °Brix sorbitol solutions during the whole gastro-intestinal tract simulation (Table 1). The survival on 30 min and 90 min were not presented, because the reductions of the probiotic viability were already

low after 60 min and even 120 min. The reductions of L. plantarum were minimal after exposure to the acidic conditions of the stomach (pH 3.0 with pepsin). Probiotic viability in these conditions could be due to the protective effect of metabolisable sugars in an acid environment as mentioned before (Corcoran et al., 2005). After exposure to bile salts at pH 7.0, a slightly decrease of L. plantarum was observed, with log reductions below 1 log-unit. Other studies have demonstrated a lower survival of L. plantarum in the presence of small intestine conditions, than at stomach conditions (Barbosa et al., 2015; Mirlohi, Soleimanian-Zad, Dokhani, Sheikh-Zeinodin, & Abghary, 2009). Cebeci and Gürakan (2003) proved the tolerance of L. plantarum HU to acid and bile salts and suggested the use of the probiotic in functional food. L. plantarum incorporated in the apple matrix survived during passage through the gastro-intestinal tract simulation and could be considered a probiotic food. Samples contained around 108 cfu/g after the OD, around 107 cfu/g after a 6 days-storage and maintained this value after the gastro-intestinal tract simulation. The probiotic apple product impregnated with L. plantarum and sorbitol could be considered a potential symbiotic product with both pro- and prebiotics properties. The synergic effect of pre-

526

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

Fig. 4. Colour changes of apple cubes during OD (24 h) with 40 or 60 °Brix solutions of sucrose or sorbitol at 37 °C and 6 days-storage at 4 °C and atmospheric pressure (a) and in vacuum of 150 mbar (b).

and probiotics has recently been investigated. Mandal et al. (2009) proved a decrease of plasma cholesterol level in Swiss albino mice with a treatment of sorbitol combined with P. acidilactici and demonstrated the in vitro positive impact of sorbitol on bacteriocin production. Nonetheless, any adverse effects of sorbitol cannot be ignored at consumption. After the 24 h-OD in 40 °Brix and 60 °Brix sorbitol solutions, the apple cubes contained, 9 and 12.5% of sorbitol, respectively. Therefore, a possible daily consumption of osmotically dehydrated apple cubes could be around 80–100 g assuming a safe consumption of 10 g sorbitol per day (Burt, 2006; Rowe et al., 2009). This amount of apple is equivalent to around 60–80 dehydrated apple cubes, which is a sufficient amount for the use in a functional food.

Table 1 Survival of L. plantarum incorporated in apple cubes after OD (24 h) with 40 or 60 °Brix solutions of sucrose or sorbitol at 37 °C and atmospheric pressure throughout a quick digestion simulation of the passage through the gastro-intestinal tract (2 h). log (N/N0)a Osmotic solution

0 min

60 minb

120 minc

40 °Brix 40 °Brix 60 °Brix 60 °Brix

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

0.00 ± 0.00
0.10 ± 0.10 0.10 ± 0.10
0.30 ± 0.30


0.50 ± 0.10
0.20 ± 0.10
0.10 ± 0.00
0.40 ± 0.20

sucrose sorbitol sucrose sorbitol

a Survival is represented as the media of the logarithmic reduction: log (N/ N0) ± the standard error of the mean, N is the cfu/ml at each sampling time, N0 is the cfu/ml at time zero. b Survival after exposure to pH 3.0 in the presence of pepsin. c Survival after exposure to pH 3.0 in the presence of pepsin and subsequent exposure to bile salts at pH 7.0.

