fermentation

Article
Valorization of Lactic Acid Fermentation of Pomegranate Juice
by an Acid Tolerant and Potentially Probiotic LAB Isolated
from Kefir Grains
Ioanna Mantzourani 1 , Antonia Terpou 2 , Argyro Bekatorou 3 and Stavros Plessas 1, *

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

68200 Orestiada, Greece; imantzou@agro.duth.gr
2 Evripos Group, Department of Agricultural Development, Agri-Food, and Natural Resources Management,
School of Agricultural Development, Nutrition & Sustainability, National and Kapodistrian University of
Athens, 34400 Athens, Greece; aterpou@agro.uoa.gr
3 Department of Chemistry, University of Patras, 26504 Patras, Greece; abekatorou@upatras.gr
* Correspondence: splessas@agro.duth.gr; Tel.: +30-255-204-1141

Abstract: The present study describes the application of an acid tolerant and potentially probiotic
L. paracasei SP3 strain, recently isolated from kefir grains, in the production of a novel functional
beverage based on the fermentation of pomegranate juice. The fermentation ability of the novel
strain was assessed during pomegranate juice fermentations at 30 ◦ C for 24 h and storage at 4 ◦ C for
4 weeks. Various parameters were assessed such as residual sugar, organic acid and alcohol levels,
total phenolics content, antioxidant activity, astringency, cell viability, and consumer acceptance.



Residual sugar was decreased by approximately 25%, while respectable amounts of lactic acid were

 determined (4.8 g/L) on the 28th day of storage, proving that the novel strain was effective at
Citation: Mantzourani, I.; Terpou, A.; lactic acid fermentation. The concentration of ethanol was maintained at low levels (0.3–0.4 % v/v)
Bekatorou, A.; Plessas, S. Valorization and low levels of acetic acid were detected (0.6 g/L). The viability of L. paracasei SP3 cells retained
of Lactic Acid Fermentation of high levels (>7 log cfu/mL), even by the 4th week. The total phenolic content (123.7–201.1 mg
Pomegranate Juice by an Acid
GAE/100 mL) and antioxidant activity (124.5–148.5 mgTE/100 mL) of fermented pomegranate juice
Tolerant and Potentially Probiotic
were recorded at higher levels for all of the studied time periods compared to the non-fermented juice.
LAB Isolated from Kefir Grains.
The employment of the novel strain led to a significant reduction in the levels of hydrolysable tannins
Fermentation 2022, 8, 142.
(42%) in the juice, reducing its astringency. The latter was further proven through sensorial tests,
https://doi.org/10.3390/
fermentation8040142
which reflected the amelioration of the sensorial features of the final product. It should be underlined
that fruit juices as well as pomegranate juice comprised a very harsh food matrix for microorganisms
Academic Editors: Chih-Yao Hou,
to survive and ferment. Likewise, the L. paracasei SP3 strain showed a significant potential, because it
Bao-Hong Lee and Ming-Kuei Shih
was applied as a free culture, without the application of microencapsulation methods that are usually
Received: 8 March 2022 employed in these fermentations, leading to a product with possible functional properties and a high
Accepted: 23 March 2022 nutritive value.
Published: 25 March 2022

Publisher’s Note: MDPI stays neutral

Keywords: pomegranate juice; tannins; phenolic content; probiotic; L. paracasei SP3; astringency;
with regard to jurisdictional claims in consumers acceptance test
published maps and institutional affil-
iations.

1. Introduction
Improvements in human health through the attunement of diet have become a
Copyright: © 2022 by the authors.
paramount strategy and target of the new Century. Awareness and employment of func-
Licensee MDPI, Basel, Switzerland.
tional food as a key tool for the accomplishment of the above target has been well estab-
This article is an open access article
lished, leading to the development of novel marketable food products [1–3]. Currently, the
distributed under the terms and
functional foods market is increasing worldwide as consumers seek not just “good” food,
conditions of the Creative Commons
but request nutritive meals of exceptional quality for “farm to fork” [4,5]. Functional foods
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
are foods that, besides the basic nutritional requirements, can provide enhanced health
4.0/).
benefits to the consumer, leading to the promotion of their health state and a reduction

Fermentation 2022, 8, 142. https://doi.org/10.3390/fermentation8040142 https://www.mdpi.com/journal/fermentation

