LWT - Food Science and Technology 134 (2020) 109851

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LWT
journal homepage: www.elsevier.com/locate/lwt

Effect of steviol glycosides as sugar substitute on the probiotic fermentation

in milk gels enriched with red beetroot (Beta vulgaris L.)
bioactive compounds☆
Tugce Ozdemir , Tulay Ozcan *
Bursa Uludag University, Faculty of Agriculture, Department of Food Engineering, Gorukle Campus, Nilufer, Bursa, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, the effects of steviol glycosides as sugar substitute on the probiotic fermentation and gelation
Probiotic properties of milk gels enriched with red beetroot (Beta vulgaris L.) bioactive compounds and natural colourants
Red beetroot were studied. As a result, the viability of Lactobacillus acidophilus was maintained within probiotic and thera­
Steviol glycosides
peutic levels (>9 log cfu mL− 1), due to the possible prebiotic effect of stevia and biologically metabolized
Bioactive compounds
compounds of red beetroot, during storage of the probiotic milk gels. In conclusion, fermented gels with total
phenolics, anthocyanins, total antioxidant capacity (DPPH and FRAP) of red beetroot bioactive components and
sweetened with steviol glycosides showed a significant increase in bacterial growth of L. acidophilus and
acceptable sensory and colour (L*, a*, b*, ΔE*, C*, h*) characteristics with enhanched textural gel matrix.
Therefore, fermented milks with stevia as an alternative non-caloric sweetener and naturally coloured red
beetroot may be an alternative for therapeutic dairy product having positive synergistic interactions providing a
rich deliver probiotic bacteria.

1. Introduction nitrate ingestion reduces blood pressure, hypertension, cardiovascular

Due to the increased awareness of the effect of gut health on meta­
bolism, the interest in functional foods containing probiotic strains and
prebiotics is constantly growing. The application of novel technologies
could make possible the production of improved functional foods con­
taining active ingredients with specific natural sweetener and colourants
(Chen et al., 2005; Ofori & Peggy-Hsieh, 2013). As the latest trend,
sensory-appreciated therapeutic foods are increasingly preferred by
consumers, where bioactive components of fruits, raw or fermented
vegetables, plants, flowers and grains are used as substrates for probiotic
bacteria (Soccol et al., 2010; Peres, Peres, Hernández-Mendoza, &
Malcata, 2012; Kurtuldu & Ozcan, 2018).
Products containing red beetroots (Beta vulgaris L.) have been in
demand in the market for years as performance-enhancing legal nutri­
tional supplements. It has been determined by clinical studies that
increased nitric oxide (NO) levels have positive effects on muscle effi­
ciency and fatigue resistance (Larsen, Weitzberg, Lundberg, & Ekblom,
2010) and improvements in time resistance tests of athletes (Murphy,
Eliot, Heuertz, & Weiss, 2012). Furthermore it has also been noted that
diseases and increased endogenous nitrosation, which can lead to the
formation of carcinogenic metabolites (Lundberg, Carlström, Larsen, &
Eddie Weitzberg, 2011; Habermeyer, Roth, Guth, & Eisenbrand, 2015).
Betalains are essential compounds found in beetroot and reduce oxida­
tive damage of lipids in humans. In addition to the anti inflammatory
effect, betacyanins also exhibit a feature that can inhibit radical scav­
enging activity, cervical ovarian and bladder cancer cells (Pedreno &
Escribano, 2000).
Using of low-calorie sweeteners and substituting sucrose with natu­
ral sugars have recently received great attention in the field of diet-
disease relations. Stevioside along with related compounds which
include rebaudioside A is the main sweet component of Stevia rebaudiana
(Bert.) leaf extract and has been applied as substitutes for sucrose to
prevent diabetes mellitus, (type II), hypertension, cardiovascular dis­
ease, hypoglycemia, dental caries, obesity (metabolic syndrome) and
diarrhoea (Geuns, 2000; Chatsudthipong & Muanprasat, 2009; Goyal &
Samsher Goyal, 2010). The sweetness of stevia is reported to be over
200–300 times that of sucrose sugar (Liu, Ren, & Shi, 2011). It has been
found by many researchers that, together with steviol glycosides, the

☆
This study is a part of Tugce Ozdemir’s M.Sc. thesis submitted to the Bursa Uludag University, Institute of Natural Sciences.
* Corresponding author. Department of Food Engineering, Bursa Uludag University, 16059, Gorukle, Bursa, Turkey.
E-mail address: tulayozcan@uludag.edu.tr (T. Ozcan).

