Access to this full-text is provided by MDPI.
Content available from Foods
This content is subject to copyright.
Citation: Moloto, M.R.; Akinola, S.A.;
Seke, F.; Shoko, T.; Sultanbawa, Y.;
Shai, J.L.; Remize, F.; Sivakumar, D.
Influence of Fermentation on
Functional Properties and
Bioactivities of Different Cowpea
Leaf Smoothies during In Vitro
Digestion. Foods 2023,12, 1701.
https://doi.org/10.3390/
foods12081701
Academic Editor: Carolina
Claudia Schebor
Received: 12 March 2023
Revised: 7 April 2023
Accepted: 15 April 2023
Published: 19 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
foods
Article
Influence of Fermentation on Functional Properties and
Bioactivities of Different Cowpea Leaf Smoothies during In
Vitro Digestion
Mapula R. Moloto 1, Stephen A. Akinola 1, Faith Seke 1, Tinotenda Shoko 1, Yasmina Sultanbawa 2,
Jerry L. Shai 3, Fabienne Remize 4and Dharini Sivakumar 1, 2, *
1Phytochemical Food Network Group, Department of Crop Sciences, Pretoria 0001, South Africa;
mapularebahlotsem@gmail.com (M.R.M.); akinolasa@tut.ac.za (S.A.A.); fayeesk@gmail.com (F.S.);
shokot@tut.ac.za (T.S.)
2Australian Research Council Industrial Transformation Training Centre for Uniquely Australian Foods,
Queensland Alliance for Agriculture and Food Innovation, Centre for Food Science and Nutrition, The
University of Queensland, Elkhorn Building (#1024), 80 Meiers Road, Indooroopilly,
Brisbane, QLD 4068, Australia; y.sultanbawa@uq.edu.au
3
Department of Biomedical Sciences, Tshwane University of Technology, Arcadia, Pretoria 0001, South Africa;
shailj@tut.ac.za
4SPO, Universitéde Montpellier, Universitéde La Réunion, Institut Agro, INRAE, 2 Place Viala,
F-34000 Montpellier, France
*Correspondence: sivakumard@tut.ac.za or dharinisivakumar@yahoo.co.uk
Abstract:
This study investigated the effects of Lactiplantibacillus plantarum 75 (LAB 75) fermentation
at 37
◦
C for 48 h on the pH, total soluble solids (TSS), colour, total titratable acidity (TTA), carotenoids,
and bioactivities of cowpea leaf smoothies from three cultivars (VOP 1, VOP 3, and VOP 4). Fermen-
tation reduced the pH from 6.57 to 5.05 after 48 h. The TTA increased with the fermentation period,
whilst the TSS reduced. Fermentation of the smoothies resulted in the least colour changes (
∆
E) in
VOP 1 after 48 h. Fermentation of cowpea smoothies (VOP 1, VOP 3, and VOP 4) improved the
antioxidant capacity (FRAP, DPPH, and ABTS), which was attributed to the increase in total phenolic
compounds and carotenoid constituents in all of the fermented cowpea smoothies. VOP 1 was further
selected for analysis due to its high phenolic content and antioxidant activity. The VOP 1 smoothie
fermented for 24 h showed the lowest reduction in TPC (11%) and had the highest antioxidant (FRAP,
DPPH, and ABTS) activity. Ltp. plantarum 75 was viable and survived the harsh conditions of the
gastrointestinal tract, and, hence, could be used as a probiotic. VOP 1 intestinal digesta showed
significantly higher glucose uptake relative to the undigested and the gastric digesta, while the gastric
phase had higher levels of α-amylase and α-glucosidase compared to the undigested samples.
Keywords:
in vitro gastrointestinal digestion; antioxidant properties; carotenoids; postharvest processing
1. Introduction
Cowpea (Vigna unguiculata) leaf consumption is very common in Africa and the
leaves have been reported to be part of the food security crops. Cowpea leaves are a very
good source of polyphenols and carotenoids [
1
,
2
]. In a previous study, digestion affected
the stability of cowpea carotenoids, namely,
α
-carotene, all-trans
β
-carotene, zeaxanthin,
9-cis-
β
-carotene, and lutein. When compared to undigested cowpea leaves, antioxidant
activity was lowered in the intestinal fraction during digestion (VOP 1, VOP 4, and VOP 3).
The strong antidiabetic properties of cowpea leaves are associated with their high levels
of expression in the glucose transporter genes in muscle cells [
1
,
2
]. Polyphenols from
cowpea leaf extract were found to have strong inhibitory properties against
α
-amylase and
α
-glucosidase [
2
]. The aforementioned enzymes are crucial in regulating obesity and blood
glucose levels because of their ability to restrict the re-absorption of glucose in the gut.
Foods 2023,12, 1701. https://doi.org/10.3390/foods12081701 https://www.mdpi.com/journal/foods
Foods 2023,12, 1701 2 of 20
The inhibition of carbohydrate hydrolysing enzymes (
α
-amylase and
α
-glucosidase) was
significantly lessened during digestion [
2
]. Zeaxanthin, all-trans-
β
-carotene,
α
-carotene,
9-cis-
β
-carotene, and lutein in cowpea cultivar leaves were linked to antioxidant properties
and an inhibitory effect on α-amylase and α-glucosidase activity [2].
Indigenous African leafy vegetables contribute significantly to Africa’s food security,
especially in rural and peri-urban settings. However, a significant number of indigenous
vegetables are lost due to improper and inadequate storage, packaging, transport, and
handling technologies [
3
,
4
]. Various postharvest preservation techniques have been devel-
oped and these include blanching, air-drying, and solar drying [
4
,
5
]. Food drying could
typically lead to product degradation from all vantage points, including sensory, physico-
chemical, and nutritional. Conventional drying techniques are more prone to mechanical
and chemical deterioration in the finished product [
6
]. As a result, it is critical to consider
other postharvest techniques for fruits and vegetables and to select appropriate conditions
that will minimize potential changes. One of the techniques that have been developed but
have received little to no attention is green vegetable fermentation. Fermentation is thought
to be a straightforward and efficient biotechnology process that aids the preservation of
vegetable safety and sensitivity and improves nutrition and shelf life. Due to its benefits
for cost-effectiveness and environmental protection, fermentation is used extensively in
food preservation around the world [
7
]. The health-promoting qualities of fermented
vegetable products include antioxidant activities, improved gastrointestinal health, antibac-
terial activities, reduced cardiovascular risk factors, and anti-inflammatory action. These
advantages of fermented vegetables could be attributed to the present carotenoids and
phenolic compounds [
8
]. Traditional green vegetables are fermented in Africa, but are less
common than cereals, tubers, meat, milk products, and alcoholic beverages [9].
Lactic acid fermentation is one of the traditional and affordable methods of food
preservation and protection in rural settings [
10
]. During the fermentation process, food
carbohydrates are converted into acid and other by-products by lactic acid bacteria (LAB).
Lactic acid fermentation improves the nutritional components and the sensory quali-
ties, such as aroma, flavour, and consumer acceptability [
11
]. Bartkiene et al. [
12
] re-
ported the effects of lactic fermentation on tomato pulp using Lactobacillus sakei KTU05-6,
Pediococcus acidilactici KTU05-7, and Pediococcus pentosaceus KTU05-8 as inoculum. The
lacto-fermentation process affected the amount and the cis/trans ratio of lycopene and
α
-carotene, resulting in a higher cis/trans lycopene ratio. The higher cis-lycopene iso-
mers are preferentially micellarized due to being less prone to crystallization, oil solubil-
ity, and quick absorption by the intestinal cells compared to the all-trans forms [
13
,
14
].
In comparison to raw nightshade leaves, the fermentation of nightshade leaves with
Ltp. plantarum 75 showed that fermentation increased the concentration of ascorbic acid
after three days, reduced colour changes, and increased the total polyphenol content and
antioxidant activity [
15
]. Managa et al. [
16
] also found that Ltp. plantarum 75 boosted the
phenolic content and antioxidant activity of chayote leaf and pineapple smoothies after
fermentation. Gao et al. [
17
] reported improved total phenol, polyphenols, and antioxidant
capacity in an indigenous vegetable Momordica charantia after lactic acid fermentation. On
the other hand, the ratio of a molecule’s availability following gastrointestinal digestion to
its availability before digestion is used to assess a molecule’s potential usefulness. Due to
oxidation or polymerization processes, the pH changes that take place during the gastroin-
testinal digestion phases result in phenolic derivatives with a high molecular weight and
poor solubility that are inaccessible for absorption [
18
]. With the help of lactic acid bacteria,
polyphenols can thus be biotransformed into molecules with improved bioavailability and
bioactivity [
19
]. Zhao et al. [
20
] found that
in vitro
digestion and fermentation by lactic
acid bacteria in tea extracts increased the antioxidant activity and cellular absorption of
phenolic components. This was also reported with pomegranate juice [
21
] and kiwi fruit
pulp [22] as well as Korean leek and cowpea leaves [23,24].
This work contributes by enhancing the low-cost fermentation method for the im-
proved utilization of cowpea leaves, which are already available in rural communities,
Foods 2023,12, 1701 3 of 20
thereby making a case for its commercialization. Furthermore, when fewer optimum
processes are used in cowpea leaf fermentation, the improved sensory qualities and health
benefits will drive consumer approval. As a result, there appears to be an opportunity to
increase efforts in Africa to study and adopt this type of biological preservation strategy for
leafy vegetables. In light of the abovementioned, the objectives of this study were to ascer-
tain the impact of the fermentation of Ltp. plantarum 75 on the physicochemical properties
of fermented cowpea leaf smoothies and to investigate the changes in the total phenol and
carotenoid components, inhibition of carbohydrate hydrolysing enzymes (
α
-amylase and
α
-glucosidase), antioxidant properties (FRAP, DPPH, and ABTS), and the bioaccessibility
of the fermented smoothies following an in vitro gastrointestinal digestion.
2. Materials and Methods
2.1. Chemicals and Reagents
Biokar Diagnostics (Solabia company, Pantin, France) and Conda Laboratories (Madrid,
Spain) supplied the culture media. The LAB strain used in this study (Ltp. plantarum 75)
was obtained from the culture collections of the microbiology laboratory at the QualiSud,
Universitéde La Réunion, France. All chemicals and reagents used in this study were of
analytical grade and were obtained from Sigma Aldrich (Johannesburg, South Africa), and
HPLC-grade reagents (≥99.8%) were used for the UHPLC analysis.
2.2. Plant Samples
Cowpea cultivars (VOP I, VOP 3, and VOP 4) were planted and propagated at Tshwane
University of Technology in Pretoria, South Africa, as detailed by Moloto [
1
]. Each cowpea
cultivar was planted in a randomized five-replicate configuration. Irrigation was carried
out at a rate of 100 mL per day. At the 8-leaf growth stage, clean leaves were plucked and
cleaned with tap water.
