Received: 3 July 2020 | Revised: 18 November 2020 | Accepted: 21 November 2020

DOI: 10.1111/jfpp.15132

ORIGINAL ARTICLE

Osmotic infusion of Lactiplantibacillus plantarum and

Lacticaseibacillus casei in cut pineapple matrix: Optimization,
survival under gastrointestinal stress, and storage stability
studies

Silpa Vijay1 | Sruthi Vikraman1 | Priyanka Rose Mary1,2 | Attar Singh Chauhan2,3 |
Mukesh Kapoor1,2

1
Department of Protein Chemistry
and Technology, CSIR-Central Food Abstract
Technological Research Institute, Mysuru, The study aimed at developing a functional food by osmotic infusion of probiotic
India
2 bacteria in pineapple matrix. Significant (p* < .05) incorporation of Lactiplantibacillus
Academy of Scientific and Innovative
Research (AcSIR), CSIR-Human Resource plantarum (L. plantarum) (8.54 ± 0.04 log CFU/g) and Lacticaseibacillus casei (L. casei)
Development Centre (CSIR-HRDC) Campus,
(8.55 ± 0.05 log CFU/g) in probiotic infused cut pineapple piece was observed after
Ghaziabad, UP, India
3
Department of Fruit and Vegetable
4 hr of dehydration in sucrose-based (50 °Brix) osmotic solution. SEM results corrob-
Technology, CSIR-Central Food orated successful infusion of probiotic cells at both surface and sub-surface levels.
Technological Research Institute, Mysuru,
India
Osmotically dehydrated pineapple pieces showed a marginal decrease in brightness,
yellowness, increased firmness, and insignificant changes in pH and aw. Under simu-
Correspondence
Mukesh Kapoor, Department of Protein
lated gastric stress, infused L. plantarum and L. casei exhibited good survival of up to
Chemistry and Technology, CSIR-Central 8.7 ± 0.04 log CFU/g. Under simulated intestinal stress, both L. plantarum and L. casei
Food Technological Research Institute,
Mysuru, India.
recorded reduction of approximately 1 log CFU/g (7.8 ± 0.07 log CFU/g). The Infused
Email: [email protected] L. plantarum and L. casei showed viability >6 log CFU/g till 10 days at −20°C.

Funding information Practical applications

Department of Biotechnology , Ministry
Increased awareness among consumers toward the intricate relationship between
of Science and Technology, Grant/Award
Number: BT/PR23266/PFN/20/1286/2017 diet and disease has rekindled interest in functional foods containing probiotics, es-
pecially the non-dairy segment. The sale of functional food snacks is projected to
reach $8.5 billion by 2020. India is among the largest producers of pineapple, which
occupies an important niche in the cut fruit market. Development of cut pineapple as
a probiotic carrier using energy saving procedure like osmotic dehydration will help
expand its reach in the functional food market. The growth and survival of probiotic
bacteria in functional food products under gastro-intestinal stress conditions is the
key to deliver the desired health benefits. Our results indicate not only efficient os-
motic infusion of L. plantarum and L. casei in the cut pineapple matrix but also good
survival under simulated gastrointestinal stress and storage conditions. Our studies
showed cut pineapple infused with probiotics by osmotic dehydration can be a suit-
able non-dairy probiotic food.

Silpa Vijay and Sruthi Vikraman have equal contributions.

J Food Process Preserv. 2021;45:e15132. wileyonlinelibrary.com/journal/jfpp © 2020 Wiley Periodicals LLC. | 1 of 10

https://doi.org/10.1111/jfpp.15132
2 of 10 | VIJAY et al.

1 | I NTRO D U C TI O N L. plantarum and L. casei in cut pineapple matrix by osmotic de-