Fermentation 2022, 8, 142 2 of 13

of disease risk [6,7]. Requirements of this kind are satisfied by various nutrients, such as
amino acids, major inorganic nutrients (calcium and iron), vitamins, and fatty acids, which
are necessary for life, growth, and tissue repair [8]. On the other hand, functional foods are
focused on beneficial improvements of one or more target functions in the body, beyond
adequate nutritional effects, and on a reduction of chronic health disorder incidences [9].
Functional foods can be mainly classified as fortified/enhanced products (amino acid,
vitamin, fatty acid, and mineral additions), natural products (fruits), and enriched products
(probiotics and prebiotics) [10–13]. In this context, probiotic foods are included in the
significant category of functional foods, as their incorporated probiotic strains interact
harmoniously with the intestinal microbiota, providing prophylactic and therapeutic effects
against various pathogens [7,14].
Probiotics are live microorganisms that, when administered in adequate amounts, confer
a health benefit on the host. Health benefits include anti-depression, anti-obesity, anti-diabetic,
and anti-cholesterol activities, as well as immunostimulant action and the secretion of func-
tional molecules [15–17]. After 2002, FAO/WHO published the “Guidelines for Evaluation
of Probiotics in Food” and established the criteria for characterizing a strain as probiotic.
These criteria deal with resistance to unfavorable conditions that the human body imposes,
epithelium adhesion ability, antimicrobial activity, and safety assessment. The relevant clinical
trials for probiotic characterization are known as placebo-controlled randomized clinical trials
(RCTs) and are performed via oral, rectal, and vaginal routes [18,19]. As far as the techno-
logical properties of the products enriched with probiotic strains are concerned, the aroma
and/or taste and/or acidity over the shelf life should not be adverse [20–22]. Additionally,
the probiotic must survive food-processing stressors, such as fermentation, freeze-drying,
variations in temperature, pH, and oxidative and osmotic stress during storage [23]. High
viabilities of the probiotic species during food storage are critical and are required. Specifically,
the minimum concentration of probiotic is approximately 106 –107 cfu/mL at the time of food
product consumption [14,20].
On the other hand, a significant matter rises as probiotic delivery has been mainly
employed through dairy products, even though they exhibit some drawbacks, such as
lactose intolerance, high fat content, and possible allergy effects [14]. These issues have
forced the research community along with the food industry to seek other food substrates
of probiotic delivery. In this vein, fruit juices seem to be a good alternative, while being
a nutritious, health-promoting, and disease-preventive source of food [24]. Fruit juices
display antioxidant, antimicrobial, and antimutagenic properties and provide consumers
with beneficial macronutrients that seems to positively be involved in genome damage
and repair [25,26]; hence, they can be considered appropriate substrates for the production
of functional food. In addition, fruit juices are rich in bioactive compounds that can
exert prebiotic properties, which enhance probiotic delivery and viability [14,27]. Apart
from the application of living probiotic cells for the production of functional beverages,
the incorporation of active biomolecules, antioxidants, and prebiotics can enhance food
functionality [28]. Significantly, the combination of both living probiotic cells and fruit
bioactive compounds can lead to the development of super foods.
In this vein, fermented fruit beverages meet functional production demands as they
are minimally processed foods with an improved nutritional value, improved sensorial fea-
tures, ameliorated shelf-life, and additional health-promoting derived components [29–31].
Specifically, pomegranate is a very good source of antioxidant phytochemicals as well as
natural antimicrobial compounds [32]. Moreover, pomegranate juice has been applied in
lactic acid fermentation, providing very promising results [31,33–36]. However, fruit juice
is a very harsh matrix as various obstacles can prevent microorganism growth. For instance,
low pH values and high viscosity are considered significant obstacles that a microorganism
should overcome in order to accomplish a juice fermentation procedure [30]. As a result,
the selection of the proper starter culture is of paramount importance and possible mod-
ifications/preparations of the fruit juice are considered critical [30,33]. Kefir grains are
Fermentation 2022, 8, 142 3 of 13

considered a very good source of probiotics, and scientific research has considering the
isolation of novel LAB strains of multifunctional probiotic characteristics [37–41].
In this frame, acid tolerant L. paracasei SP3, recently isolated from kefir grains [42] will
be applied for the first time in pomegranate juice fermentation, targeting the production
of a functional beverage. The strategy adopted in the current study is the valorization of
this novel L. paracasei SP3 in pomegranate juice fermentation in parallel with the quality
of the produced beverage, according to consumers acceptability and nutritional value
(antioxidant activity and total phenolic content). The level of astringency is also assessed
though the determination of the hydrolysable tannin content.

2. Materials and Methods

2.1. Strain Selection and Growing Conditions
The bacterial strain Lactobacillus paracasei SP3, prior isolated from traditional kefir
grains, as described before [42], was selected for the fermented pomegranate juice product
according to its potential probiotic character and tolerance to acidic environments, as well
as its potential postbiotic characteristics [43,44].
The bacterial strain was kept in the Laboratory of Food Processing of Democritus
University of Thrace. It was stored at −80 ◦ C in vials containing glycerol (50%) and
distilled sterile water (50%), and was activated in de Man, Rogosa, and Sharpe broth
(Merck, Darmstadt, Germany), and was placed overnight in a heat chamber (37 ◦ C for
18 h). The growth of L. paracasei SP3 was performed under anaerobic conditions at 37 ◦ C
for 48 h in de Man, Rogosa, and Sharpe broth (Merck, Darmstadt, Germany). The biomass
of the bacterial culture was harvested through centrifugation (5000 rpm, 10 min, 25 ◦ C)
under aseptic conditions (Sigma 3K12, Bioblock Scientific, Illkirch, France). All media were
sterilized by autoclave for 15 min (120 ◦ C, 1–1.5 atm) prior to use.

