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Received: 23 July 2019    Revised: 15 November 2019    Accepted: 22 November 2019

DOI: 10.1111/jfpp.14346

ORIGINAL ARTICLE

Survival and stability of free and encapsulated probiotic

bacteria under simulated gastrointestinal conditions and in
pasteurized grape juice

Muhammad Afzaal1  | Farhan Saeed1  | Muhammad Saeed2 | Aftab Ahmed1 |

Huda Ateeq1 | Muhammad Tahir Nadeem1 | Tabussam Tufail3

1
Institute of Home & Food Sciences,
Government College University, Faisalabad,
Pakistan
2
National Institute of Food Science &
Technology, University of Agriculture,
Faisalabad, Pakistan
3
University Institute of Diet & Nutrition
Sciences, Faculty of Allied Sciences,
University of Lahore, Lahore, Pakistan
Correspondence
Muhammad Afzaal, Institute of Home
& Food Sciences, Government College
University, Faisalabad, Pakistan.
Email: muhammadafzaal@gcuf.edu.pk
Abstract
The present study was designed to explicate the effect of encapsulation on the
stability of Bifidobacterium bifidum in pasteurized grape juice and in vitro gastroin-
testinal conditions. Purposely, hydrogel beads were prepared using sodium alginate
and K-carrageenan by internal gelation method. Free and encapsulated probiotics
were incorporated in pasteurized grape juice samples. The initial count in grape juice
samples with free probiotic cells was calculated as 9.35 log cfu/mL that reduced to
6.58 log cfu/mL after 35 days. Likewise, the probiotic count, that is, in samples con-
taining encapsulated probiotics reduced to a level of 8.51 log cfu/mL and 7.09 log
cfu/mL after the mentioned storage period. The number of free viable cells in
juice samples was lower than (107 cfu/g) the minimum recommend level. Similarly,
the free cells showed a poor survival under simulated gastrointestinal conditions.
Furthermore, encapsulation affected the physicochemical (pH, acidity, and Brix) and
sensorial characteristics of the juice samples.
Practical applications
The probiotic delivery through carrier food and under hostile gastrointestinal condi-
tions is a great challenge due to their low survival. The encapsulation technology can
be used to protect the target delivery of probiotics. The encapsulation technology is
a useful tool for delivering the probiotics for both dairy and nondairy food matrices.
Encapsulation also ensures the recommended therapeutic level (1107–108 cfu) during
transit and storage.

1 |  I NTRO D U C TI O N
Probiotics are live microbes that confer certain health benefits when
administered in adequate amounts (Hill et al., 2014). Commonly,
probiotics are used in the preparation of fermented milk prod-
ucts. Recommendations for probiotics as functional foods or even
as a medicine have been available in several fields (Gill, Franco, &
Abbreviations: GIT, Gastrointestinal tract; KC, Kepa-carrageenan; SA, Sodium alginate.
Hancock, 2015; Hempel et al., 2011; Paulina & Katarzyna, 2017).
Numerous health benefits that are related to the ingestion of live
probiotics have been stated earlier (Gómez-Gallego, Gueimonde,
& Salminen, 2018; Sanders, Merenstein, Merrifield, & Hutkins,
2018; Sanders, Merenstein, Reid, Gibson, & Rastall, 2019), which
includes improving lactose utilization, lowering blood ammonia lev-
els, and controlling intestinal infections (Griffiths, Medellin-Pena, &
Delcenserie, 2018; Nunes, 2018; Shori, Baba, & Muniandy, 2019).
On account of health-promoting benefits around the globe, food
industries are producing probiotics-supplemented foods commonly

