Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-019-09579-w

Application of Microencapsulated Synbiotics

in Fruit-Based Beverages
Camelia Rovinaru 1 & Diana Pasarin 1

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
In the last years, demand for functional products containing both prebiotics and probiotics (known as synbiotic) has increased,
which stimulated their incorporation into other food matrices than milk-based ones. Synbiotics improve gut functionality as well
as respond to the increasing demand of consumers who have become aware of the health benefits of a proper diet. The most
important criterion for preserving consumer acceptance in such products is maintaining the minimum viability and activity of
probiotics from the beginning of production to the end of shelf-life. For their viability, fixation and multiplying within the host,
several solutions have been proposed including the fortification with prebiotics and microencapsulation of prebiotics along with
probiotics. The challenge of microencapsulation is to protect the probiotic cells in foods that are not usually considered their
vehicle, such as fruit matrices. It is generally known that different prebiotics may exert different degrees of protection on the
entrapped bacteria cells. For food products, such as fruit beverages, few works exist that investigate the functionality of synbiotic
microcapsules in protecting the survivability of probiotic cells during processing and storage. This article provides an overview of
this novel trend based on a review of relevant literature. The article summarizes the synbiotic concept, challenges for synbiotic
formulation in fruit beverages, and future perspectives.

Keywords Fruit beverages . Lactic acid bacteria . Microencapsulation . Prebiotic . Probiotic . Synbiotic

Introduction According to the World Health Organization (WHO) ex-

Functional products represent a novel trend in the design of
healthy foods and the largest and growing market segment in
America, Europe, Asia (i.e., Japan) and Australia [1]. Among
functional ingredients, prebiotics and probiotics are preferred
by consumers who have become aware of the role of diet in
conferring health benefits. Prebiotics are indigestible or low-
digestible food ingredients that selectively promote the growth
and activity of one, or a limited number, of the beneficial
bacteria in the intestine. The basic criteria for prebiotic selec-
tion are resistance to digestion in the upper sections of the
gastrointestinal tract, selectively fermentation by potentially
beneficial intestinal microbiota, beneficial effect on the host’s
health, selective stimulation of growth of the probiotics, and
stability in food processing conditions [2].
* Diana Pasarin
diana.pasarin@gmail.com
1
Institutul National de Cercetare-Dezvoltare pentru Chimie si
Petrochimie, 202 Spl. Independentei, 060021 Bucharest, Romania
pert group, probiotics are defined as “live micro-organisms
which, when administered in adequate amounts, confer a
health benefit on the host” [3]. As such, the use of the word
“probiotics” can suggest a beneficial health effect on human
subjects. European national regulatory authorities forbid the
use of the word “probiotic” since it embeds a non-validated
health claim. So far, EFSA has rejected more than 500 probi-
otic health claim applications on the basis of a lack of charac-
terization of the microorganisms and because the increase in
healthy gut flora is not considered a health benefit. The
upgraded Propionibacterium freudenreichii W200 would be
the first strain with an approved health claim, implying the
first probiotic that can truly be called a probiotic [4]. Even if
the health claim incorporated in the definition is not properly
investigated, products found on the market are sold with this
premise.
Selection process of new probiotic organisms involves
safety criteria (history of safe use, human or animal origin,
precis diagnostic identification, absence of pathogenicity, in-
fectivity, and virulence) and functionality criteria (tolerance to
low pH in the stomach and to bile salts in order to reach the
gastrointestinal tract; capacity to adhere and ability to colonize