2.2. Production of Fermented Pomegranate Beverage

Pomegranate fruits were purchased from the local market of Nea Orestiada (Greece)
and fresh juice was directly produced via mechanical crushing and pressing the seeds
(after washing and peeling) for 10 min. Adjustment of the initial sugar concentration of
the fresh juice was conducted to 80 g/L, through minimal (10%) addition of sterilized,
deionized water (juice pH 3.2). Afterwards, 100 mL of the prepared pomegranate juice
solution was transferred a previously sterilized conical flask and pasteurized at 80 ◦ C for
5 min [45]. After cooling at room temperature,1 g of L. paracasei SP3 was added in 100 mL
of a fermentation substrate in biological duplicates. For comparison reasons, a conical flask
containing 100 mL of pomegranate juice with the same sugar adjustment and pH as the
fermented sample was prepared as the control sample. The samples were maintained at
30 ◦ C for 1 day and subsequently at 4 ◦ C for 28 days.

2.3. Chemical Analysis of Produced Pomegranate Beverages

The ethanol concentration and residual sugar were quantified through high-performance
liquid chromatography/HPLC (Shimadzu) using a Shimadzu HPLC system (Shimadzu,
Kyoto, Japan), as described previously [46]. The system consisted of a SCR-101N stainless
steel column (7.9 m × 300 mm i.d., Sigma, St. Louis, MO, USA), a LC-9A pump, a CTO-10A
oven set at 60 ◦ C, and a RID-6A refractive index detector. Ultra-pure water obtained by a
Milli-Q water (Millipore, Milan, Italy) purifying system (resistivity 18.2 MΩ cm−1 ) was used
as the mobile phase with a flow rate of 0.8 mL/min, while 1-butanol (0.1% v/v) was used as
the internal standard. Pomegranate beverage samples were double filtered through 0.2 µm
microfilters and 20 µL was injected directly into the column.
Organic acids were quantified through ion-exchange liquid chromatography/HPLC
(Shimadzu), as described before [47]. For the quantitative analysis, standard solutions of
acetic and lactic acid (Merk, Taufkirchen, Germany) were prepared in pure water (Milli-Q,
Merk, Taufkirchen, Germany). The determinations of the pomegranate samples were
carried out by means of standard curves.
Fermentation 2022, 8, 142 4 of 13

Folin−Ciocalteu reagent was applied for the evaluation of the total phenolic content
(TPC) of pomegranate beverages and the content was determined based on colorimetric
reduction, as described previously [45,48]. TPC was expressed as mg of gallic acid equiv-
alents (GAE/100 mL juice). The ABTS radical cation decolorization assay was used in
order to determine antioxidant activity (AA) [45,49]. AA was expressed as mg of Trolox
equivalent (TE/100 mL juice).
All of the results are presented as the means of three repetitions plus standard deviations.

2.4. Hydrolysable Tannins Content

The content of hydrolysable tannins was determined by the method applied by Hager-
man and Butier [50]. Briefly, 1 mL of pomegranate juice was mixed with 5 mL methanol. In
1 mL of the upper phase, 2 mL of an acetate buffer (pH 5.0) containing 0.1% w/v BSA and
0.99% w/v NaCl was added. Subsequently, centrifugation (Sigma 3K12, Bioblock Scientific,
France) was applied at 3000 rpm for 15 min. A reagent was produced containing 1% w/v
SDS and 5% w/v triethanolamine, and was applied (4 mL) in order to dissolve the tannins
by precipitation for 1 h at 37 ◦ C. Finally, 1 mL of reagent containing 0.27% FeCl3 6H2 O and
0.1 NHCl was added to the solution and then the whole mixture was shaken for 30 min
in a vortex (Scientific Industries, Genie-2, USA). The hydrolysable tannin content was
determined with a spectrophotometer, (510 nm) and was expressed as catechin equivalents
in mg/L by comparison with a standard curve [51]. All of the results are presented as the
means of at least three repetitions plus standard deviations.

