food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Production of a novel probiotic yogurt by

incorporation of L. casei enriched fresh apple
pieces, dried raisins and wheat grains

Loulouda A. Bosnea a,∗ , Nikolaos Kopsahelis a,b , Varvara Kokkali a ,

Antonia Terpou a , Maria Kanellaki a
a Food Biotechnology Group, Section of Analytical Environmental and Applied Chemistry, Department of Chemistry,
University of Patras, GR-26500 Patras, Greece
b Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Lactobacillus casei enriched apple pieces, dried raisins and wheat grains were incorporated
Received 4 October 2014 in yogurt in fresh and freeze dried form to produce a novel probiotic dairy product. The
Received in revised form 14 viability of L. casei cells was assessed in the yogurts during storage at 4 ◦ C for 60 days and
November 2016 the effect of the added enriched materials on physicochemical parameters, microbiological
Accepted 24 November 2016 characteristics and sensory acceptance of yogurts were evaluated. The apples, raisins and
Available online 1 December 2016 wheat grains improved the viability of embedded L. casei cells resulting at counts around
7 log cfu g−1 of yogurt after 60 days of storage at 4 ◦ C. Yogurts produced with incorporation
Keywords: of L. casei enriched raisin and wheat grains in particular, presented less syneresis due to
Probiotic their water holding capacity. The above results are encouraging for the production of novel
Lactobacillus casei yogurts with improved sensorial and nutritional characteristics in industrial and/or small
Yogurt industrial scale.
Freeze drying © 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Apple
Raisins
Wheat
Immobilization

1. Introduction though there is no clear agreement on the minimum concentration of

Nowadays, promoting health and healthy habits are of major concern
to consumers and functional foods gain in popularity and acceptance.
Lately the best known group of functional foods for gut health has been
claimed to be probiotics (de Vrese and Schrezenmeir, 2008). Probiotic
containing foods have emerged as significant part of the functional
food market, claiming to exert positive health effects for humans, e.g. in
gastrointestinal health. FAO/WHO (2002) in particular have defined pro-
biotics as ‘live microorganisms which when administered in adequate
amounts confer a health benefit on the host’.
Although potential health effects of probiotics depend on a mul-
titude of parameters, a key characteristic is the inoculum size and
survival etc. Moreover, their survival through gastrointestinal envi-
ronment is also a crucial point (Kailasapathy and Chin, 2000). Even
probiotic intake most researchers suggest that concentrations higher
than 106 cfu mL−1 are necessary to achieve beneficial effect on the host
(Gourbeyre et al., 2011). However, surveys conducted on several com-
mercially available products have shown low population levels at the
time of consumption (Shah, 2000). Several factors have been claimed
to be responsible for the reduced viability of probiotics, such as acidity
of products (pH), post acidification, level of oxygen in products, oxygen
permeation through the package, the presence of other competitive
LAB, and sensitivity to antimicrobial substances produced by bacteria
(Dave and Shah, 1997).
Fruit yogurts are generally popular among consumers. The effect of
natural fruit juices on the growth of probiotics and yogurt starter cul-
ture has been reported to be species as well as strain specific. However,
only a few studies have investigated the effect of added commercial

