Journal of Functional Foods 38 (2017) 519–528

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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 a b s t r a c t

Article history: The feasibility to incorporate Lactobacillus plantarum in apple cubes during the osmotic dehydration (OD)
Received 28 June 2017 was investigated. The effects of 40 and 60 °Brix osmotic solutions of sucrose or sorbitol on the viability of
Received in revised form 4 September 2017 L. plantarum during the OD at 37 °C and 1013 or 150 mbar was evaluated. The storage at 4 °C and a quick
Accepted 13 September 2017
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
Keywords:
for 40 °Brix solutions and it maintained the viability of 107 cfu/g during a 6 day-storage at 4 °C. L. plan-
Probiotic
Lactobacillus plantarum
tarum also survived (107 cfu/g) during the simulation of the digestion. Colour changes of the probiotic
Osmotic dehydration apple cubes occurred after OD and storage.
Sorbitol Therefore, osmotically dehydrated apple cubes incorporated with L. plantarum could be a new probiotic
Water activity food.
Colour Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Among other probiotics, the Gram-positive and aerotolerant L.

plantarum has been extensively studied and is nowadays one of
Conventional foods containing probiotics often originate from the most commonly used probiotic in the production of functional
dairy products as yoghurt and kefir. As the demand for probiotic food. Traditionally it is found in fermented plant products, such as
functional products and the preference of consumers for dairy- sauerkraut, Korean kimchi and sourdough (Lee & Lee, 2010; Vries,
free products are increasing, studies have been performed to incor- Vaughan, Kleerebezem, & Vos, 2006; Vuyst et al., 2014). Lactobacil-
porate probiotics also into several types of processing of fruits and lus plantarum 299 v is marketed for probiotic functional foods pro-
vegetables. Non-dairy probiotic products, besides supplements, duction as for example probiotic drinks and capsules (Shah, 2001;
include fruit and vegetable drinks, fruit purees and pieces, jam Tuzen, 2017; UltraFlora, 2017). Lactobacillus plantarum 299 v orig-
and powders, chocolate and even flan (Barbosa, Borges, & inates from the intestinal human mucosa and is sold as an espe-
Teixeira, 2015; Borges et al., 2016). Different fruits, e.g. banana, cially beneficial probiotic because of its claimed adherence and
strawberry, mango and many others, have been investigated for following colonization on gastrointestinal cells, which is of main
their suitability as probiotics carriers. The apple fruit matrix was importance to be considered as a probiotic bacterium (Probi AB,
shown to be highly applicable for probiotics, may be because of Sweden, 2017; Vries et al., 2006).
its high porosity and, therefore, easy incorporation of probiotics The market for fresh-cut fruits has been growing in recent years
(Espírito Santo et al., 2012). Another explanation may be associated induced by consumer demand for food products that are fresh-like,
to the cellulose in apple, which is not digested and could serve as a healthy minimally processed, nutritious and convenient (FAO,
protective matrix for probiotics through the intestinal tract 2010). To maintain the quality of these fresh-cut products tech-
(Kourkoutas, Kanellaki, & Koutinas, 2006). Rêgo et al. (2013) niques are required to preserve organoleptic characteristics and
demonstrated the compliance of apple as a fruit matrix for probi- to guarantee a reasonable shelf life. Osmotic dehydrated apple con-
otic survival over time, as they studied hot air-dried apple cubes taining probiotics would be suitable for the growing market of
with incorporated L. plantarum during 65 days of storage and found fresh-cut or minimally processed fruits. The application of fresh-
only minimal viability losses of 1 log cfu/g. cut fruits into other products brings new possibilities to the food
industry, which have been barely exploited yet.
⇑ Corresponding author. In osmotic dehydration, pre-treated sliced fruits are immersed
E-mail address: [email protected] (A.M.M. Bernardo de Morais). into an osmotic solution, for example, a sugar syrup (fruits) or a

https://doi.org/10.1016/j.jff.2017.09.021
1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
520 K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

salt solution (vegetables) for a certain period of time at a temper- pathogenic bacteria. It can also be associated with the cancer pre-
ature above room temperature and until 60 °C (Chavan & vention, the decrease of LDL-cholesterol, the improvement of the
Amarowicz, 2012). The difference of the osmotic pressure between immune system and the production of bacteriocins (Bielecka,
the food and the solution results in the diffusion of water from the Biedrzycka, & Majkowska, 2002; Mandal, Sen, & Mandal, 2009;
food into the solution, and diffusion of the solute from the solution Schley & Field, 2002). However, as sorbitol is only partly used by
to the food (An et al., 2013). The osmotic dehydrated products have the microorganisms in the gastro-intestinal tract, an elevated
a moisture content around 10–40%, an aw between 0.6 and 0.9 and intake of sorbitol may cause laxation by increasing water content
an extended shelf life (Barbosa-Cánovas, Fontana, Schmidt, & of the stool. Moreover, gastro-intestinal symptoms as diarrhea,
Labuza, 2007). For example, intermediate moisture products with abdominal pain, cramps and flatulence were reported after an
an aw from 0.75 to 0.85 are shelf stable for at least 90 days at refrig- overload consumption of sorbitol (Livesey, 2001). An acceptable
eration conditions, with an optimum temperature of 3–5 °C daily intake for sorbitol has not been estimated, because the sub-
(Nayyar et al., 2002). stance has been approved and found to be non-toxic with a laxa-
Osmotic dehydration (OD) offers many advantages compared to tion threshold of 50 g/day (Zumbe, Lee, & Storey, 2001).