https://doi.org/10.1016/j.lwt.2020.109851
Received 31 May 2020; Received in revised form 23 June 2020; Accepted 5 July 2020
Available online 30 July 2020
0023-6438/© 2020 Elsevier Ltd. All rights reserved.
T. Ozdemir and T. Ozcan
leaves of the stevia plant also contain other phyto-components such as
polyphenols, flavonoids, carotenoids, tannins, phenolic acids, chloro­
genic acids, fatty acids, amino acids, proteins and vitamins (Lemus-­
Mondaca, Vega-Gálvez, Zura-Bravo, & Ah-Hen, 2012). It has also been
noted that stevioside can hydrolyze steviol and glucose by bacteria in
the human gastrointestinal tract by glucosidases (Ozcan, Yilmaz-Ersan,
Akpinar-Bayizit, & Delikanli, 2017). Especially in recent years, there
has been an increase in fruit-vegetable-based beverage preference for
children. Among these preferences, beet juice has a relatively pleasant
taste compared to other vegetable juices due to its relatively high sugar
content. Unlike fruits, the main sugar in beets is also sucrose (Thakur &
Gupta, 2006).
Stevia has functional and sensory properties than many other
sweeteners and is likely to become a major source of high-potency
sweetener for the growing natural reduced sugar food trends in the
future (Goyal & Samsher Goyal, 2010). There is no research on the
determination of the number of probiotic microorganisms in the
composition of fermented milk products produced with the addition of
red beetroots and stevia. For this reason, it is among the purpose of this
study to determine the growth of probiotic Lactobacillus acidophilus and
viability during fermentation and storage in probiotic milk gels pro­
duced with red beetroot pulp bioactive components and stevia as a sugar
substitute and evaluate the technological properties of the dairy matrix.
2. Materials and methods
2.1. Sugar substitiues and red beetroot
Stevia or steviol glycosides as natural sugar substitiues was obtained
from PCSB (Pure Circle Sdn Bhd, Maleysia), red beetroot (Beta vulgaris
L.) and sugar/sucrose were purchased from the local market. Red
beetroot pulp was high temperature short time (HTST) pasteurized at 95
◦
C for 2 min, cooled to 10 ◦ C, wrapped in 200-mL plastic containers and
then frozen at − 18 ◦ C until time of production of fermented milk gels.
Composition of red beetroot pulp was determined and given Table 1.
2.2. Microbial cultures and inoculum preparation
Strain of freeze-dried starter cultures of Lactobacillus acidophilus La-
14 (Danisco, Sassenage, France) was prepared in 50 mL of milk 11%
(w/v) of total solids and autoclaved at 121 ◦ C for 20 min. Culture acti­
vated by sub culturing 3–4 times before use. After activation at 37 ◦ C,
3% (v/v) of the pre-culture was inoculated into 100 mL of milk. Bacterial
counts in these pre-culture was 9.16 log10 cfu mL− 1. After inoculation,
milk fermentations were performed in triplicate at 37 ◦ C up to pH 4.70
and fermentations were monitored by pH determinations for 5.5 h.
Table 1
Composition of red beetroot pulp (Beta vulgaris L.).
Constituent Value
Total Solids (%) 17.52
Ash (%) 0.86
Brix 18.20
Total Carbonhydrate (%) 5.80
Dietary Fiber (%) 1.90
Total Phenolic Content (mg GAE/100g) 32.87
Total Antioxidant Activity (FRAP) (mg Trolox/100g) 170.20
Total Antioxidant Activity (DPPH) (mg Trolox/100g) 2.03
Total Anthocyanins (mg/kg) 5.01
Acidity (acetic acid %) 0.29
pH 5.22
L* 18.64
a* 4.64
b* 1.76
2
LWT 134 (2020) 109851
2.3. Preparation of red beetroot-enriched milk gel using stevia
Reconstitued both skim milks, 11% (w/v) dry matter from low-heat
skim milk powder, were pre-heated to 45 ◦ C and optimized level of
sucrose 8.0% (w/v), or stevia 0.025% (w/v) based on primarily sensory
evaluation was added to milk and then thermally treated at 90 ◦ C for 10
min and cooled to 40 ◦ C and 10% (w/v) red beetroot added. The milk
was inoculated with 3% (v/v) L. acidophilus (37 ◦ C) and the inoculated
milk was poured aseptically into sterile 100-mL plastic containers and
incubated at 37 ◦ C until the pH reached 4.70 and then fermented milk
were immediately cooled 20 ± 1 ◦ C. Fermented milk gels with;
L. acidophilus (control L), red beetroot based probiotic gel (LR), red
beetroot based probiotic gel with sucrose (LRS) and red beetroot based
probiotic gel with stevia (LRT) were stored for 28 days at 4 ± 1 ◦ C. The
composition, physico-chemical, textural properties were assessed on day
1 (approximately 12 h post-fermentation) and after 14, and 28 days of
storage.
2.4. Counts of probiotic bacteria and viability proportion index (VPI)
The L. acidophilus strain was enumerated on selective medium DE
Man, Rogosa, and Sharpe agar (MRS, Merck, Germany) with 0.15% (w/
v) of bile (Merck, Germany) at 37 ◦ C for 72 h under anaerobic conditions
(Tharmaraj & Shah, 2003). The viability proportion index (VPI) of
L. acidophilus was calculated as follows (Kurtuldu & Ozcan, 2018):
VPI= Final cell population (log10 cfu mL− 1)/Initial cell population (log10 cfu
mL− 1)
2.5. Fermentation and post-acidification
The acidification kinetics with pH profile was recorded during
fermentation while post-acidification analyis were determined after the
fermentation, after 1, 14 and 28 days of storage at 4 ± 1 ◦ C. pH of the
milk gels was determined using a digital pH meter (WTW pH 315i/Set
Germany, AOAC Method 981.12) at 25 ◦ C. The titratable acidity (TA,
AOAC Method 947.05) were assessed by titration method according to
AOAC (2016). Syneresis was evaluated as the volume of drained whey
(Yildiz & Ozcan, 2019).
2.6. Proximate composition, radical-scavenging activity (FRAP and
DPPH) and total phenolic content
The total solid content (at 102 ± 2 ◦ C for 48 h until constant mass,
AOAC Method 990.19) and total ash (at 550 ◦ C for 48 h, AOAC Method
945.46) was determined (AOAC, 2016). The colour was based on three
colour coordinates: L* (whiteness or brightness/darkness), a* (red­
ness/greenness) and b* (yellowness/blueness) analysed using a Minolta
Colorimeter (Model CM-600d, Minolta, Osaka, Japan). The chroma
value (C*), which is the colour saturation, the colour hue angle (h*),
which indicates the colour tone, and ΔE* (colour difference) was defined
according to Mendes et al. (2019).
The total phenolic contents were determined using Folin–Ciocalteu
(FC) reagent using spectrophotometer (Shimadzu, Japan) at 725 nm.
The radical-scavenging activity was evaluated using a method utilising
the ferric-reducing antioxidant power (FRAP) measured at 595 nm
and,2-diphenyl-1- picrylhydrazyl (DPPH) radicals measured at 515 nm
as reported by Ozcan, Sahin, Akpinar-Bayizit, and Yilmaz-Ersan (2019).
Reducing sugar was assayed by phenol-free 3,5-dinitrosalicylic acid
(DNS) method (Teixeira, da Silva, Ferreira-Leitão, & Bon, 2012) at 540
nm using a spectrophotometer. The total anthocyanins of red beetroots
were estimated by a pH differential method (Wada & Ou, 2002). Brix,
total carbonhydrate and dietary fiber value of red beetroot pulp was
determined using the method described by AOAC (2016).
T. Ozdemir and T. Ozcan
2.7. Texture and sensory properties
Texture profile analysis (TPA) were performed using Texture
Analyzer TA-XT Plus (Stable Micro Systems, UK) (Patrignani et al.,
