foods

Article
Assessment of the Physicochemical, Antioxidant, Microbial,
and Sensory Attributes of Yogurt-Style Products Enriched
with Probiotic-Fermented Aronia melanocarpa Berry Juice
Stavros Plessas 1, * , Ioanna Mantzourani 1 , Antonia Terpou 2, * and Argyro Bekatorou 3

1 Laboratory of Food Processing, Faculty of Agriculture Development, Democritus University of Thrace,

193 Pantazidou Str., 68200 Orestiada, Greece; [email protected]
2 Department of Agricultural Development, Agri-Food, and Natural Resources Management,
School of Agricultural Development, Nutrition & Sustainability, National and Kapodistrian University of Athens,
Evripos Campus, 34400 Evia, Greece
3 Department of Chemistry, University of Patras, 26504 Patras, Greece; [email protected]
* Correspondence: [email protected] (S.P.); [email protected] (A.T.)

Abstract: The aim of this study was to create various formulations of yogurt enriched with freeze-
dried adjuncts, namely (i) probiotic Lactobacillus plantarum ATCC 14917 culture, and (ii) L. plantarum
ATCC 14917 fermented black chokeberry juice, along with a commercial starter culture. The goal
was to enhance functionality and optimize the nutritional value of the products. These new yogurt-
style formulations were subsequently compared with commercially produced yogurt. All products
demonstrated favorable physicochemical properties, and the probiotic strain consistently maintained
viable levels exceeding 7 log cfu/g throughout the entire storage period. The fermented milk
produced with the adjunct-free L. plantarum cells, as well as the yogurt produced with the proposed
lactobacilli-fermented chokeberry juice, exhibited the highest lactic acid production (1.44 g/100 g
yogurt by the end of storage). Levels of syneresis were observed at lower values in yogurt produced
with freeze-dried fermented chokeberry juice. Yogurts prepared with the lactobacilli-fermented
freeze-dried chokeberry juice displayed elevated total phenolic content and antioxidant capacity
(25.74 µg GAE/g and 69.05 µmol TE/100 g, respectively). Furthermore, sensory tests revealed a
Citation: Plessas, S.; Mantzourani, I.;
distinctive fruity flavor in samples incorporating fermented juice. The results demonstrate that
Terpou, A.; Bekatorou, A. Assessment
probiotic L. plantarum-fermented chokeberry juice enhances both the antioxidant capacity and the
of the Physicochemical, Antioxidant,
viability of beneficial bacteria in yogurt while it can be readily applied and commercialized, especially
Microbial, and Sensory Attributes of
Yogurt-Style Products Enriched with
in the form of a freeze-dried formulation.
Probiotic-Fermented Aronia
melanocarpa Berry Juice. Foods 2024, 13, Keywords: yogurt; probiotics; Aronia melanocarpa; bioactive; supplement; functional food
111. https://doi.org/10.3390/
foods13010111

Academic Editor: Yuyun Lu

1. Introduction
Received: 10 November 2023 Nowadays, promoting health and healthy habits are of major concern to consumers
Revised: 22 December 2023 and functional foods are gaining increased recognition, popularity, and acceptance [1,2].
Accepted: 26 December 2023 Specifically, fermented foods and their advantageous microbiota are anticipated to drive
Published: 28 December 2023
the growth of the functional food industry in the coming years [3]. Among the diverse
range of fermented food products, probiotic foods emerge as a prominent category within
the functional food industry [4–6]. Probiotics consist of living microorganisms that, when
Copyright: © 2023 by the authors.
consumed in sufficient quantities (>106 cfu/mL), offer health advantages to the host [4].
Licensee MDPI, Basel, Switzerland. Even though the potential health effects of probiotics depend on a multitude of parame-
This article is an open access article ters, a key characteristic is to retain a sufficient number of live cells during consumption.
distributed under the terms and Moreover, it is considered crucial that probiotic strains survive through the gastrointesti-
conditions of the Creative Commons nal tract and colonize the intestine to confer their beneficial health effects [4,5]. Within
Attribution (CC BY) license (https:// another perspective, probiotic cells inoculated for food fermentation can produce a wide
creativecommons.org/licenses/by/ range of bacterial metabolites depending on the food substrate [3,7]. Specifically, probiotic-
4.0/). fermented food can be a significant source of dietary postbiotics; a new term defined by the

Foods 2024, 13, 111. https://doi.org/10.3390/foods13010111 https://www.mdpi.com/journal/foods

Foods 2024, 13, 111 2 of 15

International Scientific Association for Probiotics and Prebiotics (ISAPP) as a preparation