2.3. Preparation and Fermentation of Smoothie
The VOP 1, VOP 3, and VOP 4 cowpea fresh leaves were rinsed with tap water
containing 0.01% calcium hypochlorite to remove dirt and soil before cleaning in sterile
distilled water. Leaves were allowed to drain and were dried on a paper towel before
blending into smoothies in a Russell Hobbs blender. The mixture was then pasteurized for
10 min in a water bath at a core temperature of 60
◦
C and cooled to room temperature for
2 h before fermentation [15].
2.4. Reactivation of the Ltp. plantarum 75 Cultures and Fermentation of Cowpea Smoothies
The Ltp. plantarum 75 was reactivated, and the smoothies were fermented according to
Mashitoa [
18
]. The LAB culture was reactivated in MRS broth overnight at 30
◦
C, inoculated
into fresh MRS broth, and incubated for 48 h at 30
◦
C. The broth culture was centrifuged
at 8000
×
gfor 5 min, and the cells were washed in sterile saline water. At 660 nm, the
cell population was determined in a UV-Spectrophotometer, and the cell population was
adjusted to 0.05 McFarland standard concentrations (6 Log CFU/mL). One (1) mL of the
Ltp. plantarum 75 culture (6 Log CFU/mL) was inoculated into 100 mL of smoothies and
incubated at 37
◦
C for 2 days. During the incubation period, the smoothies were withdrawn
for analysis at 2, 24, and 48 h. The fermentation of smoothies using different cultivars was
performed in triplicate.
2.5. Physicochemical Properties of Fermented and Unfermented Cowpea Smoothies
Only pasteurized and fermented smoothies had their physicochemical parameters
tested at 0, 2, 24, and 48 h of fermentation. The pH of the samples was determined
using the EUTECH pH2700 Instrument (EUTECH Instruments, Illinois, IL, USA), and the
total soluble solids (TSS) was determined using the ATAGO PAL-3 pocket refractometer
(Atago USA Inc., Tokyo, Japan). The refractive index values obtained were saved in Brix.
The total titratable acidity of the samples was evaluated using the Reddy et al. [
5
] technique.
Foods 2023,12, 1701 4 of 20
The effect of fermentation on the colour characteristics of the smoothies was measured
using a CM-3500 d spectrophotometer and spectral magic NX software (Konica Minolta,
Konica Minolta Sensing Inc., Tokyo, Japan). The samples’ degree of brightness (L*), red-
to-green component (a*), and yellow-to-blue component (b*) were all measured. The total
colour difference (∆E) was calculated following Managa [15].
2.6. Organoleptic Properties of Unfermented and Fermented Cowpea Smoothie
A quantitative descriptive analysis technique, as described by Mashitoa [
19
], was
used for the sensory evaluation of the smoothies, with some modifications. Ten trained
panellists were selected from the pool of assessors trained to identify the desired char-
acteristics of the smoothies. The panellists were composed of healthy male and female
research employees. There were two training sections adopted, and the samples were rated
using a structured scale ranging from 0 to 9 (Absent = 0, 1–3 = weak, 4–6 = moderate,
7–9 = strong). Coded samples were served chilled in white cups with lids to the panellists
under a white light-illuminated cubicle. Panellists evaluated samples based on the agreed
attributes of the smoothies, and the ratings of samples were converted into intensity scores.
The colour perception was assessed using the light and dark colour perception in green
leafy vegetables as a reference. The characteristics of aroma and flavour were assessed
using veggie aroma in a common fresh leafy vegetable, The perception of texture in the
smoothie was based on its viscosity in the mouth and was assessed using glucose syrup
as reference. The assessment of sour taste and sweetness was based on the perception of
tart and sweet taste using diluted citric acid and diluted sucrose solution, respectively,
as references. A commercial fermented vegetable smoothie was used as a reference to
determine the overall acceptability of smoothies.
2.7. Total Sugars of Cowpea Smoothies
Total sugars were evaluated using a method described by Nielsen [
25
]. The polysac-
charide was hydrolysed with concentrated sulfuric acid to yield hydroxyl methyl furfural,
which was then condensed with phenol to yield a stable yellow-gold solution. To calculate
the total carbohydrate content, 1 mL of each test or standard solution was added to a
test tube. Then, 1 mL of a 5% phenol solution was introduced. A mechanical pipet was
used to swiftly add 5 mL of concentrated sulfuric acid to the entire solution while it was
being swirled in a vortex mixer. The mixture was then immediately combined and allowed
to react for 10 min. At this point, the solution absorbance was read at 488 nm using a
microplate reader (CLARIOstar Plus BMG Labtec, Lasec, Cape Town, South Africa).
2.8. Carotenoid Extraction, Identification, and Quantification
The carotenoids were extracted using a method described by Moloto et al. [
1
]. From
each smoothie powder, 5 g of samples were mixed with 4 mL of acetone, 95% ethanol, and
0.1% butylated hydroxytoluene (w/v). The samples were centrifuged for 10 min at 2500 rpm
(Eppendorf 5804R Centrifuge, Hamburg, Germany), and the supernatant was collected.
The extraction procedure was repeated four times with a 70:30 v/vmixture of hexane and
dichloromethane containing 0.1% butylated hydroxytoluene. The extracts were freeze-
dried and kept at a temperature of 80
◦
C. An HPLC–UV–DAD system (Shimadzu, Kyoto,
Japan) was utilized to identify the individual carotenoids. The dried extracts were diluted
in methanol, methyl tert-butyl ether (50:50, v/v) containing 0.1% butylated hydroxytoluene,
before analysis. Chromatographic separation was performed on a YCM C30 carotenoid
column (3.6
×
250 mm, 3.6
µ
m) (Waters, Milford, MA, USA) maintained at 25
◦
C, with
a mobile phase consisting of 0.1% formic acid in methanol (solvent A) and 0.1% formic
acid in MTBE (solvent B). The gradient program was used and the program was run as
described: 0 min, 80%, 20 min, 75%, 30 min, 30%, 33 min, 30%, and 36 min, 80%, at the flow
rate of 0.6 mL/min. Carotenoid standard concentrations (0 to 60 ppm) of lutein, zeaxanthin,
α
-carotene, 9-cis-
β
-carotene, and trans-
β
-carotene were used to quantify the carotenoid
Foods 2023,12, 1701 5 of 20
profiles in the smoothies. The calibration curves, the limit of detection (LOD), and the limit
of quantification (LOQ) are provided in the Supplementary Data, Table S1.
2.9. In Vitro Digestion of Cowpea Smoothies
The
in vitro
digestion of the cowpea leaf smoothies was performed on the VOP 1
cultivar. The VOP 1 cultivar was selected based on its high total phenols, antioxidant
activities, and carotenoid profile after fermentation for 24 h. Using a technique previously
reported by Seke et al. [
2
], an amount of simulated salivary fluid (10 mL) at pH 7 contain-
ing 75 U mL
−1
of
α
-amylase enzyme was added to 10 g of VOP 1, and the mixture was
homogenized with a pestle and mortar for 10 s to mimic chewing before being incubated
in a shaking water bath at 170 rpm for 2 min at 37
◦
C. The simulated gastrointestinal
fluids were prepared, as described by Seke et al. [
2
]. Simulated gastric fluid (20 mL) was
added to the oral digesta, and the gastric phase was initiated by adjusting the pH to 2.5 by
adding 6 M HCl and pepsin solution (2000 U mL
−1
in 0.1 M HCl, pH 2.2). The mixture
was stirred at 170 rpm for 2 h at 37
◦
C, and 10 mL samples were collected and cooled
on ice for 10 min to stop reactions and then stored at 80
◦
C. The intestinal and dialysis
phases were initiated by adding the simulated intestinal fluid (20 mL) to the remaining
gastric digesta, and the pH was adjusted to 7.5 using 2 M NaOH. The intestinal digesta
was transferred into a dialysis tube (10 cm, MW cut-off 10–12 kDa) and 5 mL of NaCl
(0.9%) and 5 mL of NaHCO
3
(0.5 M) were added. The mixture was placed inside the flask
before the addition of 1.75 mL of pancreatin solution (800 U mL
−1
), bovine bile extract and
porcine bile extract (1:1 w/wup to 10 mM total bile salts), and 14
µ
L of 0.3 M CaCl
2
. The
mixture was kept under agitation at 37
◦
C for 2 h at 170 rpm. The collected digesta were
placed on ice, moved to a freezer at
−
80
◦
C, and freeze-dried. Later, 10 mL of each sample
was extracted from each digestion phase for additional analysis. The digested and undi-
gested smoothies were kept at
−
80
◦
C until individual carotenoid and antioxidant levels
and the inhibition of carbohydrate hydrolysing enzymes (
α
-amylase and
α
-glucosidase)
analysis were determined. Equation (1) was used to determine the bioaccessibility of
bioactive substances.
Bioaccessibility % = (BGC/BND) ×100 (1)
The BGC (mg kg
−1
) was the content of the bioactive compound in the intestinal digesta,
and BND (mg kg−1) was the bioactive compound content in the undigested sample.
2.10. Antioxidant Properties
Using the methods described by Seke et al. [
2
], the antioxidant capacity of VOP 1,
VOP 3, and VOP 4 were assessed before and after digestion. At 517 nm, the absorbance
for the DPPH test was determined. The result was expressed as the IC
50
(mg mL
−1
). The
radical scavenging ability of ABTS in smoothies was determined by measuring absorbance
at 734 nm and expressing the results as IC
50
(mg mL
−1
). A procedure outlined by Seke
et al. [
2
] was followed to calculate the ferric-reducing antioxidant potential (FRAP) of the
digested and undigested cowpea leaf smoothies. The Trolox equivalent antioxidant activity
(TEAC)/100 g of cowpea leaf smoothies was calculated from the absorbance measured
at 593 nm.
2.11. Inhibition of Carbohydrate Hydrolysing Enzymes (α-Amylase and α-Glucosidase)
The
α
-glucosidase inhibitory activity was determined using a microplate reader
(CLARIOstar Plus BMG Labtec, Lasec, Cape Town, South Africa), as described by Moloto
et al. [
1
]. The enzyme inhibitory activity was calculated and expressed as a percentage of
α
-glucosidase inhibition [
26
]. As described by Moloto et al. [
1
], the
α
-amylase inhibition
of the extracts from the cowpea leaf smoothies was assessed using a microplate reader
(CLARIOstar Plus BMG Labtec, Lasec, Cape Town, South Africa) monitored at 580 nm. The
percentage of
α
-amylase inhibition was used to calculate enzyme inhibitory activity. Due to
its efficiency, safety profile, favourable cardiovascular and metabolic effects, and capacity
Foods 2023,12, 1701 6 of 20
to be coupled with other antidiabetic drugs, metformin was used as a positive control in
the study, being the first glucose-lowering drug of choice for treating persons with type
2diabetes mellitus.
