LWT - Food Science and Technology 116 (2019) 108501

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LWT - Food Science and Technology

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Encapsulation of Lactobacillus casei ATCC 393 in alginate capsules for T

probiotic fermented milk production
⁎
Dimitra Dimitrelloua, , Panagiotis Kandylisa,b, Steva Levićc, Tanja Petrovićc, Sanjin Ivanovićd,
Viktor Nedovićc, Yiannis Kourkoutasa
a
Laboratory of Applied Microbiology and Biotechnology, Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, 68100, Greece
b
Department of Food Science and Technology, School of Agriculture, Aristotle University of Thessaloniki, P.O. Box 235, Thessaloniki, 54124, Greece
c
Institute of Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade, Belgrade-Zemun, 11080, Serbia
d
Institute of Agricultural Economics, Faculty of Agriculture, University of Belgrade, Belgrade-Zemun, 11080, Serbia

A R T I C LE I N FO A B S T R A C T

Keywords: The aim of the present study was to investigate encapsulation of probiotic Lactobacillus casei ATCC 393 on
Extrusion alginates using the extrusion technique and to evaluate cell survival under simulated gastrointestinal conditions
Gastrointestinal tolerance and during fermented milk production and storage at 4 °C for up to four weeks. Encapsulation of probiotic
Aroma profile bacteria led to significantly higher viabilities compared to free cells under simulated gastrointestinal conditions,
Dairy
as well as after storage for 28 days in fermented milk (7.13 log cfu g−1). In addition, the effect of encapsulated L.
GC-MS
casei ATCC 393 cells on aroma volatile compounds formation during milk fermentation was studied using the
SPME GC/MS technique. The fermented milks presented improved aroma, containing characteristic compounds
produced usually by L. casei in fermented dairy products. The results showed that the alginate matrix has a great
potential as encapsulation material for probiotics and application of the encapsulated cells in fermented dairy
products may result in effective maintenance of cell survival during refrigerated storage and improvement of
sensory characteristics. From an economical point of view and under certain conditions, the application of
encapsulated probiotic cells is a sustainable process for the production of fermented milk.

1. Introduction
Nowadays there is a growing interest in the food industry to con-
tinuously develop innovative dairy products containing probiotic mi-
croorganisms, such as bifidobacteria and lactic acid bacteria, with a
human health potential (Champagne, da Cruz, & Daga, 2018;
Ranadheera, Naumovski, & Ajlouni, 2018). Dairy products and espe-
cially fermented milk are ideal carriers for the delivery of probiotic
bacteria in the gastrointestinal tract (GIT). However, the challenge in
preparation of such a probiotic delivery vehicle is to ensure sufficient
number of viable cells until the time of consumption (Mani-López,
Palou, & López-Malo, 2014).
In order to exhibit the health benefits, probiotic microorganisms
should be able to survive the harsh conditions during food processing
and storage and during passage through the human GIT (Thomas,
2016). However, these requirements are not always fulfilled. Therefore,
encapsulation may provide a solution for the protection of sensitive
probiotic bacteria by increasing their stability and viability in acidic
products, such as yogurt and fermented milk, and by achieving target
delivery in human GIT conditions (De Prisco, Maresca, Ongeng, &
Mauriello, 2015; Dimitrellou et al., 2016; Huq et al., 2017; dos Santos
et al., 2019). Furthermore, encapsulation, by spray drying, has been
used for the production of ready to eat dried dairy products by just
adding water like kefir microcapsules using maltodextrin/arabic gum
mixes (Nale, Tontul, Arslan, Nadeem, & Kucukcetin, 2018).
The most widely used encapsulation matrix, for both food-grade and
non-food applications, is alginate (Manojlović, Nedović, Kailasapathy,
& Zuidam, 2010). It is an ionic polysaccharide originated from brown
seaweeds and commonly used in conjunction with calcium ions as
cross-linker for the creation of capsules containing sensitive material.
The main advantage in using such polymers in encapsulation by the
extrusion technique is the fact that cells are entrapped, during the
formation of the spheres, within the matrix, avoiding, thus, their release
into the product, as it usually happens with other methods like freeze-
drying, spray-drying and fluidized bed drying (Krasaekoopt, Bhandari,
& Deeth, 2003; Nedović, Kalušević, Manojlović, Petrović, & Bugarski,
2013). An additional benefit of the extrusion method is the mild con-
ditions applied during capsule formation, which facilitate maintenance

⁎
Corresponding author.
E-mail address: dimitrellou@gmail.com (D. Dimitrellou).