other techniques of fruit preservation. With the applied low tem- Nevertheless, consuming more than 20 g/day of this polyol is not
perature, organoleptic characteristics of the product, including fla- recommended and an intake of only 7–14 g/day can already exert
vour and colour, are retained. The activity of PPO and, therefore, adverse effects in some individuals (Burt, 2006; Rowe, Sheskey, &
the enzymatic browning are inhibited because the products are Quinn, 2009).
immersed in the solution, not exposed to O2. In addition, the incor- The objective of this study was to investigate if probiotics can
porated sugar limits PPO activity. After OD, a sweeter, lower acid be incorporated during the OD of fruits, focussing on L. plantarum
product, with the same shape and less volume, is obtained. A and apple as the fruit for OD. In addition, the impact of sucrose
sweeter product may be favourable for consumers and a decreased and sorbitol as osmotic agents on the incorporation was also stud-
volume reduces further costs of processing, storage and transport. ied. The third purpose was the examination of the viability of L.
The entire process is simple and more economic than the conven- plantarum in the apple cubes during storage and to determine
tional hot air drying (Chavan & Amarowicz, 2012). quality changes in terms of colour during the OD and storage.
Huerta-Vera et al. (2017) used OD with 40, 50 and 60 °Brix The viability of the probiotic was also evaluated after a quick sim-
sucrose solutions at 35 °C to enrich banana slices with Lactobacillus ulation of the digestion of probiotic apple cubes through the
rhamnosus encapsulated in a double emulsion. These authors used gastro-intestinal tract.
a vacuum pulse of 50 mbar at the beginning of the osmotic process
and L. rhamnosus got incorporated and survived at levels above 107
2. Materials and methods
cfu/g in the osmodehydrated bananas. Flores-Andrade et al. (2017)
also studied the effect of vacuum at the same temperature on the
2.1. Probiotic strains and growth conditions
impregnation of these L. rhamnosus microcapsules in apple slices
using OD with the same sucrose solutions, and they found a higher
The probiotic culture L. plantarum 299 v were purchased from
impregnation with an initial vacuum pulse of 20 min. In addition,
Probis Probiotika (Lund, Sweden). The bacteria strains were grown
the survival of probiotics decreased with increasing osmotic pres-
aerobically on de Man, Rogosa and Sharpe (MRS) agar (Lab M, Bury,
sure of the solution. The number of viable cells in the osmodehy-
UK) at 37 °C for 24 h and stored at
80 °C in MRS broth (Pronadisa,
drated apple was in the range 106–108 cfu/g d.b. Genevois, de
Madrid, Spain) containing 30% v/v glycerol. Before use, each strain
Escalada Pla, and Flores (2017) studied the effects of simultaneous
was sub-cultured twice in 10 mL MRS broth (Lab M).
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 2.2. Sample preparation
incorporation.
In an osmotic dehydration process with fruits, normally a 2.2.1. Apple sample
sucrose solution is used as the osmotic agent. Another option can Apples, variety ‘Royal Gala’, were graciously provided from
be a sorbitol solution, which has been studied recently by Assis, Campotec, Portugal and stored at 4 °C. The fruits (3–5 aleatory
Morais, and Morais (2017). In the performed study, the initial apples) were washed for 5 min in an aqueous 7500 ppm active
osmotic dehydration rate of apple samples with a sorbitol solution chlorine solution and then cut in cubes (12  12  12 mm) with
was higher compared to a sucrose solution with the same soluble a vegetable cutter (Secret de Gourmet, France). The apple cubes
solids content (Assis et al., 2017). Sorbitol has 60% of the sweetness were immersed in sterile Ringer’s solution (Merck, Darmstadt, Ger-
of sucrose and 2.4 kcal/g compared with sucrose, which contains many) for 3 min to prevent enzymatic browning and residual
4 kcal/g (SPI Polyols, 2017). sodium solution was then removed from the surface of the samples
Sorbitol is a low-cariogenic substance compared to sucrose, by drying the cubes carefully with tissue paper.