hydration using sucrose and to analyze the microbiological prop-
Development of novel functional foods enriched with probiotics, erties, survival under gastrointestinal stress, and storage stability
prebiotics, and dietary fiber has become one of the key research pri- (3 weeks at −20°C) of the probiotic bacteria in the developed
orities in food industries. This is due to increased awareness among product together with physicochemical characteristics of the pro-
consumers toward the intricate relationship between diet and dis- biotic infused pineapple pieces.
ease (Makki et al., 2018). As a consequence, the functional food
snacks market is estimated to generate revenue of about $8.5 bil-
lion by 2020 (https://www.ift.org/). The consumption of probiotics 2 | M ATE R I A L S A N D M E TH O DS
like lactic acid bacteria provides various health benefits to the host
such as antimicrobial and anticarcinogenic effects, amelioration of 2.1 | Chemicals
gastrointestinal diseases, control of blood sugar, reduction of serum
cholesterol levels, and anti-inflammatory activities (Ashaolu, 2020; MRS (broth and agar) and citric acid were purchased from Hi-Media
Cheng et al., 2019). laboratories Pvt. Ltd, India. Pepsin, pancreatin, and bile salts were
Research and development of probiotic foods has tradition- purchased from Sigma-Aldrich (St. Louis, MO, USA). NaCl and KCl
ally focused on fermented dairy products like yogurt, kefir (Wang were purchased from Merck India Pvt. Ltd. Table sugar and pineap-
et al., 2020), and recently on products such as ice cream (Panwar ples were purchased locally. Pineapples were selected on the basis
et al., 2019). However, lactose intolerance in around 75% of world's of color (semi-ripened, green-yellow), uniformity, and absence of any
population, milk protein allergy, high cholesterol content, and life- physical injury or infestation. L. plantarum (NCIM 2372) and L. casei
style choices such as vegetarianism and veganism represent some (NCIM 2126) were procured from NCIM, CSIR-NCL, Pune, India. All
of the limiting factors impeding the growth of dairy-based probiotic other chemicals and reagents used in the present study were of ana-
foods (Heenan et al., 2004; Prado et al., 2008). lytical grade.
Fruits and vegetables are reservoirs of many nutritionally im-
portant compounds like vitamins, minerals, dietary fiber, sugars
(mainly fructose, glucose, and sucrose) apart from antioxidant capac- 2.2 | Sample preparation
ity and these attributes makes them a suitable matrix to support and
stabilize the growth of probiotics (Cassani et al., 2020). Therefore, 2.2.1 | Pineapple processing
nowadays, demand for non-dairy based probiotic foods like fresh cut
fruit and vegetables is rising dramatically (Emser et al., 2017; Prado Pineapples were washed under running tap water followed by rins-
et al., 2008; Rascón et al., 2018). ing with reverse osmosis (RO) purified water. The washed pineapples
Osmotic dehydration of fresh-cut fruits and vegetables by im- were peeled and cut into small triangular pieces weighing ~1 g each.
mersion in a hypertonic solution has emerged as an energy con- Thereafter, pineapple pieces were blanched by dipping them into hot
serving preservation method to improve the sensorial (Ferrari RO water (90°C) for 1 min. The excess water was drained off by fil-
et al., 2010), nutritional (Cvetković et al., 2019), and functional tering using sterile cheese cloth.
(Emser et al., 2017) properties of food and reducing microbial con-
tamination (Tylewicz et al., 2020). Few researchers have attempted
incorporation of probiotics like Lactobacillus sp. in fruit matrices such 2.2.2 | Inoculum preparation
as apple (Emser et al., 2017; Flores Andrade et al., 2017) and banana
(Huerta-Vera et al., 2017) using osmotic dehydration to evaluate L. plantarum and L. casei were inoculated in MRS broth (5 ml) and
their suitability as probiotics carriers. incubated at 37°C under static conditions for 16 hr to prepare the
Pineapple (Ananas comosus L.) is the only edible member of primary inoculum. 100 μl of primary inoculum was added to the
the family Bromeliaceae. It is majorly cultivated in India, Costa secondary culture (MRS broth; 10 ml) and incubated at 37°C under
Rica, Brazil, Philippines, Thailand, and Indonesia. In 2018, 1.706 static conditions for 16 hr. The viable cell count of the secondary
million tons of pineapple was harvested in India (http://www.fao. culture was determined by serial dilution and plating onto MRS agar
org/faost​at/en/#data/QC). Pineapples are a good source of an- plates. Thereafter, the secondary culture was centrifuged (7,000 rpm
tioxidants, vitamins, fiber, polyphenols, and minerals (Hossain & [6311× g], 10 min, 4°C) and the pellet obtained was washed twice
Rahman, 2011). Owing to its large size and difficult processing, (7,000 rpm [6311× g], 10 min, 4°C) with sterile phosphate buffer sa-
pineapple forms an integral part of the fresh-cut fruit market line (PBS). The pelleted cells were re-suspended in 1 ml of sterile
(Dávila-Aviña et al., 2020). PBS.
The development of fresh cut pineapple as a probiotic carrier The viable cell count of L. plantarum or L. casei inoculum was de-
by osmotic dehydration using sucrose; along with studies on sta- termined as 9.33 log CFU/ml and 9.40 log CFU/ml, respectively, and
bility of infused probiotic under gastrointestinal stress have not maintained at similar levels by appropriate dilution in all the subse-
been explored yet. The present study was aimed at infusion of quent experiments.
VIJAY et al. | 3 of 10