https://doi.org/10.1016/j.lwt.2019.108501
Received 7 April 2019; Received in revised form 9 August 2019; Accepted 10 August 2019
Available online 10 August 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
D. Dimitrellou, et al.
of high cell viability (80–95%) (Krasaekoopt et al., 2003). Several
studies have shown that alginate-based encapsulation enhanced Lacto-
bacillus cells' viability after storage and under simulated gastric condi-
tions (Ding & Shah, 2007; Li, Chen, Sun, Park, & Cha, 2011; Nazzaro,
Fratianni, Coppola, Sada, & Orlando, 2009).
Lactobacillus casei is among the most studied species due to its
commercial, industrial and applied health potential (Hill et al., 2018). It
has been used in several dairy products, such as cheese, ice-cream,
fermented milk, etc, to improve flavor and texture (Balthazar et al.,
2018; García, Bautista, Rendueles, & Díaz, 2019; Silva et al., 2018).
Several strains of L. casei are considered to be probiotics. Among them,
L. casei ATCC 393 is a well-known probiotic strain with many appli-
cations in the food production. It has been successfully used as starter or
adjunct culture for the production of several dairy products, such as
fermented milk, yogurt and cheese (Abdel-Hamid et al., 2019;
Dimitrellou, Kandylis, Kourkoutas, & Kanellaki, 2017; Marinaki,
Kandylis, Dimitrellou, Zakynthinos, & Varzakas, 2016). In addition, it
has been associated with several health promoting activities like reg-
ulation of intestinal microbiota (Sidira et al., 2010), tumor-inhibition,
pro-apoptotic and anti-proliferative effects (Choi et al., 2006; Tiptiri-
Kourpeti et al., 2016) and recently with the production of bioactive
peptides in fermented milk (Abdel-Hamid et al., 2019).
The extrusion technique was employed for alginate-based en-
capsulation of L. casei ATCC 393 cells and the aim was to investigate the
viability of encapsulated cells in the Ca-alginate capsules under GIT
simulated conditions and during the production and storage of fer-
mented milk. The effect of encapsulation on the microbiological and
physicochemical characteristics was evaluated, in addition to the effect
on volatile compounds, which potentially might affect the sensory
properties of the final product. Sampling and analyses were performed
over a storage period of 28 days at 4 °C, in order to present a complete
study for possible industrial applications. To the best of our knowledge,
this is the first report on Ca-alginate encapsulated L. casei ATCC
393 cells used in fermented milk production and especially on evalua-
tion of their effect on aroma compounds. Finally, an economic analysis
of the microencapsulation technology for potential industrial applica-
tion was carried out.
2. Materials and methods
2.1. Strains
The thermophilic starter CH-1 (Chr. Hansen, Hørsholm, Denmark)
consisting of Streptococcus thermophilus and Lactobacillus delbrueckii spp.
bulgaricus in freeze dried form along with Lactobacillus casei ATCC 393
(DSMZ, Braunschweig, Germany) were used in the present study. CH-1
culture was activated by adding a 50 U sachet to 500 mL of sterile
140 g L−1 skim milk.
2.2. Encapsulation process
Alginate powder [sodium alginate from Macrocystis pyrifera of
medium viscosity (viscosity of 2% solution at 25 °C was approx. 3,500
cps)] was dispersed in distilled water to produce solutions of
0.015 g mL−1. Bacterial biomass was mixed with alginate and extruded
through 0.7 mm blunt stainless still needle using a syringe pump (Pump
11, Harvard Apparatus, SAD) under constant flow rate of 40 mL h−1.
The spherical droplets were formed by combined action of electrostatic
force and gravity. Electrostatic potential (7.0 kV) was formed by elec-
trostatic encapsulation unit VAR V1 (Nisco, Switzerland). The col-
lecting solution was calcium chloride (0.015 g mL−1). The distance
between the needle tip and the collecting solution was 2.5 cm. Of note,
the whole procedure was carried out under sterile conditions. The
diameter of the obtained Ca-alginate capsules was determined micro-
scopically using light microscope DMLS (Leica, Germany) equipped
with a DC 300 camera (Leica, Germany). ImageJ and image processing
2
LWT - Food Science and Technology 116 (2019) 108501
program was used for capsule measurement.
2.3. Survival of encapsulated L. casei cells
0.1 g of the samples were suspended in 9.9 mL sodium citrate so-
lution (0.06 mol L−1), stirred for complete homogenization and serial
diluted in ¼ strength Ringer's solution. Finally, the survival of en-
capsulated cells was determined after plating on de Man, Rogosa, and
Sharpe (MRS) agar and incubation at 37 °C for 72 h under anaerobic
conditions.
2.4. Survival of encapsulated L. casei cells in simulated gastric juice and bile
conditions