∗
Corresponding author. Fax: +30 2610 997105.
E-mail addresses: bosnea@upatras.gr, louloudabosnea@gmail.com (L.A. Bosnea).
http://dx.doi.org/10.1016/j.fbp.2016.11.010
0960-3085/© 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71
fruit preparations on the growth and survivability of probiotic bacteria
(Kailasapathy et al., 2008; Vinderola et al., 2002).
The importance of probiotic bacteria and the problems associated
with maintaining their viability encourages researchers to continu-
ously develop novel products (Molina et al., 2012; Noorbakhsh et al.,
2013). Fresh and dried fruits and cereal grains are essential compo-
nents of the human diet and their consumption is associated with
numerous health and nutritional benefits. Also, dairy products, such
as fresh milk, fermented milk, yogurt and cheese, have been targeted
as good carrier foods for probiotic microorganisms primarily because
many LAB are of dairy origin, and thus fit to survive better in dairy prod-
ucts and moreover because their consumption is widespread, making
them a good vehicle to reach many consumers (Fondén et al., 2003;
García de Fernando, 2016; Lourens-Hattingh and Viljoen, 2001). Using
fresh or dried fruit and cereals as a carrier of functional ingredients
are a relatively new concept. The beneficial synergies between fruit
and cereals and some bioactive components like probiotic bacteria and
incorporation in dairy products have the potential to lead to a new era in
functional food innovations (Rößle et al., 2010; Sun-Waterhouse, 2011).
Lactobacillus casei ATCC 393 strain has been extensively incorpo-
rated into food products due to its technological properties (Bosnea
et al., 2009; Kourkoutas et al., 2006; Schoina et al., 2014; Sidira et al.,
2014). In vitro and preliminary in vivo studies have demonstrated that
L. casei ATCC 393 displayed several probiotic properties such as removal
of cholesterol (Lye et al., 2010) and activity against cancer cell prolifer-
ation (Choi et al., 2006) and reduction of pathogens in parallel with
distinct adhesion in rat intestinal mucosa (Saxami et al., 2012; Sidira
et al., 2010, 2013).
The purpose of the present study was to test the feasibility of
producing a novel probiotic yogurt product by incorporating L. casei
enriched fresh apple piece, dried raisins and wheat grains. In addition
the enriched apples, raisins and wheat have been freeze dried (FD) aim-
ing to produce a more stable product. These freeze dried biocatalysts
can provide a ready to use, stable probiotic culture that can be used by
all dairy industries by their addition in yogurt or yogurt like dairy prod-
ucts. The effect of the incorporation of the enriched fruits and cereals
on growth of foodborne pathogens (Staphylococcus aureus), the yogurt
microflora, and spoilage microorganisms (yeasts and molds) was eval-
uated along with some yogurt’s physicochemical properties (e.g. yogurt
syneresis).
2. Materials and methods
2.1. Bacterial strains and growth conditions
L. casei ATCC 393 (DSMZ, Germany) was grown at 37 ◦ C for 48 h
on MRS Broth (Oxoid Ltd., Hampshire, UK) under anaerobic
conditions (AnaeroGen TM, Oxoid, Ltd., Hamphire, UK). L. casei
cells were harvested by centrifugation at 4165 × g for 10 min
at 20 ◦ C (SIGMA 3K12, Bioblock Scientific, Laborzentrifugen
GmbH, Osterode, Germany).
2.2. Preparation of fresh apples, dried raisins and
wheat grains
Apples (cultivar Granny Smith) were purchased in a local
supermarket, washed in water and cut with a stainless-steel
knife into wedges while precautions were taken to avoid cross
contamination. Wheat grains (hard wheat, variety Thivas,
commercial brand “Agrino”) and dry raisins (Corinthian type)
were also purchased in a local supermarket. Prior use, wheat
grains and raisins were sterilized at 121 ◦ C for 15 min (Bosnea
et al., 2009), but no treatments were carried out for apple
pieces. Sterilization of supports was important in order to
reduce the initial microbiological load of raw materials.
63
2.3. Production of L. casei enriched fresh and dried
fruit and grains
L. casei enriched Fruits and grains enriched with Lactobacillus
casei cells were separately produced by mixing 500 g of every
the support (fresh apple pieces, dried raisins and wheat grains)
along with 8 g biomass (wet weight) of L. casei cells in 1 L of
MRS broth (Oxoid Ltd., Hampshire, UK) and every mixture was
allowed to ferment at 37 ◦ C for 48 h without agitation. When
enrichment (immobilization of L. casei on each carrier in their
porous support) was complete, the liquid was decanted and
every enriched material was washed twice with sterile Ringer’s
solution for removal of the free cells.
2.4. Freeze-drying of immobilized L. casei cells
The prepared enriched supports were freeze-dried. More
specifically, 175 g of every enriched material was cooled to
−40 ◦ C at 3 ◦ C min−1 in a controlled rate freezer (BioCool,
FTS Systems, NY, USA). Then the samples were freeze-dried
overnight at 135.10−3 mbar and at −45 ◦ C in a Freeze Drying
System, Freezone 4.5 (Labconco, Kansas City, Missouri, USA).
No cryoprotectant medium was used during freeze-drying
(Bosnea et al., 2009).
2.5. Yogurt production
A commercial homogenized, standardized and pasteurized
cow’s milk containing 3.7% fat and 13.2% total solids was heat
treated to 90 ◦ C for 10 min, cooled down to 45 ◦ C and divided
into 8 equal portions (100 mL each). The first batch (C: control)
was inoculated with yogurt culture (5% inoculum, Streptococ-
cus thermophilus and Lactobacillus delbruecki subsp. bulgaricus,
1:1 proportion), the second (FLC) was inoculated with yogurt
culture plus L. casei ATCC 393 free cells (5% inoculum), the third
(WIA) and fourth (FIA) with yogurt culture (5%) and enriched
apple pieces (35 g per 100 mL milk) fresh and freeze dried,
respectively. The fifth (WIR) and six (FIR) batches were pre-
pared with yogurt culture and enriched raisins (35 g per 100 mL
milk) fresh and freeze dried, respectively. Finally, the seventh
(WIW) and eighth (FIW) were prepared using yogurt culture
and enriched L. casei cells on wheat grains (35 g per 100 mL
milk), in fresh and freeze dried form. All batches were poured
into plastic cups (100 mL) and incubated at 42 ◦ C until a pH of
4.6–4.8 was reached. The yogurts were then transferred to cold
storage (4 ◦ C ± 1) and stored up to 60 days. Samples were col-
lected and analyzed from each batch at several time intervals
after production. The whole yogurt production was repeated
in triplicate.
2.6. Physico-chemical measurements
All samples were homogenized prior to analysis. The pH of
all yogurts was measured using a digital pH meter by direct
immersion of the electrode (EPI-BION SENTRON pH-System
1001).