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

The resulting probiotic food could be applicable in different food products and would be suitable for consumers that eat dairy-free or are lactose-intolerant. In addition, combining the osmotically dehydrated probiotic apple cubes with yoghurt, which nowadays contains Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei or Bifidobacterium bifidum, would result in a functional food with different beneficial probiotics and could have synergic effects for consumers. 4. Conclusions OD proved to be a good method for the production of a probiotic fresh fruit product. Lactobacillus plantarum 299v (107–108 cfu/g) was successfully incorporated in apple cubes during the osmotic dehydration process (24 h) at 37 °C and normal atmosphere, as well as in vacuum, using 40 and 60 °Brix sucrose and sorbitol solutions. Osmotic solutions with lower soluble solids content seemed more adequate for the incorporation. Both sucrose and sorbitol proved to be suitable as osmotic agents. Lactobacillus plantarum, incorporated in apple cubes, survived over a storage period of 6 days at 4 °C maintaining constant values of 107 cfu/g in the apple cubes. In addition, the viability of L. plantarum did not decrease during a quick simulation of the passage through the gastro-intestinal tract (2 h), which is essential for the beneficial effect of a probiotic. Colour changes were observed after OD and storage, as browning of the apple cubes occurred. Colour changes were lower in samples from the 60 °Brix solutions, but the viability of L. plantarum was lower for higher soluble solids content (60 °Brix). Priority was set on the incorporation and survival of the probiotic and, so, 40 °Brix osmotic solutions were found to be more appropriate. Therefore, the quality aspects, such as colour need to be improved. After OD, the osmotic solutions, which are rich in nutrients from apple, could be used to grow probiotics and recycled to be used in other OD processes. Therefore, apple cubes resulting from OD, as well as after a 6 day-storage, can be considered a functional food, with a sufficient number of probiotics to exert health benefits for consumers. The functional product can be further processed or applied in other products in food industry. Regarding the probiotic apple cubes as a potential probiotic food, it would be important to examine the acceptance of consumers in terms of sensorial characteristics. In order to be implemented in industry, the duration of the OD process would need to be optimized. Acknowledgments This work was supported by National Funds from FCT – Fundação para a Ciência e Tecnologia through project UID/ Multi/50016/2013 and by the European M.Sc. in Food Science, Technology and Business (BiFTec). J. Barbosa acknowledges the support provided by the post-doctoral fellowship SFRH/ BPD/113303/2015 (FCT). The authors also acknowledge Campotec for graciously supplying the apples for this study. References An, K., Li, H., Zhao, D., Ding, S., Tao, H., & Wang, Z. (2013). Effect of osmotic dehydration with pulsed vacuum on hot-air drying kinetics and quality attributes of cherry tomatoes. Drying Technology, 31, 698–706. Assis, F. R., Morais, R. M. S. C., & Morais, A. M. M. B. (2017). Mathematical modelling of osmotic dehydration kinetics of apple cubes. Journal of Food Processing and Preservation, 43. https://doi.org/10.1111/jfpp.12895. Barbosa, J., Borges, S., & Teixeira, P. (2015). Pediococcus acidilactici as a potential probiotic to be used in food industry.. International Journal of Food Science and Technology, 50, 1151–1157. Barbosa-Cánovas, G. V., Fontana, A. J., Jr., Schmidt, S. J., & Labuza, T. P. (2007). Water activity in foods: Fundamentals and applications. Blackwell Publishing.