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referred as functional foods. Furthermore, it is suggested that pro-
biotics not only lowers the serum cholesterol level but also have a
beneficial effect on the immune system. (Liong & Shah, 2005; Nazir,
Hussain, Abdul Hamid, & Song, 2018). As probiotics provide a series
of health benefits, the assimilation of probiotics in a variety of foods,
that is, cheese, yogurt, drinks, and fruit juices, etc., has increased
(Khaneghah & Fakhri, 2019; Ong, Henriksson, & Shah, 2006).
Fruit juices contain high amounts of sugars which could encour-
age probiotic growth and could easily be monitored using a refrac-
tometer. Fruit and vegetable juices are considered as an ideal carrier
for the delivery of probiotics as they contain notable levels of vi-
tamins and antioxidants, considered suitable for lactose intolerant
people (Vodnar et al., 2019). Pineapple found to be a good alterna-
tive as a probiotic carrier due to its nutritional composition poten-
tial (Krasaekoopt & Kitsawad, 2010). Moreover, they are a suitable
medium for probiotics as they can survive in them during storage.
(Rúa et al., 2018). For this purpose, grape juice can be a potential
carrier of probiotics owing to its nutritional profile (Mokhtari, Jafari,
& Khomeiri, 2019; Panghal et al., 2018).
To achieve the most beneficial effects at a target site, probiotics
reflect resistance toward the hostile gastrointestinal tract (Rokka &
Rantamäki, 2010; Zanjani, Ehsani, Tarzi, & Sharifan, 2018). Protection
of probiotics by microencapsulation improves their viability in func-
tional foods. Microencapsulation improves the resistance and survival
of probiotics under various food processing conditions and under hos-
tile human digestive system (Zanjani et al., 2018). Encapsulation by in-
ternal gelation through alginate encapsulating material may consider as
the appropriate methods for the encapsulation of probiotics. Among
other potential ingredients, alginate biopolymer is the most studied
and examined for bioencapsulation (Vejan, Abdullah, Khadiran, &
Ismail, 2019). However, there is a limited literature available on the use
of encapsulated probiotic in fruit juices.
Therefore, this study was conducted to evaluate the physicochem-
ical, sensory, and storage stability of pasteurized grape juice as a non-
dairy probiotic carrier of free and gums encapsulated probiotics.
2 |  M ATE R I A L S A N D M E TH O DS
Good quality raw grapes (variety Flame, seedless grapes) were
purchased from an indigenous farm and fresh grape juice was ex-
tracted. Probiotic culture Bifidobacterium bifidum (ATTC-4356) was
purchased from the National Institute of Biotechnology and Genetic
Engineering (NIBGE). K-carrageenan and sodium alginate were pur-
chased from the scientific store. Further research was carried out at
Food Safety and Biotechnology lab, Government College University,
Faisalabad.
2.1 | Culture activation
Freeze-dried culture of Bifidobacterium bifidum (ATTC-4356) in
pure form was purchased from NIFSAT, University of Agriculture
Faisalabad, Pakistan. The identification of culture was carried out ac-
cording to the method of Shah and Lankaputhra (Vejan et al., 2019).
Probiotic cells were activated by inoculating it for 24 hr in MRS (Man
Rogosa Sharpe) broth at 37°C. Afterward, the cells were centrifuged
in a centrifuge machine (75,005,276 EA, Thermo Fisher Scientific
Inc. USA) and the acquired probiotic cells were encapsulated for fur-
ther studies.
2.2 | Microencapsulation of probiotic culture
Microencapsulation of B. bifidum was carried out with a little modifi-
cation in the internal gelation method according to Mokhtari, Jafari,
and Khomeiri (2018). Shortly, 60 ml of sodium alginate and k-car-
rageenan was mixed with 10 ml of cell solution. The solution ob-
tained with both encapsulating materials was then dispersed using
a 5 ml syringe into a beaker that contained germ-free oil and 5 g of
tween-80 solution as an emulsifier and the mixture was stirred at
150 rpm with a help of magnetic stirrer. Calcium chloride was added
dropwise generally till the emulsion broke. The prepared microbeads
were washed with double distilled water on Whatman filter paper
and stored at a refrigerated temperature.
2.3 | Encapsulation yield
Encapsulation yield was calculated by following the method of
Iqbal, Zahoor, Huma, Jamil and Ünlü after some modifications
(2019). For the purpose, microbeads were randomly chosen from
both types of encapsulation media. The beads were crumbled in a
stomacher with phosphate buffer solution and 0.1 M sodium citrate
solution at a pH of 6.3. The coated viable cells were calculated by
the pour plate technique and yield was calculated by the following
formula:
Number of cells released (sodium alginate bead and k − carrageen bead)
EY = × 100
Number of cells added (sodium alginate and k − carrageenan solution)
2.4 | Preparation of probiotic grape juice
The grape juice concentrate (Brix 70) was diluted in water until its
Brix decreased to 15. According to dos Santos Lima et al. 1 L grape