2.3 | Infusion of Lactobacillus sp. into pineapple Thereafter, washed cell pellets of L. casei (9.40 log CFU/ml) and
pieces by osmotic dehydration L. plantarum (9.33 log CFU/ml) were added separately to each os-
motic suspension (initial inoculum level: 8.6–8.7 log CFU/g) and
2.3.1 | Preparation of sucrose-based incubated under vacuum conditions (200 mbar, 2 hr) in a flash
osmotic solutions evaporator. Afterwards, three random pineapple pieces (weighing
~1 g each) were removed from the suspension, rinsed with sterile
Sucrose-based osmotic solutions were prepared by adding table sugar RO water, to remove the surface adhered sugar solution and ana-
in RO water and the solutions were heated under continuous stirring lyzed for the efficiency of probiotic bacteria infused in pineapple
until the sugar dissolved completely. The °Brix of each solution was matrix as described in Section 2.3.3.
measured using a digital refractometer (H196801 Hanna instruments,
USA). Thereafter, citric acid (0.1% w/v) was added to each solution. The
prepared solutions were filtered using a sterile cheese cloth. 2.4 | Survival and viability of L. plantarum and L.
casei infused in pineapple matrix under simulated
gastrointestinal conditions
2.3.2 | Optimization studies
2.4.1 | Preparation of simulated gastric juice
Pineapple pieces (25; total weight ~25 g) were immersed separately (SGJ) and simulated bile juice (SBJ)
in each sucrose-based (10–60 °Brix) osmotic solution at 1:1 mass to
volume ratio. Thereafter, washed cell pellets of L. casei (9.40 log CFU/ SGJ was prepared by dissolving sodium chloride (2 g/L) and pepsin
ml) and L. plantarum (9.33 log CFU/ml) were added separately to each (3.2 g/L) in deionized water and pH was adjusted to 2.5 with 1 M
osmotic suspension (Initial inoculum level: 8.6–8.7 log CFU/g) and in- HCl. SBJ was prepared by dissolving pancreatin (1mg/ml) and bile
cubated at 37°C for up to 24 hr under static conditions. Afterwards, salts (4.5 mg/ml) in deionized water and pH was adjusted to 8 with
three random pineapple pieces (weighing ~1 g each) were removed 0.1 M NaOH. Both SGJ and SBJ were used after filter (0.22 µm)
from the suspension, rinsed with sterile RO water, to remove the sur- sterilization.
face adhered sugar solution and analyzed for the efficiency of probi-
otic bacteria infused in pineapple matrix as described in Section 2.3.3.
2.4.2 | Survival and viability studies

2.3.3 | Measurement of probiotic viability and The survival of L. plantarum and L. casei infused in pineapple pieces
infusion efficiency was studied under quick sequential simulated gastrointestinal stress
as described earlier (Barbosa et al., 2015) with certain modifications.
Probiotic viability was estimated as per Rodrigues et al., 2012. Briefly, ~1 g of osmotically dehydrated pineapple piece was placed
Briefly, three random probiotic infused pineapple pieces weighing in 9 ml of SGJ (pH 2.5) and incubated at 37°C for 1 hr under shaking
~1 g each were removed from a given sucrose-based osmotic so- conditions (70 rpm). Samples were withdrawn at stipulated time in-
lution. Thereafter, each pineapple piece was washed and crushed tervals (0 min [time of inoculation], 30 min and 60 min [quick gastric
in 1 ml of sterile PBS buffer separately, serially diluted and plated transit]). The conditions of small intestine were simulated by trans-
on MRS agar by the drop plate method (Herigstad et al., 2001). The ferring the gastric acid transit exposed samples to 9 ml of SBJ (pH 8)
plates were incubated at 37°C for 48 hr and colony forming units per and incubating at 37°C for 1 hr under shaking conditions (70 rpm).
gram (CFU/g) were enumerated. Samples were withdrawn in the subsequent 30 and 60 min. The
The infusion efficiency was calculated as per Alfaro-Galarza withdrawn samples were washed, crushed, and dissolved in sterile
et al. (2020) using the following formula: PBS buffer and cell viability was estimated by the drop plate method
(Herigstad et al., 2001).
N
IE % = × 100 (1)
N0