2007) with back extrusion prob. Firmness, cohesiveness and viscosity
index parameters were evaluated. Sensory descriptive analysis was
performed using 5-point hedonic scale (1 = strongly disliked; 2 =
moderately disliked; 3 = indifferent; 4 = moderately liked; and 5 =
strongly liked) by a trained panel in the examination of fermented dairy
products, consisting of 11 member panel (eight females, three males;
ages 25–45 years among graduate students and scientists) by method
Yang, Lu, and Hu (2014) and Vianna et al. (2017). Morever, the pur­
chase intent of panelists on each of sample was assessed varying from
“certainly would not buy” and “certainly would buy”. Samples were
served to panellist in plastic cups coded with random three-digit
numbers and with bread and mineral water to clean their palate.
2.8. Statistical analysis
Statistical analysis was assessed using Statistica 7.0 software (Stat­
Soft, Inc., Tulsa, OK, USA) and Tukey test (p < 0.05, 0.01). The multi­
dimensional scaling analysis (MDS) was applied as pattern recognition
classification method by Cruz et al. (2013).
3. Results and discussion
3.1. Microbial fermentation and milk gel properties
The acidification profiles of the red beetroot enriched milk gel were
shown in Fig. 1 pH values recorded every 30 min. Although acidification
was faster in the L and LR samples than others, the pH of all the fer­
mented milk gels did not vary significantly. Table 2 shows growth of
L. acidophilus and viability proportion index (VPI) of red beetroot pulp-
enriched milk gel using stevia. When the VPIs were examined, it was
found that the results were almost similar in all samples except for the
V1− 14 viability range and the highest growth rate was determined in
VPI0− 1. It is a specific feature of these microorganisms that the probiotic
bacteria should be remain viable until the probiotic dairy products are
consumed. As a result of microbiological analysis, the number of pro­
biotic bacteria determined in the probiotic milk gels with L. acidophilus
culture containing red beetroot, sucrose and stevia was given in Table 3
(p < 0.01). Total viable count of all milk gels obtained ranging from 8.82
to 9.37 log10 cfu mL− 1, were higher than recommended range of 106 to
108 cfu mL− 1 at the end of storage period which were reported by Kai­
lasapathy & Chin (2000); Sumalapao, Mesina, Cabrera, & Gloriani
(2017).
It is thought that the difference in the growth factor and metabolic
Fig. 1. pH profile of different milk gels during fermentation
L, control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik
gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
3
LWT 134 (2020) 109851
activity of L. acidophilus in milk gels are due to the difference in the
composition of the samples with sucrose, steiva and red beetroot. The
number of L. acidophilus in milk gels was higher in samples containing
red beetroot (LR, LRS, LRT) than control (L) (Table 3). It is concluded
that the addition of red beetroots extracts and phenolics, stevia and
sucrose promote the growth of L. acidophilus. Eroglu and Ozcan (2019)
showed that the number of probiotic bacteria (B. animalis spp. lactis and
L. acidophilus) in stevia-added yoghurts was higher than the control
samples. In addition, Ozcan, Yilmaz-Ersan, Akpinar-Bayizit, and
Delikanli-Kiyak (2017) stated that the addition of stevia supported the
development of L. casei in vitro conditions. Malik, Bora, and Sharma
(2019) reported that the development of L. plantarum, L. acidophilus, and
L.casei increased due to the total phenolic and flavonoid content in the
lactic fermentation of beet juices.
The probiotic bacteria count reached its maximum at the 1st day after
the fermantation in both milk gel types, thereafter started to decline
(Tables 2 and 3). This can be explained by the accumulation of acid and
hydrogen peroxide. By the way, during the 28-day storage period in
probiotic gels, the number of probiotic bacteria in all samples; over the
minimum level required to see the therapeutic effect (≥106 cfu mL− 1,
FAO/WHO, 2002). Studies on the use of fruit and vegetable extracts in
fermented milks have also been reported to increase the number and
activity of lactic strains at the beginning of the storage period for utilized
phenolic compounds and dietary fibers (Martins et al., 2013; Barat &
Ozcan, 2018; Yildiz & Ozcan, 2019). During the food manufacturing
process and storage there are many factors affecting the survival of
probiotics such as pH, oxidative stress and storage temperature (Nual­
kaekul, Salmeron, & Charalampopoulos, 2011). Malik et al. (2019)
stated that L. acidophilus is less resistant due to the pH value of fer­
mented juices is generally around 4.5, and this possible decrease in the
number of living cells is due to the rapid increase in the acidity of the
juice.
Red beet contains high levels of biological compounds (Singh &
Singh Hathan, 2014; Babarykin et al., 2019). It is stated by some re­
searchers that phenolic compounds, dietary fibers,
fructo-oligosaccharides, non-starch carbohydrates etc., contained in
fruits/vegetables and beetroot extracts can be hydrolyzed by probiotic
bacteria and used as a source of prebiotics (Yoon, Woodams, & Hang,
2005; Moraru, BlancaI, & Segal, 2007; Buruleanu et al., 2009; Soccol
et al., 2010; Malik et al., 2019). Olson and Aryana (2012) reported that
L. acidophilus can use various oligosaccharides and polysaccharides as
substrates. They point out those changes in specific growth rates of
different L. acidophilus strains depend on the use of appropriate types of
prebiotics when preparing synbiotic formulations.
pH values and titratable acidity of probiotic gels during storage are
presented in Table 3. In the first day of storage, samples showed greater
(P < 0.01) pH values and low TA than the end of cold storage that as
expected, as the pH values decrease during storage period while the TA
values increased. Furthermore, LR, and LRT showed a decrease (P <
0.01) in their pH values and increase TA during the storage period.
Moreover, in overall, probiotic fermented gels manufactured with stevia
and red beetroot exhibited similar TA and pH values.
It is known that the addition of fiber based plants to fermented milk
gels decrease syneresis and increases the water-holding capacity. Syn­
ersis decreased with the addition of beetroot pulp in samples (Table 3).
Probiotic gels were found to be decreased in syneresis value up to 14 and
28 days of storage. However, lowest value of synersis was found in milk
gel with red beetroot and stevia (LR and LRT). In this study, addition of
beet fiber could be possible reasons of decreased synersis value in
samples.
Table 3 shows changes in texturel properties of beetroot-enriched
milk gels using stevia during storage. A high significant value of firm­
ness, viscosity index and cohevisiness were obtained for fermented gel
with red beetroot milk gel. Gel properties of both samples increased
significantly (P < 0.05, P < 0.01) with increasing storage period is
caused by stronger casein networking. The addition of vegetable fibers
T. Ozdemir and T. Ozcan LWT 134 (2020) 109851