of inanimate [dead] microorganisms and/or their components that confers a health benefit
on the host [8].
Fermentation is a bioprocess that involves controlled microbial activity and growth,
commonly used for preserving foods such as milk. Yogurt is a commonly fermented
dairy product, rich in various nutrients and minerals, while its consumption has increased
globally due to its nutritional value and ease of digestion [9]. Specifically, yogurt has an
increasing compound annual growth rate (6.9%), while its global market value is expected
to reach USD 51 billion by year 2024 [10]. Therefore, innovation comprises one of the most
significant challenges of the dairy industry to address adapting to consumer demands,
while maintaining sustainable development. Likewise, fermented beverages are widely
manufactured in the modern food industry as they provide specific organoleptic properties
and unique bioactive compounds conferring health benefits [11–16].
Starter cultures play a crucial role in the production of fermented foods and beverages,
contributing to nutritional value, functionality, flavor, and preservation. The advancements
in modern microbiology have allowed the identification and application of specific starter
cultures to achieve desirable attributes in food products [17]. The main application form
of starter cultures in food fermentation is the lyophilized (freeze-dried) form, allowing
for direct incorporation into food formulations [18]. This direct-to-set dried culture form
is highly popular from an industrial perspective, as it eliminates in-plant subculturing,
reduces the cost of bulk culture preparation, and protects against bacteriophage infection [4].
For example, the probiotic strain Lactobacillus plantarum has been recognized as a strain
capable of enhancing the beneficial value of foods by providing the enhanced levels of
bioactive compounds [19,20]. This capability positively influences the nutritional, sensory,
and shelf-life attributes of fermented food products [4,11].
Yogurt is traditionally produced by milk fermentation with the bacterial strains Lac-
tobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains as the starter
cultures, and represents one of the major dairy products for probiotic transfer [9]. Generally,
yogurt can be fortified with a variety of bioactive compounds, offering a wide array of
possibilities to support and enhance health, including strengthening the immune system,
alleviating allergy symptoms, delivering antioxidant properties, averting cardiovascular
conditions, increasing energy levels, and promoting cognitive functions [1,21]. Natural
bioactive additives, such as fresh and dried fruits and fruit juices, are well known for their
high nutritional value and bioactive content. They provide essential components for human
nutrition, and their consumption is associated with numerous health benefits [11]. A recent
trend involves combining probiotics and natural bioactive additives in yogurts to enhance
their safety and nutritional value [22]. Examples of specific components added to yogurt
for improved bioactive and functional properties include natural plant extracts like eugenol
and cinnamaldehyde nanoemulsions [23], beta-carotene/soy protein isolate-containing
lipid microparticles [24], green tea [25], bioactive peptides, gamma-aminobutyric acid [26],
and fruits such as sea buckthorn berries [22] and black mulberries [27].
The beneficial synergies between fruit juices and bioactive substances, including probi-
otic bacteria and postbiotics, and their incorporation into dairy products, have the potential
to initiate a new era of advancements in functional foods. For instance, probiotic cells
added either as starter or adjunct cultures for food fermentation have been reported to
produce phenolic compounds and short-chain fatty acids enhancing a product’s nutritional
value [28]. Also, the effect of fruit and fruit juices on the growth of probiotics has been
reported to be species as well as strain specific [11,12,15,22]. For instance, the fermentation
of pomegranate juice by immobilized Lactobacillus paracasei on wheat bran resulted in the
production of good-quality, potentially synbiotic beverages. This process led to an increase
in total phenolic content (TPC) and the aromatic volatile profile [12]. Additionally, probiotic
strains of L. plantarum and L. acidophilus demonstrated the ability to survive and utilize fruit
juices as substrates for their cell synthesis, showing promise for probiotic-fortified fruit
juice production [13]. In another study, the fermentation of strawberry juice by L. plantarum
Foods 2024, 13, 111 3 of 15

and L. acidophilus led to improvements in color, phenolics, and antioxidant properties in

the resulting beverage [14]. Furthermore, the fermentation of jujube-wolfberry juice by
L. plantarum enhanced its antioxidant activity [16]. Lastly, a novel potentially probiotic
strain, L. paracasei, isolated from kefir grains, demonstrated high cell survival during fermen-
tation and storage when evaluated for chokeberry juice fermentation. The fermented juice
maintained its aroma complexity and exhibited increased TPC and antioxidant capacity
compared to the non-fermented juice [15].
The utilization of freeze-dried fermented juices as carriers of functional components is
a relatively recent concept, and there is ample room for further exploration into their impact
on yogurt starter cultures and the nutritional value of yogurt. Recent studies have demon-
strated improvements in the sensory properties and enhanced functionality of yogurts
during fermentation and storage by incorporating freeze-dried mulberry juice [29], freeze-
dried powders of maqui (Aristotelia chilensis) and murra (Rubus ulmifolius) ultrasound-
assisted extracts [30], freeze-dried soybean and mung bean peel powders [31], among
others. However, there are a lack of reported assessments regarding the physicochemical,
antioxidant, microbial, and sensory attributes of yogurts enriched with probiotic-fermented
Aronia melanocarpa (black chokeberry) juice. Black chokeberries, also known as aronia
berries, are recognized for their diverse health benefits attributed to their high polyphenol
levels and significant antioxidant capacity [15]. Additionally, it is noteworthy that Aro-
nia berries are rich in anthocyanins, which exhibit health-promoting properties such as
antioxidative, anti-inflammatory, and antibacterial activity, along with prebiotic effects [32].
Given the aforementioned research, the primary focus of this study was to evaluate the
potential applications of the probiotic strain L. plantarum ATCC 14917, along with fermented
chokeberry juice using the same strain, as freeze-dried adjuncts for the development of
various yogurt formulations. The quality of the resulting yogurts was evaluated based
on several parameters, including physicochemical properties (acidity, TPC, antioxidant
activity), sensory attributes, as well as microbiological stability during storage.

2. Materials and Methods

2.1. Microbial Starter Cultures
The commercial ready-vat bacterial culture consisting of S. thermophilus and
L. delbrueckii ssp. bulgaricus at a 2:1 proportion (Lyofast Y 436 A, Sacco Systems, Cadorago,
Italy) was applied as starter culture for yogurt production [33]. The starter culture was
activated by incubation in 10 mL of sterile skim milk at 43 ◦ C for 1 h according to the
instructions of the manufacturer. The probiotic strain L. plantarum ATCC 14917 obtained in
lyophilized form (LGC Standards, Middlesex, UK), was activated and grown at 37 ◦ C in
de Man-Rogosa-Sharpe (MRS) liquid medium (Merck Darmstadt, Germany) in agitated
(180 ± 5 rpm, of atmosphere approx. 5% CO2 ) conical flasks for 24 h according to the man-
ufacturer instructions. All media were sterilized prior to use by autoclaving at 121 ◦ C for
15 min (1–1.5 atm). The obtained wet cell mass was harvested by centrifugation (5000 rpm
for 10 min, Sigma 3K12 centrifugation system, Bioblock Scientific, Illkirch Cedex, France),
and subsequently frozen to −44 ◦ C at a cooling rate of 5 ◦ C/min for minimum cell viability
loss [12]. Then, the harvested cell mass was freeze-dried for 48 h without cryoprotection
(FreeZone 4.5 Freeze-Drying System, Labconco, Kansas City, MO, USA).