juice was pasteurized to 85°C for 60–65s (2015). Afterward, the
juice sample was added with various types of probiotics as men-
tioned in Table 1 and juices were kept at 4°C for four weeks.
2.5 | Physicochemical analysis of grape juice
The pH of the grape juice was determined by a digital pH meter
according to AOAC (2006). The acidity of the grape juice was de-
termined by the titration method according to AOAC (2006). Total
AFZAAL et al.
soluble solids (Brix) were determined using a refractometer (Abbe
RefrectometerWYA-2S).
2.6 | Viability/Survival of free and encapsulated B.
bifidum in grape juice
The viability of B. bifidum in grape juice was determined at different
intervals during storage. The grape juice was mixed with peptone
6
water (0.1%) and diluted up to 10 serial dilutions. The viability of
the probiotics was determined after incubating it for 24–48 hr in
an incubator under anaerobic conditions according to Praepanitchai,
Noomhorm and Anal (2019).
2.7 | Sensory evaluation
The sensory evaluation of the prepared juices was conducted ac-
cording to the procedure adopted by Kardum et al. (2017) with little
modification. Experienced faculty members of the department were
nominated to take part in sensory evaluation. After every 7  days,
the panelists tasted the grape juice and observed it for color, fla-
vor, mouthfeel, and overall perception during the storage of over
35 days. For sensory evaluation, 9-point hedonic scale was used (0
extremely undesirable and 9 extremely desirable).
2.8 | Survival/Viability of B. bifidum in simulated
gastrointestinal conditions
The survival of B. bifidum in simulated gastrointestinal conditions
was determined according to the method as previously described
Damodharan, Palaniyandi, Yang, and Suh (2017). The simulated gas-
trointestinal conditions were prepared using different chemicals
and the pH of the simulated gastric condition was adjusted using
5M HCL. The microencapsulated and free cells were then added in
the test tubes and incubated at 37°C under continuous agitation.
TA B L E 1   Treatment plan for preparation of grape juice samples
Trails Type
G1 Control (without any probiotics)
G2 Free cells (B. bifidum)
G3 Encapsulated (Sodium alginate)
G4 Encapsulated (K-carrageenan)
TA B L E 2   Encapsulation efficiency
Type of materials
Sodium alginate (encapsulated)
K-Carrageenan (encapsulated)
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Survival of free and encapsulated cells was recorded at prede-
fined intervals, that is, 0, 30, 60, 90, and 120 min.
2.9 | Statistical analysis
All the data recorded in this research were subjected to analysis
of variance (ANOVA) to observe the level of significance (p < .05)
among the calculated values. Each value was determined after tak-
ing the mean of three replicates.
3 | R E S U LT S A N D D I S CU S S I O N S
3.1 | Encapsulation yield
Constituents of wall medium have a direct effect on the yield of
encapsulation. The encapsulation yield also affects the survival of
simulated digestive conditions (Bora, Li, Zhu, & Du, 2018). Both en-
capsulating materials showed a difference in encapsulation yield.
Alginate coating gave a higher yield as compared to carrageenan
coating as shown in Table 2
3.2 | Physicochemical analysis of grape juice
3.2.1 | pH
The pH of all the grape juice samples showed a decrease as shown
in Figure 1. A sudden decrease in pH was observed in G2 grape juice
treatment (with free B. bifidum) after 35 days. The pH decreased from
3.71 to 2.93 in G2 juice treatment after storage for over 35 days.
The least reduction in pH was observed in G3 (encapsulated with
alginate coating material) from 3.74 to 3.24 after 35 days of storage
under 4°C. The probiotics may utilize the sugars to produce organic
acids (Goderska, Nowak, & Czarnecki, 2008). Bujna, Farkas, Tran,
Sao Dam, and Nguyen (2018) also suggested in one of his studies
that a decrease in pH of fruit juices that contains Bifidobacterium
lactis, Lactobacillus paracasei, and Lactobacillus rhamnosus could be
owing to acid production and bacterial growth.
In a study conducted by Kailasapathy, it was suggested that
yogurt storage with nonencapsulated and encapsulated showed
a decrease in pH; however, encapsulated probiotics showed a de-
crease in pH as compared to free cells (Pinto, Fritzen-Freire, Dias,
& Amboni, 2019). Similar results have been reported earlier by Ding
and Shah (2008).
Number before Number after
encapsulation encapsulation Efficiency (%)
8.96 ± 0.04 8.72 ± 0.05 97
8.25 ± 0.02 7.47 ± 0.07 90
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F I G U R E 1   Effect of free
(unencapsulated) and encapsulated
(with sodium alginate and carrageenan)
on pH of pasteurized grape juice during
storage intervals (0, 7, 14, 21 28, and
35 days) compared with control. Every
bar represents a mean value for pH
of treatments. G1 (Control without
the addition of probiotics), G2 (Free/
unencapsulated cells), G3 (Probiotics
encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with
K-carrageenan)