For lactose and lactic acid determination, five grams of
yogurt sample were diluted with water to a total volume of
200 mL, mixed well and centrifuged at 4125 × g for 10 min (Shi-
madzu Application news, No L213), and then the solution was
used for lactose and lactic acid determination.
Lactose was determined by high performance liquid
chromatography, using a Shimadzu chromatograph with a
SCR-101N stainless steel column, a LC-9A pump, a CTO-10A
64 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71
oven at 60 ◦ C and a RID-6A refractive index detector. Three
times distilled water was used as mobile phase with a flow
rate of 0.8 mL min−1 and 1-butanol was used as an internal
standard. Samples of 0.5 mL and 2.5 mL of a 1% (v/v) solution
of 1-butanol were diluted to 50 mL and 40 ␮L were injected
directly to the column. Lactose concentrations were calculated
using standard curves.
Lactic acid was determined by high performance liquid
chromatography, using a Shimadzu chromatograph with a
Shim-pack IC-A1 stainless steel column, a LC-10A pump, a
(Farnsworth et al., 2006) CTO-10A oven at 40 ◦ C and a CDD-
6A conductivity detector. A solution of 2.5 mM phthalic acid
and 2.4 mM tris (hydroxymethyl) aminomethane (pH 4.0) in
three times distilled water was used as mobile phase with a
flow rate of 1.5 mL min−1 . Samples of 0.25 mL were diluted to
25 mL and 50 ␮L were injected directly to the column. Lactic
acid concentrations were calculated using standard curves.
The total solids of samples were determined by drying
samples at 110 ◦ C overnight to constant weight. Ash was deter-
mined according to AOAC. The fat and protein contents were
determined by the Gerber and Kjeldahl method respectively.
Syneresis was measured according to (Farnsworth et al.,
2006) with modifications. More specifically, the samples were
centrifuged at 350 × g for 10 min at 4 ◦ C in triplicate. The
syneresis percentage was calculated by the milk whey mass
separated from the gel network during centrifugation divide
by the initial yogurt mass, multiplied by 100.
2.7. Enumeration of L. casei ATCC 393 in enriched
yogurts
On each test day, ten-gram portions of yogurt samples were
diluted to 100 mL with sterilized Ringer solution ¼ strength
and mixed with a stomacher (Bagmixer 400, Model VW,
Interscience). Subsequent dilutions were made in sterilised
Ringer’s solution ¼ strength. Viable L. casei counts were deter-
mined on MRS agar–vancomycin agar (Fluka Analytical) after
at 37 ◦ C incubation anaerobically for 48–72 h, according to
Tharmaraj and Shah (2003). All plates were incubated aerobi-
cally for 48–72 h at 37 ◦ C followed by enumeration. All samples
were serially diluted and plated in duplicates. The original
count in the sample was expressed as log cfu per gram of
yogurt.
2.8. The effect of the incorporation of the enriched
fruits and cereals on growth of foodborne pathogens, the
yogurt microflora, and spoilage microorganisms
On each test day (1, 7, 15, 30, 45, 60) ten-gram portions of each
yogurt sample (C, FLC, WIA, FIA, WIR, FIR, WIW, FIW) were
diluted to 100 mL with sterilized Ringer solution ¼ strength
and mixed with a stomacher (Bagmixer 400, Model VW,
Interscience). Subsequent dilutions were made in sterilised
Ringer’s solution ¼ strength. Viable counts for streptococci,
lactobacilli, moulds and yeasts, coliforms, enterobacteria,
Staphylococcus were performed in duplicate by pour plat-
ing on the selective media for each species and according
to instructions given by manufacturer. Coliform counts were
enumerated on Violet red bile agar (Fluka Analytical) after
incubation at 30 ◦ C for 24 h, total Enterobacteriaceae were enu-
merated on Violet red bile glucose agar (Fluka Analytical) after
incubation at 37 ◦ C for 24 h. Streptococcus thermophilus was
determined on M-17 agar (Fluka Analytical) following incu-
bation at 30 ◦ C for 72 h, L. delbrueckii subsp. bulgaricus was
determined on MRS agar acidified at 5.2 pH. Staphylococcus
counts were determined on Baird Parker agar (Fluka Analyt-
ical) after incubation at 37 ◦ C for 48 h and yeasts and molds
were determined by plating on Potato Dextrose agar (Fluka
Analytical) after incubation at 30 ◦ C for 3–5 days.
2.9. Preliminary sensory evaluation
Yogurt samples with immobilized L. casei cells on natural
supports (apple, raisins, and wheat) were tested for their
sensory characteristics and compared with yogurt produced
with S. thermophilus and L. bulgaricus and yogurt produced
with free L. casei cells and S. thermophilus and L. bulgaricus.
Samples (∼25 g) of yogurts produced the day before were pre-
sented. Sensory evaluation was conducted by 10 laboratory
members previously trained using locally approved proto-
cols. The panel was asked to give scores on a 0–10 scale
(0 = unacceptable, 10 = exceptional) for attributes grouped into
3 categories: aroma, taste, and flavor. Panelists used water
to clean their palates between samples and were blinded of
the samples they tasted (samples were labeled with codes for
identification).
2.10. Statistical analysis
Results were analyzed using one-way analysis of variance
(ANOVA). The treatments were compared among each other
in the same storage period, and the samples of the same
treatment were compared during the time. Duncan’s multi-
ple range tests were used to determine significant differences
among results (coefficients, ANOVA tables and significance)
were computed using SPSS v.8.5. A P value <0.05 was consid-
ered statistically significant for all analyses.
3. Results and discussion
3.1. Production of novel probiotic yogurt supplemented
with L. casei enriched fresh apple pieces, dried raisins or
wheat grains
L. casei enriched apple pieces, raisins and wheat grains were
used in yogurt production. Yogurts were prepared using the
traditional set method because it is simple and it favors the
better survival of probiotic microorganisms in comparison
with methods where adjuncts are added later in production
(Gardini et al., 1999; Hull et al., 1984). Regarding the need for
careful selection of potential probiotic strains in yogurts like
products, L. casei ATCC 393 strain was selected based on the
in vitro screening of the survival in simulated GI tract condi-
tions (Choi et al., 2006) as well as in the in vivo survival of the
strain (Foligne et al., 2007). Preparations of yogurts with free L.
casei cells and fresh and freeze dried immobilized cells were
made for comparison reasons.
Apple, raisins and wheat grains were used as immobi-
lization carriers based among other constituents, on their
prebiotic character. The combination of probiotic and pre-
biotic (symbiotic) is of major importance due to the fact
that prebiotics encourage the growth of probiotic bacteria by