2.2. Black Chokeberry Juice Production and Fermentation

Black chokeberries (Aronia melanocarpa) were sourced fresh from a local organic farm-
ing producer in Nea Orestiada, located in the northeastern part of Evros, Thrace, Greece.
The black chokeberries were carefully selected and thoroughly washed with a sterile
1/4 ringer solution. Subsequently, the berries were crushed and homogenized by blending
for 10 min in a common household blender [34]. The pulp was removed by a sterile cloth
strainer and centrifuged (3000 rpm, 10 min). The primary chemical composition of Aronia
melanocarpa juice was as follows, as determined in prior research: malic acid 4.71 g/L ± 0.05,
sorbitol 20.0 g/L ± 0.2, glucose 11.05 g/L ± 0.2 and fructose 9.13 g/L ± 0.3 [15]. After
Foods 2024, 13, 111 4 of 15

extracting the juice, sterilized deionized water was added to adjust the initial sugar con-
centration (approximately 40 g/L), and the juice was then pasteurized (80 ◦ C, 10 min) [35].
After cooling the black chokeberry juice to room temperature, 1 g (dry weight) of freeze-
dried L. plantarum was suspended per 100 mL of juice. The viability of the L. plantarum
strain was assessed to be 8.3 log cfu/mL through selective media counting [36] at the onset
of fermentation. Subsequently, the juice underwent fermentation at 30 ◦ C for 48 h, with
simultaneous pH maintenance at 4.0 ± 0.2 achieved by adding Na2 CO3 solution at various
time intervals. Subsequently, each fermented juice was subjected to freeze-drying [12].

2.3. Novel Yogurt-Style Production

Yogurt-style products were prepared from pasteurized homogenized cow’s milk of
Greek origin (pH 6.8 ± 0.1, 3.7% fat, 13.0% total solids) as a fermentation media. The milk
was initially heated at 90 ◦ C for 5 min, cooled at 40 ◦ C, and subsequently divided into
5 equal portions of 100 mL each in sterile glass containers [33]. Five different formulations
were prepared, namely: CY (commercial yogurt used as control sample) inoculated with
yogurt starter culture (5% inoculum, S. thermophilus and L. bulgaricus); LPCY inoculated
with yogurt starter culture and L. plantarum (5% inoculum, 1:1); LPY inoculated only with
L. plantarum (5% inoculum); PDCY inoculated with yogurt starter culture and chokeberry
juice fermented with L. plantarum (5% inoculum); and PDY inoculated with chokeberry
juice fermented with L. plantarum (5% inoculum). The samples were incubated (40 ± 1 ◦ C
for approx. 5–5.5 h) and the acidification was monitored (Consort D130 system, Turnhout,
Belgium) until a pH drop at 4.6 ± 0.1. When a pH drop was achieved, each fermented
sample was placed for cold storage at 4 ◦ C for 28 days.

2.4. Physicochemical Analysis

Compositional analysis and pH were performed for each of the fermented milk sam-
ples after the 1st day of cold storage. Specifically, the samples were analyzed for total solids
(method 990.19), ash (method 945.46), and fat (method 989.05) and using the methods
of AOAC (2005). Fat and protein contents were determined by the Soxhlet and Kjeldahl
methods, respectively. The pH values of samples were determined by direct immersion of
the electrode using a digital pH meter (Hanna HI99161).
High-performance liquid chromatography (HPLC) was used for sugar and organic
acid quantification. In brief, lactose was determined on an HPLC system (Shimadzu, Kyoto,
Japan) with an SCR-101N stainless steel column, an LC-9A pump, a CTO-10A oven (60 ◦ C),
and an RID-6A index detector. Lactic acid was determined on an HPLC system (Shimadzu,
Kyoto, Japan) with a Shim-pack IC-A1 stainless-steel column, a LC-10A pump, a CTO-10A
oven (40 ◦ C) and a CDD-6A detector. For quantitative analysis, standard solutions of sugars
and acids (Saint Louis, Misouri, USA, Sigma-Aldrich Ltd.) were prepared in ultrapure
water (Milli-Q, Darmstadt, Germany, Merk) at various concentrations [33].
Syneresis (S%) was assessed on the first day of production to analyze the potential
effects of supplements on the yogurt-style products during refrigerated storage. Samples
weighing 10 g were placed in 15 mL Falcon tubes, and were then centrifuged (350× g,
20 min, 4 ◦ C) and the separated serum was weighed. The level of syneresis was calculated
using the following equation:

weight volume o f supernatant

S% = × 100
Weight o f f ermented yogurt − style sample

2.5. Microbiological Assessment and Viable Probiotic Cell Count

Microbiological analysis was conducted at various time intervals during the 28 days
of cold storage (4 ◦ C) according to the literature with small modifications [22]. Specifically,
10 g of each yogurt-style product was thoroughly homogenized in 90 mL of sterile Ringer
solution (LABM, Heywood, Bury, UK) and serially diluted in sterile 0.1% (wt/vol) peptone
water (Oxoid Ltd, Hampshire, UK). The microbial loads of each sample were determined by
Foods 2024, 13, 111 5 of 15

plating on selective media. Specifically, S. thermophilus was plated on M17 agar containing
1% lactose and incubated aerobically (40 ◦ C, 72 h); L. bulgaricus was plated on de Man,
Rogosa, Sharpe (MRS) agar with 10% sorbitol and incubated anaerobically (37 ◦ C, 48 h);
yeasts and molds were plated on Potato Dextrose Agar and incubated aerobically (30 ◦ C
for 72 h); coliforms were plated on Violet Red Bile agar after and incubated anaerobically
(30 ◦ C, 24 h); staphylococci were plated on Baird Parker agar after and incubated aerobically
(37 ◦ C, 24 h), and the petri dishes remained for another 24 h to assess the viability of
S. aureus strains (black or grey colonies) [37] according to the manufacturer instructions
(LABM, UK).
Viable bacterial counts of L. plantarum were determined by plating on MRS agar with
10 mg/L of vancomycin antibiotic that promotes its growth against L. bulgaricus [38] and
incubating aerobically (37 ◦ C, 48 h).
All the above media were sterilized (121 ◦ C, 15 min) before use. Cell counts were
expressed as log cfu/g [33].

2.6. Antioxidant Capacity

Free radical-scavenging activity was determined using the free radical DPPH
(2,2 diphenyl-1-picrylhydrazyl) method [39]. The DPPH radical scavenging activity was
determined according to the following equation (Ac: the absorbance of the control solution,
As: the absorbance of the test solution):

Ac − As
DPPH scavenging activity% = × 100
Ac
Total phenolic content (TPC) was determined by the Folin–Ciocalteu method as-
sessed accordingly to the literature [40]. Concentrations are expressed in Gallic Acid
Equivalents (GAE).

