fermentation
Article
Efficacy of Ultrasound-Assisted Lactic Acid Fermentation and
Its Effect on the Nutritional and Sensory Quality of Novel
Chickpea-Based Beverage
Nana Adwoa Nkuma Johnson 1,* , Selorm Yao-Say Solomon Adade 1,2, John-Nelson Ekumah 1,3, Yanshu Li 1,
Garba Betchem 1, Eliasu Issaka 4 and Yongkun Ma 1,*
1 Department of Food Science and Engineering, School of Food and Biological Engineering, Jiangsu University,
301 Xuefu Road, Zhenjiang 202013, Jiangsu, China; sysadade@st.ug.edu.gh (S.Y.-S.S.A.);
nelsonjokum@ujs.edu.cn (J.-N.E.); sysadade@gmail.com (Y.L.); garbabetchem1@gmail.com (G.B.)
2 Department of Nutrition and Dietetics, Ho Teaching Hospital, 00233, Ho P.O. Box MA 374, Ghana
3 Department of Nutrition and Food Science, College of Basic and Applied Sciences, University of Ghana,
Legon P.O. Box LG 134, Ghana
4 School of Environmental Science and Engineering, Jiangsu University, 301 Xuefu Road,
Zhenjiang 202013, Jiangsu, China; qemmy17@gmail.com
* Correspondence: nana.anjohnson@yahoo.com (N.A.N.J.); mayongkun@ujs.edu.cn (Y.M.)
Abstract: Ultrasound-assisted fermentation is a promising approach for improving food products’
nutritional and sensory qualities. This study compared the effects of ultrasound-assisted fermentation
on chickpea-based beverages’ nutritive and sensory components. Proximate, phytochemical, and
microstructural data were examined to evaluate the effectiveness of the processing techniques. The
processed samples demonstrated significantly higher (p < 0.05) nutritional and sensory outcomes
compared to the unfermented sample. The unfermented sample had a total phytochemical level of
55.41 g/mL, whereas the fermented and ultrasound-assisted fermented samples had phytochemical
contents of 66.71 and 64.51 µg/mL, respectively. With values of 8.37 ± 0.11 and 7.21 ± 0.06, respec-
tively, on a 9-point hedonic scale, the sensory evaluation revealed that the traditionally fermented
Citation: Johnson, N.A.N.; Adade, sample had a greater acceptability rate than the ultrasound-treated sample. The presence of organic
S.Y.-S.S.; Ekumah, J.-N.; Li, Y.; acids in the final product was shown to be significantly impacted by fermentation, with propanoic
Betchem, G.; Issaka, E.; Ma, Y.
having the highest concentrations of 1.79 ± 3 × 10−5a, 5.56 ± 2 × 10−5b, and 3.50 ± 0.00344c mg/mL
Efficacy of Ultrasound-Assisted
for the unfermented, fermented, and ultrasound-assisted fermented beverages, respectively. Ac-
Lactic Acid Fermentation and Its
Effect on the Nutritional and Sensory cording to these results, using ultrasound-assisted fermentation with reduced fermentation time can
Quality of Novel Chickpea-Based improve chickpea-based beverages’ nutritional and functional benefits.
Beverage. Fermentation 2023, 9, 495.
https://doi.org/10.3390/ Keywords: chickpea; ultrasound-assisted fermentation; chemical structures; physical characteristics;
fermentation9060495 sensory profile
Academic Editor: Giacomo Zara
Received: 18 April 2023
Revised: 16 May 2023 1. Introduction
Accepted: 20 May 2023 A good source of fibre, minerals, vitamins, minerals, and energy, chickpeas may also
Published: 23 May 2023 include phytochemicals that have health benefits [1,2]. Many health advantages, such as
the prevention of diabetes, hypertension, cancer, cholesterol and heart disorders, obesity,
and enhanced gut health, are brought on by these nutritional and bioactive components [3].
Copyright: © 2023 by the authors. Functional foods are products formulated using scientific research and are recognized to
Licensee MDPI, Basel, Switzerland. provide additional physiological benefits beyond their fundamental nutritional functions.