providing a protective effect during passage through the gas-
trointestinal tract because prebiotics are non hydrolyzed or
absorbed in the stomach or small intestine. Moreover, immo-
bilized bacteria on carriers appear to be more active than
free ones. Immobilized cells combine with compounds of
the carriers (e.g. proteins, polysaccharides) to produce a vis-
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71
6,8
6,6
6,4
6,2
6,0
5,8
5,6
pH
5,4
5,2
5,0
4,8
4,6
4,4
4,2
0 50 100 150 200 250
Time (min)
Fig. 1 – Acidification kinetics during yogurt production.
Error bars represent the standard deviation of means.
C: Yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus (), FLC: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + free L. casei cells (), WIA:
yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus + fresh L. casei enriched apple pieces (), FIA: yogurt
with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze
dried enriched L. casei apple pieces (), WIR: yogurt with S.
thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched
L. casei raisins (♦), FIR: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + freeze dried enriched L. casei
raisins (), WIW: yogurt with S. thermophilus + L. delbrueckii
subsp. bulgaricus + fresh enriched L. casei wheat (), FIW:
yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus + freeze dried enriched L. casei wheat (䊉).
cous and elastic material (bio-film). Microorganisms undergo
diverse and profound changes during their transition from
suspended in solution to immobilized form. Bio-film protects
microorganisms from variable environmental conditions and
so immobilized cells present higher productivity, increased
tolerance, higher resistance to inhibitors and others.
3.2. Effect of added enriched materials on
physicochemical characteristics of yogurts
Acidification trend in yogurt fermentation is presented in
Fig. 1. The lowest pH values were observed for yogurts with free
L. casei cells. As shown in Table 1 during fermentation lactose
is decreased and lactic acid is increased. Until the 15th stor-
age day the yogurt with L. casei (FLC) had the lowest pH value
(3.88). At the 30th storage day all yogurts had similar pH values
without significant difference (P < 0.05) and ranged from 4.01
to 4.05. Postacidification effect was observed in all yogurts.
However, L. casei has a pH-resistant character and usually can
survive during post-acidification effects. In case of yogurts
produced with the incorporation of enriched materials there
was a delay in decrement of pH, lactic acid production and
acidity increment during the first week of storage. Especially
in the case where raisins were used the post acidification was
significantly delayed the first 15 days of storage. After 60 days
of storage, there was no significant difference (P > 0.05) in the
amount of lactic acid between yogurt. The results presented
in this work agree with the aforementioned data (Corrieu and
Béal, 2016).
65
In yogurts produced with the incorporation of L. casei
enriched natural materials higher unfermented lactose con-
centration was generally observed than that in yogurt with
yogurt culture (C) and FLC yogurts. Generally, apples, raisins
and wheat contain sugars part of which is extracted to the pro-
duced yogurts. This phenomenon is more obvious in yogurts
produced with the incorporation of raisins that have high sug-
ars content and are easily extracted to milk. Since glucose
is the sugar that is mostly preferred by microorganisms as
energy source because it is more easily metabolized (Samona
and Robinson, 1994), it is probably preferred by microorgan-
isms over lactose or other sugars.
Protein content of the produced yogurts ranged from 3.33 to
4.49% at 1st day after production (Table 2). The higher protein
content was determined for yogurt containing immobilized
L. casei cells on wheat grains. The addition of immobilized
L. casei cells had significant effect on protein content at all
supports (P < 0.05) while there was not any significant differ-
ences between samples C and FLC (P > 0.05). Freeze drying
had no effect on protein content of yogurts when com-
pared with yogurts produced with fresh immobilized L. casei
cells (P > 0.05), except in case of immobilized cells on wheat
(P < 0.05). Since the same milk was used for all yogurts and pro-
teolysis is unlikely, the differences in protein content must be
due to added materials. Therefore, the differences in protein
level between prepared yogurts could be attributed to the pro-
teins and nitrogen compounds of the materials, especially in
case of yogurts produced with L. casei enriched wheat, where
the elevated protein level can be attributed to wheat proteins
(e.g. gluten).
Total solid contents in all yogurts were in the range of
16.00–17.64% (Table 2). The higher values were determined in
yogurts with L. casei cells enriched wheat. The state (fresh or
freeze dried) of enriched materials did not seem to affect total
solid content (P > 0.05) whereas the type of material (apple,
raisin or wheat) affected it significantly (P < 0.05). In most
countries it is common commercial practice to fortify the
solids content to ca. 15%. This improve the body of the final
yogurt reduces susceptibility to syneresis and slightly reduces
acid production during fermentation resulting in the less acid
product (Varnam and Sutherland, 1994). The TS content of pro-
duced yogurts although within appropriate limits is higher
than those reported by Oneil et al. (1979) due to the plant
material added.
Ash of produced yogurts ranged from 0.55 to 0.89% (Table 2),
where the higher ash content was determined for yogurt con-
taining L. casei enriched wheat grains, while there was not any
significant differences between samples C and FLC (P > 0.05).
Fat content of produced yogurts ranged from 3.59 to 3.92%
(Table 2), where the higher fat content was determined for
yogurt containing immobilized L. casei cells on wheat grains.
The addition of L. casei enriched materials did not seem to
affect fat content (P > 0.05), except in case of yogurts produced
with incorporation of wheat (P < 0.05) that could be attributed
in wheat grain matrix, since contains approximately 2.2% of
lipids.
3.3. Effect of added L. casei enriched apple pieces,
raisins and wheat grains on yogurt syneresis
Syneresis is a common defect in fermented dairy product
processing and it refers to the appearance of liquid on the
milk gel surfaces and gel shrinkage, with consequent milk
whey loss (Lucey and Singh, 1997). The evaluation of synere-
 66
Table 1 – pH, lactose, lactic acid and total acidity (TA) during yogurt refrigerated storage at 4 ◦ C for 60 days.
Analysis Storage time C FLC WIA FIA WIR FIR WIW FIW
(days)