because, when consumed in low amounts, it does not decrease In order to determine the soluble solids content of the apples,
the pH of the plaque enamel, which may lead to demineralisation around 100 g fresh apple was mixed with a handmixer and the
(Burt, 2006). In addition, sorbitol is considered a low-digestible resulting juice was measured with a hand refractometer (Atago,
carbohydrate and low-glycemic, because it is more slowly Guangzhou, China) before carrying out the OD. The soluble solids
absorbed in the small intestine and, consequently, not fully content of the apple was 15.3 ± 1.3 °Brix.
digested. With the resulting lower caloric value, sorbitol could be
a helpful component in diets for consumers suffering from diabetes 2.2.2. Preparation of inoculum
or trying to reduce and stabilize weight (Livesey, 2003). Sorbitol From MRS agar incubated at 37 °C for 24 h, one colony of each
may also play a role as prebiotic. Prebiotics are indigestible poly- probiotic was transferred to 10 mL of MRS broth and incubated
and oligosaccharides, which are the main substrate for beneficial in the same conditions. For the final inoculum, 0.1 mL of the last
microorganisms and selectively promote their growth, composi- culture was transferred to 10 mL of MRS broth (1:100) and incu-
tion and activity in the gastrointestinal tract (Roberfroid, 2007). bated at 37 °C for 24 h to reach stationary phase. The probiotic cul-
As prebiotic, sorbitol has a positive impact on the general bowel ture was centrifuged for 10 min at 7000 rpm and 20 °C (Hettich
function by stimulating the growth of probiotics and preventing Zentrifugen Rotina 35R, Tuttlingen, Germany). The supernatant
 K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528 521

was discarded and the cell pellets were washed twice in sterile placing approximately 3.5 g of apple sample in the measuring con-
Ringer’s solution (Merck, Darmstadt, Germany) and centrifuged tainer. The aw of the initial apple cubes was 0.993 ± 0.003.
under the same conditions. The pelleted cells were re-suspended The moisture content and the dry matter of the apple samples
in 10 mL Ringer solution. were determined by placing them in an oven (Binder, Germany)
at 105 °C for 24 h (until constant weight). The moisture content
2.3. Osmotic dehydration of the initial apple was 87.3 ± 1.0%.
Two measurements were performed for each duplicate.
2.3.1. Osmotic solutions
Osmotic solutions of 20, 40 and 60 °Brix were prepared with 2.6. Storage
commercial sucrose and sorbitol (Fagron Iberica, Barcelona, Spain)
in deionized water. Each osmotic solution was autoclaved (JSM, After 24 h-OD, around 6 g apple samples were taken from each
Model JSM 75L PL, Portugal) at 121 °C during 15 min in glass flasks solution and stored in individual petri dishes sealed with parafilm
before being used. (Bemis Company Inc., Neenah, Wisconsin, USA) at 4 °C for 6 days to
study the L. plantarum survival in the samples and the colour
2.3.2. Osmotic dehydration with L. Plantarum in the osmotic solution changes.
The inoculum of Lactobacillus plantarum 299v was prepared as Enumeration of L. plantarum was performed on days 4 and 6 as
described above, but instead of re-suspending the pelleted cells described above (Section 2.4). Water activity and moisture content
in Ringer solution, all the pellet was re-suspended in the same vol- were determined at the end of storage.
ume of sterile osmotic solution to obtain the maximum concentra-
tion of inoculum in the solution. 2.7. Colour determination
Apple cubes, prepared as described above, were immersed into
solutions of 40 and 60 °Brix of sucrose and sorbitol (with re- The colour of fresh and 24 h-osmotically dehydrated apple sam-
suspended probiotic) with a mass ratio of apple to the solution of ples was evaluated with a colorimeter (Minolta. Chromameter CR-
1:4. These conditions were based on a previous study, which had 300, Osaka, Japan). The colour of the latter samples was also eval-
identified the mass ratio of sample to solution of 1:4 as an alterna- uated on the 4th and 6th days of storage. Lightness (L⁄), Redness
tive to 1:10 in the OD at the atmospheric pressure, as lower quan- (a⁄) and Yellowness (b⁄) were measured. The total colour difference
tities of osmotic solution and, therefore, solute were required to (DE) was calculated by the following equation:
carry out the OD process to the same level of dehydration (Assis qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
  2
ðL0
L Þ þ ða0
a Þ2 þ ðb0
b Þ
2
et al., 2017). DE ¼ ð1Þ
Each sample (approx. 25 and 100 g of apple cubes, for experi-
ments at atmospheric and vacuum pressures, respectively) was the index ‘‘0” indicating the sample before OD (t = 0).