where N is the number of viable cells (log CFU) released from the pine- 2.5 | Storage stability of L. casei and L. plantarum
apple pieces and N0 is the number of viable cells (log CFU) in the initial infused in osmotically dehydrated pineapple pieces
inoculum solution.
Storage stability of infused L. casei and L. plantarum in osmotically
dehydrated pineapple pieces stored at −20°C for 20 days was esti-
2.3.4 | Vacuum-assisted osmotic dehydration for mated. At time intervals of 5, 10, and 20 days, samples were with-
infusion of Lactobacillus sp. into pineapple pieces drawn for the measurement of probiotic viability. The withdrawn
samples were washed, crushed, and dissolved in sterile PBS buffer
Pineapple pieces (25; total weight ~25 g) were immersed in su- and cell viability was estimated by the drop plate method (Herigstad
crose-based (40 and 50 °Brix) osmotic solution in 1:1 mass ratio. et al., 2001).
4 of 10 | VIJAY et al.

The kinetics of viability of probiotic bacteria over the storage pe- The browning index for pineapple pieces both before and after
riod was determined by fitting first-order reaction kinetics model to osmotic dehydration was calculated as per the following formula:
the plot between logarithmic of the relative viability (Log Nt/N0) ver-
sus storage time (t, days) according to González-Ferrero et al. (2018) 100 (x − 0.31)
BI = (4)
0.17
using the following equation:
a + 1.75L
Nt x= (5)
log = kt t (2) 5.645L + a − 3.012b
N0

where Nt represents the total viable bacteria (in CFU/g) at particular 2.6.4 | Texture and pH analysis
storage period, N0 represents the number of viable cells (in CFU/g) at
the beginning of storage, t is the storage time (in days), and kt is the The texture of fresh and osmotically dehydrated pineapple pieces
specific rate of viability loss at −20°C on a given assay day. was determined using universal texture measuring system (Model
no. LR5K, Lloyd Instruments). Warner-Bratzler attachment was used
to measure the shear force required to cut the pineapple at a speed
2.6 | Physical and physiochemical rate of 50 mm/min. pH was measured in accordance with AOAC
characterization studies method 981.12 (AOAC International, 2016). To measure the pH, un-
treated and osmotically dehydrated pineapple pieces were crushed
2.6.1 | Scanning electron microscopy (SEM) in sterile RO water and pH was determined using a digital pH meter.

The distribution of infused L. casei and L. plantarum in pineapple ma-

trix was analyzed by SEM. The pineapple pieces were cut along the 2.7 | Statistical analysis
longitudinal axis under aseptic conditions and mounted on a clean
glass cover slip followed by air drying. Thereafter, the cover slip was All the experiments were performed in triplicate unless otherwise
mounted on silver tape and sputter-coated with gold (Plaron SEM stated and values obtained are expressed as mean ± SD. Data were
coating system, Watford, England, model E5100) and examined analyzed using one-way analysis of variance (ANOVA) and Tukey's
under a scanning electron microscope (LEO 435 VP, Cambridge, UK). test for statistical significance at p* < .05 using the SPSS Statistics
20.0 (2012, SPSS Inc., Chicago, USA).