2.7. Sensory Attributes

All yogurt-style samples (LPCY, LPY, PDCY, PDY) underwent sensory evaluations
conducted by 10 adults (laboratory members; priorly trained) familiar with the consump-
tion of dairy products and were compared to yogurt made using the commercial starter
culture [33]. Approximately 50 g of samples produced the day before the evaluation were
provided to the assessors. The assessors, both males and females aged 25 to 45 years who
were non-smokers, were involved in the evaluation process. The samples were served
in 50 mL transparent plastic cups, each numbered randomly with 3-digit codes. The sen-
sory evaluation session took place in individual booths. They were provided with bread
and low-mineral content water to cleanse their palate after each tasting. The panel was
instructed to assign scores on a 0–10 scale (0 = unacceptable, 10 = exceptional) for attributes
falling under the following main categories: aroma, fruit flavor, intensity of white color,
texture, firm body, sour taste, overall flavor, and overall acceptability. At the conclusion of
the rating scale, assessors were invited to provide any additional comments.

2.8. Statistical Analysis

All fermentation experiments were carried out in triplicate. The results were analyzed
using one-way analysis of variance (ANOVA). The different treatments were compared at
the same storage period, and samples from the same treatment were compared during the
same time. Duncan’s multiple range tests were applied in order to determine significant
differences (coefficients, ANOVA tables, and significance); p value < 0.05 was considered
statistically significant for all analyses.
Foods 2024, 13, 111 6 of 15

3. Results and Discussion

3.1. Impact of Incorporated Enriched Materials on the Physicochemical Characteristics
of the Products
The results pertaining to the pH, lactic acid, and lactose content of the fermented
milk samples during cold storage (4 ◦ C) for 28 days are detailed in Table 1. Overall, a
decrease in pH values was noted across all samples during storage. The reduction in pH
during lactic acid milk fermentation is attributed to the breakdown of lactose into lactic
acid, where a higher production of lactic acid corresponds to a lower pH value [33,41].
The pH values of samples containing the probiotic strain were detected at significantly
lower levels compared to the control sample (CY). Furthermore, the incorporation of the
probiotic strain in all respective fermented milk samples (LPCY, LPY, PDCY, and PDY)
increased lactic acid production, resulting in lower pH values after fermentation and during
cold storage, compared to the control (CY). This result is consistent with previous studies
indicating that yogurt production with adjunct probiotic cultures shows heightened lactic
acid production and lower pH values compared to commercial yogurt, attributable to the
enhanced accumulation of the probiotic strain [33,42,43]. Milk fermentation by lactic acid
bacteria involves the conversion of milk lactose, mainly to lactic acid (0.6–1%), along with
other metabolites. Thus, a decline in pH during the initial periods of storage can be expected
as a result of post-acidification. The most significant decrease in lactose, accompanied by a
decline in pH, was noted in PDCY and PDY samples. Consequently, the trend toward lower
pH is a result of increased lactic acid production and potentially other organic compounds,
such as formic acid, acetaldehyde, and acetic acid [33,43,44].

Table 1. Means and standard deviations of pH, lactose, and lactic acid in fermented milk samples
during refrigerated storage at 4 ◦ C for 28 days.

Storage Time (Days) CY LPCY LPY PDCY PDY

1 4.61 a ± 0.08 4.59 a ± 0.09 4.58 a ± 0.12 4.57 a ± 0.05 4.56 a ± 0.05
7 4.56 a ± 0.10 4.51 a ± 0.17 4.49 a ± 0.10 4.48 a ± 0.10 4.39 a ± 0.08
pH 14 4.53 b ± 0.05 4.45 b ± 0.05 4.44 ab ± 0.11 4.42 ab ± 0.07 4.31 a ± 0.10
21 4.51 b ± 0.11 4.42 b ± 0.08 4.39 ab ± 0.10 4.33 ab ± 0.05 4.26 a ± 0.05
28 4.48 c ± 0.03 4.38 bc ± 0.05 4.35 ab ± 0.08 4.27 ab ± 0.09 4.24 a ± 0.05
1 2.27 a ± 0.08 2.25 a ± 0.09 2.31 a ± 0.09 2.45 a ± 0.11 2.43 a ± 0.11
Lactose 7 1.93 a ± 0.11 1.99 a ± 0.07 2.05 a ± 0.11 2.03 a ± 0.10 2.12 a ± 0.10
(g/100 g of 15 1.90 a ± 0.13 1.38 bc ± 0.11 1.69 b ± 0.08 1.47 bc ± 0.08 1.77 a ± 0.14
product) 21 1.84 b ± 0.09 1.18 a ± 0.07 1.30 a ± 0.08 1.11 a ± 0.12 1.19 a ± 0.13
28 1.72 b ± 0.05 0.99 a ± 0.05 1.12 a ± 0.07 0.95 a ± 0.09 1.05 a ± 0.10
1 0.85 a ± 0.04 0.87 ab ± 0.11 0.91 a ± 0.05 0.88 a ± 0.05 0.99 ab ± 0.05
Lactic acid 7 1.12 ab ± 0.05 1.15 ab ± 0.05 1.04 ab ± 0.05 1.14 a ± 0.03 1.05 ab ± 0.03
(g/100 g of 15 1.19 ab ± 0.05 1.22 ab ± 0.05 1.11 ab ± 0.06 1.25 ab ± 0.05 1.10 a ± 0.04
product) 21 1.23 b ± 0.04 1.34 b ± 0.06 1.18 ab ± 0.05 1.31 b ± 0.05 1.13 a ± 0.05
28 1.31 b ± 0.05 1.44 b ± 0.05 1.24 a ± 0.03 1.44 b ± 0.03 1.17 a ± 0.04
The results are expressed as mean (n = 3) ± standard deviations; CY: yogurt with commercial starter culture; LPCY:
fermented milk with commercial yogurt starter culture and L. plantarum; LPY: milk fermented with L. plantarum;
PDCY: fermented milk with commercial yogurt starter culture and chokeberry juice fermented with L. plantarum;
PDY: fermented milk with fermented chokeberry juice. Different superscript letters in rows indicate statistically
significant differences at the same time of analysis for each starter culture (MF-ANOVA with Tukey’s HSD multiple
range test).