placed in the osmotic solution with L. plantarum in a hermetic con- Tenfold measurements were performed.
tainer, which were kept in a water-bath at 37 °C and 50 rpm, dur-
ing 24 h. The osmotic dehydration was carried out at atmospheric 2.8. Gastro-intestinal tract simulation
pressure and under vacuum (150 mbar). The impregnation of the
probiotic took place during OD. 2.8.1. Inoculum
All experiments were performed in duplicate. After a 24 h-OD at 37 °C and atmospheric pressure and a 4 days-
storage at 4 °C, apple cubes incorporating L. plantarum were used
2.4. Probiotic enumeration as inoculum in the experiment of a simulation of the gastro-
intestinal tract.
Enumeration was performed in the apple samples and osmotic
solutions, in order to know if L. plantarum were incorporated in 2.8.2. Simulated gastro-intestinal conditions
apple samples or, if not, if they were viable in the osmotic solution A simulation of the gastro-intestinal tract was performed fol-
after OD. lowing the method described by Barbosa et al., 2015, with some
To enumerate probiotics in the apple samples, one apple cube modifications. One apple cube (approx. 1 g) from each sample
(approx. 1 g) was washed in sterile deionized water and blotted osmotically dehydrated with 40 or 60 °Brix sucrose or sorbitol
gently with tissue paper, and added to 9 mL of sterile Ringer’s solu- solutions at atmospheric pressure was triturated in a stomacher
tion in a stomacher bag. After trituration of the apple sample in a for 120 s and placed in a glass flask with 49.0 mL of Buffered Pep-
stomacher (BagMixerÒ 400 P, Interscience, France) for 4 min, serial tone Water (BPW, Lab M) adjusted to pH 3.0 with Hydrochloric
decimal dilutions were performed in sterile Ringer’s solution. From Acid (1 M HCl, Pronalab) and with 1000 units/mL of a filter steril-
osmotic solutions, 1 mL was added to 9 mL of sterile Ringer and ized solution of pepsin (Sigma) to simulate the stomach conditions.
serial decimal dilutions performed. Samples were taken at time 0 (time of inoculation) and after
Samples of the apple cubes and the osmotic solution were taken 30 min and 60 min (quick gastric transit simulation). To simulate
at t = 0, 6 and 24 h (for vacuum, only at t = 0 and 24 h). All samples the conditions of the small intestine, a sterile solution of bile salts
of apple and solution were treated as described previously. was added (final concentration of 0.3% (w/v), Pronadisa), after
Each sample and respective dilutions were plated on MRS agar, increasing the pH from 3.0 to 7.0 with a sterile solution of Sodium
in duplicate, by the drop count technique (Miles, Misra, & Irwin, Hydroxide (1 M NaOH, Pronalab). Again, samples were taken at
1938). Colony counting was performed after incubation at 37 °C time 0 (time of bile salts addition) and every 30 min for a total of
for 24 h. 60 min (quick digestion simulation). Enumeration of survivors
The experiments were performed in duplicate. 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 tempera-
ture of 21 ± 1 °C, before and after OD, with a hygrometer (Aqualab The statistical analysis was performed using IBM SPSSÒ Statis-
Series 3, Decagon Devices Inc., Pullmam, Washington, USA), by tics 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 cubes after the 24 h-OD in sucrose or sorbitol solutions were
data of water activity, moisture content, colour and enumeration around 1 log-unit and 2 log-units, respectively. Therefore, survival
of probiotics at the different conditions. The Levene’s test was used of L. plantarum in apple cubes osmotically dried in sucrose solu-
to verify the homoscedasticity, equality of variances. When the tions was improved in comparison with sorbitol solutions and
normality and the homoscedasticity was verified, ANOVA was used should be a better choice for an OD in vacuum. There were no sig-
to detect significant differences between the conditions. Tukey’s nificant differences in the L. plantarum survivals between 40 and
multiple range test was used to determine statistically significant 60 °Brix solutions for both osmotic agents used.
differences. A significance level of 5% was assumed. Other authors have investigated the impact of carbohydrates,
All results are presented as the average ± standard deviation including sucrose and sorbitol, on the survival of lactobacilli.