This article is an open access article These foods can lower the chance of developing chronic diseases and improve gut health
distributed under the terms and when included in a regular diet [4,5]. Because they are derived from plants rather than
conditions of the Creative Commons animals and have a high nutritional value, chickpea milk and soymilk are great alternatives
Attribution (CC BY) license (https:// to animal milk [6]. They also serve as a functional beverage due to their high nutritional
creativecommons.org/licenses/by/ and health benefits. To produce plant milk or beverages, chickpea is mixed with other food
4.0/). items, such as yam [7], wheat [8], lupin [9], and coconut [10]. Plant milk is an emulsion
Fermentation 2023, 9, 495. https://doi.org/10.3390/fermentation9060495 https://www.mdpi.com/journal/fermentation
Fermentation 2023, 9, 495 2 of 24
obtained when legumes, cereals, pseudocereals, nuts, and oilseeds are blended with water
to resemble cow’s milk. Plant-based milk is becoming increasingly popular due to consumer
interest sparked by concerns about health (including lactose intolerance and animal milk
allergies), the environment, and long-term sustainability [11].
Again, chickpeas are becoming increasingly popular as a plant-based substitute for
beverages derived from dairy and soy because of their high protein and fibre, low fat, and
potential health benefits. They also provide many phytochemicals such as polyphenols,
flavonoids, and carotenoids, all of which have a variety of benefits for one’s health [12].
However, the processing of chickpea-based beverages may impact their nutritional value,
sensory appeal, and shelf life, which may limit their acceptability and marketability. For
instance, using conventional processing methods such as heating might result in the
denaturation of proteins, the loss of phytochemicals, and changes to the sensory properties
of the final product [13]. In addition, the thermal processing of chickpea drinks reduced the
quantity of important amino acids, particularly lysine, histidine, and methionine, according
to [9]. According to [14], conventional food processing methods such as heating, drying,
and smoking can lead to changes in the sensory properties of the food. More specifically,
smoking can lead to the formation of polycyclic aromatic hydrocarbons (PAHs), which can
affect the flavour and aroma of the food. Heating and drying can also lead to changes in
the sensory properties of food.
Since ancient times, the food and beverage industries employed fermentation as
an effective and affordable way to preserve, enhance flavour, and improve the nutri-
tional value of items, generally having a major impact on the product’s physicochemical
attributes [15,16]. Recent studies focused on plant-based fermented milk’s antioxidant qual-
ities and process optimization [17]. In 2017, the US food market’s fastest-growing segment,
with a market value of USD 1347 billion, was fermented beverages. The importance of
probiotic-rich fermented beverages in human diets across the globe can be seen in the fact
that fermentation is a practical, cost-effective process that plays a crucial role in preserving
and enhancing food’s nutritional and sensory qualities [5]. Lactic acid bacteria potentially
enhance fermented beans’ biological and anti-oxidant qualities [2]. High levels of antiox-
idants found in soymilk, a historically fermented food, were associated with antiradical
properties. Many health advantages of fermented soymilk were demonstrated, including
anticancer activity, digestive function, and B vitamin levels [18]. Due to the rise in allergic
responses, nonallergenic chickpea milk is a superior alternative to soymilk [2]. In addition
to improving the nutritional and bioactive properties of food substrates, fermentation is a
preservation method. It is claimed by [19] that fermentation involves using microorganisms
and enzymes to break down complex organic substances and remained an essential step in
developing many food products. Furthermore, fermentation of legumes using LAB was
recorded to improve their total phenolic acid [20], including antifungal phenolic acids [21],
organoleptic properties [22], and reduced anti-nutritional factors [20]. Plant-based products
benefit from this technique by having better safety, nutritional value, and shelf life.