2.5. Microbiological Analysis

From each pomegranate beverage, a sample of 10 mL was collected aseptically at
various time intervals (0/T0 : the day of production, and days 1, 7, 14, 21, and 28) right after
fermentation and during storage at 4 ◦ C. In addition, a sample just before fermentation
was collected in the same manner. Pomegranate beverage samples were serially diluted
under aseptic conditions (open flame, sterilized prior in a Laminar cabinet) in 90 mL
1/4 strength Ringer’s solution and placed in sterile bag-mixers and homogenized in a
stomacher (Bagmixer 400, Model VW, Interscience).
Viable cell counts of each species—yeasts and fungi, enterobacteria, coliforms, Staphy-
lococci, and the selected L. paracasei strain—were enumerated by pouring plating (0.1 mL or
1 mL) of appropriate dilutions on the selective media under aseptic conditions, considering
the instructions given by the manufacturer (Merck, Germany). In brief, the yeasts and
fungi were enumerated on Sabouraud Chloramphenicol Agar (incubation at 30 ◦ C for 72 h),
enterobacteria were enumerated on Violet Red Bile Glucose Agar (VRBGA; incubation at
37 ◦ C for 24 h), coliforms were enumerated on Violet Red Bile agar (VRBA; incubation at
30 ◦ C for 24 h), and Staphylococci were enumerated on Baird Parker agar (BP; incubation
at 37 ◦ C for 48 h) [52].
Viable counts of the selected strain L. paracasei SP3 were enumerated on acidified MRS
agar (Merck, Germany) at 37 ◦ C for 72 h, anaerobically (Anaerobic jar, Anerocult C, Merck,
Germany). All of the media were sterilized by autoclaving at 120 ◦ C and at 1–1.5 atm for
15 min, and were then cooled before use.
In addition, the survival rate (SR%) of the selected starter culture (L. paracasei SP3)
was assessed according to the following equation, aiming to evaluate the significance of
viability rates:
N
SR% = × 100 (1)
No
The equation was based on cell counts (log CFU/g) of the selected Lactobacilli strain
enumerated in each pomegranate beverage sample: No represents the viable cell counts
(log CFU/g) from the day of production (T0 ) and N represents the viable cell counts (log
CFU/g) of each day tested during cold storage [52]. The results of the survival rate were
expressed as % of L. paracasei viability.
Fermentation 2022, 8, 142 5 of 13

2.6. Assessors Acceptance Test: Preliminary Sensory Evaluation

The sensory attributes of the fermented pomegranate beverage were compared with
the produced unfermented pomegranate juice after the end of the juice fermentation and at
the final day of cold storage (4 ◦ C) [38]. A panel of 25 (age average 22–58 years old) frequent
consumers of fruit juices was conducted. The samples were placed in panel booths and
served directly from refrigerated storage in glass containers usually applied for wine tasting
(glasses of 155 mm height, total volume of 215 mL). Each glass was covered with a sterile
Petri dish and filled with approximately ~25 mL of juice [53]. Each sample was coded with
a different three-digit number and was served in a randomized order. Initially, the panel
was asked to assess the intensity of the aroma and taste based on a 0–10 preference scale
(0 represents an unacceptable product, while 10 represents a product of superior quality).
Likewise, astringency was also evaluated. Finally, the panel was asked to comment on
whether they would prefer one of the two beverages, and if they noticed any significant
difference between the two.

2.7. Statistical Analysis

All experiments were repeated three times and the results are expressed as mean ±
standard deviation. Microbial populations were expressed as Log10 CFU/mL. The data
obtained from the ethanol, organic acids, and residual sugar concentrations; total phenolic
content; antioxidant activity; and L. paracasei SP3 cell viability of the non-fermented and
fermented pomegranate juice were analyzed for their mean differences using the analysis
of variance (ANOVA) procedure, followed by Duncan’s post hoc multiple range test, in
order to highlight the explicit differences between the various treatments. Analysis was
performed by using IMB SPSS v20 (IBM Corp., Armonk, NY, USA) at an alpha level of 5%.

3. Results and Discussion

L. paracasei SP3 previously isolated from kefir grains with potential probiotic prop-
erties [42] was examined in pomegranate juice fermentation for an evaluation of its tech-
nological abilities with a satisfied outcome [54]. Fruit juices such as pomegranate juice
have gained a lot of interest the last years as a substrate for probiotic delivery versus dairy
products [12,55–57]. As the most important drawback of fruit juices is their low pH values,
more and more potential probiotic or certain probiotic strains have been applied in these
fermentations. The main reason for this is that these strains are considered pH tolerant, as
one of the in vitro tests that should succeed is the examination of their resistance to low
pH [37,55]. Therefore, L. paracasei SP3 can be considered as an acid tolerant strain because
through in vitro tests, it exhibits satisfactory levels of viability (7.1~8.5 log cfu/mL) with
pH values between 2.0 and 4.0 [43], and it could be appropriate for fruit juice fermentation,
where the pH values are usually very low.