The decrease in pH in all samples during storage, most likely due to the production
of organic acids, is a result of the activity of lactic acid bacteria (LAB), which seem to
remain active even at low temperatures. Notably, the samples containing the adjunct
probiotic culture exhibited lower pH values, while the samples with the dried supplements
displayed even lower pH levels and the highest lactic acid production. Specifically, by
the end of storage period, the sample PDY recorded the lowest pH (4.24) followed by the
PDCY sample (pH 4.27) with the other samples having a pH in the range of 4.48–4.35. The
Foods 2024, 13, 111 7 of 15

distinct acidification observed in fermented milk samples produced with the addition of
fermented chokeberry juice with L. plantarum may be attributed to the stimulating effect of
the phenolic compounds found in chokeberries on the metabolic activity of LAB. However,
it should be noted that the pH of these samples remained within acceptable values. This
result aligns with recent studies that have confirmed the positive influence of phenolic
compounds on lactic acid fermentation [14,45].
The protein, fat, total solids, and ash content from the first day of production are
presented in Table 2 and ranged between 3.3–3.6 (%), 3.5–3.6 (%), 16.6–16.9 (%), and
0.5–0.7 (%), respectively. The slight reduction in fat content in all samples could be ascribed
to the lipolytic activity of microorganisms [46,47]. The protein content exhibited variation
among the samples. Specifically, the control sample (CY) displayed a protein content
similar to that of LPCY and LDY (3.5%), while the PDY sample had a slightly lower protein
content (3.3%). In contrast, the PDCY sample had the lowest protein content among the
samples, measuring at 3.0%.

Table 2. Physicochemical properties of fermented milk samples (first day of production).

Protein Total Solids Ash Fat Syneresis TPC DPPH

Fermented Milk
(% wt) (% wt) (% wt) (% wt) % (µg GAE/g) (µmol TE/100 g)
CY 3.52 c ± 0.05 15.90 b ± 0.11 0.57 a ± 0.08 3.57 a ± 0.11 23.31 c ± 0.28 15.18 a ± 0.76 49.12 a ± 0.35
LPCY 3.54 c ± 0.07 15.89 b ± 0.15 0.61 a ± 0.05 3.69 a ± 0.07 24.13 d ± 0.14 16.20 a ± 0.51 61.40 b ± 0.19
LPY 3.48 c ± 0.05 15.77 b ± 0.11 0.52 a ± 0.03 3.65 a ± 0.05 24.19 d ± 0.10 15.24 a ± 0.87 51.72 a ± 0.31
PDCY 3.09 a ± 0.07 15.99 b ± 0.10 0.76 b ± 0.05 3.61 a ± 0.05 22.11 a ± 0.10 25.74 b ± 1.13 69.05 c ± 1.07
PDY 3.30 b ± 0.06 15.51 a ± 0.14 0.56 a ± 0.04 3.57 a ± 0.04 22.51 b ± 0.10 15.81 a ± 0.99 68.42 c ± 0.95
The results are expressed as mean (n = 3) ± standard deviations; TPC: total phenolic content; CY: yogurt with
commercial starter culture; LPCY: yogurt with commercial starter culture and L. plantarum; LPY: milk fermented
with L. plantarum; PDCY: Milk fermented with commercial yogurt starter culture and chokeberry juice fermented
with L. plantarum; PDY: milk fermented with chokeberry juice fermented with L. plantarum. Different superscript
letters in columns indicate statistically significant differences at the same parameter of analysis for each starter
culture (MF-ANOVA with Tukey’s HSD multiple range test).

Serum separation in yogurt and yogurt-style products is a critical factor that impacts
the appearance and physical characteristics [48]. Yogurt as well as sour milk syneresis
denotes the separation of whey on the surface of the product, which can happen either
upon opening a yogurt container or in a sealed container. This separation is primarily
attributed to (i) the higher concentration of whey protein compared to casein, and (ii) the
lower concentrations of total solids, as well as (iii) changes in organic acids produced
by viable LAB during storage [49]. According to the results of the current study, synere-
sis was influenced by the initial starter culture and acidification method, as well as the
added powdered supplements (Table 2). The addition of chokeberry juice fermented with
L. plantarum (PDCY, PDY) can enhance the total solid content providing higher consistency
in fermented milk compared to commercial yogurt samples. Furthermore, as indicated by
previous studies, the addition of fruits or supplements with high antioxidant activity has
the potential to reduce serum separation and enhance the storage stability of fermented
milk products [27,50].

3.2. Antioxidant Activity and Phenolic Content

Lactic acid fermentation is known to significantly enhance the antioxidant capacity,
phenolic content, and flavor of fresh juice when lactic acid bacteria are applied for fer-
mentation, as indicated by recent studies [11,51,52]. Likewise, the results of this study
indicate that the addition of powdered supplements combined either with commercial
starter culture or fermented milk (PDY, PDCY) boosted the levels of the antioxidant activity
(approximately 69 µmol TE/100 g). In general, all samples fermented with the adjunct
probiotic L. paracasei strain showed higher antioxidant capacity in contrast to plain yogurt,
which reached a DPPH radical scavenging activity of 49 µmol TE/100 g. The results in-
dicated that the probiotic strain increased the antioxidant capacity of the fermented milk
products, while chokeberry components can also significantly enhance the antioxidant con-
Foods 2024, 13, 111 8 of 15

tent either by the direct contribution of antioxidant compounds or thought bioconversion

derived from LAB activity.
The TPC appeared also enhanced in the samples produced with the chokeberry juice
fermented with L. plantarum (PDY, PDCY) compared to all other samples. According to
the literature, in terms of the contribution sources of bioactive polyphenols (phenolic acids,
flavonols, anthocyanins, proanthocyanidins) included in chokeberry, the total antioxidant
activity mainly depends on the contribution of free polyphenols [53,54]. As a result, it is
evident that incorporating chokeberry as a supplement can contribute to the development
of functional food by providing a natural source of polyphenols.