F I G U R E 2   Effect of free (unencapsulated) and encapsulated (with sodium alginate and carrageenan) on the acidity of pasteurized
grape juice during storage intervals (0, 7, 14, 21 28, and 35 days) compared with control. Every bar represents a mean value for the acidity
of treatments. G1 (Control without the addition of probiotics), G2 (Free/unencapsulated cells), G3 (Probiotics encapsulated with sodium
alginate), and G4 (Probiotics encapsulated with K-carrageenan)

3.2.2 | Acidity 3.2.3 | Brix

The results from the acidity of pasteurized grape juice are shown
in Figure 2. As the pH of the juice decreases its acidity increases.
A continuous increase in the acid was observed in the treatment
G2 (with free probiotics). Its acid content increases from 3.13 to
7.37 mg/L. However, a minimum increase in the acid content of
the pasteurized grape juice was observed in G3 (encapsulated
with sodium encapsulating material). The initial acid content in G3
was 3.17 mg/L at Day 0 of storage which increases to 6.28 mg/L
after 35 days of storage. From the detailed results, it can be pre-
dicted that the acid production in the juice is the result of sugar
consumption by bacteria, as free bacteria have access to utilize
sugars present in the juice and convert it into acid (Ding & Shah,
2008). Goderska et al. evaluated that probiotics produce lac-
tic acid in the product accompanied with different saccharides
(2008). As fruit juices contains large levels of sugar components
which probiotics can consumed, and as a result can alter the sen-
sory profile of juice, therefore, encapsulation of probiotics can
prevent these defects and limits the acid production in fruit juices
(Mokhtari et al., 2018).
The detailed results regarding total solids (Brix) are shown in
Figure 3. From the results, it can be concluded that the Brix in all
grape juice treatments decreased due to a decrease in the sugar con-
tent. However, in treatments containing encapsulated probiotics (G3
and G4), Brix decreased gradually as the probiotics have an indirect
access toward the soluble sugars. The increase in acidity and decline
in pH and Brix are related to the activity of bacteria of the product
which is controlled in products containing coated probiotics (Bujna
et al., 2018; Mokhtari et al., 2018).
3.3 | Survival of probiotic microorganisms
The results regarding the probiotic survival in pasteurized juice are
shown in Figure 4. The initial population of B. bifidum was 9.5 log
cfu/mL. A sudden decrease in the growth of B. bifidum was noted
in G2 (containing free probiotics) after 35 days. B. bifidum reduces
from 9.5  log cfu/mL to 5.46  log cfu/mL. A 4.04  cfu/mL log reduc-
tion was observed for G2 treatment (containing free probiotics).
AFZAAL et al. |
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F I G U R E 3   Effect of free
(unencapsulated) and encapsulated (with
sodium alginate and carrageenan) on
Brix of pasteurized grape juice during
storage intervals (0, 7, 14, 21 28, and
35 days) compared with control. Every
bar represents a mean value for the
Brix of treatments. G1 (Control without
the addition of probiotics), G2 (Free/
unencapsulated cells), G3 (Probiotics
encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with
K-carrageenan)