3.1. Ethanol, Organic Acids and Residual Sugar Concentrations

The levels of sugars, organic acids (lactic acid and acetic acid), and ethanol in pomegranate
juice during fermentation (30 ◦ C for 24 h) and storage (4 ◦ C for 4 weeks) are presented in
Table 1.
The concentration of lactic acid increased (statistically significantly) in all of the studied
time periods, reaching a final value of 4.8 g/L, while acetic acid was only slightly produced
(0.6 g/L). The total sugars were decreased significantly, reaching (25.4% reduction) their
lowest value in the 4th week of storage (58.4 g/L), while alcohol was marginally detected
(0.4 g/L) at the end of storage. All of the above results demonstrate that L. paracasei SP3
was effective in the lactic acid fermentation of pomegranate juice, even during cold storage
for 4 weeks. Respectable amounts of lactic acid were produced, while the low alcohol
content (<1% v/v) met the standards set for low or non-alcoholic beverages [58]. Acetic acid
was produced in low concentrations, probable due to the enzymatic degradation of citric
acid naturally present in pomegranate juice, as other researchers have already reported
in the lactic acid fermentation of fruit juices [59]. The pH value of the fermented juices
Fermentation 2022, 8, 142 6 of 13

was monitored in all of the studied periods. No significant alteration was recorded. In
particular, the initial pH value was 3.2 and varied between 3.2 ± 0.2 for all of the time
periods, due to the high buffering capacity of the fermented juices, as other researchers
have reported in similar experiments [60].

Table 1. Analysis of sugars, organic acids, and ethanol in pomegranate juices fermented by L. paracasei
SP3 after fermentation (24 h at 30 ◦ C) and during 4 weeks of storage at 4 ◦ C.

Sugars Lactic Acid Acetic Acid Ethanol

Time
(g/L) (g/L) (g/L) (% v/v)
24 h 78.3 a ± 0.5 1.4 e ± 0.5 ND ND
Week 1 74.8 b ± 0.6 2.6 d ± 0.3 ND 0.3 a ± 0.1
Week 2 70.5 c ± 0.4 3.2 c ± 0.3 0.3 c ± 0.1 0.3 a ± 0.1
Week 3 63.3 d ± 0.5 4.0 b ± 0.4 0.6 b ± 0.1 0.4 a ± 0.1
Week 4 58.4 e ± 0.5 4.8 a ± 0.2 0.6 a ± 0.1 0.4 a ± 0.1
Similar superscript letters in columns denote no significant differences at an alpha = 0.05 (ANOVA and Duncan
Post Hoc Multiple Comparisons), ND: not detected (<0.1 g/L).

3.2. Microbial Stability and Starter Culture Survival Rate

Microbial stability is crucial in fruit juices, as spoilage microorganisms (especially
yeast and fungi) may alter a product’s characteristics and subsequently alter the nutritional
value of the juice [61].
In the present study, an initial thermal conditioning was employed to pomegranate
juices aiming to minimize any effects of yeast or fungi, which are well known as natural
fruit microflora [62]. Thus, an evaluation of the possible spoilage by yeasts, fungi, and
coliforms was carried out in all pomegranate beverages (data not shown). The results
showed that the initial thermal treatment was sufficient as no yeast or fungi were detected
after pomegranate juice fermentation or during the four weeks of storage at 4 ◦ C. Moreover,
possible coliform, Staphylococci, or enterobacteria that could appear through processing
(e.g., water treatments/human source) were not detected in any stages of production and
storage [63].
The levels of viability of L. paracasei SP3 after fermentation and during the four weeks
of cold storage (4 ◦ C) are presented in Figure 1. Initially, the viability of the potentially pro-
biotic L. paracasei SP3 was 9.2 log CFU/mL and was maintained at high levels throughout
4 weeks of storage (7.2 log CFU/mL at the 4th week of storage). These recorded viability
values were above the limit of 6–7 log cfu/mL, which is required for probiotic products [14].
It is worth mentioning that no adjustment in the pH value of the juice was applied, while
in the low pH juice environment, the survival rates of the starter culture remained high
(Figure 2). This is a significant outcome, because it highlights that the novel strain can sur-
vive the harsh environment of pomegranate juice (low pH and antimicrobial compounds)
and retain high viability rates for approximately 1 month of cold storage. In particular, the
decline in the L. paracasei SP3 viability rate was determined to be approximately 20% during
cold storage, which was a decisive issue of the industrialization potential, as minimal
processing treatments are required for the production of pomegranate beverages [3].
The preservation of the cell viability of potential probiotic strains has been reported by
other researchers during the lactic acid fermentation of fruit juices, even in cold storage con-
ditions. Specifically, other potentially probiotic L. paracasei strains applied for pomegranate
juice and cornelian cherry juice fermentation have achieved high viability levels during cold
storage [45,64]. This finding can be attributed to two main reasons, namely: (i) L. paracasei
SP3 is an acid tolerant strain [42] and (ii) components of a plant origin may possess prebiotic
properties and, consequently, ameliorate the growth of LAB [59,60]. For example, black
rice is known to exhibit a prebiotic activity by promoting the growth of Bifidobacteria and
Lactobacilli [65]. In addition, it has been reported that ellagitannins appear to be favorable
for the viability of LAB [66]. Likewise, the composition of pomegranate juice with high
levels of ellagitannins could enhance LAB growth and the survival rate.
Fermentation 2022, 8, x FOR PEER REVIEW 7 of 1