A potentially beneficial technique for improving the nutritional value, phytochem-
ical and organic acid content, colour, and sensory profile of a variety of food products
recently emerged: ultrasound-assisted fermentation. Ultrasound attracted much interest
as a method of promoting enzyme activities, metabolic performance, and microorganism
growth during food fermentation [23,24]. To improve the extraction and release of intracel-
lular components such as proteins, carbohydrates, and phytochemicals and to encourage
the growth of probiotic microorganisms, the benefits of fermentation are combined with
those of ultrasound application to enhance the nutritional and functional qualities of the
finished product [25,26].
Additionally, it was demonstrated that ultrasound-assisted fermentation improves
the nutritional value of fermented products. Ref. [27] found that ultrasound-assisted
fermentation increased black soybeans’ antioxidant activity and total phenolic content. In
a 2021 study, Ref. [28] looked into the impact of ultrasound on the stability of fermented
milk. They found that during storage, ultrasound-assisted fermentation led to a lower pH
Fermentation 2023, 9, 495 3 of 24
and a higher titratable acidity, which boosted the fermented milk’s stability during storage
by providing unfavourable conditions for microbial growth. Therefore, it is feasible that
using ultrasound to encourage fermentation in chickpea-based beverages could offer a
novel and sustainable method for creating useful and enticing drinks that offer more health
advantages than those provided by conventional drinks.
According to Refs. [29,30], ultrasound can accelerate a variety of chemical processes,
including the Maillard reaction (MR), oxidation, esterification, and proteolysis, as fer-
mented foods mature. When compared to foods produced by conventional fermentation,
ultrasound-assisted fermentation is anticipated to enhance the quality of fermented foods
simultaneously with favourable texture, colour, flavour, and taste [31,32]. According to
research by Refs. [32–34], numerous elements of food products, including proximate con-
tent, microstructure, phytochemical and organic acid content, colour, and sensory profile,
were demonstrated to benefit from the use of ultrasound-assisted fermentation. Fermented
milk, soybeans, and grape pomace are a few examples of food items that were researched
in this regard. The precise effects of ultrasound-assisted fermentation on chickpea bev-
erages are yet to be studied. Therefore, the goal of this study is to examine how the
nutritional, sensory, and consumer approval of beverages made from chickpeas are affected
by ultrasound-assisted fermentation.
The main objective was to assess any variations in the beverages’ nutritional com-
position, such as changes in the amounts of proteins, carbs, and other crucial elements
that might take place throughout the fermentation process. High-performance liquid
chromatography (HPLC) was used to evaluate these alterations. Furthermore, the impact
of ultrasound-assisted fermentation on the general sensory profile of beverages made
from chickpeas was evaluated. The study measured the scent and colour changes in the
beverages using colourimetry and electronic nose (eNose) technology. The acceptance of
the beverages among customers, investigations on consumer taste, and acceptability were
also carried out. The use of ultrasound-assisted fermentation in this study may help create
unique chickpea-based drinks with better nutritional and sensory qualities. Ultimately,
this research may open the door to creating novel, nutrient-dense, and consumer-pleasing
functional food products.
2. Materials and Methods
2.1. Formulation of Ultrasound-Assisted Chickpea Fermented Milk
2.1.1. Chickpea Milk Preparation
As Ref. [7] demonstrated, fresh boiled Cicer arietinum (chickpea) beverage was made
with some minor adjustments. In a nutshell, 200 g of chickpeas were measured out, followed
by overnight soaking in NaHCO3, after which the chickpeas were dehusked after being
removed from the water. The dehulled chickpeas were blanched in a solution containing
0.5% NaHCO3 for 30 min at a temperature of 100 ◦C, washed in clean water from the tap,
and then blended at high speed for 5 min with sufficient hot distilled water at a temperature
of 60 ◦C to obtain a solid content of 16%. The obtained chickpea milk was pasteurized in a
water bath at 60 ◦C for 30 min.
2.1.2. Ultrasound-Assisted Chickpea Milk Preparation
Fermentation of chickpea beverages was achieved based on previous experiments
carried out to establish optimal ultrasonic and fermentation parameters (under review).