The survival of encapsulated L. casei cells was evaluated after im-
mersing 0.1 g of encapsulated L. casei cells in simulated gastric or bile
solution (Li et al., 2011), as described in a previous study (Dimitrellou
et al., 2016).
2.5. Fermented milk production
Fermented milks using encapsulated L. casei cells (10 g wet capsules
L−1 of milk corresponding to 8.85 log cfu L−1 milk) and the activated
CH-1 culture (0.3% v/v) were prepared (LCFM) following the proce-
dure described in a previous study (Dimitrellou et al., 2016). Fermented
milk, without L. casei, using only the CH-1 yogurt culture was also
produced for comparison reasons (CFM).
2.6. Analyses
2.6.1. Determination of culture viability
The viability of S. thermophilus, L. bulgaricus and L. casei was mea-
sured using fermented milk samples (10 g diluted to sodium citrate
solution (0.06 mol L−1) and following the procedure described in a
previous study (Dimitrellou et al., 2016; Vinderola & Reinheimer,
2000). In all cases, the survival rate (%) was calculated as cfu g−1 of
fermented milk after storage multiplied by 100 and divided by cfu g−1
of fermented milk at 0 day.
2.6.2. Titratable acidity and pH
A pH-meter was used for pH determination of fermented milks,
while titratable acidity was determined by milk titration with
0.1 mol L−1 NaOH.
2.6.3. Solid-phase microextraction GC-MS analysis
After 28 days of refrigerated storage, 7 g of fermented milk samples
were used for solid-phase microextraction (SPME) GC-MS analysis in
order to determine the volatile composition, as previously described
(Dimitrellou, Kourkoutas, Koutinas, & Kanellaki, 2009).
2.6.4. Sensory evaluation
The sensory evaluation was conducted by 20 panelists, familiar with
the consumption of fermented milk. The sensory panel consisted by 10
females and 10 males aged 20–45 years. All samples were presented in
uniform plastic cups at 15 °C, coded with random three digit numbers.
Water was provided between samples to cleanse the palate. The fer-
mented milks were evaluated for color, sweet odor, sourness, smooth-
ness, sweetness, viscosity, aftertaste and overall acceptability, using a 9-
point hedonic scale (Amaral et al., 2018; Souza et al., 2019) ranging
from 1 (“dislike extremely”) to 9 (“like extremely”).
2.6.5. Economic analysis of encapsulation process for fermented milk
production
Economic evaluation was based on partial budgeting approach
which was used to determine changes in variable costs, fixed costs and
profit caused by introduction of encapsulation in fermented milk
D. Dimitrellou, et al.
Fig. 1. Ca-alginate beads with encapsulated L. casei ATCC 393.
production. Cost-profit analysis resulted in determination of break-even
point for different increase of selling price.
2.7. Experimental design and statistical analysis
All experiments were carried out in triplicate. Significance was es-
tablished at P < 0.05. The results were analyzed for statistical sig-
nificance with ANOVA, and Tukey honest significant difference (HSD)
test was used to determine significant differences between the results;
coefficients, ANOVA tables, and significance (P < 0.05) were com-
puted using Statistica version 5.0 (StatSoft Inc., Tulsa, OK).
3. Results and discussion
In the present study, capsules containing L. casei ATCC 393 cells
were prepared by the extrusion method using Ca-alginate as a carrier
(Fig. 1). The diameter of the beads were 597.46 ± 58.66 μm and the
viable cells count of L. casei ATCC 393 in the capsules was 7.85 ± 0.10
log cfu g−1, levels that are similar to previous studies using alginate
based microencapsulation (Li, Chen, Cha, Park, & Liu, 2009; Li, Chen,
Liu, Liu, & Xue, 2008; Li et al., 2011). The size of capsules were sig-
nificantly smaller compared to other studies, using Ca-alginate based
capsules of probiotics (Prasanna & Charalampopoulos, 2018, 2019).
The smaller size may be an advantage for the application on several
dairy products, like fermented milks and especially during sensory
evaluation. Survival of the encapsulated cells was subsequently tested
in simulated GI conditions, as well as in experimental production of
fermented milk.
3.1. Survival of encapsulated L. casei in simulated gastrointestinal
conditions
3.1.1. Survival in simulated gastric juice
It has already been reported that L. casei ATCC 393 free cells present
low viability under simulated GI conditions (Dimitrellou et al., 2016; Li
et al., 2011; Sidira et al., 2010). Therefore, the use of protective ma-
terial is a necessity. Encapsulation in Ca-alginate capsules provided a
significant (P < 0.05) cell protection in the simulated gastric juices
(Table 1). More specifically, the reduction in their viability after ex-
posure to pH 2 and pH 3 for 3 h was only 1.55 and 0.53 log cfu g−1,