(SD). Ferreira et al. (2005) observed a 60% higher survival of L. sakei dur-
ing spray drying when it had previously been grown in the pres-
ence of sucrose. However, Linders, De Jong, Meerdink, and Van’t
3. Results and discussion Riet (1997) claimed that, although sorbitol and sucrose were pro-
tective for L. plantarum during fluidized bed drying, sucrose was
3.1. Viability of Lactobacillus plantarum after OD less successful. Also, in Perdana et al.’ study (2014), low-
molecular weight carbohydrates like sorbitol provided better sta-
The 60 °Brix solutions were used in the OD in order to have a bilization of L. plantarum during spray drying. Corcoran, Stanton,
higher process rate (Assis et al., 2017). Keeping in mind any Fitzgerald, and Ross (2005) demonstrated an enhanced survival
adverse effect of an excess intake of sorbitol, it was aimed to avoid of L. rhamnosus in gastric acid juice with metabolisable sugars,
osmotically dehydrated apple cubes with too high sorbitol concen- such as sucrose. In a more acid apple environment, sucrose could
tration and, therefore, 40 °Brix solutions were also tested. The ratio support the survival of lactobacillus. In the OD process, not only
of 1:4 had proved to be an efficient ratio for OD of apple cubes the carbohydrate solution, but also the apple matrix may benefit
(Assis et al., 2017). the survival of L. plantarum.
At the time of incubation (t = 0), counts of L. plantarum in the 40 Vacuum impregnation of probiotics showed to be an efficient
and 60 °Brix solutions were 9.9 ± 0.1 log cfu/mL. This initial value used method for the fortification of fruit matrices, possibly because
was considered N0. The probiotic showed viability in both the solu- of a fast introduction of external liquids into the fruits (Betoret
tions and in the apple cubes where it was incorporated during the et al., 2003; Zhao & Xie, 2004). Krasaekoopt and Suthanwong
OD. In Fig. 1 the log-unit percentage differences (%Dlog) between (2008) applied a pressure of 50 mbar for 5–15 min in the vacuum
the osmotic solution and the apple cubes are shown, for OD at impregnation of L. casei in papaya and guava pieces and noted an
37 °C at normal pressure (Fig. 1a) and in vacuum (Fig. 1b). irreversible destruction of the porous fruit matrix and a decreased
At atmospheric pressure, Dlog of 15.7 ± 3.9% were found for incorporation of L. casei, after only 10 min in vacuum. Interestingly,
apple cubes at t = 6 h, for both osmotic agents and both solution both the counts in the osmotically dehydrated apple and the osmo-
concentrations used (Fig. 1a), meaning that the counts of L. plan- tic solutions after the 24 h-OD were not significantly different
tarum were around 8 log cfu/mL. There does not seem to be a clear (p > 0.05). Since no measurement was performed at 6 h in vacuum,
tendency for the viability after the 6 h-OD. The differences it may be speculated that an equilibrium was reached of L. plan-
between the solutions (at t = 0) and the apple cubes after the tarum in the solution.
24 h-OD ranged from 13.9 ± 1.1% Dlog, for 40 °Brix sorbitol, to Small differences were also shown between OD performed with
22.6 ± 5.1% Dlog, for 60 °Brix sucrose. After this OD time, L. plan- the two different solutes, but the use of sucrose did ameliorate sur-
tarum could survive better in a lower concentrated solution, i.e. vival compared to sorbitol. Especially noteworthy is the OD in vac-
40 °Brix. Krasaekoopt and Suthanwong (2008) showed that lower uum, as even after a 24 h-OD, no significant differences (p > 0.05)
incorporation of L. casei into guava and papaya pieces was obtained were detected in the viability of probiotic from sucrose solutions
with 30 °Brix solutions compared to 15 °Brix solutions and between the osmotic solutions at t = 0 and apple cubes after
explained the inhibition of bacteria by the high sugar content. In 24 h-OD, as already mentioned. The reason could be the higher
fact, the degree of protection afforded by a given solute during dry- resulting water content in relation to the correspondent experi-
ing processes is species- and strain-dependent (Carvalho et al. ments with sorbitol (Fig. 2). The probiotic L. plantarum most prob-
2004). The duration of 6 h of the OD could be sufficient for the ably suffered from osmotic stress and, therefore, did not survive as
incorporation of L. plantarum into apple cubes, because the longer well in an apple cubes with reduced water content and aw. Ribeiro
time of 24 h decreased the viability of L. plantarum in apple cubes et al. (2014) demonstrated a 1-log-unit reduction of L. plantarum
for more concentrated solutions. In relation to the osmotic agent, incorporated in strawberry and banana, as well as a 3-log unit
there was no significant difference in the viability of L. plantarum reduction of the probiotic in kiwi after an air-drying process, and
in the apple cubes osmotically dehydrated in sucrose or sorbitol suggested that the bacteria counts decreased probably due to the
solutions. These findings go along with Assis et al.’s, who did not reduced water content.