2.12. Glucose Uptake Assay
A method described by Chauke et al. [
27
] was followed to measure cellular glucose
uptake. Muscle cells were placed in 96-well plates, cultivated for 4 days and incubated
for a further 1, 4, and 6 h at 37
◦
C. The glucose test kit (KAT Medicals, Johannesburg,
South Africa) was used to determine the amount of glucose in the medium. Insulin
(100 M) was used as the positive control, and untreated cells as the negative control.
The absorbance was then recorded at 540 nm, using a Multiskan GO (Thermo Scientific,
Waltham, MA, USA). The results were presented as a percentage of the total amount of
glucose consumed (mmol/L).
2.13. Statistical Analysis
The experiments were performed in triplicate and repeated twice, and the data were
analyzed using one-way ANOVA in the statistical program Statistica data analysis software
system (10) (Statsoft, Inc., Tulsa, OK, USA). The Fisher LSD test was performed to discover
significant differences at p-values of 0.05. The linear correlations between phenolics and
bioactivities were established using regression correlation coefficients. The nonlinear
regression “dose-response inhibition” was used to determine the IC
50
for DPPH and
ABTS activities.
3. Results and Discussion
3.1. Physicochemical Properties of Cowpea Smoothies Fermented Using Ltp. plantarum 75
Food acidification is the key process involved in the lactic acid fermentation of food
for preservation and safety by limiting the growth of spoilage and harmful bacteria in
fermented foods. After 48 h of fermentation, the pH value of the cowpea smoothies
decreased from 6.28 (VOP 1), 6.57 (VOP 3), and 6.51 (VOP 4), to 5.15 (VOP 1), 5.12 (VOP 3),
and 5.05 (VOP 4) (Table 1). The pH decline observed in this study supports the findings
by Managa. [
15
], who reported a pH drop in fermented chayote and pineapple smoothie
after two days of fermentation. The reduction in the pH value of the smoothies was a
key indicator of the fermentation progress, which relates to the production of organic
acids [
28
]. Lowering the pH during LAB fermentation could hinder the growth of spoilage
and pathogenic microorganisms, thereby aiding food preservation.
The total titratable acidity (TTA) increased gradually from 0.69 mg/mL (VOP 3),
0.75 mg/mL (VOP 4) and 0.78 mg/mL (VOP 1) to 1.98 mg/mL (VOP 1), 2.07 mg/mL
(VOP 3) and 2.22 mg/mL (VOP 4) (Table 1). The gradual increase in total titratable acidity
could be due to the metabolization of carbohydrates into organic acids, such as lactic
acid being the predominant metabolic product in lactic acid bacteria fermentation [
18
].
Lower pHs during the fermentation of Ltp. plantarum-fermented emmer-based beverages
supplemented with fruit juices have been reported, indicating Ltp. plantarum 75 is a
vigorous heterofermenter that can thrive at low pH [29].
Total soluble solids (TSS) are significant quality markers concerning sweetness, often
known as the sugar index (Magwaza and Opara [
30
]). The TSS content of smoothies
fermented for 48 h significantly decreased in all of the cowpea smoothies (Table 1). The
initial TSS content of the fermented cowpea smoothies was 1.70 (VOP 1), 1.51 (VOP 3),
and 1.53 (VOP 4)
◦
Brix but decreased to 0.82 (VOP 1), 0.63 (VOP 3), and 0.61 (VOP 4)
◦
Brix in the fermented smoothies after two days (Table 1). The decrease in TSS levels
during fermentation indicated the use of sugars in smoothies for metabolism, cellular
development, and bioconversion into organic acid. This observation corroborates the claim
of decreased TSS in Ltp. Plantarum-fermented beet juice after 72 h [
31
]. The same trend
was also observed in mango juice [
32
]. However, an increase in TSS of the smoothies,
as observed at 2 h of fermentation for all accessions, before an actual decrease might be
Foods 2023,12, 1701 7 of 20
attributed to polysaccharide breakdown into monosaccharide and oligosaccharide [
33
].
Furthermore, the total sugar content significantly decreased with increasing fermentation
period across all cowpea accessions (Table 1). The decrease in the sugar content could,
therefore, suggest that the fermented cowpea leaf smoothies could be a nutritional option
for managing diabetic conditions.
Table 1.
Changes in physicochemical properties of Ltp. plantarum 75-fermented cowpea
(Vigna unguiculata) leaf smoothies from three different accessions.
Cowpea Cultivars Treatment h pH TTA (mg/mL) TSS (◦BRIX) TS (30 mg/100 g)
VOP 1 Unfermented 0 6.28 ±0.21 a0.78 ±0.05 c1.30 ±0.28 c1.58 ±0.16 b
VOP 3 Unfermented 0 6.57 ±0.44 a0.69 ±0.02 c1.35 ±0.15 c1.35 ±0.11 b
VOP 4 Unfermented 0 6.51 ±0.12 a0.75 ±0.01 c1.43 ±0.47 c1.96 ±0.23 a
VOP 1 LAB 75 2 6.18 ±0.33 a1.05 ±0.04 c1.70 ±0.08 b1.01 ±0.27 b
VOP 3 LAB 75 2 6.4 ±0.41 a1.11 ±0.08 c1.51 ±0.11 c0.96 ±0.13 c
VOP 4 LAB 75 2 6.4 ±0.31 a1.12 ±0.05 c1.53 ±0.17 a1.66 ±0.27 b
VOP 1 LAB 75 24 5.66 ±0.82 b1.29 ±0.08 b1.01 ±0.11 d0.62 ±0.02 c
VOP 3 LAB 75 24 5.33 ±0.10 b1.50 ±0.10 b1.16 ±0.05 d0.37 ±0.01 d
VOP 4 LAB 75 24 5.21 ±0.61 b1.80 ±0.10 b1.00 ±0.08 d0.12 ±0.01 e
VOP 1 LAB 75 48 5.15 ±0.03 c1.98 ±0.10 a0.82 ±0.11 e0.33 ±0.04 d
VOP 3 LAB 75 48 5.12 ±0.01 c2.07 ±0.19 a0.63 ±0.15 e0.22 ±0.01 d
VOP 4 LAB 75 48 5.05 ±0.02 c2.22 ±0.29 a0.61 ±0.05 e0.09 ±0.01 e
LSD * 0.63 ** 0.80 ** 0.37 *** 0.30 **
Values are mean
±
standard error of means; means followed by a different letter within the column are significantly
different * p
≤
0.05, ** p
≤
0.01, and *** p
≤
0.001. TTA = titratable acidity, TSS = total soluble solids, TS = total
sugars, LAB 75 = Ltp.Plantarum 75, VOP 1, VOP 3, and VOP 4 = cowpea cultivar leaf smoothies, and LSD = least
significant difference.
3.2. Effect of Fermentation on the Ascorbic Acid Content of Three Different Cowpea Cultivar
Leaf Smoothies
Table 2shows the ascorbic acid (AA) concentration of fermented and unfermented
cowpea leaf smoothies. An extended length of fermentation with Ltp. plantarum 75 resulted
in a substantial rise in AA concentration. The unfermented samples had the lowest AA,
whereas the two-day fermented smoothies had the highest AA. The AA content of the
unfermented smoothies varied from 4.30 to 6.02 mg/100 g, whereas it ranged from 15.67
to 17.67 mg/100 g after two days of fermentation (Table 2). The results from this current
research confirm a study that found an increase in AA when a vegetable–fruit beverage
was fermented with Ltp. plantarum [
34
]. Several studies have investigated the effect of
lactic acid fermentation on the vitamin C content of fruit and vegetable juices. Up to this
point, their findings have indicated that the fermentation process has a variable impact
on the ascorbic acid level of fermented foods. Predefined phases of fermentation as well
as some lactic acid bacteria strains had a positive effect on AA content. After 12 h of
fermentation of cashew apple juice [
35
] with Ltp. plantarum and Lb. casei, vitamin C levels
increased and then remained constant after 48 h of fermentation. This observation could be
attributed to microorganisms synthesizing ascorbic acid. Citrus juice fermented for 12 h
had a vitamin C level that is comparable to conventional pasteurized juice [
36
]. Similarly,
Znamirowska et al. [
37
] proposed that lactic acid bacteria could increase the vitamin C
content of fermented dairy.
In contrast, the utilization of Lb. acidophilus and Lb. casei as inoculum for fermentation
culminated in a small decrease in vitamin C levels after 48 h of fermentation [
37
]. Following
a few hours of fermentation, the ascorbic acid concentration of fermented juices from
sweet lemon [
38
], prickly pears [
39
], and papaya [
40
] increased. The decrease in AA
levels after some period of fermentation could be attributed to the increased activity of the
ascorbate oxidase enzyme produced during lactic acid fermentation. It is not unexpected
that bacteria that do not degrade ascorbic acid frequently have a protective effect, since
ascorbic acid oxidizes in an aqueous solution, resulting in the creation of dehydroascorbic
Foods 2023,12, 1701 8 of 20
acid and hydrogen peroxide. Therefore, the observed increase in ascorbic acid may be due
to decreased oxygen in the medium, and increased acidity since ascorbic acid is stabilized
in acidic environments [41,42].
Table 2.
Ascorbic acid contents of Ltp. plantarum 75-fermented and unfermented cowpea leaf
smoothies from different cultivars.
Cowpea Cultivar h Treatment AA (mg/100 g)
VOP 1 0 Unfermented 6.02 ±0.01 e
VOP 3 0 Unfermented 4.30 ±0.48 g
VOP 4 0 Unfermented 5.20 ±0.35 f
VOP 1 2 LAB 75 6.33 ±0.50 e
VOP 3 2 LAB 75 4.32 ±0.01 g
VOP 4 2 LAB 75 5.52 ±0.57 f
VOP 1 24 LAB 75 16.10 ±0.01 b
VOP 3 24 LAB 75 12.38 ±0.01 d
VOP 4 24 LAB 75 15.33 ±5.48 c
VOP 1 48 LAB 75 17.67 ±0.48 a
VOP 3 48 LAB 75 15.67 ±1.96 c
VOP 4 48 LAB 75 16.34 ±0.48 b
LSD * 1.65 ***
Values are mean
±
standard error of means; means followed by a different letter within the column are significantly
different * = p
≤
0.05, *** p
≤
0.001. AA = ascorbic acid, h = hour, LAB 75 = Lactiplantibacillus plantarum 75,
LSD = least significant difference, and VOP 1, VOP 3, and VOP 4 = cowpea cultivars leaf smoothies.