Fermentation 2022, 8, 142

potential, as minimal processing treatments are required for the production 7ofofpomegran
13

ate beverages [3].

Figure1. 1.
Figure Viability
Viability of paracasei
of L. L. paracasei
SP3 SP3
(log (log CFU/mL)
CFU/mL) after after production
production (D0during
(D0 ) and ) and during cold storage (
cold storage
°C),
◦ similar superscript letters in bars denote no significant differences at an alpha = 0.05 (ANOVA
(4 C), similar superscript letters in bars denote no significant differences at an alpha = 0.05 (ANOVA
and Duncan Post Hoc Multiple Comparisons).
and Duncan Post Hoc Multiple Comparisons).

Figure2. 2.
Figure Survival
Survival raterate
(SR(SR %)the
%) of ofstarter
the starter culture
culture (L. paracasei
(L. paracasei SP3)
SP3) after after manufacture
manufacture and durin
and during
coldstorage
cold storage(4 (4
◦ C)°C)
forfor 28 days.
28 days.

3.3. Total Phenolic Content and Antioxidant Activity

The preservation of the cell viability of potential probiotic strains has been reporte
The total
by other phenolicsduring
researchers content the
(TPC) andacid
lactic antioxidant activityof
fermentation (AA) ofjuices,
fruit fermented
even(FJ)
in and
cold storag
unfermented (UFJ) juice was also monitored, and the results are presented in Table 2. The
conditions. Specifically, other potentially probiotic L. paracasei strains applied for pome
TPC value of freshly prepared pomegranate juice (fermentation time 0) was approximately
granate juice and cornelian cherry juice fermentation have achieved high viability level
101 ± 10 mg GAE/100 mL. UFJ was also studied in the same period until the 4th week at
4during coldto
◦ C, in order storage
display[45,64]. This
the effect finding
of juice can be attributed
fermentation to two
by L. paracasei SP3.main reasons, namely
(i) L.Atparacasei SP3 is an acid tolerant strain [42] and (ii) components
the beginning of fermentation with the starter L. paracasei SP3, the values of a plant origin ma
of TPC
possess
were prebiotic
boosted properties
and were and, consequently,
always statistically significantameliorate the growth
higher compared of LAB
to the TPC [59,60]. Fo
of UFJ.
Specifically,
example, blackthe TPC
riceofisFJknown
reachedto itsexhibit
highest avalue in the activity
prebiotic 3rd weekby of promoting
storage (201.1 mg
the growth o
GAE/100 mL), while the TPC of UFJ continuously decreased, reaching its lowest
Bifidobacteria and Lactobacilli [65]. In addition, it has been reported that ellagitannins ap value in
the
pear4thtoweek of storagefor
be favorable (58.4
themg GAE/100
viability mL). The
of LAB [66].same outcome
Likewise, thewas observed in
composition ofthe
pomegran
case of the antioxidant activity (AA) determination of FJ and UFJ. Specifically, the AA of the
ate juice with high levels of ellagitannins could enhance LAB growth and the survival rate
freshly prepared pomegranate beverage was about 107 ± 10 mg TE/100 mL (fermentation
time 0). The values of AA of FJ were statistically significantly higher compared to the AA of
Fermentation 2022, 8, 142 8 of 13

UFJ in all of the studied periods. The highest value for AA of FJ was observed in the 1st week
of storage (148.5 mg TE/100 mL), while the AA of UFJ decreased, reaching its lowest value
in the 4th week of storage (53.4 mg TE/100 mL). Enhancements in the total phenolic content
could be attributed in the enzymatic transformation of the initial complex phenolics by
LAB, through decarboxylation and reduction reactions to other simpler compounds, which
increased the quantitative amount of TPC [30,67–69]. Specifically, the metabolic activities
of the L. paracasei strains seemed to effectively modify the levels and the composition of
the bioactive phenolic compounds of pomegranate juice, due to certain enzymes that they
contained (β-glucoside and β-galactoside), as others researchers have reported in similar
studies [36,69,70].