find significant differences in the sugar gain (around 16%), there- Even though lactobacilli can adapt easily to changes in their
fore, in the total soluble solids of apple cubes osmotically dehy- environments, the solute concentration in the environment should
drated in the two types of solution with 60 °Brix and at 40 °C, be relatively constant (Poolman & Glaasker, 1998). An increased
using the same mass ratio of sample to solution, i.e., 1:4 (Assis osmolarity in the environment, which is the case of the sorbitol
et al., 2017). The incorporation of the probiotic could have followed and sucrose solutions, leads to osmotic stress, in consequence of
a pattern similar to the solute gain during OD. The water loss in a water flow from the inside of the cell to the environment, and,
that study was not significantly different after OD in both solutions therefore, a reduction of the cell turgor, a shrinkage of cells and
either (Assis et al., 2017). an intracellular increase of fruit acids (De Angelis & Gobetti,
In vacuum, counts of L. plantarum in apple cubes after the 24 h- 2004; Ribeiro et al., 2014). To some extent, L. plantarum can bal-
OD in 40 and 60 °Brix sucrose solutions were not significantly dif- ance intra- and extracellular concentrations of sucrose and lactose,
ferent (p > 0.5) than their osmotic solutions at t = 0 and, so, % Dlog but osmotic stress can also be detrimental (Jordan, Hutchings, &
relative to these conditions were low (Fig. 1b). However, the same Mascher, 2008; Van de Guchte et al., 2002). Santivarangkna,
behaviour does not occur in 40 and 60 °Brix sorbitol solutions. Dif- Kulozik, and Foerst (2006) found that sorbitol has a positive effect
ferences between survivals in the solution (at t = 0) and the apple 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 higher aw, % Dlog presented a tendency to be lower for the OD with
extent than without any addition and probably suffered osmotic 40 °Brix sucrose at atmospheric pressure and in vacuum, this
stress. The viability of the probiotic during OD may also depend meaning that L. plantarum survived better. Following this pattern,
on the structure of the fruit. Ribeiro et al. (2014) found that straw- apple cubes osmotically dehydrated in 60 °Brix sorbitol solutions,
berry had the highest cell count after immersion in L. plantarum with the lowest water content and aw, tended to contain smaller
suspension and attributed this fact to the high porosity of this fruit, amounts of L. plantarum. Exceptions were apple cubes osmotically
which probably promoted the adherence of the probiotic to the dried in the 60 °Brix sucrose solution at 37 °C and at normal atmo-
fruit. Apple is also a high porous fruit. sphere, which showed similar viability, in spite of the moisture
Considering the OD time, it can be considered that longer OD content and the aw being higher than in apples from OD in the 60 °-
times cause higher viability losses of L. plantarum inside the apple Brix sorbitol solution. Also, L. plantarum in apple samples dehy-
cubes. drated under vacuum presented a tendency to survive less in the
After the 24 h-OD in normal atmosphere and in vacuum, the 40 °Brix sorbitol solution than in the 60 °Brix sucrose solution,
counts of bacteria in 60 °Brix sorbitol solutions decreased signifi- although the aw and moisture content of osmotically dried apple
cantly (p < 0.05), while for sucrose solutions no changes were from 40 °Brix sorbitol was significantly higher (p < 0.05). As dis-
found. cussed before, an explanation could be a better viability of the pro-
biotic with sucrose than with sorbitol.
From this point of view, it cannot be claimed that either the
3.2. Relation between the moisture content and water activity and the
sucrose or sorbitol, or 40 or 60 °Brix osmotic solutions are more
viability of Lactobacillus plantarum in osmotically dehydrated apple
recommended for the incorporation of L. plantarum into apple
cubes
cubes, but the osmotic stress could play an important role in the
survival of L. plantarum, while OD and the resulting water content
Fig. 2 shows the viability of L. plantarum in apple cubes in rela-
in the apple matrix may also be crucial.
tion to the moisture content and the aw after 24 h-OD. No signifi-
cant differences (p > 0.05) were found among % Dlog
corresponding to the different values of moisture content and aw 3.3. Storage of apple cubes incorporated with Lactobacillus plantarum
of samples osmotically dehydrated with 40 and 60 °Brix sucrose after OD
or sorbitol solutions either at atmospheric pressure or in vacuum.