food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71

pH 1 4.35 ± 0.0 4.27 ± 0.5 4.41 ± 0.09 4.45 ± 0.01 4.65 ± 0.3 4.60 ± 0.28 4.35 ± 0.14 4.45 ± 0.1
7 3.98 ± 0.73 3.88 ± 0.13 4.08 ± 0.21 4.07 ± 0.18 4.21 ± 0.11 4.34 ± 0.1 4.08 ± 0.16 4.07 ± 0.2
15 3.97 ± 0.14 3.88 ± 0.02 4.03 ± 0.03 4.02 ± 0.03 4.30 ± 0.2 4.23 ± 0.03 4.03 ± 0.2 4.06 ± 0.02
30 4.02 ± 0.03 4.01 ± 0.09 4.03 ± 0.05 4.05 ± 0.09 4.05 ± 0.03 4.03 ± 0.02 4.01 ± 0.02 4.02 ± 0.4
45 4.02 ± 0.02 4.02 ± 0.03 3.98 ± 0.02 4.01 ± 0.12 4.00 ± 0.14 3.98 ± 0.07 3.98 ± 0.06 4.03 ± 0.1
60 4.01 ± 0.08 4.03 ± 0.1 4.01 ± 0.3 4.02 ± 0.21 4.03 ± 0.05 3.96 ± 0.1 4.01 ± 0.05 4.04 ± 0.02
Lactose (g/100 g 1 2.14 ± 0.1 2.23 ± 0.1 2.73 ± 0.4 1.93 ± 0.33 3.60 ± 0.05 2.12 ± 0.1 1.96 ± 0.02 1.96 ± 0.4
yogurt) 7 1.72 ± 0.1 1.94 ± 0.4 2.43 ± 0.73 1.76 ± 0.05 1,89 ± 0.16 2,08 ± 0.1 1,93 ± 0.4 1,95 ± 0.02
15 1,26 ± 0.23 1,16 ± 0.05 1,73 ± 0.16 1,16 ± 0.1 1.82 ± 0.35 1.87 ± 0.4 1.47 ± 0.09 1.87 ± 0.1
30 1.1 ± 0.03 0.94 ± 0.1 1.52 ± 0.4 1.11 1.11 ± 0.4 1.33 ± 0.05 1.13 ± 0.05 1.53 ± 0.02
45 0.83 ± 0.18 0.80 ± 0.02 1.31 ± 0.1 1.04 ± 0.4 1.09 ± 0.02 1.25 ± 0.1 1.0 ± 0.12 1.14 ± 0.02
60 0.53 ± 0.73 0.64 ± 0.09 1.14 ± 0.05 0.83 0.94 ± 0.01 1.01 ± 0.09 0.99 ± 0.05 1.32 ± 0.09
Lactic acid (g/100 g 1 0.91 ± 0.02 0.93 ± 0.04 0.89 ± 0.02 0.85 ± 0.06 0.83 ± 0.08 0.81 ± 0.03 0.94 ± 0.05 0.85 ± 0.14
yogurt) 7 1.05 ± 0.02 1.1 ± 0.14 0.99 ± 0.04 1.01 ± 0.09 0.95 ± 0.09 0.99 ± 0.02 1.11 ± 0.73 0.99 ± 0.03
15 1.14 ± 0.1 1.9 ± 0.4 1.13 ± 0.05 1.21 ± 0.1 0.97 ± 0.1 0.92 ± 0.09 1.21 ± 0.1 1.05 ± 0.09
30 1.25 ± 0.09 1.31 ± 0.03 1.24 ± 0.22 1.23 ± 0.4 1.22 ± 0.03 1.12 ± 0.02 1.33 ± 0.03 1.22 ± 0.1
45 1.29 ± 0.20 1.35 ± 0.1 1.26 ± 0.14 1.31 ± 0.05 1.12 ± 0.73 1.32 ± 0.16 1.41 ± 0.02 1.25 ± 0.02
60 1.32 ± 0.18 1.44 ± 0.02 1.33 ± 0.03 1.39 ± 0.03 1.41 ± 0.02 1.44 ± 0.1 1.39 ± 0.02 1.32 ± 0.05

C: Yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus, FLC: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + free L. casei cells, WIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh L.
casei enriched apple pieces, FIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei apple pieces, WIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei
raisins, FIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei raisins, WIW: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei wheat, FIW: yogurt
with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei wheat.
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71 67

sis is of importance particularly during storage (Zare et al.,

C: Yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus, FLC: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + free L. casei cells, WIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh L.
casei enriched apple pieces, FIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei apple pieces, WIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei
raisins, FIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei raisins, WIW: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei wheat, FIW: yogurt
3.92 ± 0.041–3,5,6
19.21 ± 0.171–6,8
2011). The most important causes for syneresis in fermented

17.64 ± 0.021,6
4.49 ± 0.021–6

0.89 ± 0.051–4
products include the use of high temperatures of incubation,
low solids content or inadequate storage temperatures. In our

FIW
study, no significant differences in syneresis was detected in
yogurts produced only with the addition of S. thermophilus
and L. bulgaricus subsp. delbrueckii (C), yogurt produced with
starter culture and free L. casei cells (FLC), and yogurts pro-

3.88 ± 0.021–3,5,6
20.84 ± 0.091–6,8
duced with starter culture and enriched apple pieces in fresh
17.52 ± 0.071–6
1–6

0.85 ± 0.031–4
and freeze dried form (WIA, FIA) (Table 2). Nevertheless, differ-
4.41 ± 0.08

ences were observed in yogurts produced with starter culture

WIW

and enriched wheat and raisins.

Initially, syneresis in all yogurts was observed macroscop-
ically during storage however, neither of the observed yogurts
presented syneresis during the first 15 days of storage. After
17.20 ± 0.011–4,7,8

19.89 ± 0.551–4,7,8
1–4,7–8

the 15th storage day, control yogurt as well as yogurts pro-

3.72 ± 0.034,7,8
0.88 ± 0.061–4

duced with free L. casei cells and with immobilized L. casei cells
3.78 ± 0.03

on apple started to present syneresis. On the contrary yogurts

containing enriched raisin and/or wheat grains delayed to
FIR

present syneresis. The physical form of the added material

significantly affected the syneresis, since less syneresis was
observed in yogurts with freeze dried enriched fruits and
wheat grains, may be because some of the serum was used
17.24 ± 0.071–4,7,8

20.12 ± 0.251–4,7,8
1–4,7–8

for rehydration of the matrixes. Moreover, raisins contain

0.82 ± 0.041–4
3.64 ± 0.021–3
3.74 ± 0.05

polysaccharides that are extracted to the medium thus form-

ing more stable gels and since raisins are dry products may
WIR

absorb water from the medium resulting in less syneresis.