3.3. Microbial Stability of Yogurts-Style Products during Cold Storage

Microbial stability is crucial in preserving the physicochemical and organoleptic
characteristics, as well as the safety of foods during storage, especially dairy products [55].
Microbial counts (log cfu/g) of possible spoilage microorganisms in the fermented milk
samples were monitored throughout cold storage for 28 days. As noted, no spoilage
or possible pathogenic microorganisms such as staphylococci, coliforms, enterobacteria,
yeasts, or molds were detected during cold storage for 28 days in samples with the adjunct
probiotic strain (Table 3). On the contrary, yeasts and molds were detected in the commercial
yogurt samples after the 21st day of storage. This result is most likely due to the slightly
higher pH values of the commercial yogurt, as well as the antagonistic effect of L. plantarum
observed in all other samples (LPCY, LPY, PDCY, PDY).

Table 3. Microbial counts of samples during cold storage (4 ◦ C) for 28 days.

Storage Time S. thermophilus L. bulgaricus Yeasts and Molds

Fermented Milk
(Days) (log cfu/g)
1 8.43 a ± 0.16 8.77 ab ± 0.12 nd
7 8.14 a ± 0.25 8.44 b ± 0.23 nd
CY 14 7.66 b ± 0.11 8.19 b ± 0.11 nd
21 7.21 b ± 0.10 7.74 b ± 0.14 nd
28 7.11 b ± 0.10 7.26 b ± 0.11 1.06 a ± 0.10
1 8.45 a ± 0.23 8.56 ab ± 0.11 nd
7 8.07 a ± 0.19 7.17 a ± 0.09 nd
LPCY 14 7.12 a ± 0.14 6.64 a ± 0.28 nd
21 6.42 a ± 0.11 6.57 a ± 0.10 nd
28 6.06 a ± 0.08 6.34 a ± 0.12 nd
1 nd nd nd
7 nd nd nd
LPY 14 nd nd nd
21 nd nd nd
28 nd nd nd
1 8.85 b ± 0.11 9.41 b ± 0.13 nd
7 8.82 b ± 0.09 9.32 c ± 0.11 nd
PDCY 14 8.86 c ± 0.12 9.45 c ± 0.21 nd
21 8.63 c ± 0.13 8.92 c ± 0.15 nd
28 8.49 c ± 0.07 8.67 c ± 0.09 nd
1 nd nd nd
7 nd nd nd
PDY 14 nd nd nd
21 nd nd nd
28 nd nd 1.10 a ± 0.14
The results are expressed as mean (n = 3) ± standard deviations; nd: not detected; CY: yogurt with commercial
starter culture; LPCY: fermented milk with commercial yogurt starter culture and L. plantarum; LPY: milk
fermented with L. plantarum; PDCY: fermented milk with commercial yogurt starter culture and chokeberry juice
fermented with L. plantarum; PDY: fermented milk with chokeberry juice fermented with L. plantarum. Different
superscript letters in columns indicate statistically significant differences at the same time of analysis for the
comparison of each starter culture (MF-ANOVA with Tukey’s HSD multiple range test).
 commercial starter culture; LPCY: fermented milk with commercial yogurt starter culture and L.
plantarum; LPY: milk fermented with L. plantarum; PDCY: fermented milk with commercial yogurt
starter culture and chokeberry juice fermented with L. plantarum; PDY: fermented milk with choke-
berry juice fermented with L. plantarum. Different superscript letters in columns indicate statistically
Foods 2024, 13, 111 significant differences at the same time of analysis for the comparison of each starter culture (MF-
9 of 15
ANOVA with Tukey’s HSD multiple range test).

Microbial counts of S. thermophilus, L. bulgaricus (Table 3), and L. plantarum (Figure 1)

wereMicrobial
also monitored. of S.counts
counts The thermophilus,
of lacticL.acid
bulgaricus (Table
bacteria and L. plantarum
3), during
(LAB) (Figure
cold storage 1)
are in-
were also monitored. The counts of lactic acid bacteria (LAB) during cold storage are
tricately linked to nutritional and environmental factors [4]. As shown in Table 3, the in-
intricately linked to nutritional and environmental factors [4]. As shown in Table 3, the
corporation of chokeberry juice fermented with L. plantarum resulted in an elevation of
incorporation of chokeberry juice fermented with L. plantarum resulted in an elevation of
LAB counts in the PDCY sample during cold storage, contrasting with the commercial
LAB counts in the PDCY sample during cold storage, contrasting with the commercial
yogurt sample. More specifically, this resulted in higher counts of S. thermophilus and L.
yogurt sample. More specifically, this resulted in higher counts of S. thermophilus and
bulgaricus, exceeding 8 log cfu/mL on the 28th day of storage, whereas the control yogurt
L. bulgaricus, exceeding 8 log cfu/mL on the 28th day of storage, whereas the control yogurt
exhibited significantly lower viability at 7 log cfu/mL. This outcome can be attributed to
exhibited significantly lower viability at 7 log cfu/mL. This outcome can be attributed
the enrichment of nutrients and prebiotic oligosaccharides provided by the adjunct fer-
to the enrichment of nutrients and prebiotic oligosaccharides provided by the adjunct
mented chokeberry juice [22,56,57]. This finding is consistent with previous studies sug-
fermented chokeberry juice [22,56,57]. This finding is consistent with previous studies
gesting that prebiotic ingredients can enhance LAB viability in dairy products and gut
suggesting that prebiotic ingredients can enhance LAB viability in dairy products and
microenvironment [4,33,44,58]. Moreover, the S. thermophilus and L. bulgaricus viable cell
gut microenvironment [4,33,44,58]. Moreover, the S. thermophilus and L. bulgaricus viable
counts
cell werewere
counts found at aatsignificantly
found lower
a significantly concentration
lower concentrationin sample
in sampleLPCY, compared
LPCY, compared to
the commercially produced yogurt samples (Table 3). This could be attributed
to the commercially produced yogurt samples (Table 3). This could be attributed to the to the an-
tagonistic effect
antagonistic ofof
effect thetheprobiotic
probioticL.L.plantarum
plantarumculture
cultureagainst
againstother
other bacteria
bacteria during
during milk
milk
fermentation [33,41]. In addition, the effect of low temperature on S.
fermentation [33,41]. In addition, the effect of low temperature on S. thermophilus thermophilus and L.
and
bulgaricus can affect their viability as they belong to thermophilic bacteria
L. bulgaricus can affect their viability as they belong to thermophilic bacteria (optimum(optimum tem-
perature 40–45
temperature °C).◦ C).
40–45

Figure 1. Viable cell counts (log cfu/g)

Figure cfu/g) of
ofL.
L.plantarum.
plantarum. LPCY:
LPCY: yogurt
yogurt with
with commercial
commercial starter culture
and L. plantarum; LPY: milk fermented with L. plantarum; PDCY: fermented
and L. plantarum; LPY: milk fermented with L. plantarum; PDCY: fermented milk milk with
with commercial
commercial
yogurt starter culture and chokeberry juice fermented with L. plantarum; PDY: milk fermented
yogurt starter culture and chokeberry juice fermented with L. plantarum; PDY: milk fermented with with
fermented chokeberry juice. Different superscript letters in the bars indicate statistically significant
fermented chokeberry juice. Different superscript letters in the bars indicate statistically significant
differences at the same time of analysis for the comparison each starter culture (MF-ANOVA with
Tukey’s HSD multiple range test).