Table 2
The viability proportion index (VPI) of L. acidophilus at the end of fermentation and during storage (log10 cfu mL− 1) a‡
Probiotic Milk Gel Viability proportion index (VPI)

Days

0b V1 V14 V28 VPI 0− 1

VPI 1− 14
VPI 14− 28
VPI 1− 28

L 9.16 9.59 8.54 8.34 1.05a 0.89c 0.98ab 0.87b

LR 9.16 9.48 9.61 9.01 1.03ab 1.01a 0.94b 0.95ab
LRS 9.16 9.45 9.17 9.32 1.03ab 0.97ab 1.02a 0.99a
LRT 9.16 9.67 9.13 9.32 1.06a 0.94b 1.02a 0.96ab
Pvalue – – – – * ** ** **

VPI = Final cell population (log10 cfu mL− 1)/Initial cell population (log10 cfu mL− 1).
‡Significance level: significant at P < 0.01 (**) and P < 0.05 (*), ns (non-significant) different superscript letters on the same column indicate significant differences. L,
control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
a
Values are means of triplicates.
b
Culture before the fermantation.

Table 3
Bacterial growth and gel properties of red beeroot enriched milk gels during post-acidificationa, ‡
Probiotic Milk Gel L. acidophilus (log10 cfu mL− 1) Titratable pH Syneresis (mL25g) Firmness (g) Viscosity Cohesiveness (g)
Acidity (%) Index (g.s)

L 8.82b 0.72c 4.62a 8.66a 223.80d − 216.10b − 125.13b

LR 9.37a 0.95a 4.37c 7.00cd 340.25a − 240.15a − 159.19a
LRS 9.31a 0.79b 4.56b 7.50b 244.78c − 232.62a − 121.39b
LRT 9.37a 0.92a 4.41c 7.26c 299.11b − 229.74ab − 147.60a
Storage Time (Day)
1 9.48a 0.74c 4.65a 7.81a 251.35b − 214.75b − 126.73a
14 9.12b 0.85b 4.50b 7.75a 283.75ab − 243.63a − 137.23a
28 9.01b 0.94a 4.31c 7.26b 311.09a − 240.95a − 132.25a

ANOVA
Sample (S) ** ** ** ** ** * *
Storage Time (D) ** ** ** ** ** ** ns
S*D ** ** ** ** ** ** ns

‡Significance level: significant at P < 0.01 (**) and P < 0.05 (*), ns (non-significant) different superscript letters on the same column indicate significant differences. L,
control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
a
Values are means of triplicates.

to dairy matrix increases the water-holding capacity with improving the
texture and structure (Yildiz & Ozcan, 2019). It is known that repulsive
or relational interactions or phase separation reactions occur due to
protein charge. Mende, Rohm, and Jaros (2016) state that the interac­
tion between the polysaccharide molecules and the protein network is
affected by the acidity of the food matrix, because the polysaccharides
directly connected to the protein network are charged and charge of the
protein molecules changes due to the food acidity. The addition of su­
crose increased viscosity index while decreasing cohesiveness. However,
the firmness was lower in these samples, with heterogeneity caused by
sucrose and lactic acid bacteria fermenting the sugar and lactose
(Table 3). Fernandez-Garcia, McGregor, and Traylor (1998) stated that
viscosity and textural properties of milk increase with the addition of
sucrose (from 2% to 6%) is related to the higher water-binding capacity
of hydrolyzed monosaccharides compared to disaccharides.
3.2. Proximate composition, radical-scavenging activity (FRAP and
DPPH) and total phenolic content
Fruit and vegetable purees provide therapeutic benefits not only with
their sensory properties, but with biologically active compounds such as
vitamins, minerals, antioxidants that are effective on health as well

Table 4
Proximate composition, radical-scavenging activity (FRAP and DPPH) and total phenolic content of red beeroot enriched milk gelsa, ‡
Probiotic Milk Gel Total Solids (%) Ash (%) Reduced Sugar (%) Total Antioxidant Activity (FRAP) Total Antioxidant Activity (DPPH) Total Phenolic
(mg Trolox/100g) (mg Trolox/100g) Content (mg GAE/100g)

L 11.17c 1.06a 3.13d 37.47c 1.48b 12.04b

LR 11.79c 1.08a 3.62c 60.94a 1.54a 14.67a
LRS 17.74a 1.10a 4.01b 55.17b 1.55a 14.16a
LRT 12.24b 1.11a 4.39a 61.81a 1.58a 14.49a
Storage Time (Day)
1 13.35a 1.05a 3.78a 53.09b 1.46c 13.82a
14 13.33a 0.99b 3.78a 54.27ab 1.54b 13.77b
28 13.04b 1.02a 3.80a 54.19b 1.62a 13.63c

ANOVAa
Sample (S) ** ns ** ** ** **
Storage Time (D) * * ns ** ** **
S*D ** ** ns ** ** **

‡Significance level: significant at P < 0.01 (**) and P < 0.05 (*), ns (non-significant) different superscript letters on the same column indicate significant differences. L,
control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
a
Values are means of triplicates.