3.3. Colour Changes in Cowpea Leaf Smoothies after Fermentation
Table 3shows the impact of fermentation on the colour of cowpea leaf smoothies.
The luminosity (L*) value of the fermented and unfermented cowpea smoothies varied
from 17.80 to 27.61, and a significant (p< 0.05) rise in the fermented smoothies (Table 3)
was observed. The redness to greenness (a*) values of all of the accessions (VOP 1, VOP 3,
and VOP 4) decreased significantly during fermentation, with a* values for the fermented
and unfermented smoothies ranging from
−
6.91 to
−
3.90. The fermenting LAB metabolic
activities could have caused enzymatic oxidation throughout the fermentation process
resulting in the greenness-to-redness colour features in the fermented smoothies. The blue-
to-yellow (b*) values increased significantly in the unfermented to the fermented smoothies
after two days (Table 3). The
∆
E relates to the colour difference of the fermented cowpea
smoothies. After 48 h, the
∆
E varied from 1.32 to 3.89. VOP 1 (1.32) had the lowest
∆
E
and was substantially different from VOP 3 (2.67) and VOP 4 (3.89) fermented smoothies.
The fermentation duration had a significant effect on the colour parameter values for all
smoothies. The lesser colour change in the VOP 1-fermented smoothie at 48 h may be due
to the slower fermentation taking place in the cultivar compared to others, as shown by a
lower TTA (Table 1). This might be due to the presence of polysaccharides in the cultivar
that needed to be broken down into simple sugars before utilisation by Ltp. plantarum 75,
thus reducing enzymatic degradation in fermented smoothies. Ltp. plantarum has been
reported as a powerful facultative heterofermenter of food substrates [
43
]. However, the
increased
∆
E levels (VOP 3 and VOP 4) in fermented smoothies might be attributed to the
auto-oxidation of the polyphenolic components [44].
3.4. Microbial Counts in Fermented and Unfermented Cowpea Leaf Smoothies
To evaluate the variations in microbial quality in the fermented smoothies, the yeast
and mould count, total viable bacteria count, and lactic acid bacteria count were evalu-
ated. As shown in Figure 1A, the total viable bacteria counts were lowest in the unfer-
mented but pasteurised smoothies from all cultivars and were not significantly different
(p
≥
0.05). The total bacteria counts of fermented smoothies from different cultivars were
not significantly different to each other at 2, 24, and 48 h of fermentation, except VOP 4 at
48 h of fermentation (p
≤
0.05). The yeast count was highest in the unfermented smoothies
Foods 2023,12, 1701 9 of 20
and lowest in the 2 h-fermented smoothies (Figure 1B). However, the yeast and bacte-
rial counts were within the acceptable limits (Log 6 CFU/mL) for beverages [
18
]. No
pathogens, such as E. coli,Salmonella spp. and Staphylococcus aureus, were detected in the
smoothies (Table S2). Presumptive lactic acid bacteria count ranged from 7 Log CFU/g
after 2 h to 10 Log CFU/g after 48 h (Figure 1C). As expected, there was an increase in the
presumptive LAB count in the fermented cowpea leaf smoothie after 48 h in all cultivars
(VOP 1, VOP 3, and VOP 4). The increase in the LAB count might be due to the evolution of
Ltp. plantarum 75 through the utilisation of organic sugars in smoothies causing fermenta-
tion. Ltp. plantarum prefers glucose and lactose as carbon sources and can adapt to a variety
of environments, which explains its versatility in fermentation [45].
Table 3.
Colour changes in fermented and unfermented cowpea leaf smoothies from three different
cowpea cultivars.
Accession h Treatment L* a* b* ∆E
VOP 1 0 Unfermented 17.80 ±0.24 i−5.81 ±0.13 b11.66 ±0.02 c
VOP 3 0 Unfermented 22.16 ±0.92 e−6.25 ±0.29 a12.15 ±0.10 b
VOP 4 0 Unfermented 23.97 ±0.19 e−6.91 ±0.32 c10.88 ±0.21 d
VOP 1 2 LAB 75 20.33 ±0.37 b−5.55 ±0.06 e10.03 ±0.21 c1.03 ±0.62 d
VOP 3 2 LAB 75 23.86 ±0.23 f−6.09 ±0.30 a12.03 ±0.35 b1.05 ±0.58 d
VOP 4 2 LAB 75 25.14 ±0.05 a−6.88 ±0.29 d10.11 ±0.23 d1.00 ±0.10 d
VOP 1 24 LAB 75 21.90 ±0.08 g−4.07 ±0.18 c12.69 ±0.05 b3.83 ±0.24 b
VOP 3 24 LAB 75 25.73 ±0.50 c−5.98 ±0.30 c10.92 ±0.26 d2.44 ±0.85 c
VOP 4 24 LAB 75 27.64 ±0.22 b−5.70 ±0.31 b11.78 ±0.58 b5.75 ±0.65 a
VOP 1 48 LAB 75 21.70 ±0.90 g−4.62 ±0.23 a9.96 ±0.36 e1.32 ±0.08 d
VOP 3 48 LAB 75 24.57 ±0.44 d−3.68 ±0.24 a12.55 ±0.68 b2.67 ±0.29 c
VOP 4 48 LAB 75 27.61 ±0.14 b−3.90 ±0.08 a12.88 ±0.09 a3.89 ±0.48 b
LSD * 1.26 ** 2.71 *** 1.31 *** 0.22 **
Values are mean
±
standard error of means; means followed by a different letter within the column are significantly
different at * p
≤
0.05, ** p
≤
0.01, *** p
≤
0.001, and ns = not significant. LSD = least significant difference;
L* = degree of lightness; a* = red-to-green component; b* = yellow-to-blue component;
∆
E = total colour change,
h = hour; LAB 75 = Lactiplantibacillus plantarum 75; VOP 1, VOP 3, and VOP 4 = cowpea cultivar leaf smoothies.
3.5. Sensory Evaluation of Unfermented and Fermented Cowpea Leaf Smoothies
Figure 2shows how the organoleptic properties of fermented and unfermented cow-
pea leaf smoothies were obtained from different cultivars. The colour perception of the
unfermented and fermented smoothies varied from browning leafy vegetable colour, (2.70)
in VOP 4 at 48 h, to dark-green leafy vegetable colour, (8.75) in VOP 3 after 2 h. There
were no significant differences between the unfermented and 24 h-fermented smoothies;
however, the overall acceptability of smoothies fermented for 48 h was considerably lower
than the 2 and 24 h counterparts. VOP 3 and VOP 4 were the least acceptable. The de-
clining green colour of leaf smoothies may be due to the oxidation of the samples [
46
]
during acidity and fermentation. Lactic acid fermentation makes use of the oxidation
and reduction processes in the transformation of molecules found in substrates. Typically,
during fermentation, pyruvates are reduced to lactate by oxidizing NADH to NAD+ [
10
].
When compared to the other samples, the VOP 1 fermented smoothie after 24 h (8.20)
had a highly acceptable flavour, while the VOP 4 fermented smoothie after 48 h had the
least (3.10), as shown in Figure 2. The unfermented VOP 1 smoothie had a considerably
better taste in terms of acceptability than the other unfermented samples, whereas the
48 h-fermented VOP 4 had the lowest acceptable taste (Figure 2). As fermentation pro-
gressed, the perception of flavour diminished. This could be due to the utilization of the
sugar substrates that resulted in the fast depletion of sugars in the smoothies, thereby
leaving the Ltp. plantarum 75 to utilise phenolics as a substrate for its bioconversion.
Lactic acid bacteria have been reported to hold the potential as probiotics by utilising
metabolites such as phenolics for their metabolic activities [
10
]. The aroma of VOP 1 fer-
mented for 24 h was found to be substantially more acceptable than the other fermented
Foods 2023,12, 1701 10 of 20
smoothies. Overall, the 24 h-fermented VOP 1 smoothies can be recommended regarding
organoleptic acceptability.
Figure 1.
The microbial load in fermented and unfermented cowpea leaf smoothies. (
A
) Total viable
bacterial count, (
B
) total fungal count, and (
C
) lactic acid bacteria count. The data presented on the
graphs consist of average quantities
±
SD of three independent samples. Different letters on the bars
represent statistically significant differences (p< 0.05). VOP 1, VOP 3, and VOP 4 are different cowpea
leaf cultivar smoothies, and units are shown as colony-forming units per millilitre (CFU/mL).
Figure 2.
Sensory evaluation of fermented and unfermented cowpea leaf smoothies. VOP 1,
VOP 3, and VOP 4 = cowpea cultivar leaf smoothies; LAB 75 = Ltp. Plantarum 75; VOP 1_0 h,
VOP 3_0 h, and VOP 4_0 h = pasteurized and unfermented smoothies; VOP 1_2 h, VOP 3_2 h, and
VOP 4_2 h = 2 h-fermented smoothies; VOP 1_24 h, VOP 3_24 h, and VOP 4_24 h = 24 h-fermented
smoothies; VOP 1_48 h, VOP 3_48 h, and VOP 4_48 h = 48 h-fermented smoothies.
Foods 2023,12, 1701 11 of 20
3.6. Total Phenolic Content (TPC) and Antioxidant Activities of Unfermented and Fermented
Cowpea Leaf Smoothies
The total phenolic content of the unfermented and fermented smoothies from
different cowpea leaf cultivars is shown in Table 4. The TPC was noted to be signif-
icantly higher in the unfermented (249.80 mg/100 g DW) and 2 h-fermented VOP 1
smoothie (249.90 mg/100 g DW), and was lowest in the 48 h-fermented VOP 3 smoothie
(172.23 mg/100 g DW). The VOP 3 and VOP 4 fermented smoothies had a highly sig-
nificant (p< 0.05) reduction in TPC at 48 h at 19.7% and 15.25%, respectively (Table 4),
whilst there was an 11.11% reduction in the TPC of the VOP 1 smoothie after 48 h of
fermentation (Table 4). The period of fermentation had a significant effect on the TPC
of the cowpea smoothies. The reduction in the TPC of the fermented smoothies is con-
sistent with the findings of Hashemi et al. [
10
], based on sweet lemon juice fermented
by Ltp. plantarum. Similarly, Yang and co-workers noted a decrease in the TPC of a
vegetable–fruit beverage with an increase in the fermentation period [
34
]. The decrease
could be attributed to phenolic component degradation due to the low sugar content
of the smoothies (Table 1). Some lactic acid bacteria may degrade phenols to continue
growing; this action may result in a decrease in overall phenolic concentration [34,47].
Table 4.
Antioxidant properties and total phenol content of fermented and unfermented cowpea leaf
smoothies obtained from three cultivars.