respectively. These results confirmed those of previous studies, re-
porting the protecting effect of beads based on Ca-alginate on L. casei
and bifidobacteria viability under similar conditions (Prasanna &
Charalampopoulos, 2018, 2019; Sandoval-Castilla, Lobato-Calleros,
García-Galindo, Alvarez-Ramírez, & Vernon-Carter, 2010; Li et al.,
2008, 2009, 2011).
3
LWT - Food Science and Technology 116 (2019) 108501
3.1.2. Survival in bile salts solutions
Probiotic bacteria should have the ability to survive at conditions of
human small intestine and more specifically at high bile concentrations
encountered there. The interaction of bile salt with the free cells may
negatively affect cell wall integrity, leading to leakage of intercellular
materials and finally resulting in cell death (Bron et al., 2004). The
possible protective effect of Ca-alginate encapsulation on survival of L.
casei in bile salts solution (0.5% and 1%) is presented in Table 1. A
significant effect (P < 0.05) by encapsulation on the viability of L.
casei cells in bile salts solutions was recorded. In our previous experi-
ments, we have already presented that free cells demonstrated a re-
duction of 3.05 log cfu g−1 and 4.03 log cfu g−1 in 0.5% and 1% bile
salts, respectively (Dimitrellou et al., 2016). In contrast, the corre-
sponding reduction for Ca-alginate encapsulated cells was 0.97 log cfu
g−1 and 1.35 log cfu g−1 , respectively. The protective effect of en-
capsulation was more obvious in the highest concentration of bile so-
lution (1%) and after 6 h, where loss for free cells was 5.96 log cfu g−1,
while for Ca-alginate encapsulated cells the loss was only 2.20 log cfu
g−1 (Table 1). Noticeably, extrusion technique provided increased cell
protection in comparison to spray-drying microencapsulation
(Dimitrellou et al., 2016) or cell immobilization on apple pieces (Sidira
et al., 2010).
3.2. Fermented milk production
In order to evaluate the suitability of encapsulated cells for dairy
industry applications, fermented milks were produced using en-
capsulated L. casei cells, along with the traditional yogurt starter culture
(Streptococcus thermophilus and Lactobacillus bulgaricus) used for the
production of fermented milks.
Τhe physicochemical characteristics and cell survival during re-
frigerated storage of fermented milks for 28 days are presented in
Table 2. The storage time affected significantly (P < 0.05) titratable
acidity and pH (P < 0.05). A continuous increase in titratable acidity
up to 0.9 g/100 g was detected, while pH was reduced during storage,
in correlation with our previous study using spray dried L. casei ATCC
393 cells (Dimitrellou et al., 2016).
Although, probiotic products are not considered a new entry in the
world market, consumers retain their interest and the global market of
probiotic foods and beverages is still growing. On the other hand, there
is an increased confusion among consumers, who are no longer aware
which products, even yogurts, contain probiotic strains, since they are
now usually mass produced using standard starter cultures, which are
not necessarily associated with probiotic properties or able to survive
the gut (Thomas, 2016). Therefore, it is very important to evaluate the
survival of probiotic and starter cultures during storage of the potential
probiotic foods. In the present study, storage affected (P < 0.05) the
viability of all microbial strains (Table 2). Encapsulation provided
sufficient protection to L. casei cells during the fermented milk pro-
duction and storage. This result is very important since probiotic strains
added to foods need to remain alive at reasonable numbers over the full
shelf life of the product, in order to provide their health benefits
(Thomas, 2016). After 3 and 4 weeks of storage, survival rates of 57.5%
and 47.9% were monitored, respectively. Interestingly, these values
were significantly (P < 0.05) higher than those obtained in our pre-
vious investigation with spray dried microencapsulated L. casei cells
under the same experimental conditions (Dimitrellou et al., 2016). The
effective protection of Ca-alginate capsules on probiotic viability in
dairy products like yogurt, has been reported in several recent studies
(Prasanna & Charalampopoulos, 2018, 2019). Of note, levels of the
encapsulated Lactobacillus casei cells in the fermented milk during
production and refrigerated storage (up to 28 days) remained above the
minimum requirement (6 log cfu g−1) for adjunct cultures (FAO/WHO,
2003) and for probiotic foods, according to US FDA and food industry
recommendations (Tripathi & Giri, 2014), suggesting that the con-
sumption of only 100 g of products may fulfill the daily intake of 109 of
D. Dimitrellou, et al. LWT - Food Science and Technology 116 (2019) 108501