3.4. Viability of L. casei during storage

22.25 ± 0.321–3,5–8
16.65 ± 0.061,2,5–8

Utilizing food as a vehicle to deliver probiotics has been an

1,2,4–8

0.77 ± 0.021,2,5–8
3.59 ± 0.041–3

interest to food scientists for decades due to their purported

3.33 ± 0.01

health-associated benefits to customers. Maintaining viable

Table 2 – Protein content, total solids, ash and fat of yogurts at 1st day after production.

probiotic cell counts at high level by the end of the expiration

FIA

date is required for most health benefits. The results clearly

showed that L. casei cells survive for 60 days a period that is
higher than usual yogurt self life (Smit, 2003). Table 3 shows
the starter’s populations L. bulgaricus ssp. delbrueckii and S.
23.51 ± 0.121,2,4–8
16.58 ± 0.071,2,5–8

with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei wheat.
1,2,4–8

0.75 ± 0.011,2,5–8

thermophilus as well the population of the adjunct L. casei dur-

3.71 ± 0.034,7,8

ing yogurt storage at 4 ◦ C for 60 days. L. casei was added into

3.39 ± 0.06

the yogurt as free and/or on enriched apple, raisin and wheat

grains during yogurt production at 45 ◦ C. In all cases, at the
WIA

beginning of the experiment yogurts showed counts of starters

higher than 109 CFU g−1 .
L. casei population level in all produced yogurts with added
24.02 ± 0.231,3–8

enriched fruits and grains, at the first storage day ranged

16.13 ± 0.173–8

3.66 ± 0.044,7,8
0.66 ± 0.033–8
3–8

from 8.2 to 8.7 log cfu g−1 , while after 15 days of storage at
3.57 ± 0.25

4 ◦ C ranged in 7.8–8.2 log cfu g−1 and after 60 days of storage

ranged in 7.2–7.8 log cfu g−1 . The respective populations for
FLC

the adjunct added as free in the yogurt were 9.3, 7.2 and
6.1 log cfu g−1 at the 1st, 15th and 60th day of storage. Among
the supports, raisin and wheat grains presented better results
16.01 ± 0.023–8

25.21 ± 0.312–8
3.68 ± 0.034,7,8

than those for apple regarding viable counts of L. casei cells

3–8

0.55 ± 0.093–8
3.55 ± 0.06

(Fig. 2). More specifically, the viable counts of yogurts with

fresh enriched apple pieces are generally lower than that of
the respective raisin or wheat grains in all studied storage time
C

intervals (1, 7, 15, 30, 45 and 60 days).

As regards yogurts with freeze dried enriched wheat grains,
those from 15th storage day showed higher viable counts than
Total solids (%)

freeze dried apples pieces. Generally, the highest viable counts

of L. casei after 60 days of storage at 4 ◦ C was observed in
Protein (%)

Syneresis
Analysis

Ash (%)

those produced with the addition of freeze dried wheat. At

Fat (%)

some cases, like in wheat biocatalyst, freeze drying signifi-

cantly affected viability (P < 0.05) while no significant effect
68 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71

Table 3 – Total aerobic, Streptococcus thermophilus, L. delbrueckii subsp. bulgaricus and L. casei counts in produced yogurts
during storage in 4 ◦ C for 60 days.
Yogurt Storage time Streptococcus L.delbrueckii L. casei Yeasts
(days) thermophilus subsp. bulgaricus (log CFU g−1 ) (log CFU g−1 )
(log CFU g−1 ) (log CFU g−1 )