It has been reported that phenolic compounds at relatively high concentrations can
inhibit the growth of bacteria [59]. In this study, higher viable counts of both S. thermophilus
and L. bulgaricus were found in the PDCY sample from the first till the last days of stor-
age, although these samples were enriched in phenolic compounds from the fermented
chokeberry juice. On the other hand, samples produced with the commercial starter culture
(CY and LPCY) showed significantly lower viability after the first days of production
compared to PDCY samples. This may be attributed to the high phenolic content of PDCY
samples, which, in line with previous studies, can promote the growth of S. thermophilus
and Lactobacillus spp. during milk fermentation [25].

3.4. Viability of L. plantarum and Possible Beneficial Effects

Figure 1 illustrates the population of the probiotic strain L. plantarum in samples LPCY,
LPY, PDCY, and PDY during cold storage (4 ◦ C) at various time intervals (days 1, 3, 7,
10, 14, 18, 21, 28). Well-documented, unique health benefits may arise from novel and
unconventional sources of probiotics, which can produce bioactive compounds contribut-
Foods 2024, 13, 111 10 of 15

ing to well-being, and therefore, it is crucial to maintain the strains viability through-
out storage [4,60]. It is essential to note that the initial viability of L. plantarum was
recorded at 8.8 log cfu/mL after juice fermentation (48 h), with a slight decrease in viability
(8.6 log cfu/mL) observed after freeze-drying of the fermented juice. This result is possibly
attributed to the harsh conditions of freeze-drying to which the fermented juice was sub-
jected without the use of cryoprotectants. Regarding the viability of the probiotic strain
in fermented milk samples, it is important to note that the L. plantarum strain consistently
maintained levels above 8 log cfu/g throughout the storage period in all samples.
All yogurt-style products meet the criteria to be considered probiotic, aligning with
the recommended levels of viable probiotic cell counts at the time of consumption [4]. This
result is noteworthy, as prior studies suggest that the L. plantarum strain is not as well
suited for milk fermentation as other probiotic strains since genomic and milk fermentation
test verified low-level lactic acid production in this particular strain [19]. Conversely, it
was demonstrated to be highly suitable for chokeberry juice fermentation, and its adjunct
addition in milk alongside chokeberry was proven to be highly successful. In relation to
the above observation, a significant decline (p < 0.05) in probiotic cell counts was observed
at the end of the storage period in samples produced with L. plantarum as the starter cul-
ture (LPY) and in samples produced with both L. plantarum and the commercial yogurt
culture (LPCY), compared to samples produced with chokeberry juice fermented with
L. plantarum (PDCY). This decrease in cell counts can be attributed to the acidic environ-
ment of fermented milk samples, which adversely affects the viability of free bacterial
cells, leading to their reduction [33]. Furthermore, this decrease may be attributed to the
deficiency of LacZ/LacLM genes, as confirmed by previous studies [19]. This deficiency
can result in the incapacity of L. plantarum to ferment lactose, especially during cold storage,
thus leading to a loss of viability. In contrast, samples produced with chokeberry juice and
L. plantarum (PDCY), as well as milk fermented with chokeberry juice and L. plantarum
(PDY), demonstrated remarkable stability in viable probiotic cell counts throughout the
entire storage period (4 ◦ C, 28 days). This result may be attributed to the increased phenolic
content of samples produced with the powdered supplements, as anthocyanins provided
by chokeberry may promote the growth of lactic acid bacteria (LAB) due to their prebiotic
activities [57,61].
In the recent literature, it is also suggested that prebiotics may contribute to the
protection of probiotic cells against acidic and harsh environmental conditions during
the production and storage of dairy products [5]. Likewise, in this study, the viability of
L. plantarum could be enhanced by prebiotic ingredients present in the dried chokeberry
juice providing a synbiotic supplement [62]. Importantly, prior studies have proposed
a synergistic interaction between phenolic compounds and probiotic bacteria, resulting
in an improved antioxidant capacity and the colonization of probiotic cells within the
gastrointestinal tract [63]. These synergistic interactions may also be associated with
postbiotic effects, which involve the production of small metabolites by bacteria during
their life cycles. These metabolites play a crucial role in regulating bacterial growth and
cell communication. Additionally, they contribute to the growth of beneficial bacteria and
offer protection against various stresses [64].
Building on these findings, the present study suggests a blend of probiotic bacteria and
chokeberry juice, known for its phenolic compounds, as a powdered supplement for yogurt-
style products’ manufacturing. This strategy, incorporating the components of chokeberry
juice, is envisioned to contribute to the sustained high survival rates of probiotics in the
resultant dairy products. Additionally, previous research has highlighted the diverse
functional properties of L. plantarum strains in the food industry. These properties include
enhancing nutritional quality, flavor characteristics, antioxidant and antimicrobial activities,
and extending the shelf life of foods, while also reducing undesirable compounds [65].
However, it is important to highlight that for populations with allergies or intolerances to
dairy-based products, the fermentation of fruit juices with Lactobacillus isolates may serve
as an alternative. Probiotic-fortified fruit juices, such as chokeberry juice, can be explored
Foods 2024, 13, 111 11 of 15

as functional and healthy beverages delivering probiotics, as demonstrated in previous

studies [13,15].