However, a tendency can be derived from both the moisture con- Factors as levels of oxygen, pH, storage temperature, the pres-
tents as well as the aw values. For higher moisture contents and ence of competing microorganisms, among others, determine the
524 K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528

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 sur-
vival 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 suffi-
cient for further handling in food industry as the osmotically dried
apple cubes are intermediate moisture products meant to be fur-
ther 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 addi-
tionally 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
Fig. 2. Moisture content and water activity of apple cubes incorporated with L. pieces over 65 days with a decrease of only 1 log cfu/g. However,
plantarum after OD (24 h) with 40 or 60 °Brix solutions of sucrose or sorbitol at in these two latter studies the products presented a much lower
37 °C and atmospheric pressure (a) and in vacuum of 150 mbar (b) (Dlog (%) = (log aw than the products of the present study.
(N0,solution)
log(N))/log(N0,solution) * 100).

shelf life of a probiotic product, i.e., the viability of probiotics in the 3.4. Colour during OD and storage
food matrices (Coman et al., 2012). The storage temperature of 4 °C
has been proven to be the best temperature to maintain the viabil- The colour changes (DE) of apple cubes during OD at 37 °C and
ity of probiotic in a fruit matrix over time in several studies at atmospheric pressure as well as in vacuum are depicted in
(Barbosa et al., 2015; Borges et al., 2016; Ribeiro et al., 2014). In Fig.4a and b. The colour tended to change in all samples from t =
the present work, the viability of L. plantarum during storage at 0 to the end of the OD (t = 24 h), while over the storage period
4 °C was high (Fig. 3a and b). lower changes were observed.
After a 6 day-storage at 4 °C the log reductions were higher for After 4 days of storage, no significant differences (p > 0.05) in
L. plantarum in apple samples from 40 °Brix sucrose solutions with DE were observed among samples osmotically dehydrated with
0.9 log-unit reduction, compared with the 4 day-storage. Lacto- 40 and 60 °Brix sucrose or sorbitol solutions either at atmospheric
bacillus plantarum presented tendency to increase in apple cubes pressure or in vacuum, and DE was maintained relatively constant.
from 60 °Brix solutions (Fig. 3a). The influence of the soluble solids may also have played an
In Fig. 3b the behaviour during storage of L. plantarum in apple important role. Lower DE values were obtained for apple samples
cubes from the OD at 37 °C in vacuum is depicted. Overall, after 4 dehydrated in 60 °Brix solutions (Fig. 4a and b). This could be
days, the reductions observed in the viability of the probiotic were due to the protective effect of incorporated sugar, which prevents
all inferior to 1 log-unit. A reduction of 0.8 log-units was found for browning reactions. Krokida, Karathanos, and Maroulis (2000)
the dehydrated apple samples from the 40 °Brix solutions and a studied the colour changes during the OD of apple samples and
reduction of 0.7 log-units was also observed in apple cubes from noticed that the infusion of sugar had positive effect on stability
60 °Brix solutions. No reduction of L. plantarum occurred in apple of L⁄, a⁄ and b⁄ values.
samples osmotically dehydrated with sorbitol. After 6 days, log L⁄ of fresh apple was 71.02 ± 3.30, a⁄ was
3.86 ± 0.96 and b⁄
cfu/mL of L. plantarum increased in sucrose solutions, but not in 29.96 ± 2.40 The browning process led to a lower L⁄ (indicating a
sorbitol solutions compared to storage after 4 days. No consistent less light colour), higher a⁄ (a redder product) and lower b⁄ (a less
tendency can be drawn from analysing the storage of apple cubes yellow product) values over time. L⁄ values decreased less in apple
from the different treatments. samples from 60 °Brix solutions and b⁄ values were constant or
Differences in the viability of the probiotic among samples were even increased. (L⁄ 51.35 ± 3.19, b⁄ 30.36 ± 3.01). Increased b⁄-
irrelevant. Therefore, L. plantarum survived without any significant values were observed for more yellowish apple samples from 60 °-
log reductions (p > 0.05) in all samples during a 6 day-storage at Brix solutions after OD and storage. A decrease in yellowness with
4 °C. a b⁄ = 24.00 ± 3.52 was observed for apple cubes from 40 °Brix
Other authors claimed a positive effect of carbohydrates during solutions, after a 6 days storage. L⁄ was 45.08 ± 2.97 after storage
the storage of L. plantarum. Carvalho et al. (2002) found that 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 low after 60 min and even 120 min. The reductions of L. plan-
cubes was not studied. Randazzo et al. (2013) had not find any tarum were minimal after exposure to the acidic conditions of
relation between added L. rhamnosus to peach jam and the colour the stomach (pH 3.0 with pepsin). Probiotic viability in these
parameters. conditions could be due to the protective effect of metabolisable
sugars in an acid environment as mentioned before (Corcoran
3.5. Gastro-intestinal tract simulation et al., 2005). After exposure to bile salts at pH 7.0, a slightly
decrease of L. plantarum was observed, with log reductions
A functional product can only be claimed as probiotic, if it con- below 1 log-unit. Other studies have demonstrated a lower sur-
tains about 106 – 107 colony forming units of viable probiotic cells vival of L. plantarum in the presence of small intestine condi-
in 1 gram of product upon ingestion. During digestion, this number tions, than at stomach conditions (Barbosa et al., 2015;
of viable microorganisms needs to be constant and, therefore, the Mirlohi, Soleimanian-Zad, Dokhani, Sheikh-Zeinodin, & Abghary,
survival of probiotics through gastro-intestinal tract is indispens- 2009). Cebeci and Gürakan (2003) proved the tolerance of L.