2.6.2 | Water activity (aw)

3 | R E S U LT S A N D D I S CU S S I O N
Water activity was determined as per Emser et al. (2017).
Measurement of water activity was done by placing ~4 pineapple 3.1 | Optimization of L. plantarum and L. casei
pieces (weighing ~1 g each) in the container of water activity meter infusion into cut pineapple pieces by osmotic
(Novasina, Lachen, Switzerland). Measurements were carried out at dehydration
27°C both before and after osmotic dehydration.
Osmotic dehydration involves partial removal of moisture from plant
tissues after immersion in a concentrated solution due to the non-
2.6.3 | Color determination destructive osmosis phenomenon (Rastogi et al., 2014). Counter dif-
fusion of solutes from the osmotic solution into the plant tissue also
The surface color of fresh and osmotically dehydrated pineap- occurs simultaneously with the loss of moisture. This process helps
ple pieces was evaluated by placing ~5 pineapple pieces (weighing to retain the color, flavor, and organoleptic characters of the osmoti-
~1 g each) in the measuring container of colorimeter (CM-5 Konica cally dehydrated fruits (Emser et al., 2017; Rastogi et al., 2014).
Minolta, Tokyo, Japan). D-65 illumination lamp was used to obtain To optimize the effect of °Brix of sucrose-based osmotic solution
the coordinates of L* (measure of lightness), a* [varies from green (−) and time on the osmotic infusion of probiotics in cut pineapple ma-
to red (+)], and b* [varies from blue (−) to yellow (+)] and the observer trix, an initial inoculum containing 8.63 log CFU/g of L. plantarum and
was kept at 10°. 8.70 log CFU/g of L. casei was used (Figure 1). Significantly (p* < .05)
The total color difference (ΔE) was calculated according to Emser higher infusion of L. plantarum (8.38 log CFU/g) and L. casei (8.39
et al. (2017) as per the following equation: log CFU/g) was observed in sucrose-based (50 °Brix) osmotic solu-
tion (Figure 2). High number of infused bacteria in sucrose-based (50
√
E=
(
L0∗ − L ∗
)2 )2 (
+ a0∗ − a ∗ + b0∗ − b ∗
( )2
(3) °Brix) osmotic solution could be due to the higher osmotic gradient
between hypertonic solution and core of pineapple matrix when
The subscript “0” indicates the sample before osmotic dehydra- compared with other sucrose-based (10–40 °Brix) osmotic solutions.
tion at time t = 0. The marginal reduction in the number of infused bacteria at higher
VIJAY et al. | 5 of 10

F I G U R E 1 Flow chart for the osmotic

infusion of Lactiplantibacillus plantarum
and Lacticaseibacillus casei in cut pineapple
matrix

TA B L E 1 Effect of time on infusion of probiotic bacteria by

10 osmotic dehydration under atmospheric pressure conditions
c,d,e

d,e,f
d,e,f
b,c,d

a
b,c,d

Lactiplantibacillus Lacticaseibacillus
e,f
f

b,c

8
b

plantarum casei
g
log CFU/g

6 Time (h) log CFU/g

a
1 8.47 ± 0.20 8.34 ± 0.20a
4 2 8.50 ± 0.08a 8.49 ± 0.09a
a
3 8.53 ± 0.10 8.51 ± 0.06b
2
4 8.54 ± 0.04a 8.55 ± 0.05b
b
24 8.71 ± 0.07 8.75 ± 0.05c
0
10 20 30 40 50 60 Note: Values are mean ± standard deviation; Means with different
superscript (a–c) in each row denote significant differences (p* < .05).
οBrix