Cultivars h Treatment Total Phenols Loss FRAP DPPH ABTS
(mg/100 g DW) (%) (mmol TEAC/100 g DW) (IC50 µg/mL) (IC50 µg/mL)
VOP 1 0 Unfermented 249.80 ±68.26 a171.79 ±30.25 b1.14 ±0.06 b30.41 ±3.05 a
VOP 3 0 Unfermented 214.49 ±62.12 b167.32 ±30.08 b1.50 ±0.05 a24.52 ±2.85 b
VOP 4 0 Unfermented 205.17 ±52.32 b163.38 ±40.55 b1.38 ±0.08 a15.47 ±1.25 d
VOP 1 2 LAB 75 249.90 ±61.59 a0.04 170.54 ±35.26 b1.15 ±0.02 b20.20 ±2.56 c
VOP 3 2 LAB 75 211.78 ±58.12 b1.26 160.54 ±52.32 b1.48 ±0.05 a2.96 ±0.59 g
VOP 4 2 LAB 75 205.45 ±45.28 b0.13 160.25 ±45.36 b1.39 ±0.05 a10.95 ±2.45 f
VOP 1 24 LAB 75 223.97 ±43.92 b10.34 315.59 ±45.13 a1.03 ±0.08 b13.78 ±4.25 e
VOP 3 24 LAB 75 201.94 ±64.88 b5.85 165.59 ±33.32 b1.20 ±0.04 b10.33 ±1.56 f
VOP 4 24 LAB 75 183.29 ±45.97 c10.66 124.00 ±24.25 c0.87 ±0.01 c9.89 ±1.28 f
VOP 1 48 LAB 75 222.03 ±34.98 b11.11 300.41 ±45.05 a0.07 ±0.01 e0.53 ±0.01 i
VOP 3 48 LAB 75 172.23 ±48.10 c19.70 81.61 ±14.24 d0.44 ±0.01 d1.69 ±0.21 h
VOP 4 48 LAB 75 173.87 ±43.80 c15.25 83.69 ±9.05 d0.39 ±0.01 d2.80 ±0.18 g
LSD * 25.89 *** 41.88 ** 0.18 * 4.20 ***
Values are mean
±
standard error of means; means followed by a different letter within the column are
significantly different at * p
≤
0.05, ** p
≤
0.01, and *** p
≤
0.001. LSD * = least significant difference;
LAB 75 = Lactiplantibacillus plantarum 75; VOP 1, VOP 3, and VOP 4 = cowpea cultivar leaf smoothies.
Table 4presents the FRAP antioxidant activity of cowpea leaf smoothies before and
after fermentation. Fermentation by Ltp. plantarum 75 increased the antioxidant activity of
the cowpea leaf smoothies to 300.41, 81.61, and 83.69 mmol TEAC/100 g DW, in VOP 1,
VOP 3, and VOP 4, respectively, compared to the unfermented cowpea leaf smoothies.
VOP 3 showed the lowest antioxidant activity while VOP 1 had the highest FRAP, DPPH,
and ABTS antioxidant activity after fermentation. The FRAP, DPPH, and ABTS activities
positively correlate with the TPC, with coefficient values of R
2
= 0.98, R
2
= 0.49, and
R2= 0.74, respectively (Table S3).
3.7. Effect of Fermentation on the Carotenoid’s Profiles in Cowpea Leaf Smoothies
The main free xanthophyll in the fermented cowpea smoothies was lutein, and it
was significantly higher in concentration than the carotenoids during the fermentation
period (Table 5). The content of lutein decreased with increasing fermentation period
in all of the cowpea cultivars; VOP 1 (33.48 to 42.70%), VOP 3 (38.48 to 46.77%) and
VOP 4 (41.03 to 46.49%) after fermentation for 48 h (Table 5). A similar trend was noted
with zeaxanthin,
α
-carotene, 9-cis-
β
-carotene, and all-trans
β
-carotene. The decrease in
carotenoid content after fermentation was in the order VOP 4 > VOP 3 > VOP 1 (Table 5).
Foods 2023,12, 1701 12 of 20
In general, carboxylic acids created during fermentation are reported to provide an acidic
environment in which carotenoids are considered to be unstable due to their potential
hydrolyzation into free xanthophylls and fatty acids, which could be further converted
or degraded [
27
]. The unfermented smoothies indicated the highest carotenoid content
in VOP 1 (148.71 mg/100 g), VOP 3 (123.29 mg/100 g), and VOP 4 (107.09 mg/100 g).
The lowest degradation rate of total carotenoid content was noted in VOP 1 fermented
for 24 h. However, the highest percentage loss was noted in VOP 3 with 41.96% at 24 h
of fermentation, which increased to 50.26% after 48 h of fermentation. A degradation
of total carotenoids during fermentation in sweet potato [
46
], vegetable juice [
48
], and
yellow lantern pepper soup [
27
] fermented with Ltp. plantarum have been reported. The
degradation of total carotenoids suggests fermentation with Ltp. plantarum 75 could im-
pact carotenoid stability. Therefore, the reduction in carotenoids during fermentation
could be attributed to bacterial metabolism that results from the change in substrate tem-
perature, pH, and produced metabolites [
48
]. However, the degree of metabolism by
fermenting microorganisms is determined by the strain utilized, the substrate, and the
fermentation conditions [48].
Table 5.
The carotenoid profile of Ltp. plantarum 75 fermented and unfermented cowpea leaf
smoothies from different cultivars (mg/100 g).
Cultivars Treatment Hours Lutein Zeaxanthin α-Carotene 9-cis-β-
Carotene
All-Trans
β-Carotene
Total
(Carotenoids)
VOP 1
Unfermented
0 99.88 ±12.36 a2.19 ±0.15 a4.63 ±1.05 a3.76 ±0.78 a38.27 ±4.25 a148.71 ±40.01 a
VOP 3
Unfermented
085.04 ±9.28 b2.22 ±0.25 a4.55 ±1.85 a3.64 ±0.65 a27.84 ±3.95 b123.29 ±23.58 b
VOP 4
Unfermented
0 70.38 ±7.68 c2.29 ±0.29 a5.07 ±1.90 a3.12 ±0.72 ab 26.23 ±2.86 b107.09 ±19.08 c
VOP 1 LAB 75 24 66.46 ±8.01 d2.17 ±0.26 a3.03 ±1.02 b2.96 ±0.52 b21.75 ±3.58 b96.39 ±14.02 d
VOP 3 LAB 75 24 52.32 ±4.32 e2.12 ±0.30 a2.21 ±1.26 c2.48 ±0.48 b12.43 ±2.96 c71.56 ±10.58 e
VOP 4 LAB 75 24 41.50 ±5.09 e2.04 ±0.21 a3.13 ±0.98 b2.23 ±0.59 b10.48 ±2.54 c59.38 ±8.36 f
VOP 1 LAB 75 48 57.23 ±4.18 e1.64 ±0.15 b2.19 ±0.19 b2.66 ±0.60 b19.47 ±1.95 b82.91 ±11.25 e
VOP 3 LAB 75 48 45.27 ±5.00 e1.84 ±0.17 b1.53 ±0.20 c2.24 ±0.54 b10.44 ±2.47 c61.32 ±8.96 f
VOP 4 LAB 75 48 37.66 ±4.89 f1.90 ±0.18 b2.45 ±1.28 b1.80 ±0.48 c9.75 ±1.05 c54.2 ±7.49 f
LSD * 14.28 ** 0.25 * 1.35 ** 0.44 ** 9.80 *** 10.20 **
Values are mean
±
standard error of means; means followed by a different letter within the column are significantly
different at * p
≤
0.05, ** p
≤
0.01, and *** p
≤
0.001; LAB 75 = Lactiplantibacillus plantarum 75; LSD = least significant
difference; VOP 1, VOP 3, and VOP 4 = cowpea cultivar leaf smoothies.
3.8. Effect of In Vitro Digestion on Total Phenolic Content and Antioxidant Activity of VOP 1
Fermented Cowpea Smoothie
Table 6indicates the effect of gastrointestinal digestion on total phenolic compounds
of the VOP 1 cowpea leaf smoothie fermented for 24 h. The VOP 1 cultivar was selected
based on its high antioxidant activity, retained carotenoid profile and total phenolic content
after 24 h of fermentation. The TPC of the undigested smoothie (223.97 mg/100 g DW)
was significantly higher than that of the digested smoothie. The TPC of the gastric digesta
in the fermented smoothies was reduced significantly when compared to the equivalent
undigested smoothie, with a bioaccessibility of 86.07%. (Table 6). The TPC levels in
the intestinal phase (335.25 mg/100 g DW) were greater than the undigested smoothie
(223.97 mg/100 g DW) and had the highest bioaccessibility (149.57%). The bioaccessibility
at the dialysis phase was 30.68%. TPC decreased significantly throughout the dialysis
phase when compared to the undigested, gastric, and intestinal digesta. A similar pattern
was observed in digested apple cultivars, such as Jonaprinz, Jonagold, and Golden, with
total phenol levels in the intestinal phase being higher but less conspicuous than in the
gastric phase [
49
]. Similarly, during the
in vitro
digestion of fermented chayote leaf and
pineapple smoothies, the intestinal phase had higher levels of total polyphenol content
than the gastric digesta [
15
]. The observed increase in the TPC of the intestinal digesta
compared to the gastric digesta may be due to an increased release of phenolics bound to
the matrix due to the activity of the intestinal digestive enzyme (pancreatin), or due to the
Foods 2023,12, 1701 13 of 20
phenolic interaction with cell wall carbohydrates such as pectin present in the smoothies,
thereby obstructing the phenolic compound solubilization during gastric digestion.
Table 6.
Antioxidants and total phenol activity of
in vitro
digested and fermented cowpea leaf
smoothies from VOP 1 cowpea cultivar.
Total Phenols Bioaccessibility FRAP DPPH ABTS
(mg/100 g DW) % (µmol TEAC/100 g) (IC50 µg/mL) (IC50 µg/mL)
Undigested 223.97 ±43.92 b320.78 ±39.14 b1.03 ±0.08 c30.78 ±4.25 c
Gastric 192.78 ±35.68 c86.07 ±11.86 b304.89 ±48.21 c5.49 ±0.44 b32.28 ±11.48 b
Intestinal 335.25 ±65.32 a149.57 ±28.21 a345.46 ±36.76 a0.94 ±0.01 c31.09 ±9.23 c
Dialysis 68.70 ±5.90 d30.68 ±1.67 c156.13 ±24.92 d16.97 ±3.85 a46.69 ±10.58 a
LSD * 30.87 *** 28.58 *** 15.68 *** 4.95 ** 1.25 *
Values are mean
±
standard error of means; means followed by a different letter within the column are significantly
different at * p
≤
0.05, ** p
≤
0.01, and *** p
≤
0.001; LAB 75 = Lactiplantibacillus plantarum 75; LSD = least significant
difference; VOP 1, VOP 3, and VOP 4 = cowpea cultivar leaf smoothies; FRAP = Ferric-reducing antioxidant
power; DPPH = 2,2-diphenyl-1-picrylhydrazyl; ABTS = 2,2
0
-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid);
TEAC = Trolox equivalent antioxidant capacity; and IC50 = the concentration of a drug or inhibitor needed to
inhibit a biological process or response by 50%.