able (FAO/WHO, 2002). In this work, a quick digestion in a simu- plantarum HU to acid and bile salts and suggested the use of
lated gastro-intestinal tract was performed of the dehydrated the probiotic in functional food.
apple cubes containing L. plantaru, according to the method pro- L. plantarum incorporated in the apple matrix survived during
posed by Barbosa et al. (2015). No significant changes (p > 0.05) passage through the gastro-intestinal tract simulation and could
in the viability of L. plantarum were found in apple cubes dehy- be considered a probiotic food. Samples contained around 108
drated in either 40 or 60 °Brix sucrose solutions, as well as 40 or cfu/g after the OD, around 107 cfu/g after a 6 days-storage and
60 °Brix sorbitol solutions during the whole gastro-intestinal tract maintained this value after the gastro-intestinal tract simulation.
simulation (Table 1). The probiotic apple product impregnated with L. plantarum and
The survival on 30 min and 90 min were not presented, sorbitol could be considered a potential symbiotic product with
because the reductions of the probiotic viability were already 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).

Table 1
and probiotics has recently been investigated. Mandal et al. (2009) Survival of L. plantarum incorporated in apple cubes after OD (24 h) with 40 or
proved a decrease of plasma cholesterol level in Swiss albino mice 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).
with a treatment of sorbitol combined with P. acidilactici and
demonstrated the in vitro positive impact of sorbitol on bacteriocin log (N/N0)a
production. Osmotic solution 0 min 60 minb 120 minc
Nonetheless, any adverse effects of sorbitol cannot be ignored at
40 °Brix sucrose 0.00 ± 0.00 0.00 ± 0.00
0.50 ± 0.10
consumption. After the 24 h-OD in 40 °Brix and 60 °Brix sorbitol 40 °Brix sorbitol 0.00 ± 0.00
0.10 ± 0.10
0.20 ± 0.10
solutions, the apple cubes contained, 9 and 12.5% of sorbitol, 60 °Brix sucrose 0.00 ± 0.00 0.10 ± 0.10
0.10 ± 0.00
respectively. Therefore, a possible daily consumption of osmoti- 60 °Brix sorbitol 0.00 ± 0.00
0.30 ± 0.30
0.40 ± 0.20
cally dehydrated apple cubes could be around 80–100 g assuming a
Survival is represented as the media of the logarithmic reduction: log (N/
a safe consumption of 10 g sorbitol per day (Burt, 2006; Rowe et al., N0) ± the standard error of the mean, N is the cfu/ml at each sampling time, N0 is the
2009). This amount of apple is equivalent to around 60–80 dehy- cfu/ml at time zero.
b
drated apple cubes, which is a sufficient amount for the use in a 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
functional food.
exposure to bile salts at pH 7.0.
 K. Emser et al. / Journal of Functional Foods 38 (2017) 519–528 527

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This work was supported by National Funds from FCT – Fun-
Science, 42, 723–731.
dação para a Ciência e Tecnologia through project UID/ Krokida, M. K., Karathanos, V. T., & Maroulis, Z. B. (2000). Effect of osmotic
Multi/50016/2013 and by the European M.Sc. in Food Science, dehydration on color and sorption characteristics of apple and banana. Drying
Technology, 18, 937–950.
Technology and Business (BiFTec). J. Barbosa acknowledges the
Lee, K., & Lee, Y. (2010). Effect of Lactobacillus plantarum as a starter on the food
support provided by the post-doctoral fellowship SFRH/ quality and microbiota of kimchi. Food Science and Biotechnology, 19, 641.
BPD/113303/2015 (FCT). The authors also acknowledge Campotec Linders, L. J. M., De Jong, G. I. W., Meerdink, G., & Van’t Riet, K. (1997). Carbohydrates
for graciously supplying the apples for this study. and the dehydration inactivation of Lactobacillus plantarum: The role of
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