transfer of mass (probiotic bacteria) to surface and sub-surface of

F I G U R E 2 Optimization of °Brix of sucrose-based osmotic
solution for the infusion of Lactiplantibacillus plantarum (■) and pineapple tissue during osmotic dehydration process requires less
Lacticaseibacillus casei (■) in cut pineapple matrix. Means with time (Flores-Andrade et al., 2017). It has been shown earlier that the
different superscript (a–g) denote significant differences (p* < .05) osmotic driving force carrying the probiotic cells from the surround-
ing hypertonic solution to the dilute sap of the fresh fruit is highest
at the beginning of the process (Flores-Andrade et al., 2017). Fruits
Brix concentration (60 °Brix) might be due to the inhibitory effect like pineapple (porosity: 0.16–0.25), apple (porosity: 0.18–0.22) and
of sugars on the cell growth. Similar to our findings, Krasaekoopt strawberry (porosity: 0.47) have porous tissue and thin cell walls,
and Suthanwong (2008) reported lesser incorporation of L. casei into thereby offering little resistance to the mass transfer of water and
guava and papaya pieces at 30 °Brix solutions when compared to 15 soluble solids during osmotic dehydration (Fernandes et al., 2019).
°Brix solutions. Other researchers have reported appreciable incor- Though the increase in the numbers of infused bacteria after
poration of probiotic bacteria at 40 and 60 °Brix degrees in apple 24 hr of incubation in the sucrose-based osmotic solutions was sig-
(Emser et al., 2017) and banana (Flores-Andrade et al., 2017; Rascón nificant (p* < .05) in comparison to 4 hr of incubation but the cell
et al., 2018), respectively. multiplication was not appreciable enough. This could due to the in-
Studies on the effect of time required for optimal infusion of L. hibitory effect of high sugar concentration on the growth of bacte-
plantarum and L. casei during osmotic dehydration showed maximum ria. The count of probiotic bacteria incorporated in pineapple pieces
incorporation in 24 hr (8.71–8.75 log CFU/g). However, shorter in- corresponded well with the values shown previously for other fruits
cubation periods (1–4 hr) also showed comparable numbers of in- incorporated with probiotic bacteria (Emser et al., 2017; Rascón
fused bacteria (8.34–8.55 log CFU/g) (Table 1). This showed that et al., 2018).
6 of 10 | VIJAY et al.

F I G U R E 3 Scanning electron
micrographs of osmotically infused (a and
b) Lactiplantibacillus plantarum and (c and
d) Lacticaseibacillus casei in cut pineapple
matrix

TA B L E 2 Infusion of probiotic bacteria by osmotic dehydration many researchers as an efficient method for the incorporation of
under vacuum conditions (200 mbar) probiotics in fruit matrices (Emser et al., 2017; Flores Andrade et al.,

Lactiplantibacillus Lacticaseibacillus 2017).

plantarum casei Vacuum impregnation of sucrose-based (40 and 50 °Brix) osmotic
°Brix of sucrose-based
solutions containing L. plantarum and L. casei at 200 mbar pressure
osmotic solution log CFU/g
showed infusion of 7.30–7.69 and 8.04–8.27 log CFU/g, respectively
a
40° 7.30 ± 0.08 7.69 ± 0.04b (Table 2). The efficiency of vacuum impregnation of probiotic cells
50° 8.04 ± 0.06 c 8.27 ± 0.07d was marginally less (L. plantarum 8.2%–12.9%; L. casei 1.4%–4.2%)
Note: Values are mean ± standard deviation; Means with different than that reported under the atmospheric pressure conditions. This
superscript (a–d) in each row denote significant differences (p* < .05). could be due to the highly porous structure of the pineapple matrix
which offers less resistance to the osmotically driven movement of
SEM micrographs showed that surface of pineapple pieces was fluids (Fernandes et al., 2019). Our results are in contrast with other
continuous, smooth, and homogeneous. L. plantarum and L. casei had researchers who have shown higher infusion of cells by vacuum im-
rod shape with an approximate size of 3.2 μm and 2.2 μm, respectively. pregnation in apple and banana when compared with atmospheric
The pineapple matrix was found to retain most of the infused bac- conditions (Emser et al., 2017; Huerta-Vera et al., 2017). Therefore,
terial population within the core (sub-surface infusion) (Figure 3b,d) energy consuming vacuum impregnation does not appear as a suit-
while some bacteria remained on the surface (Figure 3a,c). Both L. able method for probiotic infusion in the cut pineapple matrix.
plantarum and L. casei were found to infuse approximately at equal
numbers without any strain to strain variation as can also be seen in
Table 1. Sub-surface infusion could improve the bacterial viability 3.2 | Survival of infused L.
when exposed to adverse gastrointestinal stress environment. There plantarum and L. casei under quick sequential
were no evident pores on the surface of pineapple pieces and this simulated gastrointestinal transit
would prevent any subsequent cell leakage.
The beneficial effects of functional foods enriched with probiotic
bacteria can only be realized when the probiotic bacteria remains
3.1.1 | Vacuum-assisted osmotic dehydration for viable under the harsh gastrointestinal stress conditions (Cassani
infusion of Lactobacillus sp. into pineapple pieces et al., 2020; FAO/WHO, 2001).
In SGJ, both L. plantarum and L. casei infused in pineapple pieces
Vacuum impregnation results in replacement of the internal gas or exhibited good survival of up to 8.6 and 8.7 log CFU/g, respectively,
liquid, which is occluded in open pores, with the osmotic solution after 60 min of incubation (Figure 4). Presence of metabolizable
when the atmospheric pressure is restored. Overall, it results in sugars in acidic environment might render protective effect to the
rapid entry of extraneous fluids into the fruits (Betoret et al., 2003). probiotic strains in simulated gastric stress conditions (Corcoran
Vacuum impregnation of probiotics has been shown previously by et al., 2005). Tolerance and sustenance of probiotic strains under
VIJAY et al. | 7 of 10