The FRAP activity was significantly high (p< 0.05) in the intestinal digesta
(345.46
µ
mol TEAC/100 g) compared to the undigested samples (320.78
µ
mol TEAC/100 g)
and the gastric digesta (304.89
µ
mol TEAC/100 g) (Table 6). Furthermore, the ABTS and
DPPH radical scavenging activities were found to be significantly high in the undigested
and the intestinal digesta (Table 6). Tagliazucchi et al. [
50
] made a similar report on phenolic-
rich grape extracts that offered better protection and high ABTS antioxidant capacity at the
intestinal phase than in the gastric phase. The observed tendency could be attributed to the
higher phenolic contents of the undigested sample and intestinal digesta [
50
]. According to
reports, pH fluctuations between the gastric and intestinal phases also altered antioxidant
activity by increasing the antioxidant capacity of phenolics [
51
]. Phenols can function as
reducing agents, hydrogen donors, and singlet oxygen-reducing agents due to their redox
properties, all of which increase their capacity as natural antioxidants. Additionally, the
quantity and placement of hydrogen-donating hydroxyl groups on the aromatic ring of
phenol molecules affect their ability to scavenge free radicals and act as antioxidants [52].
3.9. Effect of In Vitro Digestion on the C2C12 Glucose Uptake of Fermented VOP 1 Cowpea
Leaf Smoothies
The glucose uptake of the VOP 1 fermented smoothies at 1 h, 4 h, and 6 h indicated an
improved glucose uptake activity (Figure 3). The intestinal digesta indicate a significantly
high glucose uptake relative to the undigested samples and the gastric digesta, with a
trend of 50
µ
g/mL > 25
µ
g/mL > 12.5
µ
g/mL, and 6.25
µ
g/mL. However, the glucose
uptake was not significantly different between 6.25 and 12.5
µ
g/mL (p
≥
0.05) except in the
undigested and intestinal fractions at 1 h and 6 h of glucose uptake, respectively (p
≤
0.05).
The insulin treatment of the skeletal muscle cells resulted in an increase that was equal to
the intestinal digesta (Figure 3). The enhanced translocation and movement of GLUT4 to the
plasma membrane are primarily responsible for the stimulation of glucose absorption [
53
].
Moloto et al. [
1
] observed that leaf extracts of cowpea cultivar VOP 1 dramatically elevated
the GLUT4 gene to a level comparable to insulin therapy. This could be an indicator of
increased glucose absorption by C
2
C
12
cells stimulated by the pool of phenolic chemicals
in the VOP 1 fermented smoothies. The antioxidant and anti-inflammatory effects of
polyphenols have been linked to the ability to reduce the risk of developing diabetes by
inhibiting tyrosine phosphatase. This activates tyrosine phosphorylation and suppresses
hepatic glucose output through processes that interact with the cell membrane receptors.
Green tea epigallocatechin gallate (EGCG) has been shown to regulate the activity of the cell
surface receptor, tyrosine kinases (RTK), such as insulin receptors (InsR) and insulin-like
growth factor receptors (IGFR) [
54
,
55
]. In addition, some polyphenols such as flavonoids
Foods 2023,12, 1701 14 of 20
are known for their capacity to chelate metals, which inhibits the generation of free radicals
that are catalysed by metals [56].
Figure 3.
The effect of the
in vitro
digestion of VOP 1 cowpea leaf smoothies fermented by
Ltp. plantarum 75 on muscle cell C
2
C
12
glucose uptake at 1, 4, and 6 h. The data presented on
the graphs consist of average quantities
±
SD of three independent samples. The different letters on
the bars represent statistically significant differences per treatment (p< 0.05). (
A
) 1 h C
2
C
12
glucose
uptake (%), (B)4hC2C12 glucose uptake (%), and (C)6hC2C12 glucose uptake (%).
Foods 2023,12, 1701 15 of 20
3.10. Effect of In Vitro Digestion on the α-Glucosidase and α-Amylase Inhibitory Capacity of
Fermented VOP 1 Cowpea Leaf Smoothies
The major enzymes for digesting carbohydrates,
α
-amylase and
α
-glucosidase, have
been identified as therapeutic targets for the management of postprandial hyperglycaemia,
which manifest in type 2 diabetes mellitus. The
α
-glucosidase and
α
-amylase inhibitory
activity of the digested and undigested VOP 1 fermented smoothie are presented in Figure 4.
The inhibitory capacity of
α
-glucosidase was greater in the intestine phase than in the
undigested, gastric, and dialysis phases (Figure 4). The diabetic-lowering abilities of
polyphenols have been linked to their anti-inflammatory and antioxidative properties, in
addition to their excellent insulin signalling abilities [57].
Figure 4.
The effect of the
in vitro
digestion of Ltp. plantarum 75 fermented VOP 1 cowpea leaf
smoothie on
α
-glucosidase and
α
-amylase inhibition using muscle cells C
2
C
12
for 24 h. The data
presented on the graphs consist of average quantities
±
SD of three independent samples. Different
letters on the bars represent statistically significant differences per treatment (p< 0.05).
A comparable study has linked the high polyphenol content after digestion to a strong
α
-glucosidase inhibition capacity observed in the intestinal phase compared to the gas-
tric phase [
58
]. Pomegranate extracts have been noted to inhibit
α
-glucosidase activities
in the intestinal phase [
59
]. Similarly, Rusak et al. [
59
] reported the abilities of matcha
tea and sencha green teas in inhibiting
α
-glucosidase in the intestinal phase. The TPC
positively correlates with the
α
-glucosidase inhibition capacity (R
2
= 0.95) in the intesti-
nal phase (Table S2), indicating that an increase in phenolics consequently increased the
α
-glucosidase inhibition. Similarly, the
α
-amylase was significantly inhibited by the intesti-
nal digesta (p< 0.05) compared to the undigested, gastric, and dialysed digesta (Figure 4).
With a trend of 50
µ
g/mL > 25
µ
g/mL > 12.5
µ
g/mL > 6.25
µ
g/mL at different concentra-
tions of
α
-glucosidase and
α
-amylase, glucose absorption was promoted in the intestinal
compartment at high concentrations. Consequently, blocking the enzymes may help to
lessen the rate of glucose absorption and ameliorate postprandial hyperglycaemia [
60
,
61
]. A
study reported a significant inhibitory effect of
α
-glucosidase and
α
-amylase in fermented
vegetable juice [
62
]. Koh et al. [
63
] demonstrated a significantly higher
α
-glucosidase
inhibition activity in an Lb. mali-fermented pumpkin-based beverage.
Foods 2023,12, 1701 16 of 20
3.11. Evolution of Lactic Acid Bacteria in Fermented VOP 1 Cowpea Leaf Smoothie after Simulated
Gastrointestinal Digestion
The recommended characteristics of a probiotic microbe include gut survival, persis-
tence in the host, and proof of safe human consumption [
62
]. The surviving LAB population
across digestion phases was investigated. LAB counts ranged from 6.3 Log CFU/mL in
the undigested sample to 6.8 Log CFU/mL in the gastric phase, which had a significantly
high population of LABs (p< 0.05) (Figure 5). Many consumed microorganisms die due
to the harsh acidity conditions in the stomach; however, acid-resistant strains, such as
Lactobacillus spp., Bifidobacterium spp., and Streptococcus spp., can survive [
64
]. A similar
result regarding the significantly high surviving LAB count in the Lb. acidophilus-fermented
beverage subjected to gastric digestion [65] supports the observation in this study.
Figure 5.
Surviving LAB count in simulated gastrointestinal digesta from fermented VOP 1 cowpea
leaf smoothie. The data presented on the graphs consist of average quantities
±
SD of three indepen-
dent samples. The different letters on the bars represent statistically significant differences (p< 0.05);
Log CFU/mL = logarithmic colony-forming unit per millilitre of smoothies.
Furthermore, lactic acid bacteria in orange juice with a pH of 3.80 were found to be
more resistant to the gastrointestinal environment [
65
]. According to Chen et al. [
66
], the
Ltp. plantarum PM153 strain showed the best adhesion ability and high survival in gastric
fluids. Similarly, it was found that fermented beverages made from chickpeas and coconut
had a considerably greater LAB survival rate in the gastric phase [
67
]. The acid tolerance
ability among the Lactobacillus genus could be due to the presence of a continuous gradient
between their extracellular and intracellular pH [
68
]. Moreover, the presence of glucose in
an acid environment has been shown to improve Lactobacillus probiotic life by providing
the needed ATP pool, thus allowing optimal H
+
extrusion via F0F1-ATPase, and, therefore,
boosting the capacity to survive in simulated gastric juice [
69
]. The cause of the decline
in bacterial survival in the simulated intestinal digesta could be attributed to bile salts,
which are components of bovine bile, and the pancreatin solution, which most impairs the
viability of microorganisms. Bile salts can modify cellular homeostasis and macromolecular
stability by affecting phospholipids and membrane proteins. A study by Mesquita et al. [
67
]
demonstrated the reduction in Ltp. plantarum viability after exposure to pancreatic juice.
4. Conclusions
This study demonstrated that the fermentation of cowpea smoothies from VOP
1, VOP 3, and VOP 4 cultivars with Ltp. Plantarum 75 improves the AA and antioxi-
dant capacity (FRAP, DPPH, and ABTS) due to an increase in the total phenolic com-
pounds and carotenoid constituents after fermentation. VOP 1 smoothies fermented
for 24 h had acceptable sensory properties. Ltp. Plantarum 75 was viable and could
survive the harsh conditions of the gastrointestinal tract; hence, it could be used as
a probiotic in cowpea leaf smoothies. VOP 1 cowpea leaf smoothies have bioaccessi-
ble polyphenols and antidiabetic effects at gastric and intestinal phases, as shown by
Foods 2023,12, 1701 17 of 20
its increased glucose uptake by the C
2
C
12
cells. Moreover, VOP 1 digesta had higher
α
-glucosidase and
α
-amylase inhibition activity and could be effective in managing dia-
betes. Thus, the lactic acid bacteria fermentation of cowpea leaf smoothies could improve
the bioaccessibility of carotenoids, phenolic compounds, and antioxidants, and improves
its potential as a vehicle for nutraceuticals to manage pathological conditions in the body.