some sites within the gastrointestinal epithelium, reducing
pathogenic adherence; the capability to survive, multiply,
and maintain the metabolic activity; resistance to acids, hydro-
gen peroxide, and bacteriocins produced by endogenous in-
testinal pathogens) [4–6].
The biological effects of probiotics are strain-specific. It is
shown that probiotic strains have unique genetic traits that
determine particular phenotypes. As such, the properties of a
genus or species could not be extrapolated to their strains, and
consequently, the characteristics of a probiotic are determined
at a strain level. The strain can be identified using novel mo-
lecular technologies (16S r RNA gene sequence analysis,
DNA-DNA hybridization) [7].
In the last years, demand for functional products containing
both prebiotics and probiotics (known as synbiotic) has in-
creased, which stimulated their incorporation into other food
matrices than milk-based ones [8]. Therefore, the food indus-
try has an ever-growing interest in the development of non-
dairy synbiotic products to improve gut functionality, as well
as to respond to the increasing customer demand for new
flavors. Fruits, cereals, vegetables, and soybeans have been
proposed as new matrices supporting synbiotics delivery in
humans. Adding probiotic strains to fruit matrices rich in car-
bohydrates, dietary fibers, vitamins minerals, and antioxidants
offers specific health benefits [9, 10]. These matrices affect the
delivery of the total number of viable cells mL−1 product at the
moment of consumption, due to high level of organic acids
and a low pH, typically 2.5–3.7. The most important criterion
to preserve the consumer acceptance in such probiotic prod-
ucts is maintaining the quantity and the minimum viability
and activity of probiotics during processing, storage as well
as at the end of shelf-life [9]. It is known that the major prob-
lem for dairy probiotic products is the low survival rate of
probiotic bacteria during storage. The minimum recommend-
ed level of probiotic count in the food at daily intake, neces-
sary to provide a therapeutic effect, is usually 10 6 –
108 cfu mL−1. In different products in Japan, the minimum
viable level of 107 cfu g−1/cfu mL−1 of product is imposed,
which corresponds approximately to 109 cfu per portion of
100 g of probiotic food/day. From a technological and cost-
effective point of view, the food industry respects the recom-
mended probiotic bacteria level of 106 cfu g−1 at the time of
consumption, concentration at which beneficial health effects
in humans are visible [11, 12].
In the purpose of enhancing the viability of probiotics in
the product and their fixation and multiplying within the host,
several solutions have been adopted, among which we men-
tion the fortification with prebiotics and microencapsulation
of pre- and probiotics. By adding prebiotics, a favorable ad-
aptation of the probiotic to the prebiotic substrate before
consumption is developed; this leads to an increase of lactic
acid bacteria and bifidobacteria activity [13–15].
Microencapsulation technology provides a matrix that
Probiotics & Antimicro. Prot.
protects bacterial cells against the damage caused by low
pH, bile acids, and digestive enzymes, and increases their
stability and viability during processing and storage [16].
The main challenge of microencapsulation is to protect probi-
otic cells in foods usually not considering their vehicle, such
as fruit beverages, bakery, fermented meat products, and choc-
olate [17]. For such food products, few works investigate the
functionality of microcapsules in protecting cells. As a result,
non-dairy foods still represent only a niche in the market of
probiotic foods.
The present review focused on the practicality of develop-
ing microencapsulated synbiotic fruit beverages, the survival
aspect of probiotic bacteria during storage, as well as chal-
lenges in the synbiotic formulation of beverages and future
perspectives.
Overview of Synbiotics
In recent years, consumers have expectations for the develop-
ment of food products which offer not only beneficial nutri-
ents, but other innovative ingredients, such as prebiotics,
probiotics, or synbiotics. The synbiotic concept, introduced
by Gibson and Roberfroid, refers to the association and syn-
ergism between prebiotics and probiotics that induce favor-
able effects on the host “by improving the survival and im-
plantation of live microbial dietary supplements in the gastro-
intestinal tract, by selectively stimulating the growth and/or by
activating the metabolism of one or a limited number of
health-promoting bacteria, thus improving host welfare”
[18]. Synbiotics are much more than just a mixture of pre-
and probiotic. FAO recommends the term “synbiotic” to be
approached carefully only if the net health benefit is synergis-
tic. The microorganism must be selectively targeted by the
prebiotic compound for the synergism to exist. Even in a com-
petitive environment, the intended bacteria will be the most
likely to use the prebiotic.
Probiotics are sensible to stomach acidity, the length of
exposure to acid, and bile salts [5]. Prebiotic provides the
specific substrate to the probiotic for nourishment and protec-
tion. It supports the survival of the probiotic bacteria during
the passage through the upper intestinal tract in a sufficient
number to provide and modulate metabolic function, to im-
plant in the colon and growth and with little or no stimulation
of other microorganisms [19]. In the development of
synbiotics, the most important property is for the probiotic
to exert a greater effect in the presence of a prebiotic than in
its absence [20]. Due to this specific synergism, controlled
release of probiotics in the gut-specific site as well as the
production of short-chain fatty acids (SCFA) in the most distal
region of the colon, known to be the site of origin of several
chronic diseases, the term “integrated synbiotics” is preferred
instead of synbiotics alone [21].