12

10

b
b

b
b

a
8

a
log CFU/g
6

2
F I G U R E 4 Survival of
Lactiplantibacillus plantarum (■) and
Lacticaseibacillus casei (■) osmotically 0
infused in cut pineapple matrix through 0 20 40 60 80 100 120 140
quick digestion simulation of the
gastrointestinal tract conditions. Means Simulated gastric transit Time (min)
with different superscript (a and b) denote
significant differences (p* < .05) Simulated bile transit

bile stress is important for successful colonization of gut epithelial observed during storage at −20°C after 20 days. Both L. plantarum
lining of the host (Ranadheera et al., 2012). In SBJ, both L. casei (up and L. casei showed survival higher than 6 log CFU/g till 10 days of
to 7.8 log CFU/g) and L. plantarum (7.8 log CFU/g) recorded reduction refrigerated storage. These results were within the required thera-
of approximately 1 log CFU/g after incubation for 120 min (Figure 4) peutic minimum level of intake (6–9 log CFU/g or ml) recommended
but maintained a viability of >6 log CFU/g which is required to exert for probiotic-supplemented foods before consumption (Kemsawasd
positive health benefits (Kemsawasd et al., 2016). et al., 2016).
It has been shown previously that food matrix plays a significant The first-order reaction kinetics model of viability of probiotic
role in protecting the probiotic bacteria from gastrointestinal stress bacteria over the storage period showed R 2 values between .897
(Casarotti et al., 2015). Therefore, pineapple matrix having a dietary and .950. L. plantarum showed a lower rate (k = −0.09) of viability
fiber-rich, porous structure acted as a good support to adhere and loss when stored at −20°C over a period of 20 days, in compari-
protect the probiotic bacteria during the simulated gastrointestinal son to L. casei (k = −0.07) (Figure 5b). Previously, González-Ferrero
transit (Borges et al., 2016). et al. (2018) have reported the viability loss of k = −0.151 and
k = −0.096 for L. plantarum and L. casei-free cell suspensions, re-
spectively, at 25°C.
3.3 | Evaluation of storage In general, sugars promote the viability of probiotic bacte-
stability of L. plantarum and L. casei infused in ria during storage as shown earlier in fermented cereal extracts
osmotically dehydrated pineapple matrix (Charalampopoulos & Pandiella, 2010). However, the gradual decline
in the number of Lactobacillus sp. under frozen conditions might be
In probiotic enriched food products, viability of probiotic bacteria due to the cold shock leading to cell death (Homayouni et al., 2008).
during the storage period depends on several parameters such as Moreover, water activity (aw > 0.25) and the presence of atmo-
pH, growth factors, presence of competing microorganisms, com- spheric oxygen might also contribute to the reduction in viable cell
position of food matrix, storage temperature, and levels of oxygen counts (Anal & Singh, 2007).
(Buriti et al., 2010; Coman et al., 2012).
The pineapple matrices were osmotically infused with ap-
proximately 8.6–8.7 log CFU/g of L. plantarum and L. casei cells. 3.4 | Physical and physiochemical
Thereafter, all samples were stored at −20°C for 20 days and ex- characterization studies
amined for the viability of infused probiotic bacteria (Figure 5a).
Infused L. plantarum showed a log reduction of 2.8 (from 8.4 to 5.6 Consumer preference for functional food products is majorly gov-
log CFU/g) during storage at −20°C after 20 days. Whereas a log re- erned by the perception of sensory attributes such as color (Balthazar
duction of 2.62 (from 8.39 to 5.77 log CFU/g) of infused L. casei was et al., 2017; Panwar & Kapoor, 2019). The L*, a*, and b* values of the
8 of 10 | VIJAY et al.