F I G U R E 4   Survival and stability of

free (unencapsulated) and encapsulated
(Sodium alginate and carrageenan)
in pasteurized grape juice during
storage intervals (0, 7, 14, 21 28, and
35 days) compared with control. Every
bar represents a mean value for the
survival/viability of probiotics. G2 (Free/
unencapsulated cells), G3 (Probiotics
encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with
K-carrageenan)

Comparatively, a slow reduction in the population of B. bifidum was
observed in G3 and G4. The minimum decrease in the probiotic
growth was observed in G3 treatment (encapsulated with sodium en-
capsulating material). The probiotic growth after 35 days of storage
was 8.27 log cfu/mL. A 1.23 log cfu/mL log reduction was observed.
G2 treatment contained B. bifidum in free form; therefore, due to
an increase in the acidic content in juice the chances for the survival
of B. bifidum lessens which results in a rapid decline in its population.
According to Mokhtari et al. (2018), B. bifidum may not survive the
acidic conditions of the grape juice that supports the results of pres-
ent study. To increase the chances of its viability of probiotics, these
were encapsulated with two different encapsulating material so-
dium alginate and k-carrageenan. Sodium coated probiotics showed
a better resistance as compared to carrageenan coated probiotics. It
was found by Praepanitchai et al. (2019) that survival of L. plantarum
was detected in simulated digestive conditions when coated with
alginate coating. Terpou et al. (2019) predicted that microencapsu-
lation improves the viability of probiotics in different carrier foods.
Similar results were previously reported by (Azarkhavarani, Ziaee, &
Hosseini, 2019).
3.4 | Simulated gastric conditions
The sustainability of probiotics in gastrointestinal conditions is es-
sential to get the desired therapeutic benefits. The results from
the simulated gastric conditions are shown in Figure 5. B. bifidum
in encapsulated and nonencapsulated form were exposed to acti-
vated gastric juice. A rapid decrease in the number was observed
in free probiotic bacteria with a log reduction of 4.55, while in the
case of encapsulated cells (sodium alginate coating and k-carra-
geenan coating), a gradual decrease was observed. Cells encap-
sulated with S.A showed a log reduction of 1.68; however, in the
case of K.C, a log reduction of 2.77 was noted. The encapsula-
tion of cells affected the survivability of B. bifidum significantly
(p < .05). From the results it can be concluded that encapsulation
enhances the survival of cells. Similar results were also observed
by Afzaal et al. (2006).
3.5 | Simulated intestinal conditions
The viability of probiotics is very essential in intestinal condition.
Free/nonencapsulated and encapsulated cells were exposed to
simulated intestinal conditions for a specific time interval. A rapid
decline in the population of free cells was noted as compared to
encapsulated cells at 7.6 pH as shown in Figure 6. A slow log re-
duction in probiotics encapsulated with S.A and K.C was observed
that showed the protection effect of encapsulation. The results are
similar to the findings of Iqbal et al. (2019) and Afzaal et al. (2006).
Results showed that encapsulation has a shielding effect against
simulated conditions.
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F I G U R E 5   Survival of free
(unencapsulated) and encapsulated
(sodium alginate and carrageenan) under
simulated gastric conditions with intervals
of (0, 30, 60, 90, and 120) minutes.
Every bar represents a mean value for
the survival of probiotics G2 (Free/
unencapsulated cells), G3 (Probiotics
encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with
K-carrageenan)

F I G U R E 6   Survival of free
(unencapsulated) and encapsulated
(sodium alginate and carrageenan) under
simulated intestinal conditions with
intervals of (0, 30, 60, 90, and 120 min).
Every bar represents a mean value for
the survival of probioticsG2 (Free/
unencapsulated cells), G3 (Probiotics
encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with
K-carrageenan)