These parameters were obtained using response surface methodology (RSM) coupled
with Box–Behnken design (BBD). The LAB used, Lacticaseibacillus paracasei S2601-8, was
isolated from a bacteria compound obtained from Danyang Yihe Food Co., Ltd. (Danyang,
China) and cultured in MRS broth. A 3% volume of the culture was then centrifuged
and washed in saline water twice. The washed cells were then suspended in 1% glucose
solution for 30 min, after which the solution was added to the prepared beverage; thus, an
inoculum size of 3% Lacticaseibacillus paracasei S2601-8 was added to a chickpea beverage
that had 16% solid content. Before the ultrasonication process, the beverage was allowed to
Fermentation 2023, 9, x FOR PEER REVIEW 4 of 25 
 
isolated from a bacteria compound obtained from Danyang Yihe Food Co., Ltd. (Dan-
yang, China) and cultured in MRS broth. A 3% volume of the culture was then centri-
fuged and washed in saline water twice. The washed cells were then suspended in 1% 
Fermentation 2023, 9, 495 glucose solution for 30 min, after which the solution was added to the prepared beve4roafg2e4 ; 
thus, an inoculum size of 3% Lacticaseibacillus paracasei S2601-8 was added to a chickpea 
beverage that had 16% solid content. Before the ultrasonication process, the beverage was 
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in20t0h erpbmev.e Trahgee pprroecpeasrsa itniovnoilsvecdap itnu rtehde ibneFviegruargee 1p. rTehpearuantfioernm ise nctaepdt,ufreermd ein tFeidg,uarned 1U. TAhFe 
cuhnicfkeprmeaebnetevde,r afgerems wenetredd, iaspnedn UseAdFin cthoisctkeprielae cboenvteariangeers (w50e0rem dLisepaecnhs)efdo rinsttor asgteerialte 4c◦oCn-
ptraiionretros a(5n0a0l ymseLs .each) for storage at 4 °C prior to analyses. 
 
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22.2.2..P PrrooxximimaatteeA Annaalylyssisis 
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mdouirsetsu: rme ocoisntuterne tc, otnotteanl ts,o tloidtasl, sfoalti,dps,r ofatet,i np,roatsehi,n,a nasdh,c arnbdo hcayrdbroahteysd.raTthese. fTohrem fuolrameufloare 
ofbotra oinbitnagintihneg pthroex pimroaxtiemcaotnet econntsteanretsd aersec driebsecdribbeeldo wbe. low. 
TThheem mooisistuturreec coonntetenntto offt hthee𝑀c ch𝑜hi𝑖ci𝑠ck𝑡k𝑢pp𝑟ee𝑒aa𝑐b b𝑜e𝑛evv𝑡e𝑒er𝑛raag𝑡geessw waassd dee1tet0errmminineeddu ussininggt htheef oformrmuulala: : %  W2=− W3%Moisture content = 0 (1)
W2 − × 100 (1)W1
where W1 = weight of empty crucible, W2 = weight of crucible and sample, and W3 = 
wwheeirgehtW of1 cr=ucwibelieg ahntdo sfamempplet yaftcerru dcirbylien,gW in2 th=e owveeing.h t of crucible and sample, and
W3 = Twheei gahmt oofucnrtu ocifb sleolaidn dbsya mdipffleereanftceer dwraysi ncgominptuhteeodv beny. deducting the moisture per-
centTaghee farmomou onnt eo fhsuonliddrbeyd𝑇 d(𝑜1if𝑡0f𝑎0e𝑙r):e𝑠 n𝑜c𝑙𝑖e𝑑w𝑠 as comput𝑀ed𝑜𝑖b𝑠y𝑡𝑢d𝑟e𝑒d𝑐u𝑜c𝑛ti𝑡n𝑒g𝑛𝑡the moisture percentagefrom one hundred (100):  = 100 − %   (2)
Total solids = 100 − %Moisture content (2)
The ash content of the chickpea beverages sample was determined using the for-
mulaT:h e ash content of the chickpe𝐴a 𝑠bev𝑐e𝑜r𝑛a𝑡g𝑒e𝑛s𝑡sample wa1s0d0etermined using the formula:% ℎ W3=− W1  (3)
%Ash content = × 100 (3)
W2
where W1 = weight of empty crucible, W2 = weight of crucible and sample, and
W3 = weight of crucible with lid and ash.