Probiotics & Antimicro. Prot.
These bacteria strains must have the ability to ferment spe-
cific prebiotics (oligosaccharides and polysaccharides) [22].
Unfortunately, probiotics are not usually combined with an
appropriate prebiotic [23]. If in a product, for example,
fructo-oligosaccharides (oligofructose, FOS) prebiotic is
blended with probiotic Bifidobacterium spp., this combination
is considered a synbiotic, but if the same prebiotic is combined
with probiotic Lactobacillus casei the resulted product is not
synbiotic [24]. The known inulin fiber, in contrast to soybean
oligosaccharides (SOS), isomalto-oligosaccharides, or
lactulose, supports total bacterial counts, but populations of
Bifidobacterium spp. and Lactobacillus spp. strains are lower.
On the other hand, inulin, as compared to glucose, improves
stress resistance in Lactobacillus plantarum, whose adhesion
to the enterocytes and mucosal cells surface through self-
assembly and co-aggregation is about 10 times higher [25].
FOS sustains the growth of Streptococcus spp. populations
better than galacto-oligosachharides (GOS) or SOS.
Lactobacillus rhamnosus prefers monosaccharides and disac-
charides as a growth substrate and Bacillus lactis prefers tri-
saccharide and tetrasaccharide fractions. Finding synergic
pairs to develop new synbiotics involves long-term screening,
that consists of tests in vitro and in vivo, both in animal and
human, to demonstrate not only the effect of the prebiotic on
the probiotic, but also the efficacy of the combination on the
host versus if they are taken separately [26].
The widely used and well-characterized probiotic strains in
synbiotic formulations include Lactobacillus spp. and
Bifidobacterium spp., predominant and subdominant organ-
isms of the human gastrointestinal microbiota, used for many
years as starter cultures in the production of milk and its de-
rivative food products, as well as Streptococcus thermophilus,
Saccharomyces boulardii, and Bacillus coagulans, classified
as generally safe (GRAS) microorganisms. Most currently
marketed probiotics (Table 1) suit these requirements.
Depending on the health of humans, the biological activity
of consumed probiotics may be altered and, therefore, they
must be transient in the host, which implies the need for re-
peated administration for health effects [28].
Several studies have confirmed that using multistrain
probiotics has a positive effect on health, due to the synbiosis
among strains. Among multispecies probiotic combinations
used commercially there are mentioned: L. acidophilus +
L. casei; L. rhamnosus + L. reuteri; VSL 3 (mixture of
S. thermophilus + L. acidophilus, L. plantarum, L. casei,
L. delbrueckii subsp bulgaricus + B. breve, Bifidobacterium
infantis, B. longum); L. acidophilus + Bifidobacterium bifidum;
L. helveticus + L. rhamnosus; Bacillus clausii strains [5].
The major prebiotics used are oligosaccharides like
FOS, GOS, xylose-oligosaccharides (XOS), SOS, raffi-
nose and stachyose, polysaccharides like inulin and re-
sistant starch, and some new emerging prebiotics like
ulvan (from green alga Ulva rigida) [29], β-glucans from
Pleurotus mushrooms (pleuran), oligosaccharides from
yacon root [30], and dragon fruit [31]. Some of the pre-
biotics available in the international market are presented
in Table 2.
There are probiotics that do not have to be combined with a
prebiotic to function. A probiotic such as Ganeden BC30
(Bacillus coagulans GBI-30, 6086) delivers the same health
benefits even if prebiotic is not present (http://www.
ganedenbc30.com/).
Health Promoting Effects of Synbiotics
The intake of a synbiotic food leads to a modulation of com-
mensal microbiota composition and gut metabolic activities.
The representative metabolites whose change is associated
with the ingestion of synbiotics are SCFAs (especially acetic
and valeric acids), methylketones (2-pentanone, 2,3-
butanedione), carbon disulfide and methyl acetate. The in-
crease of these metabolites suggests potential implications of
the synbiotic food in health maintenance [35].
The health benefits claimed by synbiotics consumption by
humans include increasing levels of strains of lactobacilli and
bifidobacteria and gut microbiota balance, improving liver
function (in cirrhotic patients) and immune-modulating abili-
ty, prevention of bacterial translocation and decreased suscep-
tibility to pathogens in surgical patients [36, 37].
According to a report in Clinical Infectious Diseases the
benefic effects of probiotics is based on three mechanisms,
namely, antimicrobial effects by inhibitory substances
(H2O2, bacteriocins, organic acids), enhancement of mucosal
barrier integrity, and immune response modulation due to the
probiotics’ ability to metabolize complex carbohydrates and
to produce lactic acid and SCFAs, which are further used by
the host organism as an energy source. As supported by recent
evidence, prebiotics are also able to increase the production of
SCFAs (such as butyrate, acetate, propionate), compounds
which possess anti-inflammatory, antimicrobial and anti-
carcinogenic effects [38].
Also, synbiotics can be used as a prophylactic therapeutic
strategy for goal 3 of the UN Sustainable Development Goals
(SDGs). They have potential clinical indications such as infant
diarrhea and metabolic and inflammatory diseases in adults,
because they act through competitive exclusion of pathogens
and/or the secretion of metabolites that influence host physi-
ology [39]. All these favorable effects occur when bacteria
that reach the intestine are alive and in high quantity, which
shows dependence on their metabolic activity. Following re-
cent studies conducted on relevant bacterial species, some
soluble factors named postbiotics were isolated and character-
ized. They are by-products resulting from prebiotic fermenta-
tion by the probiotic. Postbiotics include organic acids, en-
zymes, and carbon substrates and have been proposed as a
 Probiotics & Antimicro. Prot.