Table 1
Effect of the exposure to simulated gastric juice (pH 2 and 3) and bile salts (0.5% and 1%) on the survival of microencapsulated L. casei cells.
Survival amount (log cfu g−1)

Time (h) 0 0.5 1 2 3

a b bc cd
Simulatedgastricjuice pH 2 7.85 ± 0.10 7.30 ± 0.08 6.88 ± 0.06 6.46 ± 0.07 6.30 ± 0.05d
pH 3 7.85 ± 0.10a 7.60 ± 0.08ab 7.45 ± 0.07ab 7.36 ± 0.07b 7.32 ± 0.09b

Time (h) 0 3 6

Bile salt 0.5% 7.85 ± 0.10a 6.88 ± 0.10b 6.00 ± 0.09cd

1% 7.85 ± 0.10a 6.88 ± 0.10b 5.65 ± 0.11d

a-d
Means within a row with different superscripts differ significantly (P < 0.05).

probiotic cells (Knorr, 1998). Likewise, S. thermophilus retained high
viable counts during the storage period. Similar observation has already
been reported in previous studies (Dimitrellou et al., 2016; Kudełka,
2010; Varga, Süle, & Nagy, 2014). L. bulgaricus demonstrated the
highest reduction in counts, during the storage period. Ηowever, the
total sum of S. thermophilus and L. bulgaricus was above the minimum
requirement of 107 viable cells/g (FAO/WHO, 2003).
3.3. Aroma related compounds of fermented milks
Τhe effect of Ca-alginate capsules containing L. casei cells on the
volatile compounds of fermented milk was studied by the SPME GC-MS
technique and compared with the results of the control sample (fer-
mented milk produced with the commercial starter only). The en-
capsulated L. casei cells resulted in no significant differences on the
qualitative composition of volatile compounds, in accordance to a re-
cent study, reporting that probiotics, when used in combination with
traditional yogurt starters, do not affect the profile of major aroma
compounds, but have rather an effect on their concentration (Chen
et al., 2017). The compounds identified in the fermented milks are
listed by chemical group in Table 3.
From a quantitative point of view, the results demonstrated that
there were no significant differences (P > 0.05) in the content of esters
and alcohols (Table 3). On the contrary, the use of encapsulated L. casei
cells significantly (P < 0.05) increased the concentrations of organic
acids, aldehydes, ketones and total volatile compounds, compared to
the control sample, probably due to the relatively higher viability of
encapsulated L. casei during the storage period, which resulted in
higher metabolic activity, leading to increased concentration of volatile
compounds.
A total of 4 esters were identified in the volatile fraction from the
fermented milk produced with an adjunct encapsulated culture.
However, ethyl octanoate and ethyl decanoate were not detected in the
control sample (Table 3). The presence of esters even in low con-
centration is essential for the formation of “fruity and floral” aromas
and flavors, but is also associated with extended storage (Cheng, 2010).
Another well-represented class of volatile compounds is organic
acids. Acetic, butanoic, hexanoic, octanoic, nonanoic and decanoic
acids were detected in the tested samples and are also frequently found
in dairy products (Dan et al., 2017; Dimitrellou et al., 2017; Gurkan &
Hayaloglu, 2017; Tian, Shen, Yu, He, & Chen, 2017). The addition of L.
casei in dairy products has been well correlated with the formation of
organic acids (Dimitrellou, Kandylis, Sidira, Koutinas, & Kourkoutas,
2014; Tian et al., 2017; Zaręba, Ziarno, Ścibisz, & Gawron, 2014), and
associated with the significantly (P < 0.05) higher concentrations of
the majority of organic acids in samples containing the encapsulated L.
casei cells.
Ethanol is a common fermentation product of lactic acid bacteria,
having an agreeable ethereal odor, mild flavor note, and is also present
in fresh milk and many dairy products. The 2-ethyl-1-hexanol content
was significantly (P < 0.05) increased in the products containing the
encapsulated L. casei. Previously, it was shown that ethanol contributed
to the flavor and aroma of dairy product produced by L. casei in yogurt
and cheese samples (Dimitrellou et al., 2014; Tian et al., 2017).
Aldehydes are also an important group of volatile compounds re-
sponsible for the formation of the characteristic aroma profile of dairy
products. The use of encapsulated L. casei resulted in significantly
(P < 0.05) higher concentrations of octanal and nonanal, which are
known to possess strong fruity odor, and (E,E)-2,4-heptanedial and (E)-
2-nonenal. Likewise, the highest concentrations of volatiles found in the
new products compared to the control sample was due to increased
(P < 0.05) amounts of ketones. Several methyl ketones, which are
known for their fruity, floral and musty notes were detected, such as 2-
butanone, 2-pentanone, 2-heptanone, 2-octanone, 2-nonanone, 2-un-
decanone and 2-tridecanone (Molimard & Spinnler, 1996). Μethyl ke-
tones have been already characterized as significant volatile organic
compounds in dairy samples fermented with L. casei (Tian et al., 2017).
Significantly (P < 0.05) higher concentrations of 3-hydroxy-2-buta-
none (acetoin), as constant metabolic products of citrate metabolism
that affect the creamy and butter-like flavor, as well as δ-dodecalactone
with a positive effect on flavor formation, was recorded in the sample
with encapsulated L. casei cells. γ- and δ-dodecalactones, detected in
both fermented milks, are considered important compounds for the
final aroma of dairy products (Curioni & Bosset, 2002; Dimitrellou
et al., 2014, 2017).
3.4. Sensory evaluation of fermented milks
Apart from the main physicochemical and aroma characteristics of
fermented milks with Ca-alginate encapsulated L. casei cells, it is im-
portant to conduct a sensory evaluation, in order to have a better un-
derstanding of the effect of capsules on the sensory properties of