(a) (b) Time ( Days)

0 5 10 15 20 25
0.00
LP
10
LC
-0.04

Log (Nt/N0)
8
-0.08
log CFU/g

LJ сͲϬ͘ϬϬϳdž
Zϸ с Ϭ͘ϴϵϳ
6 -0.12

LP
-0.16 LC LJ сͲϬ͘ϬϬϵdž
4 Zϸ с Ϭ͘ϵϱϬ
Fit
-0.20
0 5 10 15 20 25
Days

F I G U R E 5 Storage stability of Lactiplantibacillus plantarum (●) and Lacticaseibacillus casei (○) osmotically infused in cut pineapple matrix
under frozen conditions (−20°C) for 20 days. (B) Kinetics of viability loss of infused probiotic bacteria [Lactiplantibacillus plantarum (●) and
Lacticaseibacillus casei (■)] under storage conditions (−20°C) for 20 days

TA B L E 3 Physical and physiochemical

Lactiplantibacillus plantarum Lacticaseibacillus casei
analysis of osmotically dehydrated
Before After Before After pineapple infused with probiotic bacteria
Analysis Parameter treatment treatment treatment treatment

Water activity (aw) 0.958 ± 0.02a 0.951 ± 0.05a 0.955 ± 0.05a 0.949 ± 0.03a
Color
L* (brightness) 65.41 ± 0.3a 61.43 ± 0.7b 65.41 ± 0.7a 61.43 ± 0.4b
a a a
a* (red-green) −0.62 ± 0.04 −0.6 ± 0.06 −0.62 ± 0.05 −0.6 ± 0.05a
b* (yellow-blue) 47.85 ± 0.3a 42.8 ± 0.5b 47.85 ± 0.4a 42.82 ± 0.7b
ΔE 0 6.4 0 6.4
BI 115.957 107.169 115.957 107.169
Texture (hardness)
Maximum 7.789 ± 0.81a 10.75 ± 0.63b 7.789 ± 0.74a 10.75 ± 0.08b
load applied
(N) (speed of
50 mm/min)
pH 4.2 ± 0.02a 4.4 ± 0.02a 4.2 ± 0.01a 4.5 ± 0.02a

Note: Values are mean ± standard deviation; Means with different superscript (a and b) in each
column denote significant differences (p* < .05).

osmotically dehydrated pineapple pieces infused with L. plantarum et al., 2007). Similar results have been reported for osmotically dried
and L. casei were measured and the results are presented in Table 3. apple slices infused with probiotic bacteria using hypertonic solu-
Infusion of soluble solids in pineapple led to a marginal decrease in tions of sucrose and sorbitol (Emser et al., 2017).
the brightness and yellowness of the product. However, redness (b*) In food matrices such as fruit and vegetables, water activity (aw)
values remained unchanged. Accordingly, a low ΔE value (6.4) was can serve as a useful indicator for changes in microbiological and
obtained for both L. plantarum and L. casei infused pineapple pieces physicochemical parameters (Maltini et al., 2003). Water activity of
after 4 hr of osmotic dehydration in 50 °Brix solutions. The brown- fresh cut pineapple pieces after osmotic dehydration treatment was
ing index for the infused pineapple pieces after osmotic dehydra- tested to analyze the changes in aw values. Table 3 shows a very mild
tion (107.169) was significantly lesser (p* < .05) when compared with reduction (up to 0.007) in the water activities of osmotically dehy-
pineapple pieces before osmotic dehydration (115.957) (Table 3). drated pineapple pieces infused with either L. plantarum or L. casei.
Decrease in L* values and rise in a* values are commonly used indi- The negligible change observed in the water activity might be due
cators of browning (Rojas-Graü et al., 2006). to higher initial solid content of the fruit, action of solute on water
On the basis of ΔE and browning index values, our results suggest sorption behavior, or both, during the dehydration process in con-
a protective effect of osmotically impregnated sugar in preventing centrated sucrose solutions (Rastogi et al., 2014). Earlier, apple cubes
excessive browning reactions in the cut pineapple pieces (Krokida osmotically dehydrated in 60 °Brix solution at atmospheric pressure
VIJAY et al. | 9 of 10

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PR23266/PFN/20/1286/2017; GAP No. 523) funded by the DBT, rived Lactobacillus plantarum in fermented cereal extracts during re-
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