Future research should focus on the bioaccessibility of phenolic compounds in CaCo
2
cells
and the prebiotic potential of polyphenols in cowpea leaves should be investigated.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods12081701/s1, Table S1: The limit of detection (LOD), the
limit of quantification (LOQ), and regression equations for carotenoid identification and quantifi-
cation using HPLC–DAD; Table S2: The microbial load of viable bacterial count in fermented and
unfermented cowpea leaf smoothies; Table S3: The correlation coefficient of total phenolic content
with antioxidants and
α
-glucosidase of fermented and unfermented cowpea leaf smoothies obtained
from three cultivars.
Author Contributions:
M.R.M.: formal analysis, investigation, and writing—original draft prepara-
tion; S.A.A.: conceptualization, methodology, validation, data curation, and writing—review and
editing; F.S.: visualization and writing—review and editing; T.S.: software and data curation; Y.S.,
J.L.S. and F.R.: resources and conceptualization; D.S.: conceptualization, supervision, project admin-
istration, and funding acquisition. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was supported and funded by the National Research Foundation (Grant
No. 98352) for the Phytochemical Food Network to Improve Nutritional Quality for Consumers.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data supporting this research will be made available upon request.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Moloto, M.R.; Phan, A.D.T.; Shai, J.L.; Sultanbawa, Y.; Sivakumar, D. Comparison of phenolic compounds, carotenoids, amino
acid composition,
in vitro
antioxidant and anti-diabetic activities in the leaves of seven cowpea (Vigna unguiculata) cultivars.
Foods 2020,9, 1285. [CrossRef] [PubMed]
2.
Seke, F.; Moloto, M.R.; Shoko, T.; Sultanbawa, Y.; Sivakumar, D. Comparative study of the functional compounds and antioxidant
properties of different cowpea (Vigna unguiculata) leaf cultivars after
in vitro
digestion. Int. J. Food Sci. Technol.
2022
,58, 1089–1097.
[CrossRef]
3.
Kitinoja, L.; Saran, S.; Roy, S.K.; Kader, A.A. Postharvest technology for developing countries: Challenges and opportunities in
research, outreach and advocacy. J. Sci. Food Agric. 2011,91, 597–603. [CrossRef] [PubMed]
4.
Guiné, R. The drying of foods and its effect on the physical-chemical, sensorial and nutritional properties. Int. J. Food Eng.
2018,2, 93–100. [CrossRef]
5.
Kasangi, D.M.; Shitandi, A.A.; Shalo, P.L.; Mbugua, S.K. Effect of spontaneous fermentation of cowpea leaves (Vigna unguiculata)
on proximate composition, mineral content, chlorophyll content and beta-carotene content. Int. Food Res. J. 2010,17, 721–732.
6.
Calín-Sánchez, Á.; Lipan, L.; Cano-Lamadrid, M.; Kharaghani, A.; Masztalerz, K.; Carbonell-Barrachina, Á.A.; Figiel, A.
Comparison of traditional and novel drying techniques and its effect on quality of fruits, vegetables and aromatic herbs. Foods
2020,9, 1261. [CrossRef]
7.
Tang, Z.; Zhao, Z.; Wu, X.; Lin, W.; Qin, Y.; Chen, H.; Wang, Y.; Zhou, C.; Bu, T.; Xiao, Y.; et al. A Review on fruit and vegetable
fermented beverage-benefits of microbes and beneficial effects. Food Rev. Int. 2022, 1–38. [CrossRef]
8.
Oguntoyinbo, F.A.; Fusco, V.; Cho, G.S.; Kabisch, J.; Neve, H.; Bockelmann, W.; Huch, M.; Frommherz, L.; Trierweiler, B.;
Franz, C.M.; et al. Produce from Africa’s gardens: Potential for leafy vegetable and fruit fermentations. Front. Microbiol.
2016,7, 981. [CrossRef]
9.
Franz, C.M.; Huch, M.; Mathara, J.M.; Abriouel, H.; Benomar, N.; Reid, G.; Galvez, A.; Holzapfel, W.H. African fermented foods
and probiotics. Int. J. Food Microbio. 2014,190, 84–96. [CrossRef]
10.
Fessard, A.; Kapoor, A.; Patche, J.; Assemat, S.; Hoarau, M.; Bourdon, E.; Bahorun, T.; Remize, F. Lactic fermentation as an efficient
tool to enhance the antioxidant activity of tropical fruit juices and teas. Microorganisms 2017,5, 23. [CrossRef]
Foods 2023,12, 1701 18 of 20
11.
Blana, V.A.; Grounta, A.; Tassou, C.C.; Nychas, G.J.E.; Panagou, E.Z. Inoculated fermentation of green olives with potential
probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives. Food
Microbiol. 2014,38, 208–218. [CrossRef]
12.
Bartkiene, E.; Vidmantiene, D.; Juodeikiene, G.; Viskelis, P.; Urbonaviciene, D. Lactic acid fermentation of tomato: Effects on
cis/trans lycopene isomer ratio,
β
-carotene mass fraction and formation of L (+)-and D (–)-lactic acid. Food Technol. Biotechnol.
2013,51, 471–478.
13.
Failla, M.L.; Chitchumroonchokchai, C.; Ishida, B.K.
In vitro
micellarization and intestinal cell uptake of cis isomers of lycopene
exceed those of all-trans lycopene. J. Nutr. 2008,138, 482–486. [CrossRef]
14.
Kiczorowski, P.; Kiczorowska, B.; Samoli ´nska, W.; Szmigielski, M.; Winiarska-Mieczan, A. Effect of fermentation of chosen
vegetables on the nutrient, mineral, and biocomponent profile in human and animal nutrition. Sci. Rep.
2022
,12, 13422. [CrossRef]
15.
Degrain, A.; Manhivi, V.; Remize, F.; Garcia, C.; Sivakumar, D. Effect of lactic acid fermentation on color, phenolic compounds
and antioxidant activity in African nightshade.Microorg 2020,8, 1324. [CrossRef]
16.
Managa, M.G.; Akinola, S.A.; Remize, F.; Garcia, C.; Sivakumar, D. Physicochemical parameters and bioaccessibility of lactic
acid bacteria fermented chayote Leaf (Sechium edule) and pineapple (Ananas comosus) smoothies. Front. Nutr.
2021
,8, 649189.
[CrossRef]
17.
Gao, H.; Wen, J.J.; Hu, J.L.; Nie, Q.X.; Chen, H.H.; Xiong, T.; Ning, S.; Xie, M.Y. Fermented Momordica charantia L. juice modulates
hyperglycemia, lipid profile, and gut microbiota in type 2 diabetic rats. Int. Food Res. J. 2019,121, 367–378. [CrossRef]
18.
Fujita, A.; Sarkar, D.; Genovese, M.I.; Shetty, K. Improving anti-hyperglycemic and anti-hypertensive properties of camu-camu
(Myriciaria dubia Mc. Vaugh) using lactic acid bacterial fermentation. Process Biochem. 2017,59, 133–140. [CrossRef]
19.
Mashitoa, F.M.; Akinola, S.A.; Manhevi, V.E.; Garcia, C.; Remize, F.; Slabbert, R.M.; Sivakumar, D. Influence of Fermentation
of Pasteurised Papaya Puree with Different lactic acid Bacterial Strains on Quality and bioaccessibility of phenolic compounds
during in vitro digestion. Foods 2021,10, 962. [CrossRef]
20.
Chiou, Y.S.; Wu, J.C.; Huang, Q.; Shahidi, F.; Wang, Y.J.; Ho, C.T.; Pan, M.H. Metabolic and colonic microbiota transformation may
enhance the bioactivities of dietary polyphenols. J. Funct. Food 2014,7, 3–25. [CrossRef]
21.
Zhao, D.; Shah, N.P. Lactic acid bacterial fermentation modified phenolic composition in tea extracts and enhanced their
antioxidant activity and cellular uptake of phenolic compounds following
in vitro
digestion. J. Funct. Food
2016
,20, 182–194.
[CrossRef]
22.
Valero-Cases, E.; Nuncio-Jáuregui, N.; Frutos, M.J. Influence of fermentation with different lactic acid bacteria and
in vitro
digestion on the biotransformation of phenolic compounds in fermented pomegranate juices. J. Agric. Food Chem.
2017,65, 6488–6496. [CrossRef] [PubMed]
23.
Chen, X.; Yuan, M.; Wang, Y.; Zhou, Y.; Sun, X. Influence of fermentation with different lactic acid bacteria and
in vitro
digestion
on the change of phenolic compounds in fermented kiwifruit pulps. J. Food. Sci. Technol. 2022,57, 2670–2679. [CrossRef]
24.
Yang, J.; Ji, Y.; Park, H.; Lee, J.; Park, S.; Yeo, S.; Shin, H.; Holzapfel, W.H. Selection of functional lactic acid bacteria as starter
cultures for the fermentation of Korean leek (Allium tuberosum Rottler ex Sprengel.). Int. J. Food Microbiol.
2014
,191, 164–171.
[CrossRef]
25.
Reddy, S.R.S.; Karnena, M.K.; Yalakala, S.; Saritha, V. Biological treatability of low total dissolved solids (LTDS) using SBR as a
pre-treatment for reverse osmosis. J. Water Resour. Prot. 2020,12, 135–154. [CrossRef]
26. Nielsen, S.S. Introduction to food analysis. In Food Analysis; Springer: Berlin/Heidelberg, Germany, 2017; pp. 3–16. [CrossRef]
27.
Sagbo, I.J.; van de Venter, M.; Koekemoer, T.; Bradley, G.
In vitro
antidiabetic activity and mechanism of action of
Brachylaena elliptica (Thunb.) DC. E. Based Complem. Altern. Med. eCAM 2018,2018, 4170372. [CrossRef]
28.
Chauke, A.M.; Shai, L.J.; Mogale, M.A. Plants today drugs tomorrow: Cordia Grandicalyx A possible future anti-hypoglycaemic?
J. Med. Plants By-Prod. 2022,in press. [CrossRef]
29.
Jabło´nska-Ry´s, E.; Sławi ´nska, A.; Skrzypczak, K.; Goral, K. Dynamics of changes in pH and the contents of free sugars, organic
acids and LAB in button mushrooms during controlled lactic fermentation. Foods 2022,11, 1553. [CrossRef]
30.
Dimitrellou, D.; Kandylis, P.; Kokkinomagoulos, E.; Hatzikamari, M.; Bekatorou, A. Emmer-based beverage fortified with fruit
juices. Appl. Sci. 2021,11, 3116. [CrossRef]
31.
Magwaza, L.S.; Opara, U.L. Analytical methods for determination of sugars and sweetness of horticultural products—A review.