Table 2
Physicochemical and microbiological characteristics of fermented milks during storage at 4 °C.
Days pH Acidity (% w/w lactic acid) Streptococcus thermophilus (log cfu g−1) Lactobacillus bulgaricus (log cfu g−1) Lactobacillus casei (log cfu g−1)

0 4.65 ± 0.03a 0.70 ± 0.02a 8.57 ± 0.07a 8.50 ± 0.06a 7.45 ± 0.05a
7 4.21 ± 0.02b 0.75 ± 0.01ab 8.45 ± 0.05ab 8.22 ± 0.07ab 7.39 ± 0.04a
14 4.15 ± 0.02bc 0.80 ± 0.01bc 8.30 ± 0.07ab 7.90 ± 0.06b 7.30 ± 0.03ab
21 4.06 ± 0.02cd 0.87 ± 0.02cd 8.11 ± 0.06bc 7.52 ± 0.05c 7.21 ± 0.04ab
28 4.01 ± 0.02d 0.92 ± 0.02d 7.90 ± 0.07c 7.18 ± 0.04d 7.13 ± 0.05b

a-d
Means within a column with different superscripts differ significantly (P < 0.05).

4
D. Dimitrellou, et al. LWT - Food Science and Technology 116 (2019) 108501

c
Table 3 Nd: non detected.
Major aroma-related compounds (μg kg−1) of fermented milks after 28 days of
refrigerated storage. probiotic fermented milks and therefore consumers’ acceptance
Compound Identification Fermented milkb (Prasanna & Charalampopoulos, 2019). During sensory evaluation, the
Methoda commercial fermented milk and the product produced using en-
CFM LCFM capsulated L. casei, received similar scores (Table 4), suggesting that the
application of encapsulated cells had no effect on their acceptability.
Esters
Ethyl butanoate RT, KI, MS 1.0 ± 0.2a 0.7 ± 0.1a More specifically, the results showed that there were no significant
Ehtyl hexanoate RT, KI, MS 1.2 ± 0.2a 0.5 ± 0.3a differences in the color of fermented milks (P = 0.064). Similar results
Ethyl octanoate RT, KI, MS NDc 0.8 ± 0.3 were also observed in previous studies using alginate based capsules
Ethyl decanoate RT, KI, MS ND 0.9 ± 0.2
contained Hi-MaizeTM starch (Kailasapathy, 2006) and double-layer
Organic acids
Acetic acid MS 20.1 ± 1.2a 51.4 ± 3.0b alginate microcapsules (Mousa, Liu, Chen, Zhang, & Chen, 2014), re-
Butanoic acid KI, MS 15.3 ± 2.1a 31.4 ± 3.1a flecting the advantages of sodium alginate as probiotic carrier in dairy
Hexanoic acid KI, MS 39.5 ± 3.0a 81.5 ± 5.0b products that does not affect appearance and color.
Octanoic acid RT, KI, MS 45.6 ± 3.5a 48.9 ± 3.0a It has been reported that the use of alginate as encapsulation ma-
Nonanoic acid RT, KI, MS 7.8 ± 0.9a 13.4 ± 0.8b
terial may influence the smoothness of fermented milks and yogurts,
Decanoic acid RT, KI, MS 40.1 ± 2.2a 50.2 ± 3.2a
Alcohols increasing the grittiness of coagulum by replacing sodium with calcium
Ethanol RT, KI, MS > 10,000 > 10,000 ions (Kailasapathy, 2006). However, such effect was not observed in the
1-Hexanol RT, KI, MS 2.2 ± 1.0 ND present study and all products received similar scores for smoothness
1-Heptanol KI, MS 6.4 ± 0.8a 4.1 ± 0.5a
attribute (P = 0.847). Importantly, no report on grainy texture was
1-Octanol RT, KI, MS ND 0.8 ± 0.6
1-Nonanol RT, KI, MS 1.5 ± 0.5 ND
received by the panel, in contrast to a previous study concerning ad-
3-Methyl-1-butanol RT, KI, MS ND 1.5 ± 0.2 dition of encapsulated probiotic in κ-carrageenan beads in yogurts
2-Heptanol RT, KI, MS ND 2.3 ± 0.4 (Adhikari, Mustapha, & Grün, 2003), probably due to the lower quan-
2-Nonanol RT, KI, MS 1.2 ± 0.6a 4.5 ± 0.8a tity of Ca-alginate beads added during the fermented milk production
1-Octen-3-ol RT, KI, MS 3.1 ± 0.4a 3.9 ± 0.2a
(10 g L−1) compared to the κ-carrageenan beads (100 g L−1) (Adhikari
2-Ethyl-1-hexanol RT, KI, MS 7.8 ± 0.3a 19.4 ± 1.2b
Aldehydes et al., 2003). Several studies suggest that the use of large capsules
Acetaldehyde KI, MS 15.2 ± 1.0a 17.2 ± 0.5a (larger than 1000 μm) may cause a coarseness of texture in mouthfeel
3-Methyl butanal KI, MS 0.9 ± 0.2a 1.0 ± 0.2a (Hansen, Allan-Wojtas, Jin, & Paulson, 2002). Similarly, Kailasapathy
Hexanal KI,MS 2.6 ± 0.4 ND
(2006) observed a significant change in the textural properties, parti-
Heptanal KI,MS 1.5 ± 0.4a 2.7 ± 0.9a
Octanal KI,MS 6.5 ± 0.5a 14.9 ± 1.0b