C 1 9.66 ± 0.85 9.90 ± 0.1 2.02 ± 0.1 0

7 9.34 ± 0.08 9.35 ± 0.77 0 0
15 9.03 ± 0.15 8.04 ± 0.66 0 0
30 8.65 ± 0.25 7.95 ± 0.05 0 0
45 7.03 ± 0.05 7.71 ± 0.16 0 6.98 ± 0.89
60 6.95 ± 0.16 6.23 ± 0.54 0 7.34 ± 0.45
FLC 1 9.72 ± 0.28 9.12 ± 0.16 9.36 ± 0.8 0
7 9.11 ± 0.31 9.14 ± 0.08 8.37 ± 0.12 0
15 9.15 ± 0.22 8.02 ± 1.1 7.23 ± 0.41 0
30 8.76 ± 0.8 7.67 ± 0.1 7.05 ± 0.56 0
45 7.40 ± 0.42 7.40 ± 0.16 6.92 ± 0.1 8.34 ± 0.74
60 7.05 ± 0.05 6.54 ± 0.74 6.12 ± 0.08 10.30 ± 0.2
WIA 1 9.82 ± 0.31 9.91 ± 0.18 8.23 ± 0.52 0
7 9.59 ± 0.45 9.30 ± 0.06 7.95 ± 0.3 0
15 9.25 ± 0.85 8.34 ± 0.8 7.85 ± 0.9 0
30 8.22 ± 0.16 7.42 ± 0.22 7.65 ± 0.1 0
45 7.18 ± 1.1 7.68 ± 0.16 7.43 ± 0.5 8.48 ± 0.8
60 6.96 ± 0.66 5.96 ± 0.01 7.25 ± 0.55 9.63 ± 0.11
FIA 1 9.63 ± 1.02 9.23 ± 0.56 8.55 ± 0.54 0
7 9.54 ± 0.8 9.15 ± 0.08 8.23 ± 0.1 0
15 8.95 ± 0.05 7.98 ± 1.1 7.86 ± 0.8 0
30 8.63 ± 0.45 7.05 ± 0.85 7.69 ± 0.22 0
45 7.96 ± 0.1 6.85 ± 0.44 7.35 ± 0.6 7.52 ± 1.8
60 6.85 ± 0.06 5.36 ± 0.16 7.42 ± 0.35 8.63 ± 0.61
WIR 1 9.72 ± 0.16 9.95 ± 0.31 8.46 ± 0.8 0
7 9.30 ± 0.08 9.20 ± 0.66 8.56 ± 0.54 0
15 8.32 ± 0.82 8.42 ± 1.1 8.23 ± 1.1 0
30 7.43 ± 0.22 7.09 ± 0.73 8.01 ± 0.05 0
45 6.63 ± 0.78 7.12 ± 0.16 7.56 ± 0.1 8.48 ± 0.8
60 6.85 ± 0.63 6.35 ± 0.1 7.68 ± 0.08 9.45 ± 1.1
FIR 1 9.26 ± 0.02 9.28 ± 0.44 8.73 ± 0.11 0
7 9.12 ± 0.6 9.01 ± 0.08 8.56 ± 0.8 0
15 8.78 ± 0.52 8.56 ± 0.22 8.23 ± 0.64 0
30 8.63 ± 0.38 7.964 ± 0.33 8.12 ± 0.74 0
45 7.85 ± 0.22 7.56 ± 0.1 7.99 ± 1.1 6.48 ± 0.77
60 6.98 ± 0.42 6.25 ± 0.85 7.86 ± 0.03 7.45 ± 0.35
WIW 1 9.84 ± 0.44 9.26 ± 0.35 8.55 ± 0.29 0
7 9.36 ± 1.1 8.75 ± 0.05 8.23 ± 1.1 0
15 8.00 ± 0.8 8.30 ± 0.08 8.14 ± 0.8 0
30 7.65 ± 0.73 7.34 ± 0.1 7.96 ± 0.66 0
45 6.87 ± 1.1 7.49 ± 0.16 7.83 ± 0.45 7.69 ± 0.85
60 6.65 ± 0.1 6.54 ± 0.52 7.69 ± 0.22 8.52 ± 1.1
FIW 1 9.56 ± 0.31 9.21 ± 0.16 8.26 ± 0.73 0
7 9.25 ± 0.08 8.96 ± 0.24 8.02 ± 0.8 0
15 8.98 ± 1.22 8.25 ± 1.1 8.16 ± 0.66 0
30 8.25 ± 0.56 7.56 ± 0.06 7.85 ± 1.1 0
45 7.65 ± 0.8 7.06 ± 0.05 7.78 ± 0.12 5.88 ± 0.22
60 6.12 ± 0.44 5.98 ± 0.86 7.66 ± 0.65 6.55 ± 0.05

C: Yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus, FLC: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + free L. casei cells,
WIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh L. casei enriched apple pieces, FIA: yogurt with S. thermophilus + L. delbrueckii
subsp. bulgaricus + freeze dried enriched L. casei apple pieces, WIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei
raisins, FIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei raisins, WIW: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + fresh enriched L. casei wheat, FIW: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L.
casei wheat.

was observed for immobilized cells in apples and raisins
(P > 0.05).
It is apparent that the enrichment with L. casei contributed
to its higher viable population during storage than that for
free cell adjunct. During the 60 days of cold storage the L. casei
count remained above 105 CFU g−1 . L. casei is considered an
acid tolerant lactic acid bacteria and probably the most impor-
tant adventitious non- starter LAB (NSLAB).
In the work Sidira et al. (2013) L. casei ATCC 393 cells were
immobilized on strawberry, banana and oat pieces and were
incorporated in yogurts. The results demonstrated that both
free and immobilized L. casei ATCC 393 were detected in pro-
biotic yogurts at levels required to confer a probiotic effect
(≥6 log cfu/g) after 30 days of storage at 4 ◦ C but not after
67 days of refrigerated storage.
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71 69

105 be explained by the antagonistic effect of high Lactobacil-

lus counts in all yogurt samples that create a competitive
100
environment limiting any other microflora growth data also
95 reported in recent literature (Bian et al., 2016; Delcaru et al.,
2016; Zhang et al., 2016). All the above results are encourag-
L. casei viability (%)