Table 1 Commercially available

probiotic strains [27] Bacterial strain Company Country

L. rhamnosus GG Valio Dairy, Helsinki Finland

Lactobacillus delbruekii subsp. Meiji Dairies Corporation Japan
bulgaricus 2038
Lactobacillus johnsonii Lal Nestle, Lausanne Switzerland
L. casei Shirota Yakult, Tokyo Japan
L. casei CRL-43i Gilliland (La-Mo) Chr. Hansens (Horsholm) Denmark
L. casei DN 014001 Danone Le Plessis-Robinson (Paris) France
Lactobacillus reuteri SD 2112 BioGaia, North Carolina USA
L. plantarum 299 V Probi AB, Lund Sweden
Lactobacillus acidophilus NCFM Danisco Rhodia Inc., (Madison WI) USA
Lactobacillus acidophilus SBT-2062 SnowBrand Milk Products Co.Ltd., Tokyo Japan
Bifidobacterium longum SBT-2928 Snow Brand Milk Products Co., Ltd., Tokyo Japan
Bifidobacterium longum BB536 MorinagaBifidusMilk Industry Co. Ltd., (Zama City) Japan
Bifidobacterium breve Yakult Yakult, Tokyo Japan
S. boulardii Biocodex, Seattle USA
S. thermophilus 1131 Meiji Milk Products, Tokyo Japan

safer alternative for clinical applications, especially in chronic
inflammatory conditions, where the results obtained with
synbiotics did not yield satisfactory results [40].
The review of published results on the health effect of
probiotics and synbiotics reveals that there is a lack of studies
performed until now on the development of synbiotics.
Though the results from animal studies are of great interest,
they are not always transferable to humans, due to differences
in their gut microbiome and pathophysiology of different dis-
eases. Also, the in vivo effectiveness of synbiotics has not
been intensively studied to date; there are only a few human
clinical trials [41]. Several meta-analyses suggest preliminary
promising capacities to modulate microbiota composition and
seem to be active in the amelioration of some immune-related
and obesity-associated disorders, but in most trials the pro-
and prebiotic treatment effects were not determined
independently [42]. Moreover, microbial analysis was either
absent or non-strain specific [43], which led to conflicting
observations. Future comparative studies must consider not
only the effect of prebiotic on probiotic, but also have to offer
an understanding of the mechanisms of action and changes
that occur in the host microbiota when they are used separately
or together, to elucidate how the benefits are achieved [44].
Current Research in Microencapsulated
Fruit-Based Beverages
Nowadays, worldwide, the market for synbiotic products is
dominated by milk-based products. Due to consumer concern
about the foods they ingest and their benefits on health, there
is a growing trend for the development of synbiotic food

Table 2 Food grade commercial prebiotics in the international market [32–34]