527

Betoret, N., Puente, L., Dı´az, M., Pagán, M., Garcı´a, M., Gras, M., & Fito, P. (2003). Development of probiotic-enriched dried fruits by vacuum impregnation. Journal of Food Engineering, 56, 273–277. Bielecka, M., Biedrzycka, E., & Majkowska, A. (2002). Selection of probiotics and prebiotics for symbiotic and confirmation of their in vivo effectiveness. Food Research International, 35, 139–144. Borges, S., Barbosa, J., Silva, J., Gomes, A. M., Pintado, M., Silva, C. L. M., ... Teixeira, P. (2016). A feasibility study of Lactobacillus plantarum in fruit powders after processing and storage. International Journal of Food Science and Technology, 51, 381–388. Burt, B. A. (2006). The use of sorbitol- and xylitol-sweetened chewing gum in caries control. The Journal of the American Dental Association, 137, 190–196. Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcatal, F. X., & Gibbs, P. (2002). Survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in the presence of protectants. Biotechnology Letters, 24, 1587–1591. Cebeci, A., & Gürakan, C. (2003). Properties of potential probiotic Lactobacillus plantarum strains. Food Microbiology, 20, 511–518. Charalampopoulos, D., & Pandiella, S. S. (2010). Survival of human derived Lactobacillus plantarum in fermented cereal extracts during refrigerated storage. LWT – Food Science and Technology, 43, 431–435. Chavan, U. D., & Amarowicz, R. (2012). Osmotic dehydration process for preservation of fruits and vegetables. Journal of Food Research, 1, 201–209. Coman, M. M., Cecchini, C., Verdenelli, M. C., Silvi, S., Orpianesi, C., & Cresci, A. (2012). Functional foods as carriers for SYNBIOÒ, a probiotic bacteria combination. International Journal of Food Microbiology, 157, 246–352. Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and Environmental Microbiology, 71, 3060–3067. De Angelis, M., & Gobetti, M. (2004). Environmental stress responses in Lactobacillus: A review. Proteomics, 4, 106–122. Espírito Santo, A. M., Cartolanoa, N. S., Silva, T. F., Soares, F. A. S. M., Gioielli, L. A., Peregob, P., ... Oliveira, M. N. (2012). Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts. International Journal of Food Microbiology, 154, 135–144. Ferreira, V., Soares, V., Santos, C., Silva, J., Gibbs, P. A., & Teixeira, P. (2005). Survival of Lactobacillus sakei during heating, drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate. Biotechnology Letters, 27, 249. Flores-Andrade, E., Pascual-Pineda, L. A., Alarcón-Elvira, F. G., Rascón-Díaz, M. P., Pimentel-González, D. J., & Beristain, C. I. (2017). Effect of vacuum on the impregnation of Lactobacillus rhamnosus microcapsules in apple slices using double emulsion. Journal of Food Engineering, 202, 18–24. Food and Agriculture Organization (FAO) (2010). Processing of fresh-cut tropical fruits and vegetables. A technical guide. In J. B. James, T. Ngarmsak, & R. S. Rolle (Eds.). Food and Agriculture Organization of the United Nations, Regional Office for Asia and the Pacific, Bangkok: RAP publication 2010/16. Food and Agriculture Organization/World Health Organization (2002). Guidelines for the evaluation of probiotics in food: Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. London: ON, Canada. Genevois, C., de Escalada Pla, M., & Flores, S. (2017). Novel strategies for fortifying vegetable matrices with iron and Lactobacillus casei simultaneously. LWT – Food Science and Technology, 79, 34–41. Huerta-Vera, K., Flores-Andrade, E., Pérez-Sato, J. A., Morales-Ramos, V., PascualPineda, L. A., & Contreras-Oliva, A. (2017). Enrichment of Banana with Lactobacillus rhamnosus using double emulsion and osmotic dehydration. Food and Bioprocess Technology, 10(6), 1053–1062. Jordan, S., Hutchings, M. I., & Mascher, T. (2008). Cell envelope stress response in Gram-positive bacteria. FEMS Microbiology Reviews, 32, 107–146. Kourkoutas, Y., Kanellaki, M., & Koutinas, A. (2006). Apple pieces as immobilization support of various microorganisms. LWT – Food Science and Technology, 39, 980–986. Krasaekoopt, W., & Suthanwong, B. (2008). Vacuum impregnation of probiotics in fruit pieces and their survival during refrigerated storage. Journal of Nature and Science, 42, 723–731. Krokida, M. K., Karathanos, V. T., & Maroulis, Z. B. (2000). Effect of osmotic dehydration on color and sorption characteristics of apple and banana. Drying Technology, 18, 937–950. Lee, K., & Lee, Y. (2010). Effect of Lactobacillus plantarum as a starter on the food quality and microbiota of kimchi. Food Science and Biotechnology, 19, 641. Linders, L. J. M., De Jong, G. I. W., Meerdink, G., & Van’t Riet, K. (1997). Carbohydrates and the dehydration inactivation of Lactobacillus plantarum: The role of moisture distribution and water activity. Journal of Food Engineering, 31, 237–250. Livesey, G. (2001). Tolerance of low-digestible carbohydrates: A general view. British Journal of Nutrition, 85, 7–16. Livesey, G. (2003). Health potential of polyols as sugar replacers, with emphasis on low-glycaemic properties. Nutrition Research Reviews, 16, 163–191. Mandal, V., Sen, S. K., & Mandal, N. C. (2009). Effect of prebiotics on bacteriocin production and cholesterol lowering activity of Pediococcus acidilactici LAB 5. World Journal of Microbiology and Biotechnology, 25, 1837–1841. Miles, A. A., Misra, S. S., & Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene, 38, 732–749. Mirlohi, M., Soleimanian-Zad, S., Dokhani, S., Sheikh-Zeinodin, M., & Abghary, A. (2009). Investigation of acid and bile tolerance of native lactobacilli isolated from fecal samples and commercial probiotics by growth and survival studies. Iranian Journal of Biotechnology, 7, 233–240.