4
T. Ozdemir and T. Ozcan LWT 134 (2020) 109851

(Vasudha & Mishra, 2013). Gross composition of probiotic fermented than the other samples (Table 5). The L* values of probiotic gels
milk gels during storage period was presented in Table 4. Nutrients significantly (P < 0.01) decreased during storage. It is thought that the
composition of fermented gels was partially affected by addition of degradation of anthocyanins may have caused significant changes in
sugars and red beetroot. In the fermented milk gels, the highest dry brightness during the storage of milk. It is assumed that the maximum a*
matter was found in the LRS samples as expected due to the sucrose value was observed in the LR sample due to the presence of red and
addition (P < 0.01). Ash content represents the amount of total minerals blue–purple colours of red beetroot depends on the concentration of
but showed no significant difference (P > 0.01) for the samples. In anthocyanins (Patras, Brunton, O’Donnell, & Tiwari, 2010; S’cibisz
addition to the highest reduced sugar value was found in gel with stevia et al., 2019).
and milk gels with red beetroot due to the amount of reducing sugar Anthocyanins as natural pigments are the most used in the red-pur­
(carbohydrate) present in beet root. In addition to red beet juice or ple colour range, although betalains are more resistant to pH and tem­
extract colour pigments, it consists of naturally high levels of sugar and perature, and show broad pH stability in low-acid foods, where
minerals. It is stated that carbohydrates in beet juice can be a potential colouring with anthocyanins is often not possible (Stintzing, Schieber, &
substrate for the development of probiotic microorganisms (Singh & Carle, 2003). Red beetroot enriched milk gels demonstrated a significant
Singh Hathan, 2014). increase of this parameter that most probably due to degradation of
Antioxidant capacity of milk by fermentation of L. acidophilus was anthocyanins or/and the formation of yellow and brown polymerization
shown in Table 4. Beetroots (Beta vulgaris L.) are rich in antioxidant compounds (S’cibisz et al., 2019). There was a significant effect of
compounds (Babarykin et al., 2019). In this study, the total antioxidant addition of sugar substitues and probiotic fermentation on h* and ΔE*
capacity values (FRAP and DPPH) in probiotic fermented milk gels value for beetroot enriched milk gels. However, addition of red beetroot
containing red beetroots were also high and increased during storage pulp presented almost the same ΔE*. It is known that high concentra­
because of red beetroot’s high value of nutrient composition and total tions of phenolic compounds such as phenolic acids and flavan-3-ols,
phenolic compounds and polyphenols, which are hydrolyzed by some which are interact with anthocyanins enhance the colour intensity in
microflora causing the formation of various aromatic acids (P < 0.01) beetroot enriched gels depends on pH of food matrix (Cabrita, Fossen, &
(Table 4). By the way total phenolic content of fermented milks were Andersen, 2000; Pascual-Teresa & Sanchez-Ballest, 2008). The impact of
changed during storage because of the possible effect of degradation of the production method on the C* of probiotic gel was significant for red
polyphenolic compounds by lactic acid bacteria (LAB) and interaction beetroot fermented gels had a significantly higher C* than the milk
with caseins or whey proteins for new formations (Alberto, fermented with sucrose and stevia depens on more fermantation and
Gomez-Cardoves, & Manca de Nadra, 2004; S’cibisz, Ziarno, & Mitek, degration of some flovonoids and betalains. S’cibisz et al. (2019) re­
2019). However, it was concluded that L. acidophilus and L. plantarum ported that pH of food matrix is important factor that influences the
were able to degrade more phenolic compounds (>50%) than L. casei stability and colour of the pigments.
(Alberto et al., 2004; Manach, Scalbert, Morand, Rémésy, & Jiménez,
2004). After fermentation, a further decrease in total phenolic content 3.4. Sensory properties
value was observed in the juices by Li et al. (2019) who have reported
that fermentation decreases total phenolics. It is also stated that Stevia Sensory of dairy products has a large influence over the attitude of
rebaudiana Bertoni contains have biological activity, including alkaloids, consumer for the choice of food and acceptance. The sensory evaluation
flavanoids, tannins, and phenolic compounds with potential antioxidant of red beetroot enriched fermented gels are presented in Table 6. The
activity (Lemus-Mondaca et al., 2012). sensory score obtained from the analysis showed that the all gels fer­
mented with L. acidophilus, sucrose, stevia and red beetroot were char­
3.3. Colour differences acterized with highest score in all the parameters taste, aroma, colour,
appearance, sourness, and overall impression. After 28 days of storage,
Betalains are compounds in high concentrations in red beet and sensory attributes such as colour and appearance, sourness and acetal­
consist of two subclasses: betacyanins (red-violet pigments) and betax­ dehyde and bitterness of probiotic gels decreased significantly (P < 0.05,
anthin (yellow-orange pigments)(Gandía-Herrero, Escribano, & Gar­ P < 0.01) while those of other parameters were improved. Purchase
cía-Carmona, 2010). The L* values of the probiotic mik gels changed intention showed values from 4.72 to 4.84, revealing a positive tendency
between 63.09 and 90.25. The high L* and b* value of the L sample for purchasing of the probiotic product (Table 6).
shows that its whiteness/brightness and yellowness values were higher The results show that the characteristic that has the greatest effect on
the acceptance and purchase intent of probiotic gels is taste and then
Table 5 general appreciation. Fig. 2a and b shows multidimensional scaling
Colour differences of red beeroot enriched milk gels a, ‡ analysis (MDS) that reveals the similarities, differences and typical or
Probiotic Milk Gel L* a* b* C* h* ΔE*
outlier data between the samples using both sensory variables. MDS are
a c a c c
projected on a two-dimensional surface and described by two orthog­
L 90.25 − 3.96 7.11 8.12 − 1.06 –
onal factors used as dimensions. Fermented milk samples were grouped
LR 65.59b 21.92a 4.45c 22.37a 0.20b 69.31a
LRS 63.09c 19.02ab 5.24b 19.74b 0.27a 66.12b as positive Group I (L and LRT) and negative Group II (LR and LRS)
LRT 63.36c 18.55b 5.34b 19.30b 0.28a 66.24b values of the different dimension. Group I were characterized by their
Storage Time (Day) sensory texture, bitterness and also sourness/acetaldehyde while Group
1 72.14a 14.29a 5.01b 17.57a − 0.10c 74.64a II correlated with smoothness, colour/appearance, taste and sweetness.
14 71.55a 13.95b 5.68a 17.57a − 0.08b 74.05a
28 68.03b 13.40c 5.90a 17.03b − 0.06a 70.52b
4. Conclusion
ANOVA
Sample (S) ** ** ** ** ** **
Storage Time (D) ** ** ** ** ** ** The consumption of functional and reduced sugar/fat/salt foods has
S*D ** ** ** ** ** ** been a global food trend for long due to an increase in the risk of cho­
ronic diseases such as obesity, diabetes, cardiovascular diseases and
‡Significance level: significant at P < 0.01 (**) and P < 0.05 (*), ns (non-sig­
nificant) different superscript letters on the same column indicate significant cancer. Among dairy products, fermented milk products enriched with
differences. L, control milk gel with L. acidophilus; LR, mik gel with red beetroot; dietary fibers, probiotics has been widely perceived as a healthy with
LRS, mik gel with red beetroot and sucrose; LRT, mik gel with red beetroot and therapeutic benefits. Red beetroot is good sources of fibers, natural
stevia. sugars and colourants. In addition, stevia is a safe natural sweetener for
a
Values are means of triplicates. diabetics, as it does not affect blood sugar levels and has no neurological