Prebiotic Manufacturer Product name Applications

Inulin Sensus, NL Frutafit® Beverages, infant foods, ice creamsconfectionaries, bakery products, etc.
Inulin Cosucra, Belgium Fibruline® Beverages, confectionaries, bakery products, breakfast cereals, etc.
Inulin Orafti GR, Belgium Beneo® Baked products, dairy alternatives, beverages confectionary, breakfast
cereals, soups, and sauces
FOS Sensus, NL Frutalose® Beverages, infant foods, confectionaries, ice creams, bakery products, etc.
FOS Cosucra, Belgium Fibrulose® Beverages, baby food, dairy derivatives, bakery, confectionary,
cereals, soups, sauces
FOS Beneo-Orafti, Belgium Beneo Raftilose P95 Beverages, baby food, dairy derivatives, bakery, confectionary,
cereals, soups, sauces
GOS FrieslandCampina Domo, NL Vivinal® GOS Infant formula
GOS ClasadoBioSciencesUK Bimuno® Drinks, bakery products, cereals, dietary supplements
GOS Yakult Honsha, JP Oligomate 55 N Dairy products
Probiotics & Antimicro. Prot.
products using non-dairy substrates. Fruit beverages are pre-
ferred by a significant percentage of health-centric consumers,
because of their lack of lactose and dairy allergens, soy, and
cholesterol. As raw material for the production of probiotic/
synbiotic beverages, different matrices such as mango, or-
ange, apple, grape, peach, pomegranate, and watermelon have
been investigated [45]. Synbiotic fruit-based beverages com-
mercially available are scarce, and they represent only a small
share of the market [46].
The survival of probiotics during the entire beverage-
processing operations, including storage, is a very important
issue to validate the benefits of probiotics in this type of prod-
uct. Fruit beverage manufacturers are confronted with factors
that could limit probiotic survival, such as chemical parame-
ters of juices (pH, titratable acidity, molecular oxygen, pres-
ence of salt, sugar, hydrogen peroxide, bacteriocins, artificial
flavoring, and coloring agents), processing parameters (heat
treatment, incubation temperature, cooling rate, packaging
materials, and storage methods), and microbiological param-
eters (species/strain of probiotics, rate, and proportion of in-
oculation) [13, 14, 47]. However, the low pH of fruit juices is
the main drawback for the total viable counts and activities of
probiotics [48]. The acidity of fruit juices caused by high level
of organic acids which drop the pH and affects probiotic bac-
teria. In an environment of low pH, the cells cannot survive
because, on the one hand, the energy provided by ATP for
maintenance of the intracellular pH is increased and, on the
wide side, the ATP deficit affects the other cellular functions
[49]. Also, in relation to the chemical composition of the
juices, it is mentioned that insufficient quantities of peptides
and free amino acids in juices affects metabolism of probiotic
cultures and their viability during storage [50]. In fact, the
protective effect of proteins on probiotic bacteria was con-
firmed in a study by Nualkaekul et al. [51].
It is known that probiotic viability is strain-dependent,
which means that only certain bacteria that are able to keep
their viability for an acceptable shelf-life are selected. Some
probiotics, in particular, strains of Lactobacilli (L. plantarum,
L. acidophilus, and L. casei) not only tolerate a low pH rang-
ing from 4.3 to 3.7, but also grow in the fruit matrix, resulting
in good viable counts. Bifidobacteria strains, even at a pH
about 4.6, are less tolerant [47, 52]. In any case, this behavior
differs depending on the type of probiotic.
Because the low pH of fruit juices is a great challenge,
microencapsulation in different polysaccharides and
protein-based systems is considered a way to preserve,
protect, and maintain the viability rate of probiotic cells
above the critical threshold of 106 cfu mL−1 and to en-
sure their controlled delivery in the gastrointestinal tract.
Using fruit beverages as a matrix, many experimental
studies have investigated the effectiveness of encapsula-
tion on the survival of probiotic microorganisms versus
free bacteria during gastrointestinal transit, at low-
temperature storage, as well as implications on organo-
leptic aspects [53–56].
Microencapsulation, however, does not always ensure an
increase in the viability of probiotics [57]. A successful way to
improve the viability and stability of probiotics in fruit juices
is coupling probiotic microencapsulation with prebiotics, a
process which is included in the synbiotic concept. Although
there is a great potential for the use of fruit juices as matrices
for microencapsulated synbiotics, only a few studies have
been reported; these studies focus not only on chemical and
microbiological characteristics of substrate, but also to shelf-
life and consumer acceptability.
Gandomi et al. [58] evaluated the viability of probiotic
L. rhamnosus GG encapsulated with inulin in matrix of algi-
nate and chitosan, in apple juice during 90 days storage at 4 °C
and 25 °C. The survival rate of encapsulated L. rhamnosus
GG was 4.5 times higher than that of free bacteria during
storage period of the juice at both 4 °C and 25 °C. The results
of this study suggest the protective effect of microencapsula-
tion on bacterial viability. The presence of the chitosan mem-
brane surrounding the alginate beads decreases the transition
of acids and flavonoids from the fruit juice into the beads, thus
limiting the adverse effect of said juice on encapsulated pro-
biotic bacteria. With respect to pH changes of the apple juices
containing free and encapsulated bacteria during storage at
both temperatures, there was a decrease in pH following the
addition of inulin. The pH of the apple juices containing en-
capsulated probiotic bacteria decreased, but less than pH of
the juices containing free bacteria and inulin. Inulin has a
slight acidic behavior when dissolved in water and free
probiotics release lactic acid during storage. This post-
acidification of the probiotic products during storage is an
important challenge for commercialization. This study has
shown the possibility of using apple juice as a matrix for
delivering microencapsulated probiotic bacteria to humans.
Apple juices containing encapsulated bacteria had acceptable
sensory properties.
Another study [59] investigated microencapsulation of
B. coagulans IBRC-M 10807 in a pectin (pec)-based
bionanocomposite improved with nanochitin (NC),
nanolignocellulose (NLC), and bacterial nanocellulose. The
evaluation of the survivability of probiotic under simulated
gastrointestinal conditions and in peach juice was carried out
over 5 weeks storage at 4 °C and 25 °C. In the juice containing
probiotic cells microencapsulated using pec-NC-NLC matrix
the decrease in the survivability was of ~ 2.98 ± 0.02 log
(cfu100 mL−1 juice) for juice stored at 25 °C and ~ 2.66 ±
0.04 log (cfu100 mL−1 juice) for the juice stored at 4 °C.
The highest survivability obtained by this matrix was ~ 68%
± 0.1, in comparison to the free cells (~53% ± 0.3) at the end
of storage period at both temperatures. The microscopic image
of pec-NC-NLC matrix highlights a surface morphology rel-
atively smooth coherent, compact, and wrinkled, without