90
85
80
75
70
65
0 10 20 30 40
Time (days)
Fig. 2 – L. casei survival % in yogurts during storage at 4 ◦ C
for 60 days. Error bars represent the standard deviation of
means.
FLC: yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus + free L. casei cells (), WIA: yogurt with S.
thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched
L. casei apple pieces (), FIA: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + freeze dried enriched L. casei
apple pieces (), WIR: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + fresh enriched L. casei raisins
(♦), FIR: yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus + freeze dried enriched L. casei raisins (), WIW:
yogurt with S. thermophilus + L. delbrueckii subsp.
bulgaricus + fresh enriched L. casei wheat (), FIW: yogurt
with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze
dried enriched L. casei wheat (䊉).
3.5. The effect of the incorporation of the enriched
fruits and cereals on growth of foodborne pathogens, the
yogurt microflora, and spoilage microorganisms
The addition of L. casei as free cell culture or as enriched
materials (fresh or freeze dried) did not affect the population
levels of the starter cultures in relation to the control (P > 0.05).
Hamann and Marth (1984) reported that the addition of fruits
to yogurts does appear not to affect the numbers of fermenting
organisms.
It is noteworthy that no-growth of Enterobacteriaccae and
coliforms was detected while the disappearance of staphy-
lococci after the 7th day of storage (data not shown) could
ing regarding pathogen microorganisms. Moreover, yogurt is
an inhospitable medium for pathogens which will not grow
and will not survive well because of its high acidity and low
pH (usually 3.8–4.2).
As far as yeasts are concerned, they appeared in yogurt
samples after the 30th storage day (Table 3). It is noteworthy
that yogurts samples enriched with apple biocatalyst showed
no yeast growth until the 30th day of storage even though
apples naturally provide a great diversity of yeasts (Graça et al.,
50 60
2015). Even though, yeasts are not involved in the fermenta-
tion process during yogurt production they are a major cause
of spoilage of the final product (Fleet, 1990; Howell, 2016).
Higher counts of yeast were observed in yogurts produced with
fresh immobilized L. casei cells compared with freeze dried
immobilized L. casei cells resulting that enriched yogurts give
better preserved products of high quality.
3.6. Sensory evaluation of yogurts
All produced yogurt was assessed for their aroma, flavor,
texture and overall acceptance by 10 laboratory members pre-
viously trained using locally approved protocols. Generally, all
yogurts were favorably evaluated, but significant differences
were observed between yogurts produced with immobilized
L. casei cells in wheat and raisins compared to others and
commercial yogurt examined (Table 4).
According to comments, raisin provided special aroma and
flavor to yogurt, probably due to sugars and volatile com-
pounds extracted to yogurt. Those yogurts were characterized
for their sweet flavor and pleasant coherent texture. Similar
comments were expressed for yogurts produced with immobi-
lized L. casei in wheat. However, the flavor was not as sweet as
in case of raisins and the aroma was less intense. The yogurts
produced with immobilized L. casei cells in apples, although
were generally accepted by evaluation members, they were
characterized for their fruity aroma and acid flavor. Freeze
drying does not seem to affect the organoleptic characteris-
tics of the examined yogurts (P > 0.05) even though in some
cases affected their texture.

Table 4 – Sensory evaluation results.

Type of yogurt Aroma Flavor Texture Total consumer acceptance

C 6.98 ± 0.31 7.03 ± 0.11 6.64 ± 0.06 7.12 ± 0.21

FLC 7.78 ± 0.18 8.14 ± 0.25 7.16 ± 0.05 7.89 ± 0.12
WIA 7.06 ± 0.09 6.24 ± 0.13 6.56 ± 0.3 7.23 ± 0.23
FIA 7.12 ± 0.13 6.27 ± 0.21 7.21 ± 0.09 8.02 ± 0.29
WIR 8.21 ± 0.15 8.34 ± 0.24 8.98 ± 0.31 9.21 ± 0.31
FIR 8.24 ± 0.19 8.24 ± 0.19 9.56 ± 0.18 9.88 ± 0.08
WIW 9.24 ± 0.15 9.14 ± 0.21 9.14 ± 0.21 9.13 ± 0.31
FIW 8.98 ± 0.3 9.12 ± 0.3 9.88 ± 0.12 9.89 ± 0.15

C: Yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus, FLC: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + free L. casei cells,
WIA: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh L. casei enriched apple pieces, FIA: yogurt with S. thermophilus + L. delbrueckii
subsp. bulgaricus + freeze dried enriched L. casei apple pieces, WIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + fresh enriched L. casei
raisins, FIR: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L. casei raisins, WIW: yogurt with S. thermophilus + L.
delbrueckii subsp. bulgaricus + fresh enriched L. casei wheat, FIW: yogurt with S. thermophilus + L. delbrueckii subsp. bulgaricus + freeze dried enriched L.
casei wheat.
70 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 62–71
3.7. Technological consideration
This study proposes the potential to lead to a new era in func-
tional food innovation development. The use of fresh or dried
fruit and cereals as a carrier of functional ingredients is a rel-
atively new concept. The novelty is due to the use of apple,
raisin and wheat grains supported probiotic lactic acid bacte-
ria in yogurt production which has not yet been reported in
the past. The results showed a substantial improvement of L.
casei cell viability by the presence of raisin, wheat grains and
apple which strengthens the probiotic character of yogurt. Fur-
thermore, the presence of these probiotic adjuncts in yogurt
favored the inhibition of pathogens, affected positively the
results of sensorial test and decreased syneresis. The combi-
nation of these improvements results to a novel bioprocessing
and yogurt products. Raisin, wheat and apple are common
foods of low cost and high production capacity and their incor-
poration in yogurt production is feasible.
According to the ICAP yogurt Greek exports amounted to
23.468 t in 2008, of which 92% were white yogurt, while total
exports ranging close to 23% of the total annual production
(magazine “Xrima”, 2010). While competition intensified in
the field of tenders, large dairies have turned their interest
and create new innovative products that address specific con-
sumer groups. The growing consumer preference for high
quality and value added products, such as special milk
desserts and complex yogurt (with fruit, cereals, etc.) is the
main trend in the dairy market in recent years and industries
seem to exploit.
The next step could focus on the research of in vivo tests
for probiotic potential health effects.
4. Conclusions
The production of a novel probiotic yogurt by introducing L.
casei enriched fresh and freeze dried apple piece, dried raisins
and wheat grains was assessed in the present study. The
obtained results showed that enrichment of apples, raisins
and wheat grains favored the viability of L. casei during
refrigerated storage of yogurts while in many cases affected
positively the texture of the produced yogurts since less
syneresis was observed.
Wheat and raisins are promising supports for probiotic bac-
teria since their matrix seem to protect the bacteria from the
acidic environment of yogurts and also their potential prebi-
otic character can increase viability of probiotic cultures. It
is also important that there were no significant differences
between L. casei counts in fresh and freeze dried supports and
therefore a ready to use and easily preserved freeze dried L.
casei enriched fruits and grains seems to have potential to be
used by dairy industries.
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