5
T. Ozdemir and T. Ozcan LWT 134 (2020) 109851

Table 6
Sensory properties of red beeroot probiotic milk gelsa, ‡
Probiotic Milk Colour and Texture and Smoothness Aroma Taste Sweetness Sourness and Bitterness Purchase Overall
Gel Appearance Thickness Acetaldehyde Intention Impression

L 4.92a 4.90a 5.00a 4.80a 4.72a 4.74bc 4.79a 4.85a 4.72a 4.75a
LR 4.92a 4.87a 4.96a 4.79a 4.72a 4.73bc 4.83a 4.86a 4.72a 4.77a
LRS 4.90a 4.88a 4.92a 4.79a 4.77a 4.78b 4.84a 4.83a 4.77a 4.78a
LRT 4.92a 4.90a 4.96a 4.82a 4.84a 4.90a 4.86a 4.86a 4.84a 4,82a
Storage Time (Day)
1 4.93ab 4.82a 4.95a 4.71b 4.80a 4.73a 4.70b 4.73b 4.72a 4.74a
14 4.99a 4.93a 4.99a 4.95a 4.81a 4.79a 4.94a 4.98a 4.83a 4.85a
28 4.83b 4.92a 4.95a 4.76ab 4.86a 4.86a 4.86ab 4.86ab 4.74a 4.77a

ANOVA
Sample (S) ns ns ns ns ns ** ns ns ns ns
Storage Time ** ns ns ** ns ns ** ** ns ns
(D)
SxD ns ns ns ns ns * * ns ns ns

‡Significance level: significant at P < 0.01 (**) and P < 0.05 (*), ns (non-significant) different superscript letters on the same column indicate significant differences.
L, control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
a
Values are means of triplicates.

or kidney side effects like other artificial sweeteners. Hence, in this study
red beetroot and stevia have improved the functional content and
technological properties of fermented mik with L. acidophilus. Moreover,
it is necessary to deeply understand how natural colour pigments,
phenolics and also natural sugar stevia effect gelation properties of
fermented dairy foods and growth of probiotic bacteria with potential
prebiotic activity and as in some cases how to the food matrix can
positively affect the stability of the colour pigment and nutraceutical
bioavailability and bioaccessibility to recommended intakes and iden­
tify beneficial dietary modifications.

CRediT authorship contribution statement

Tugce Ozdemir: Formal analysis. Tulay Ozcan: Writing - original

draft, Formal analysis.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was funded by Bursa Uludag University (Grant number:

QUAP (Z)-2019/11).

Rerferences

Alberto, M. R., Gomez-Cardoves, M. C., & Manca de Nadra, M. C. (2004). Metabolism of