 The amount of crude protein was obtained using titration. The % of nitrogen and
crude protein of the chickpea beverage sample were obtained using the formula:
Calculation: 1 mL of 0.01 N HCl titrated against sample = 0.0015 g;
Fermentation 2023, 9, 495 5 of 24
Titre volume = (A − B) mL;
A − B × 0.0015
%Nitrogen = × V × 100 (4)
W V1
%Crude Protein = %Nitrogen × Conversion factor (6.25) [36]
where; A = volume of 0.01 mol/L HCl titrated against sample;
B = volume of 0.01 mol/L HCl titrated against blank =1.2 mL;
W = weight of sample = 2 mL;
V = total volume of sample digest = 50 mL;
V1 = volume of sample digest distilled = 5 mL.
Using the Soxhlet equipment, the crude fat of the samples was ascertained. The
formula below was used to determine the percentage of crude fat (ether extract) removed
from the sample:
W2 − W1
%Crude fat = × 100 (5)
W1
where W1 = weight of empty extraction flask, W2 = sample, and W3 = weight of extraction
flask and fat after solvent evaporation.
Finally, the total carbohydrate content was measured by;
g
Total carbohydrate = 100 − mL (moisture + total protein + total fat + total ash)
100
2.3. Effect of Ultrasound Treatment on Chickpea Beverage Fermentation
2.3.1. Scanning Electron Microscopy (SEM) Analysis
A slightly modified version of the SEM method reported by Ref. [37] was utilized to
investigate the structures of both unfermented and fermented materials. Double-sided
adhesive tape was used to secure the raw and cultured samples to a copper sample holder.
Following this step, a conductive layer of gold powder was vacuum sprayed onto the
entire assembly (diameter: roughly 10 nm). Their structures were analyzed using an
acceleration voltage of 15 kV and a Hitachi S-3400N, which Hitachi High Technologies in
Tokyo, Japan, manufactured.
2.3.2. Atomic Force Microscopy (AFM) Analysis
The method outlined by Ref. [37] with slight modifications, was used to determine
the topography of chickpea beverages samples. The samples were dissolved in 0.01 M
saline phosphate buffer (pH 8.0) to prepare a 10 µg/mL final concentration. The solution
was heated in a thermostatic water bath (50 ◦C) for 10 min and centrifuged (4000 rpm,
10 min). Next, 5 µL aliquots of the supernatant were rapidly pipetted onto a newly cleaved
mica substrate and dried in an incubator (25 ◦C) for 12 h in Petri dishes. A multimode
microscope (Bruker, Santa Barbara, CA, USA) was used to generate the AFM images of the
samples. The lens was used in Peak Force QNM mode with a Bruker ScanAsyst needle at a
typical spring and resonance frequency of 25.1 N/m and 300 kHz, respectively.
2.3.3. Fourier Transform Infrared (FTIR) Spectroscopy
The FTIR spectroscopy approach described by Ref. [37] was utilized with minor
changes to identify the chemical structure of unfermented and fermented materials. With a
mortar and pestle (both made of agate), 4 mg of freeze-dried fermented and unfermented
(control) chickpea beverages were ground separately and well combined with 200 mg of
dried spectroscopic grade KBr (at 105 ◦C for 24 h) powder. The resulting mixture was
compressed with a hydraulic machine (10 t) in 1–2 mm-thick see-through (transparent)
glass-like pellets. The pellets were scanned at a resolution of 4 cm−1 in a wavenumber
range of 4000–400 cm−1, with 128 scans using the Nicolet IS50 equipment (Thermo Nicolet
Fermentation 2023, 9, 495 6 of 24
Corporation, Waltham, MA, USA). The blank (KBr pellet without test samples) utilized in
the parameter setup was provided as reference spectra.