3.5. Sensory Evaluation

Overall, all samples garnered high acceptance scores and were marked by a favorable
total impression, with no off-flavors detected (Figure 2). The samples prepared with
fermented chokeberry juice containing L. plantarum stood out for its exceptional aroma and
fruit flavor. This characteristic could be attributed to the abundance of phenolic compounds,
recognized as significant constituents in plant-origin food products. Phenolic compounds
are related to the significant sensory characteristics of foods such as flavor, astringency,
and color [66]. According to the results presented in Figure 2, the color of fermented milk
samples was significantly affected by the addition of the fermented chokeberry juice in
PDY and PDCY samples. The abundance of phenolic compounds (Table 2) in samples
Foods 2024, 13, x FOR PEER REVIEW 12 of 16
PDCY (Figure 2) and PDY (Figure 2), provided by the initial juice content, can contribute
to the flavor of fermented milk highlighting its unique aromatic characteristics. Similarly,
volatile phenols generated through the metabolic processes of probiotic or yogurt cultures
beneficial
can health
contribute effects.
to an Thisflavor
enriched shift is
andemphasized by thealigning
aromatic profile, increased significance
with placed
findings from on
prior
health, [66].
studies especially in the aftermath of the COVID-19 pandemic [68].

Figure2.2.Radar
Figure Radarplot
plotfor
forthe
thesensory
sensory attributes
attributes of of samples.
samples. CY:CY: yogurt
yogurt with
with commercial
commercial starter
starter cul-
culture.
ture. LPCY: milk fermented with commercial yogurt culture and L. plantarum; LPY: milk fermented
LPCY: milk fermented with commercial yogurt culture and L. plantarum; LPY: milk fermented with
with L. plantarum; PDCY: milk fermented with commercial yogurt culture and chokeberry juice fer-
L. plantarum; PDCY: milk fermented with commercial yogurt culture and chokeberry juice fermented
mented with L. plantarum; PDY: milk fermented with fermented chokeberry juice.
with L. plantarum; PDY: milk fermented with fermented chokeberry juice.
4. Conclusions
The texture of all samples received high evaluation scores, likely because the syneresis
values Freeze-dried fermented
remained within chokeberry
acceptable juiceThis
ranges. withcould
the probiotic strain to
be attributed L. plantarum ATCC
the presence of
14917metabolites,
LAB was effectively
whichintegrated in thethe
may enhance production
firmnessof offunctional fermented
the product’s texture.milk
For products.
example,
The resulting
probiotic innovative
and LAB strains yogurt-style
are recognizedproducts maintained
for their robust probiotic
exopolysaccharide culture
production, via-
which
bilities
can and an enhanced
contribute phenolic
to the texture profile throughout
of fermented a 4-week
milk, reducing storageand
syneresis period. Notably,
improving in
the
all cases where L. plantarum was incorporated, the achieved viabilities consistently sur-
passed the threshold (106–107 cfu/mL) required to confer the health benefits of a specific
probiotic food. Furthermore, the novel yogurt-style products containing the incorporated
fermented chokeberry juice exhibited microbiological safety throughout the entire storage
period. This novel supplement, exemplified by freeze-dried fermented chokeberry juice,
Foods 2024, 13, 111 12 of 15

overall mouthfeel and body of the product [67]. Similarly, it has been indicated that yogurt
texture can be enhanced through the production of exopolysaccharides, which interact
with the water in the products, creating a gel-like structure Guzel-Seydim, Sezgin, and
Seydim [67]. The elevated texture scores observed in the samples of this study may also be
attributed to other constituents, such as total solids. Notably, the slightly higher texture
scores of PDCY and PDY (yogurts with fermented chokeberry juice with L. plantarum)
compared to other samples could be associated with carbohydrates and fibers derived from
the fermented juice powder. These components could play a significant role in improving
yogurt texture. Quality characteristics like these are crucial for the food industry, as they
can have substantial effects on the viscosity and firmness of fermented milk products. More
crucially, in today’s context, consumers are increasingly receptive to trying novel flavor
combinations, particularly those integrating ingredients that can provide beneficial health
effects. This shift is emphasized by the increased significance placed on health, especially
in the aftermath of the COVID-19 pandemic [68].

4. Conclusions
Freeze-dried fermented chokeberry juice with the probiotic strain L. plantarum ATCC
14917 was effectively integrated in the production of functional fermented milk products.
The resulting innovative yogurt-style products maintained robust probiotic culture viabili-
ties and an enhanced phenolic profile throughout a 4-week storage period. Notably, in all
cases where L. plantarum was incorporated, the achieved viabilities consistently surpassed
the threshold (106 –107 cfu/mL) required to confer the health benefits of a specific probiotic
food. Furthermore, the novel yogurt-style products containing the incorporated fermented
chokeberry juice exhibited microbiological safety throughout the entire storage period. This
novel supplement, exemplified by freeze-dried fermented chokeberry juice, not only meets
the demand as a probiotic product but also merges bioactive phenolic components from
the original juice with those generated by LAB metabolism. Readily deployable functional
supplements are notably attractive to the food industry, especially in the post-pandemic
era. Hence, the groundbreaking functional yogurt-style products developed in this study
showcase substantial potential for commercialization in the dairy industry, offering a blend
of advantageous antioxidant, prebiotic, probiotic, and postbiotic attributes. Finally, future
research endeavors should focus on delving deeper into the nutritional aspects and inter-
actions posed by the expression of LAB genes in novel products. This is essential to fully
understand the metabolic compounds occurring in the final products, as well as to uncover
the potential health effects and benefits associated with the consumption of these products.

Author Contributions: Conceptualization, S.P. and A.T.; methodology, A.T. and I.M.; software, A.T.
and I.M.; validation, A.T. and I.M.; formal analysis, A.T.; investigation, I.M.; resources, A.B. and S.P.;
data curation, A.T.; writing—original draft preparation, S.P. and A.T.; writing—review and editing,
A.B. and A.T.; visualization, S.P.; supervision, S.P.; project administration, S.P. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: All participants volunteered to take part in this project.
All subjects gave their informed consent for inclusion before they participated in the study. These
materials were safe for sensory research. All the experimental procedures involving volunteers were
conducted in accordance with the Declaration of Helsinki.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data are contained within the article.
Conflicts of Interest: The authors declare no conflicts of interest.
Foods 2024, 13, 111 13 of 15

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