Table 2. Determination of the total phenolic content and antioxidant activity of unfermented (UFJ)
and fermented pomegranate juice (FJ) with L. paracasei SP3 for the first 24 h at 30 ◦ C and during
storage at 4 ◦ C for 4 weeks.

Total Phenolic Content Antioxidant Activity

Time
(mg GAE/100 mL) (mg TE/100 mL)
UFJ FJ UFJ FJ
a 124.5 a
24 h 98.5 b ± 11 123.7 ± 14 101.7 b ± 10 ± 11
Week 1 87.3 b ± 15 157.7 a ± 21 99.3 b ± 11 130.2 a ± 29
Week 2 80.2 b ± 11 201.1 a ± 16 88.7 b ± 13 144.7 a ± 12
Week 3 73.4 b ± 13 190.9 a ± 28 69.1 b ± 11 148.5 a ± 17
Week 4 70.8 b ± 17 179.3 a ± 22 53.4 b ± 18 144.8 a ± 19
Similar superscript letters in columns denote no significant differences at an alpha = 0.05 (ANOVA and Duncan
Post Hoc Multiple Comparisons).

3.4. Hydrolysable Tannins

Tannins affect sensorial properties of fruit juice, in both appearance and flavor, causing
an astringent sensation in the juice [71]. Likewise, the reduction of astringency is a very
important issue in many fruit juices, such as pomegranate. The main categories of tannins
are condensed and hydrolysable tannins [72]. Ellagitannins are the major category of
hydrolysable tannins in pomegranate juice. Their formation takes place, when ellagic acid
binds with a carbohydrate, like punicalin and punicalagin [32,73]. During the industrial
extraction process, they are introduced in high levels into the juice, due to their hydrophilic
properties [74]. The application of lactic acid fermentation in fruit juices seems to overcome
this problem to a satisfactory level [35]. This assumption was verified in the present study,
according to the results presented in Figure 3.
The levels of hydrolysable tannins were reduced statistically significantly (approxi-
mately 42%) during the whole period of fermentation and storage. Before fermentation,
the concentration of the hydrolysable tannins level was 589 mg/L and after 28 days it was
341 mg/L. It should be also underlined that a statistically significant decrease was moni-
tored in every time period examined, as well as during the fermentation of pomegranate
juice and cold storage. In addition, no significant changes were recorded regarding the
unfermented juices, where hydrolysable tannins remained almost constant during the time
period examined. These results are in accordance with other researchers, who demon-
strated that lactic acid fermentation is a significant tool for mitigating this drawback, even
though the type of strain and the fruit juice applied are critical [35]. A main explanation for
the decrease in the astringency of pomegranate is that transformed phenolic compounds
(possible produced by the degradation of tannins) do not induce astringency. On the other
hand, sensorial tests are considered crucial in order to further evaluate the level and the rate
of astringency in fruit juices, including pomegranate, because others phenolic compounds
besides tannins could provoke astringency [72,74].
Fermentation 2022,8,8,142
2022,
Fermentation x FOR PEER REVIEW 9 of 13
9 of 14

Figure 3.
Figure 3. Hydrolysable
Hydrolysabletannins
tanninsofoffermented
fermentedpomegranate
pomegranate beverages; similar
beverages; superscript
similar letters
superscript in in
letters
bars denote no significant differences at an alpha = 0.05 (ANOVA and Duncan Post Hoc Multiple
bars denote no significant differences at an alpha = 0.05 (ANOVA and Duncan Post Hoc Multiple
Comparisons).
Comparisons).
The levels
3.5. Sensory of hydrolysable tannins were reduced statistically significantly (approxi-
Evaluation
mately 42%) during the whole period of fermentation and storage. Before fermentation,
The results regarding the preliminary sensory evaluation for the evaluation of the
the concentration of the hydrolysable tannins level was 589 mg/L and after 28 days it was
produced FJ and UFJ in terms of aroma, taste, and astringency are presented in Table 3.
341 mg/L. It should be also underlined that a statistically significant decrease was moni-
toredStatistically,
in every timesignificant differences
period examined, were
as well found the
as during for fermentation
the 2nd, 3rd, of and 4th week of
pomegranate
cold storage between UFJ and FJ, where FJ was scored better
juice and cold storage. In addition, no significant changes were recorded regarding by consumers. Regarding
the
astringency,
unfermented juices, where hydrolysable tannins remained almost constant during athe
FJ was found to be more attractive by consumers and developed statis-
tically
time period examined. These results are in accordance with other researchers, who the
significant difference compared with UFJ from the 1st week of storage until
last one. This outcome
demonstrated that lacticagrees with the determined
acid fermentation contents
is a significant of the
tool for hydrolysable
mitigating tannins
this draw-
(Figure
back, even though theL.type
3). Likewise, paracasei SP3and
of strain seems
the to be juice
fruit a good alternative
applied for the
are critical fermentation
[35]. A main
of pomegranate
explanation juice,
for the leading
decrease to aastringency
in the final product with improved
of pomegranate sensorial
is that characteristics
transformed phe-
and
nolicacompounds
lower astringency.
(possibleThe latter isby
produced very
the important,
degradationasofastringency
tannins) do isnotconsidered a draw-
induce astrin-
back
gency. inOn
thethecommercialization
other hand, sensorial of some fruit
tests are juices [72,75–77].
considered Thesetovery
crucial in order hopeful
further results
evaluate
are in agreement with other researchers, who have reported that lactic
the level and the rate of astringency in fruit juices, including pomegranate, because others acid fermentation
enhances the aromatic
phenolic compounds profile
besides and flavor
tannins of pomegranate
could provoke astringencyjuice [31]. However, this phe-
[72,74].
nomenon depends on the type of strain used for fermentation and other parameters, such
3.5.fermentation
as Sensory Evaluation
temperature [78]. More research is needed in the future applying GC-MS
analysis, in order
The results to correlate
regarding the this outcomesensory
preliminary with the certain volatile
evaluation compounds
for the evaluation ofthat
the the
fermented
produced FJ juice
andpossess. Finally,
UFJ in terms the evaluators
of aroma, taste, andfound that the
astringency arefermented
presentedbeverage
in Table 3.would
be preferable as it has attributes of good aroma and a smoother taste compared to the
unfermented juice. At this stage, this outcome is quite encouraging and gives more value
to the final product.