Fig. 2. Multidimensional scaling (MDS) factor analysis performed on data from
projective mapping for a) different milk gels and b) descriptive sensory eval­
uation (Dim = dimension)
L, control milk gel with L. acidophilus; LR, mik gel with red beetroot; LRS, mik
gel with red beetroot and sucrose; LRT, mik gel with red beetroot and stevia.
gallic acid by Lactobacillus hilgardi from wine. Journal of Agricultural and Food
Chemistry, 52, 6465–6469.
AOAC. (2016). Official methods of analysis (20th ed.). USA: Association of Official
Analytical Chemists, AOAC Int Maryland.
Babarykin, D., Smirnova, G., Pundinsh, I., Vasiljeva, S., Krumina, G., & Agejchenko, V.
(2019). Red Beet (Beta vulgaris) impact on human health. Journal of Biosciences and
Medicines, 7, 61–79.
Barat, A., & Ozcan, T. (2018). Growth of probiotic bacteria and characteristics of
fermented milk containing fruit matrices. International Journal of Dairy Technology,
71(1S), 120–129.
Buruleanu, L. C., Nicolescu, C. L., Gorghiu, G., Bratu, M. G., Avram, D., & Manea, I.
(2009). Lactic acid fermentation of carrot and red beet juices by probiotic bacteria.
Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca -
Agriculture, 66, 132–139.
Cabrita, L., Fossen, T., & Andersen, Ø. M. (2000). Colour and stability of six common
anthocyanidin 3-glucosides in aqueous solution. Food Chemistry, 68, 101–107.
Chatsudthipong, V., & Muanprasat, C. (2009). Stevioside and related compounds:
Therapeutic benefits beyond sweetness. Pharmacology & Therapeutics, 121, 41–54.
Chen, T. H., Chen, S. C., Chan, P., Chu, Y. L., Yang, H. Y., & Cheng, J. T.( (2005).
Mechanism of the hypoglycemic effect of stevioside, a glycoside of Stevia rebaudiana.
Planta Medica, 7, 108–113.
Cruz, A. G., Cadena, R. S., Castro, W. F., Esmerino, E. A., Rodrigues, J. B., & Faria, J. A. F.
(2013). Consumer perception of probiotic yogurt: Performance of check all that