pores and cracks. This is due to the formation of hydrogen and
ionic bonds which connected polysaccharides of the pec-
nanofibers together in a three-dimensional uniform arrange-
ment. The lack of breaks in the matrix suggests the physical
isolation and protection of probiotics against the low pH of
peach juice. In terms of pH changes in synbiotic peach juice,
no statistically significant variation of pH was observed over
storage period. Sugars or oligofructoses from juice can be
metabolized to organic acids by B. coagulans IBRC-M
10807. But the microencapsulation matrix probably limited
the diffusion of juice sugars into the matrix. Also, prebiotics
from matrix composition are not easily metabolized. Another
important aspect relates to the micro-scale average size of the
matrix, which cannot be detected in the mouth, what is of great
importance for sensorial acceptance. It is concluded that this
bionanocomposite has the ability to protect and deliver live
probiotic cells in the harsh environment of peach juice to the
consumers.
Krasaekoopt and Watcharapoka [60] reported the addition
of GOS and inulin prebiotics during microencapsulation of
L. acidophilus 5 and L. casei 01 in alginate beads coated with
chitosan. The viability of microencapsulated probiotics with
prebiotics introduced in orange juice was determined at refrig-
erated storage for 4 weeks. Significant results were obtained
when using GOS as a prebiotic. At the beginning of storage,
cell count of microencapsulated probiotics without GOS was
8.3–9.4 log cfu g−1. Although the number of probiotics with-
out GOS was higher, the presence of GOS 1.5% enhanced the
growth of entrapped cells by approximately 0.4–1.1 logs in
orange juice until the end of the storage at 4 °C. The numbers
of L. acidophilus 5 encapsulated with GOS constantly in-
creased, the loss being only 0.2 logs versus initial load. For
L. casei 01 entrapped with GOS 0.6 logs reduction was found.
The difference in growth between the two strains of
lactobacilli could be due to the prebiotic GOS which only
stimulates the growth of L. acidophilus 5. There has also been
an increase of about 0.2% in the acidity of the orange juice. It
is assumed that probiotics use GOS, and organic acids pass
into juice. The results of this experiment highlight the protec-
tive role of GOS in microcapsules over harsh conditions of
orange juice.
Other work [61] has been carried out to develop a synbiotic
fig juice. Adding probiotic (L.delbrueckii DSMZ 15996) and
prebiotic (inulin) in fig juice resulted in a functional food
which was evaluated 4 weeks. Viability of probiotic was de-
creased with a rate lower in the synbiotic sample, where viable
cell counts from 8.5 log cfu mL−1 reached 7.49 log cfu mL−1,
compared to probiotic juice (6.59 log cfu mL−1) at the end of
cold storage. Causes of reduction in probiotic viability could
be acidity, the presence of oxygen in the juice, or the low level
of nitrogenous compounds. Past studies have shown that inu-
lin has a protective and enhancing effect on probiotic viability.
The protective role of inulin could be explained by the fact
Probiotics & Antimicro. Prot.
that inulin is an accessible source of carbon for cells activity. A
study by Takagi et al. [62] showed that L. delbrueckii grows
faster in the inulin enriched media. In the first phase, the bac-
terium imports inulin into its cells and then consumes it intra-
cellularly. Sensory evaluation assessed in the second week of
storage noticed differences in terms of odor, taste, and overall
acceptance between the synbiotic fig juices and control. The
lower score for the overall acceptance of synbiotic juice com-
pared to control is probably due to the L. delbrueckii DSMZ
15996 metabolites, including lactic acid, that have a negative
effect on the aroma and taste of the product.
Masking medicinal flavor of probiotic strains by adding
tropical fruits or by microencapsulation of synbiotics would
be alternatives for the improvement of fig juice organoleptic
attributes. Thus, fig juice could be used as a suitable matrix for
obtaining a functional synbiotic beverage [61].
Prakash et al. [63] studied the feasibility to develop a
synbiotic litchi juice containing probiotic L. casei 359 micro-
encapsulated in whey protein concentrate (WPCP) along with
prebiotics including gum acacia, inulin, or FOS. The study
was carried out to evaluate the viability, physicochemical,
and sensory analysis of synbiotic litchi juice. The survivability