cularly smoothness, of yogurts containing microencapsulated probiotic
Nonanal KI,MS 7.6 ± 0.6a 16.8 ± 1.1b bacteria. In addition, in a study using probiotic capsules of approxi-
(E,E)-2,4-Heptanedial KI,MS ND 12.3 ± 0.8 mately 2 mm in diameter, the capsules were visible to the panelists,
(E)-2-Nonenal KI,MS 4.1 ± 0.4a 8.4 ± 0.5b which is an undesirable quality parameter for yogurt (Ortakci & Sert,
Ketones
2012). In the present work, the capsules’ diameter was
Acetone KI, MS 14.5 ± 0.8a 15.1 ± 1.2a
2-Butanone KI, MS 25.2 ± 1.5a 90.2 ± 9.5b 587.46 ± 58.66 μm, significantly smaller compared to other studies,
2-Pentanone MS 14.8 ± 1.0a 70.4 ± 5.5b using Ca-alginate based capsules of probiotics (Ortakci & Sert, 2012;
2-Heptanone KI, MS 20.2 ± 2.3a 62.3 ± 4.9b Prasanna & Charalampopoulos, 2018, 2019), and therefore no such
2-Octanone MS 13.9 ± 1.1a 31.5 ± 2.2b
effect was observed. Smaller capsules provide better organoleptic
2-Nonanone KI, MS 15.8 ± 1.0a 19.2 ± 1.1a
2-Undecanone KI, MS 5.6 ± 0.5a 9.4 ± 0.8a
properties, but may result in unsatisfactory protection of the en-
2-Tridecanone MS ND 11.4 ± 1.0 capsulated bacteria (Varankovich, Khan, Nickerson, Kalmokoff, &
3-Hydroxy-2-butanone KI, MS 35.8 ± 5.5a 106.5 ± 11.0b Korber, 2015).
(acetoin) No significant differences were observed in viscosity (P = 0.164)
2,3-Butanedione (diacetyl) MS 29.4 ± 1.5a 30.4 ± 3.0a
and sweetness (P = 0.847). In a previous study, the use of alginate
2,3-Pentanedione MS 4.5 ± 2.0a 6.2 ± 1.0a
Lactones capsules in dairy products was correlated with a slight increase in bit-
γ-Dodecalactone KI, MS 1.0 ± 0.2a 1.5 ± 0.2a terness, mainly due to the addition of Ca+2 ions that provide a bitter
δ-Dodecalactone KI, MS 0.9 ± 0.2a 6.3 ± 0.7b taste (Ortakci & Sert, 2012). However, such a trend was not observed in
our study and may be attributed to the lower quantity of Ca-alginate
Total compounds
beads added during the fermented milk production and their smaller
Esters 2.2 ± 0.4a 2.9 ± 0.6a size.
Organic Acids 168.4 ± 12.9a 276.8 ± 18.1b Finally, fermented milks, produced with encapsulated cells and
Alcohols 22.2 ± 3.6a 36.5 ± 3.0a commercial sample presented significantly (P = 0.011) higher values in
Aldehydes 38.4 ± 3.1a 73.3 ± 5.0b
overall acceptability compared to the control samples, as well as those
Ketones 179.7 ± 14.7a 452.6 ± 39.2b
Lactones 1.9 ± 0.2a 7.8 ± 0.7b produced by free L. casei cells. This may be attributed to the improved
body and texture of fermented milks with Ca-alginate capsules identi-
Total 412.8 ± 34.9a 849.9 ± 66.6b fied by the consumers also in several previous studies and may be at-
a-b
tributed to reinforcement of fermented milk gel through ionic interac-
Means within a row with different lowercase superscripts differ significantly
tions between milk proteins and alginates, and to creamy texture due to
(P < 0.05).
a the presence of polysaccharides taste (Ortakci & Sert, 2012). These
RT: Positive identification by retention times that agree with authentic
compounds generated in the laboratory, KI: Tentative identification by kovats' results show the great potential for industrial application of the tech-
retention index compared to the literature (Bianchi, Careri, Mangia, & Musci, nology, due to the products’ acceptance during the sensory evaluation.
2007; Dimitrellou et al., 2009; Dimitrellou, Kandylis, Kourkoutas, Koutinas, &
Kanellaki, 2015; Gurkan & Hayaloglu, 2017), MS: tentative identification by 3.5. Economic sustainability of encapsulation process for fermented milk
mass spectra obtained from NIST107, NIST21 and SZTERP libraries. production
b
CFM = Fermented milk produced with CH-1 culture; LCFM = Fermented
milk produced with Ca-alginate microencapsulated L. casei ATCC 393 cells and Based on the encapsulation method presented above, we estimated
CH-1 culture. the economic sustainability of application of the encapsulated probiotic