6
T. Ozdemir and T. Ozcan
apply (CATA), projective mapping, sorting and intensity scale. Food Research
International, 54, 601–610.
Eroglu, E., & Ozcan, T. (2019). The use of natural sweeteners in the production of synbiotic
yogurt with reduced sugar content. VIII. Bursa: Bursa Uludag University Information
and R&D Days, 18-19 March, 2019.
FAO/WHO. (2002). Food and agriculture organization – world health organization. In
Report of a joint FAO/WHO working Group on drafting guidelines for the evaluation of
probiotics in food. iAvalable from: ftp://ftp.fao.org/es/esn/food/wgreport2.pdf.
Fernandez-Garcia, E., McGregor, J. U., & Traylor, S. (1998). The addition of oat fiber and
natural alternative sweeteners in the manufacture of plain yogurt. Journal of Dairy
Science, 81, 655–663.
Gandía-Herrero, F., Escribano, J., & García-Carmona, F. (2010). Structural implications
on color, fluorescence, and antiradical activity in betalains. Planta, 232, 449–460.
Geuns, J. M. C. (2000). Safety of stevia and stevioside. Recent Research Development in
Phytochemistry, 4, 75–88.
Goyal, S., & Samsher Goyal, R. (2010). Stevia (Stevia rebaudiana) a bio-sweetener: A
review. International Journal of Food Sciences & Nutrition, 61, 1–10.
Habermeyer, M., Roth, A., Guth, S., & Eisenbrand, G. (2015). Nitrate and nitrite in the
diet: How to assess their benefit and risk for human health. Molecular Nutrition &
Food Research, 59, 106–128.
Kailasapathy, K., & Chin, J. (2000). Survival and therapeutic potential of probiotic
organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.
Immunology & Cell Biology, 78, 80–88.
Kurtuldu, O., & Ozcan, T. (2018). Effect of β-glucan on the properties of probiotic set
yoghurt with Bifidobacterium animalis subsp. lactis strain Bb-12. International Journal
of Dairy Technology, 71(1S), 157–166.
Larsen, F. J., Weitzberg, E., Lundberg, J. O., & Ekblom, B. (2010). Dietary nitrate reduces
maximal oxygen consumption while maintaining work performance in maximal
exercise. Free Radical Biology and Medicine, 48, 342–347.
Lemus-Mondaca, R., Vega-Gálvez, A., Zura-Bravo, L., & Ah-Hen, K. (2012). Stevia
rebaudiana Bertoni, source of high-potency natural sweetener: A comprehensive
review on the biochemical, nutritional and functional aspects. Food Chemistry, 132,
1121–1132.
Li, Z., Teng, J., Lyu, Y., Hu, X., Zhao, Y., & Wang, M. (2019). Enhanced antioxidant
activity for apple juice fermented with Lactobacillus plantarum ATCC14917.
Molecules, 24, 51.
Liu, X., Ren, G., & Shi, Y. (2011). The effect of organic manure and chemical fertilizer on
growth and development of Stevia rebaudiana Bertoni. Energy Procedia, 5,
1200–1204.
Lundberg, J. O., Carlström, M., Larsen, F. J., & Eddie Weitzberg, E. (2011). Roles of
dietary inorganic nitrate in cardiovascular health and disease. Cardiovascular
Research, 89, 525–532.
Malik, M., Bora, J., & Sharma, V. (2019). Growth studies of potentially probiotic lactic
acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus
casei) in carrot and beetroot juice substrates. Journal of Food Processing and
Preservation, 43, 1–8.
Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols:
Food sources and bioavailability. American Journal of Clinical Nutrition, 79, 727–747.
Martins, E. M. F., Ramos, A. M., Vanzela, E. S. L., Stringheta, P. C., de Oliveira, P. C. L., &
Martins, J. M. (2013). Products of vegetable origin: A new alternative for the
consumption of probiotic bacteria. Food Research International, 51, 764–770.
Mende, S., Rohm, H., & Jaros, D. (2016). Influence of exopolysaccharides on the
structure, texture, stability and sensory properties of yoghurt and related products.
International Dairy Journal, 52, 57–71.
Mendes, A. H. L., Dionísio, A. P., Mouta, C. F. H., Abreu, F. A. P., Pinto, C. O.,
Garruti, D. S., et al. (2019). Sensory acceptance and characterization of yoghurt
supplemented with yacon syrup and cashew apple extract as a source of bioactive
compounds. Brazilian Journal of Food Technology, 22, Article e2018153.
Moraru, D., Blanca, I., & Segal, R. (2007). Probiotic vegetable juices. Food Technology, 4,
87–91.
Murphy, M., Eliot, K., Heuertz, R. M., & Weiss, E. (2012). Whole beetroot consumption
acutely improves running performance. Journal of the Academy of Nutrition and
Dietetics, 112, 548–552.
Nualkaekul, S., Salmeron, I., & Charalampopoulos, D. (2011). Investigation of the factors
influencing the survival of Bifidobacterium longum in model acidic solutions and fruit
juices. Food Chemistry, 129, 1037–1044.
Ofori, J. A., & Peggy-Hsieh, Y. H. (2013). Novel technologies for the production of
functional foods. In D. Bagchi, M. Bagchi, H. Moriyama, & F. Shahidi (Eds.), Bio
LWT 134 (2020) 109851
nanotechnology: A revolution in food, biomedical and health Sciences (pp. 141–162).
Chichester, West Sussex, PO19 8SQ, UK: John Wiley & Sons, Ltd, The Atrium,
Southern Gate.
Olson, D., & Aryana, K. (2012). Effect of prebiotics on Lactobacillus acidophilus growth
and resulting pH changes in skim milk and a model peptone system. Journal of
Microbial & Biochemical Technology, 4, 121–125.
Ozcan, T., Sahin, S., Akpinar-Bayizit, A., & Yilmaz-Ersan, L. (2019). Assessment of
antioxidant capacity by method comparison and amino acid characterisation in
buffalo milk kefir. International Journal of Dairy Technology, 72, 65–73.
Ozcan, T., Yilmaz-Ersan, L., Akpinar-Bayizit, A., & Delikanli-Kiyak, B. (2017). Using of
stevia as non-caloric sugar substitutes on viability of probiotic bacteria Lactobacillus
casei. In 186th International Conference on Food and Agricultural Engineering (ICFAE),
11-12 may 2017 (pp. 17–22). Lisbon, Portugal.
Pascual-Teresa, S., & Sanchez-Ballest, a M. T. (2008). Anthocyanins: From plant to
health. Phytochemistry Reviews, 7, 281–299.
Patras, A., Brunton, N. P., O’Donnell, C., & Tiwari, B. K. (2010). Effect of thermal
processing on anthocyanin stability in foods; mechanisms and kinetics of
degradation. Trends in Food Science and Technology, 21, 3–11.
Patrignani, F., Iucci, L., Lanciotti, R., Vallicelli, M., Mathara, J., Holzapfel, W. H., et al.
(2007). Effect of high-pressure homogenization, nonfat milk solids, and milk fat on
the technological performance of a functional strain for the production of probiotic
fermented milks. Journal of Dairy Science, 90, 4513–4523.
Pedreno, M. A., & Escribano, J. (2000). Studying the oxidation and the antiradical
activity of betalain from beetroot. Journal of Biological Education, 35, 49–51.
Peres, C. M., Peres, C., Hernández-Mendoza, A., & Malcata, F. X. (2012). Review on
fermented plant materials as carriers and sources of potentially probiotic lactic acid
bacteria - with an emphasis on table olives. Trends in Food Science & Technology, 26,
31–42.
Singh, B., & Singh Hathan, B. (2014). Chemical composition, functional properties and
processing of Beetroot- A review. International Journal of Scientific Engineering and
Research, 5, 679–684.
Soccol, C. R., Vandenberghe, L. P. S., Spier, M. R., Medeiros, A. B. P., Yamaguishi, C. T.,
Lindner, J. D., et al. (2010). The potential of probiotics. Food Technology and
Biotechnology, 48, 413–434.
Stintzing, F. C., Schieber, A., & Carle, R. (2003). Evaluation of colour properties and
chemical quality parameters of cactus juices. European Food Research and Technology,
216, 303–311.
Sumalapao, D. E. P., Mesina, J. A. R. T., Cabrera, E. C., & Gloriani, N. G. (2017). Viability
kinetics of Lactobacillus casei Shirota strain in a commercial fermented milk drink
during refrigerated storage. National Journal of Physiology, Pharmacy and
Pharmacology, 7, 1–5.
Ścibisz, I., Ziarno, M., & Mitek, M. (2019). Color stability of fruit yogurt during storage.
Journal of Food Science & Technology, 56, 1997–2009.
Teixeira, R. S. S., da Silva, A. S., Ferreira-Leitão, V. S., & Bon, E. P. (2012). Amino acids
interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid
assay mislead carbohydrase activity measurements. Carbohydrate Research, 36,
33–37.
Thakur, V., & Gupta, D. K. D. (2006). Studies on the clarification and concentration of
beetroot juice. Journal of Food Processing and Preservation, 30, 194–207.
Tharmaraj, N., & Shah, N. P. (2003). Selective enumeration of Lactobacillus delbrueckii
ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria,
Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria. Journal of Dairy
Science, 86, 2288–2296.
Vasudha, S., & Mishra, H. N. (2013). Non dairy probiotic beverages. International Food
Research Journal, 20, 7–15.
Vianna, F. S., Canto, A. C. V. C. S., da Costa-Lim, B. R. C., Salim, A. P. A. A., Costa, M. P.,
Balthazar, C. F., et al. (2017). Development of new probiotic yoghurt with a mixture
of cow and sheep milk: Effects on physicochemical, textural and sensory analysis.
Small Ruminant Research, 149, 154–162.
Wada, L., & Ou, B. (2002). Antioxidant activity and phenolic content of Oregon
cranberries. Journal of Agricultural and Food Chemistry, 50, 3495–3500.
Yang, X., Lu, Y., & Hu, G. (2014). Optimization of sweetener formulation in sugar free
yoghurt using response surface methodology. CyTA - Journal of Food, 12, 121–126.
Yildiz, E., & Ozcan, T. (2019). Functional and textural properties of vegetable-fibre
enriched yoghurt. International Journal of Dairy Technology, 72, 199–207.
Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2005). Fermentation of beet juice by
benefical lactic acid bacteria. LWT – Food Science and Technology, 38, 73–75.