results, after 4 weeks of storage, has shown that the WPCP in
combination with inulin has recorded the highest value of 8.12
log cfu mL−1 of litchi juice, followed by the mix between
WPCP and FOS (7.42 log cfu mL−1). The data suggest that
inulin, during the encapsulation process, acted as a
thermoprotector for the cells over the drying process and pro-
vided tolerance to probiotic cells.
The rheological properties, performed at two temperatures
10 °C and 20 °C respectively, suggest the shear-thinning be-
havior of the juice drink. FTIR spectroscopy of the probiotic
litchi juice showed that the whey protein, and all prebiotics
were retained as such in the microcapsules after spray drying.
Considering sensory scores for quality properties of litchi
juice, the sample containing inulin had a higher acceptability.
Fratianni et al. [64] have tested the ability of probiotic yeast
Saccharomyces cerevisiae var. boulardii, microencapsulated
with inulin—xanthan gum—alginate, to grow in berry juice
and survive 4 weeks at 4 °C. Microencapsulation increases
yeast cells viability after fermentation and storage, compared
to the free yeast (7.59 log cfu mL−1 versus 6.98 log cfu mL−1,
respectively). Microencapsulation of S. cerevisiae var.
boulardii with a prebiotic is considered a novelty for two
reasons. First, this yeast is not long resistance to low pH.
Secondly, this study revealed that synbiotic microcapsules ab-
sorb from the berry juice a certain amount of anthocyanins that
are cleaved into simpler molecules in the intestine, with ben-
eficial effects on human microbiota. The results obtained may
be the starting point for the development of new functional
products based on microencapsulated probiotic yeasts.
To sum up, fruit juice type selection is a key factor for the
stability of added probiotics. Maintaining the viability at least
Probiotics & Antimicro. Prot.
106–107cells mL−1 and the activity of probiotics in fruit bever-
ages at the end of shelf-life are two important criteria that must
be taken into account, knowing the low pH is a drawback. In
the formulation of the synbiotic microcapsules, it is essential to
combine the bacteria strains with the specific prebiotic for
obtaining synbiotic effect. The impact of prebiotic on the phys-
ical properties of capsules (size, integrity) must also be taken
into account. Combination of probiotics with prebiotics could
be a dietary strategy which modulates commensal microbiota
composition and metabolism with health effects.
The evaluation of sensorial profile of fruit juices supplement-
ed with microcapsules as well as details about microcapsules
size and their behavior in liquid food matrices (sedimentation,
optical residues) are, so far, found in few reports. Also, the
viability of bacteria under conditions of storage and commer-
cialization at ambient temperature must be investigated [65].
Utilization of synbiotics in fruit beverages continues to
gain interest and popularity among consumers.
Challenges for Formulation of Fruit Beverages
with Synbiotics
Formulated synbiotic fruit beverages must deliver health ben-
efices, nutrition, a sensory profile accepted by consumers, and
convenience for commercialization [66]. When designing this
new generation of functional beverages some challenges need
to be considered: (a) uses of specially selected probiotic cul-
tures, because not all lactic acid bacteria can survive in acid
environment [67]; (b) combination of probiotics with an ap-
propriate prebiotic; (c) establishment of proper bacteria level
required to have a beneficial effect and delivery system [68];
(d) in the strain selection process to take into account safety
features to avoid the potential interactions with other classes
of ingredients which produce biogenic amines. The criteria for
selection are GRAS status, desirable antibiogram profile, non-
pathogenic (even to immune-compromised hosts), and non-
inflammatory microorganisms. The European Community
proposed a system for a pre-market safety assessment of se-
lected microorganisms from a defined taxonomic group that
leads to a “Qualified Presumption of Safety (QPS)” [68]; (e) to
be considered the effect of the processing steps on viability.
The low pH value combined with long storage periods affect
the viability of most probiotic strains, especially those
representing anaerobic bifidobacteria, which need a good ox-
ygen barrier packaging material. From a technical perspective,
the probiotic strains' survival dictates the type of synbiotic