5
D. Dimitrellou, et al.
Table 4
Sensory evaluation of fermented milks produced using free or encapsulated L. casei cells in comparison with commercial and control samples.
Days control
a a
Color 7.6 ± 0.1
Sweet Odor 6.3 ± 0.2a
Viscosity 8.1 ± 0.1a
Sweetness 5.5 ± 0.1a
Smoothness 7.6 ± 0.1a
Overall acceptability 7.7 ± 0.1a
a-d
Means within a row with different superscripts differ significantly (P < 0.05).
bacteria in production of fermented milk. The basis for our calculations
was a daily dairy capacity of 10,000 L of product and adequate capacity
for production of encapsulated probiotic bacteria. Investment in
equipment required for encapsulation was 50,000 €, while its estimated
useful life was 8 years. The additional production costs related to in-
troduction of encapsulation were divided into two groups: (i) variable
(material) costs (i.e. costs of carrier materials and energy costs) and (ii)
fixed costs (i.e. depreciation, interest costs, etc.). We assumed that dairy
capacities are employed between 70% and 100% annually and all prices
used for calculations were obtained from local markets in Serbia and
Mediterranean European countries. Depreciation was evaluated using
straight-line method and average annual interest costs were determined
by applying an interest rate of 5%.
Our results showed that increase of variable costs caused by en-
capsulation process is 0.02 € L−1, whereas additional fixed costs are
estimated at around 64,000.00 € per year. Therefore, increase of total
costs depends on capacity usage and varies from 0.04 € L−1 (for 100%
capacity usage) to 0.05 € L−1 (for 70% capacity usage). On the other
hand, change of revenues is related to the increase of the selling price of
product (caused by encapsulation), as well as the level of production
volume (capacity usage). It was determined that introduction of en-
capsulation is profitable if an increase of the selling prices of the new
product ranges 0.04 and 0.05 € L−1 (depending on level of capacity
usage, i.e. production volume). Additional economic calculations are
provided in the supplementary data.
4. Conclusions
In vitro experiments revealed that Ca-alginate capsules produced by
extrusion technique can provide protection to L. casei ATCC 393 cells
against the adverse simulated conditions of human GIT. The overall loss
of viability after 3 h of exposure to pH 2 and 1% bile salts solution was
only 1.55 and 1.35 log cfu g−1, respectively. High survival rates were
also observed during the fermented milk processing and storage (up to
28 days at 4 °C), without affecting the physicochemical and sensory
characteristics of the final product. Moreover, no grainy texture in the
fermented milk with incorporated Ca-alginate beads was noticed by the
sensory panel. Although the encapsulated cells increased the con-
centration of particular volatile compounds, they had no significantly
effect on the sensory perception of the novel product. Our results in-
dicated that L. casei ATCC 393 represents a suitable strain for en-
capsulation by extrusion technique and that the encapsulated cells
might be successfully used for possible industrial application for the
production of novel functional fermented milks.
Acknowledgements
The research project was implemented within the framework of the
Action « Supporting Postdoctoral Researchers» of the Operational
Program “Education and Lifelong Learning” (Action's Beneficiary:
General Secretariat for Research and Technology), which was co-fi-
nanced by the European Social Fund (ESF) and the Greek State, Grant
No LS9 (3546). Part of the work was implemented within the project of
the Serbian Ministry of education, science and technological
LWT - Food Science and Technology 116 (2019) 108501
Free cells Encapsulated cells Commercial
a
8.1 ± 0.1 8.0 ± 0.1 8.1 ± 0.1a
6.9 ± 0.1ab 7.1 ± 0.1b 7.1 ± 0.1b
7.7 ± 0.1a 7.9 ± 0.1a 8.0 ± 0.1a
5.9 ± 0.1ab 6.2 ± 0.1b 6.1 ± 0.1b
7.5 ± 0.2a 7.6 ± 0.1a 7.7 ± 0.2a
7.8 ± 0.2a 8.6 ± 0.1b 8.7 ± 0.2b
development, Grant No 46010.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2019.108501.
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