Table 3. Preliminary sensory evaluation of fermented (FJ) and non-fermented (UFJ) pomegranate
juice after fermentation (24 h at 30 ◦ C) and over 4 weeks of storage at 4 ◦ C.

Time Substrate Aroma Taste Astringency

0h Fresh juice a
8.8 ± 0.1 a
8.8 ± 0.3 8.0 a ± 0.3
UFJ 8.4 1b ± 0.2 8.4 1a ± 0.2 7.5 1a ± 0.2
24 h
FJ 8.3 1b ± 0.2 8.3 1a ± 0.3 7.8 1a ± 0.1
UFJ 7.5 1b ± 0.2 7.8 1b ± 0.3 7.1 1b ± 0.3
Week 1
FJ 7.7 1b ± 0.1 7.7 1b ± 0.1 7.7 1a ± 0.2
UFJ 7.0 1b ± 0.1 7.1 1b ± 0.1 6.7 1c ± 0.3
Week 2
FJ 7.3 2b ± 0.1 7.5 2b ± 0.2 7.6 2a ± 0.2
Fermentation 2022, 8, 142 10 of 13

Table 3. Cont.

Time Substrate Aroma Taste Astringency

UFJ 6.5 1b ± 0.2 6.41b ± 0.2 6.3 1b ± 0.2
Week 3
FJ 7.3 2b ± 0.1 6.8 2b ± 0.1 7.4 2b ± 0.3
UFJ 6.0 1b ± 0.3 6.0 1b ± 0.2 6.1 1b ± 0.1
Week 4
FJ 6.9 2b ± 0.1 6.7 2b ± 0.1 7.1 2b ± 0.2
Similar superscript letters (comparison of FJ and UNFJ only to fresh juice) and numbers (comparison only between
FJ and UNJ for the same time period) in columns denote no significant differences at an alpha = 0.05 (ANOVA
and Duncan Post Hoc Multiple Comparisons).

4. Conclusions
The outcome of the present study showed that the novel potentially probiotic L.
paracasei SP3 was successfully applied in the fermentation of pomegranate juice (at 30 ◦ C
for 1 day and at 4 ◦ C for 28 days). Specifically, fermented pomegranate juice (i) exhibited
improved AA and TPC contents, (ii) high viability, (iii) high levels of organic acids, and
(iv) acceptable sensorial features. In addition, the fermentation of pomegranate juice
with L. paracasei SP3 significantly reduced the juice astringency, due to the lower levels of
hydrolysable tannins. It should be underlined that fruit juices comprise a difficult substrate
for fermentation, due mainly to their low pH values. In this context, the prospects of L.
paracasei SP3 seem attractive, as in the frame of the present study, it was verified as an
effective acid tolerant strain that retained its viability without the application of protective
techniques, such as encapsulation.

Author Contributions: Conceptualization, S.P. and I.M.; methodology, I.M., A.T. and S.P.; software,
A.B. and S.P.; validation, I.M., A.T. and A.B.; formal analysis, I.M. and A.T.; investigation, I.M.;
resources, A.B. and S.P.; data curation, I.M. and A.T.; writing—original draft preparation, S.P.;
writing—review and editing, S.P.; visualization, S.P.; supervision, S.P.; project administration, S.P.;
funding acquisition, 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: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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