F I G U R E 7   Effect of free (unencapsulated) and encapsulated (sodium alginate and carrageenan) on the sensory profile of pasteurized
grape juice during storage intervals (0, 7, 14, 21 28, and 35 days) compared with control. Every bar represents a mean value for the sensory
score. G1 (Control without the addition of probiotics), G2 (Free/unencapsulated cells), G3 (Probiotics encapsulated with sodium alginate),
and G4 (Probiotics encapsulated with K-carrageenan)

3.6 | Sensory analysis color of juice in all treatments at Day 0 of storage. After 35 days of
storage, no color change was observed except G2. As reported by
The mean results from the sensory analysis are shown in Figure 7. Mokhtari et al. (2018), acid production by free bacteria may affect the
Most of the evaluators found no significant difference (p < .05) in the nutrients and sensory profile of fruit juice and hence change in color
AFZAAL et al.
was observed by most of the evaluators. When mean results from the
flavor parameter were observed, results showed a significant differ-
ence. The flavor of G2 bacteria was least accepted due to high acidic
and sour flavor. However, the flavor of the control treatment (G1) and
treatment encapsulated with S.A (G3) were accepted the most.
From the data it was concluded that the treatments that contains
nonfree (encapsulated) bacteria recorded minimum about mouthfeel
of the juice. A similar result was found by Mokhtari et al. (2018).
He also suggested that optimizing bacteria may reduce this problem.
It was reported by Krasaekoopt and Kitsawad (2010) that less than
20% of consumers did not like the fruit juice that contained encap-
sulated beads that sometimes stuck in the throat or remained in the
mouth (Krasaekoopt & Kitsawad, 2010).
From the data regarding overall perception, it can be concluded
that the treatment G1 and G3 showed higher scores. Treatment G2
scored lowest because it contained free bacteria that utilizes the
sugars and produced acid that decreased the acidity and causes
sourness in the juice. Bacteria with a coating on surroundings pre-
vent acid production which increases its overall sensory perception.
In the present study, G3 treatment (sodium alginate coating material)
was the most acceptable juice treatment.
4 | CO N C LU S I O N S
Encapsulation technology is suitable in ensuring the beneficial level
(106–10 8 cfu/g) of probiotics in carrier food. The purpose of this
work was to evaluate the suitability of fruit juice as a nontraditional
carrier of probiotics. The encapsulation of probiotics with either al-
ginate or carrageenan coating material provided better resistance to
probiotics under simulated GI conditions as well as during stages’
conditions. The encapsulation with sodium alginate and carrageenan
can effectively be used to improve the viability and stability under
hostile conditions and in the product. Overall, pasteurized grape
juice is suggested as a good alternative to dairy products for the de-
livery of probiotics.
AC K N OW L E D G M E N T S
The authors are thankful to the Institute of Home and Food Sciences,
Government College University Faisalabad and National Institute of
Food Science and Technology for providing technical support and
laboratory facilities during research work.
C O N FL I C T O F I N T E R E S T
The authors have declared no conflicts of interest for this article.
E T H I C A L A P P R OVA L
This article does not contain any studies with human participants or
animals performed by any of the authors.
I N FO R M E D C O N S E N T
Ethics approval and consent to participate. For this type of study,
formal consent is not required.
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C O N S E N T FO R P U B L I C AT I O N
Not applicable.
ORCID
Muhammad Afzaal  https://orcid.org/0000-0001-9047-9075
Farhan Saeed  https://orcid.org/0000-0001-5340-4015
Tabussam Tufail  https://orcid.org/0000-0002-7632-5261
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How to cite this article: Afzaal M, Saeed F, Saeed M, et al.
Survival and stability of free and encapsulated probiotic
bacteria under simulated gastrointestinal conditions and in
pasteurized grape juice. J Food Process Preserv.
2019;00:e14346. https​://doi.org/10.1111/jfpp.14346​