product to be developed [69]; (f) determining in the product
the added bacteria population to understand their mechanism
of action, to elucidate more specifically what kind of benefit
every strain gives [68]. In order to have a competitive advan-
tage on the market, it is of great importance to confirm the
functional properties of the starter culture before incorporating
it into the product [48, 70]; (g) obtaining probiotics to be done
in a cost-effective way that will ensure probiotic viability over
shelf life. Incorporation of pre- and probiotics in diverse food
vehicles requires changes to the product and technological
process that may bear the risk of modulating the probiotic
functionality, as suggested so far mostly by in vitro testing.
Consumers are confused and skeptic about numerous probiot-
ic strains and health benefits of each. Consumers need to be
convinced that the functional beverages they buy contain the
claimed quantity of probiotic cells and prebiotic, a major issue
for a long-term profitability of synbiotic beverages [71]; (h)
changes in sensory properties of juices due to the presence of
microorganism strain in the juice, storage temperature, addi-
tion of other compounds, could negatively affect the overall
acceptance. The sensory alteration of juices can be masked by
the addition of pleasant aroma, flavors, and volatile com-
pounds [72].
Another currently challenging task is the demonstration of
probiotics beneficial health effects in humans by development
and validation of biomarkers of health and disease. For this
purpose, it is necessary to know the mechanism of action of
probiotics for identification of bioactive molecules in probiot-
ic products, to evaluate the food matrix effects on probiotic
functionality, and to set up pre-trial functional tests with pre-
dictive value. As such, it will optimize the selection of probi-
otic bacterial cells and technological parameters. All these
investigations help to undertake large and long-term animal
and human proof-of-concept trials for supporting and demon-
strating potential health claims [13, 73].
Future Perspectives
Current trends dictated by changing consumer needs and in-
creased interest towards healthier foods indicate a great oppor-
tunity for using novel, economic, and technological matrices
to develop healthy functional products [48]. These potential
heterogeneous matrices opened to innovation, require assess-
ment of efficacy, and safety for their consumer acceptance and
market launch [70]. A prominent feature in their marketing
strategy will be the approved specific health claims and con-
sumer awareness [13].
An area of emerging interest which opens up new possibil-
ities for the synbiotics application in food sector is nanotech-
nology. The application of nano-encapsulated probiotics is
currently in the development, but requires precaution regard-
ing their impact on environment and human health [74, 75].
For the technological functionality, in the last years, genome
sequencing of representative lactic acid bacteria with health-
promoting effects in consumers have been reported. The gene
expression profile is a tool for a better understanding of phys-
iological characteristics and identification of novel genes
(stress and acid tolerance genes) [76, 77].

In the future, the preventive and curative role of commer-
cially available probiotics on gastrointestinal as well as extra-
intestinal related disorders have to be demonstrated at clinical
or population level, involving large cohorts of patients, a va-
riety of bacteria sources and daily doses, and different admin-
istration periods [5, 78]. In order to standardize the mode of
administration, correct formulation in terms of the amount of
bacteria, viability, and associated growth factors is required
for clinical outcomes to be comparable [79].
Acknowledgments The authors would like to thank the financial support
of the Romanian National Authority for Scientific Research and
Innovation, CCCDI–UEFSCDI, project number PN-III-P3-3.5-EUK-
2016-0015.
Funding This work was supported by a grant of the Romanian National
Authority for Scientific Research and Innovation, CCCDI–UEFSCDI,
project number PN-III-P3-3.5-EUK-2016-0015.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Ethical Approval We state that this article does not contain any studies
with human participants or animals performed by any of the authors.
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