Scie. Hortic. 2015,184, 179–192. [CrossRef]
32.
Soibam, H.; Ayam, V.S.; Chakraborty, I. Preparation, and evaluation of wine from sugarcane and beet juice. Adv. Biol. Res.
2017,8, 216–219.
33.
Cele, N.P.; Akinola, S.A.; Manhivi, V.E.; Shoko, T.; Remize, F.; Sivakumar, D. Influence of lactic acid bacterium strains on changes
in quality, functional compounds and volatile compounds of mango juice from different cultivars during fermentation. Foods
2022,11, 682. [CrossRef] [PubMed]
34.
Bhardwaj, R.L.; Mukherjee, S. Effects of fruit juice blending ratios on kinnow juice preservation at ambient storage condition.
Afr. J. Food Sci. 2011,5, 281–286.
35.
Yang, X.; Zhou, J.; Fan, L.; Qin, Z.; Chen, Q.; Zhao, L. Antioxidant properties of a vegetable–fruit beverage fermented with
two Lactobacillus plantarum strains. Food Sci. Biotech. 2018,27, 1719–1726. [CrossRef] [PubMed]
Foods 2023,12, 1701 19 of 20
36.
Kaprasob, R.; Kerdchoechuen, O.; Laohakunjit, N.; Sarkar, D.; Shetty, K. Fermentation-based biotransformation of bioactive
phenolics and volatile compounds from cashew apple juice by select lactic acid bacteria. Process Biochem.
2017
,59, 141–149.
[CrossRef]
37.
Yu, T.; Niu, L.; Iwahashi, H. High-pressure carbon dioxide used for pasteurization in food industry. Food Eng. Rev.
2020,12, 364–380. [CrossRef]
38. Znamirowska, A.; Szajnar, K.; Pawlos, M. Probiotic fermented milk with collagen. Dairy 2020,1, 126–134. [CrossRef]
39.
Hashemi, S.M.B.; Mousavi Khaneghah, A.; Barba, F.J.; Nemati, Z.; Sohrabi Shokofti, S.; Alizadeh, F. Fermented sweet lemon juice
(Citrus limetta) using Lactobacillus plantarum LS5: Chemical composition, antioxidant and antibacterial activities. J. Food Funct.
2017,38, 409–414. [CrossRef]
40.
Panda, S.K.; Behera, S.K.; Witness Qaku, X.W.; Sekar, S.; Ndinteh, D.T.; Nanjundaswamy, H.M.; Ray, R.C.; Kayitesi, E. Quality
enhancement of prickly pears (Opuntia sp.) juice through probiotic fermentation using Lactobacillus fermentum-ATCC 9338. LWT
2017,75, 453–459. [CrossRef]
41.
Chen, R.; Chen, W.; Chen, H.; Zhang, G.; Chen, W. Comparative evaluation of the antioxidant capacities, organic acids, and
volatiles of papaya juices fermented by Lactobacillus acidophilus and Lactobacillus plantarum.J. Food Qual.
2018
,2018, 9490435.
[CrossRef]
42. Eddy, B.P.; Ingram, M. Interactions between ascorbic acid and bacteria. Bacteriol. Rev. 1953,17, 93–107. [CrossRef]
43.
Abasi Joozdani, F.A.; Taghdir, M. Evaluation of transport mechanism of ascorbic acid through cyclic peptide-based nanotubes:
A molecular dynamics study. J. Mol. Liq. 2022,349, 118136. [CrossRef]
44.
Lee, J.Y.; Kim, C.J.; Kunz, B. Identification of lactic acid bacteria isolated from kimchi and studies on their suitability for application
as starter culture in the production of fermented sausages. Meat Sci. 2006,72, 437–445. [CrossRef]
45.
Mellican, R.I.; Li, J.; Mehansho, H.; Nielsen, S.S. The role of iron and the factors affecting off-color development of polyphenols.
J. Agric. Food Chem. 2003,51, 2304–2316. [CrossRef]
46.
Takó, M.; Zambrano, C.; Kotogán, A.; Kerekes, E.B.; Papp, T.; Krisch, J.; Vágvölgyi, C. Fermentative and enzyme-assisted
production of phenolic antioxidants from plant residues. In Microbial Fermentation and Enzyme Technology; CRC Press: Boca Raton,
FL, USA, 2020; pp. 175–193.
47.
Cebeci, A.; Gürakan, C. Properties of potential probiotic Lactobacillus plantarum strains. Food Microbiol.
2003
,20, 511–518.
[CrossRef]
48.
Panda, S.H.; Ray, R.C. Lactic acid fermentation of
β
-carotene rich sweet potato (Ipomoea batatas L.) into lacto-juice. Plant Foods
Hum. Nutr. 2007,62, 65–70. [CrossRef]
49.
Do, T.V.T.; Fan, L. Probiotic viability, qualitative characteristics, and sensory acceptability of vegetable juice mixture fermented
with lactobacillus strains. Food Nutr. Sci. 2019,10, 412–427. [CrossRef]
50.
Bouayed, J.; Hoffmann, L.; Bohn, T. Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated
gastro-intestinal digestion and dialysis of apple varieties: Bioaccessibility and potential uptake. Food Chem.
2011
,128, 14–21.
[CrossRef]
51.
Tagliazucchi, D.; Verzelloni, E.; Bertolini, D.; Conte, A.
In vitro
bio-accessibility and antioxidant activity of grape polyphenols.
Food Chem. 2010,120, 599–606. [CrossRef]
52.
Shahidi, F.; Ambigaipalan, P. Phenolics and Polyphenolics in foods, beverages and spices: Antioxidant activity and health
effects–A review. J. Food. Funct. 2015,18, 820–897. [CrossRef]
53.
Ketnawa, S.; Reginio, F.C., Jr.; Thuengtung, S.; Ogawa, Y. Changes in bioactive compounds and antioxidant activity of plant-based
foods by gastrointestinal digestion: A review. Crit. Rev. Food Sci. Nutr. 2022,62, 4684–4705. [CrossRef]
54. Klip, A.; McGraw, T.E.; James, D.E. Thirty sweet years of GLUT4. J. Biol. Chem 2019,294, 11369–11381. [CrossRef] [PubMed]
55.
Shimizu, M.; Deguchi, A.; Hara, Y.; Moriwaki, H.; Weinstein, I.B. EGCG inhibits activation of the insulin-like growth factor-1
receptor in human colon cancer cells. Biochem. Biophys. Res. Commun. 2005,334, 947–953. [CrossRef] [PubMed]
56.
Kim, K.S.; Park, K.S.; Kim, M.J.; Kim, S.K.; Cho, Y.W.; Park, S.W. Type 2 diabetes is associated with low muscle mass in older
adults. Geriatr. Gerontol. Int. 2014,14 (Suppl. 1), 115–121. [CrossRef] [PubMed]
57.
Fraga, C.G.; Galleano, M.; Verstraeten, S.V.; Oteiza, P.I. Basic biochemical mechanisms behind the health benefits of polyphenols.
Mol. Asp. Med. 2010,31, 435–445. [CrossRef]
58.
Aryaeian, N.; Sedehi, S.K.; Arablou, T. Polyphenols and their effects on diabetes management: A review. Med. J. Islam. Republi.
Iran 2017,31, 134. [CrossRef]
59.
Fraisse, D.; Bred, A.; Felgines, C.; Senejoux, F. Impact of simulated gastrointestinal conditions on Antiglycoxidant and
α-glucosidase inhibition capacities of cyanidin-3-O-glucoside. Antioxidants 2021,10, 1670. [CrossRef]
60.
Les, F.; Arbonés-Mainar, J.M.; Valero, M.S.; López, V. Pomegranate polyphenols and urolithin A inhibit
α
-glucosidase, dipeptidyl
peptidase-4, lipase, triglyceride accumulation and adipogenesis related genes in 3T3-L1 adipocyte-like cells. J. Ethnopharm.
2018,220, 67–74. [CrossRef]
61.
Rusak, G.; Šola, I.; Vujˇci´c Bok, V. Matcha and Sencha green tea extracts with regard to their phenolics pattern and antioxidant and
antidiabetic activity during in vitro digestion. J. Food Sci. Technol. 2021,58, 3568–3578. [CrossRef]
62.
Alqahtani, M.S.; Alqahtani, A.; Al-Thabit, A.; Roni, M.; Syed, R. Novel lignin nanoparticles for oral drug delivery. J. Mater. Chem.
B2019,7, 4461–4473. [CrossRef]
Foods 2023,12, 1701 20 of 20
63.
Simsek, S.; El, S.N.; Kancabas Kilinc, A.K.; Karakaya, S. Vegetable and fermented vegetable juices containing germinated seeds
and sprouts of lentil and cowpea. Food Chem. 2014,156, 289–295. [CrossRef]
64.
Koh, W.Y.; Uthumporn, U.; Rosma, A.; Irfan, A.R.; Park, Y.H. Optimization of a fermented pumpkin-based beverage to improve
Lactobacillus mali survival and
α
-glucosidase inhibitory activity: A response surface methodology approach. Food Sci. Hum.
Wellness 2018,7, 57–70. [CrossRef]
65.
De Vries, M.C.; Vaughan, E.E.; Kleerebezem, M.; de Vos, W.M. Lactobacillus plantarum—Survival, functional and potential probiotic
properties in the human intestinal tract. Int. Dairy J. 2006,16, 1018–1028. [CrossRef]
66.
Elizaquível, P.; Sánchez, G.; Salvador, A.; Fiszman, S.; Dueñas, M.T.; López, P.; de Palencia, P.F.; Aznar, R. Evaluation of yogurt
and various beverages as carriers of lactic acid bacteria producing 2-branched (1, 3)-
β
-D-glucan. J. Dairy. Sci.
2011
,94, 3271–3278.
[CrossRef]
67.
Chen, Z.Y.; Hsieh, Y.M.; Huang, C.C.; Tsai, C.C. Inhibitory effects of probiotic Lactobacillus on the growth of human colonic
carcinoma cell line HT-29. Molecules 2017,22, 107. [CrossRef]
68.
Mesquita, M.C.; dos Santos Leandro, E.; Rodrigues de Alencar, E.; Botelho, R.B.A. Survival of Lactobacillus paracasei subsp.
paracasei LBC 81 in fermented beverage from chickpeas and coconut in a static
in vitro
digestion model. Fermentation
2021
,7, 135.
[CrossRef]
69.
Wu, C.; Zhang, J.; Chen, W.; Wang, M.; Du, G.; Chen, J. A combined physiological and proteomic approach to reveal lactic-acid-
induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance. Appl. Microbiol. Biotech.
2012,93, 707–722. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
Available via license: CC BY 4.0
Content may be subject to copyright.