528

K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

Nayyar, D. K., Hill, L. G., Nauth, K. R., Loh, J. P., Behringer, M., & Apel, L. (2002) Premium quality intermediate moisture vegetables and method of making. Patent No. US 6,403,134 B1. Kraft Foods Holdings Inc, Northfield, Illinois, US. Perdana, J., Martijn, B. F., Siwei, C., Boom, R. M., & Schutyser, M. A. I. (2014). Interactions between formulation and spray drying conditions related to survival of Lactobacillus plantarum WCFS1. Food Research International, 56, 9–17. Poolman, B., & Glaasker, E. (1998). Regulation of compatible solute accumulation in bacteria. Molecular Microbiology, 29, 397–407. Probi, A. B. (2016). Sweden: Probiotic platforms. Retrieved on July 11, 2017, from . Randazzo, C. L., Pitino, I., Licciardello, F., Muratore, G., & Caggia, C. (2013). Survival of Lactobacillus rhamnosus probiotic strains in peach jam during storage at different temperatures. Food Science and Technology (Campinas), 33(4), 652–659. Rêgo, A., Freixo, R., Silva, J., Gibbs, P., Morais, A. M. M. B., & Teixeira, P. A. (2013). Functional dried fruit matrix incorporated with probiotic strains: Lactobacillus plantarum and lactobacillus kefir. Focusing on Modern Food Industry, 2, 138–143. Ribeiro, C., Freixo, R., Silva, J., Gibbs, P., Morais, A. M. M. B., & Teixeira, P. (2014). Dried fruit matrices incorporated with a probiotic strain of Lactobacillus plantarum. International Journal of Food Studies, 3, 69–73. Roberfroid, M. (2007). Prebiotics: The concept revisited. Journal of Nutrition, 137, 830–837. Rowe, R. C., Sheskey, P. J., & Quinn, M. E. (2009). Handbook of pharmaceutical excipients (6th ed. p. 637). London, England: Pharmaceutical Press. Santivarangkna, C., Kulozik, U., & Foerst, P. (2006). Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying. Letters in Applied Microbiology, 42, 271–276.

Schley, P. D., & Field, C. J. (2002). The immune-enhancing effects of dietary fiber and prebiotics. British Journal of Nutrition, 87, 221–230. Shah, N. P. (2001). Functional foods from probiotics and prebiotics. Food Technology, 55, 46–53. SPI Polyols (2017). Polyols Comparison Chart. from: . SPI Polyols Inc., New Castle, Delaware, USA [Accessed on April 21, 2017]. TuzenÒ (n.d.). Stop suffering from IBS symptoms. Retrieved on May 11, 2017, from . UltraFloraÒ Intensive Care (n.d.). Retrieved on May 11, 2017, from . Van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S. D., & Maguin, E. (2002). Stress responses in lactic acid bacteria. Antonie van Leeuwenhoek, 82, 187–216. Vries, M. C., Vaughan, E. E., Kleerebezem, M., & Vos, W. M. (2006). Lactobacillus plantarum – survival, functional and potential probiotic properties in the human intestinal tract. International Dairy Journal, 16, 1018–1028. Vuyst, L. D., Kerrebroeck, S. V., Harth, H., Huys, G., Daniel, H., & Weckx, S. (2014). Microbial ecology of sourdough fermentations: Diverse or uniform? Food Microbiology, 37, 11–29. Zhao, Y., & Xie, J. (2004). Review of practical applications of vacuum impregnation in fruit and vegetable processing. Trends in Food Science & Technology, 15, 434–451. Zumbe, A., Lee, A., & Storey, D. (2001). Polyols in confectionery: The route to sugarfree, reduced sugar and reduced calorie confectionery. British Journal of Nutrition, 85(S1), S31–S45.