2.4. High-Performance Liquid Chromatography (HPLC) Analysis
2.4.1. Organic Acids
Analysis of organic acids, mainly mallic, lactic, acetic, propanoic, and citric acid, was
performed by following the method proposed by Ref. [38] with few modifications. A
standard curve was used as a reference for the various organic acids with concentrations of
0.05, 0.2, 0.5, 2, and 5 mg/mL. Subsequently, the chickpea beverages were centrifuged, and
the obtained supernatant was filtered using a 0.45 mm membrane filter. Next, 20 mL of the
filtered sample was injected into an Agilent1100 HPLC system with an Agilent ZORBAX
SB-Aq column (250 mm × 4.6 mm), and quantification was conducted using a diode array
detector (DAD). The conditions were: mobile phase A: 0.1% phosphoric acid aqueous
solution; mobile phase B: methanol; detection wavelength: 210 nm; column temperature
40 ◦C; flow rate 1 mL/min; and sample volume 10 uL.
2.4.2. Phytochemicals
The quantitative identification of phytochemicals was conducted using HPLC-MS. The
sample extraction was carried out using a Superclean LC-18 cartridge (Supelco, Bellefonte,
PA, USA) after it was conditioned with 10 mL methanol and deionized water. The samples
(5 mL), each with pH adjusted to 2 using a 7.2 M HCL, were separately dispensed into
the Supelclean LC-18 cartridge. The phenolic acids, flavonols, and anthocyanins were
separately eluted using 3.0 mL each of methanol, acetonitrile (20%, pH 2.5), and methanolic
HCl (0.01%), respectively. The eluents were transferred into dark sample vials and stored,
awaiting filtration and subsequent instrumental analysis.
Polyphenol testing was conducted according to Ref. [38] with some modifications.
Quickly, 2 g of the chickpea sample was weighed into a 10 mL centrifuge tube containing
5 mL of 70% methanol solution, oscillated and mixed, ultrasonicated for 30 min, and
centrifuged. The supernatant was collected and put in a liquid phase vial for testing,
using Agilent 1100; triple quadrupole mass spectrometer API4000 with Agilent Poroshell
120 EC-C18 2.7µm column (3 × 50 mm). The conditions set for the chromatography were
mobile phase A: 0.5% formic acid in water; mobile phase C: acetonitrile solution, flow rate:
0.6 mL/min, injection volume: 10 µL, and column temperature: 35 ◦C.
Mass spectrometry was used to quantify the polyphenols present in conjunction with
the quantitative identification of phytochemicals using HPLC-MS, set as spray voltage
4.5–5.5 kv: desolvation temperature 500 ◦C; and desolvation gas (N2) 1000 L/h. The
mass spectrometry scanning conditions were also set as ESI+ mode: spray voltage 5.5 kv;
desolvation temperature 500 ◦C; desolvation gas (N2) 1000 L/h, and ESI-mode: spray
voltage 4.5 kv; desolvation temperature 500 ◦C; and desolvation gas (N2) 1000 L/h.
2.5. Microbiological Analysis of Chickpea Beverage Samples
The microbial quality of the chickpea beverage samples was evaluated with standard
methods. The samples were diluted four times before plating. The colonies enumer-
ated after the incubation period were expressed as the log colony-forming unit as in
Equation (6) [39].
( ( )Colony − forming unit )log cfu mL−1
Number o f colonies countered (6)= Volume o f sample plated ÷ (Dilution f actor) )
The mesophilic aerobic bacteria enumeration of the chickpea beverage samples was
determined by pour plate technique, culturing 1 mL of serial dilutions on plate count agar
(PCA) (M0024, Hangwei, Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China) [39].
The plates were counted and expressed as log cfu mL−1. Additionally, the Staphylococcus
Fermentation 2023, 9, 495 7 of 24
aureus count was carried out by spread plating 0.1 mL of appropriate dilutions on Baird
Parker agar (BPA) (BW, Shanghai Bio-way Technology Co., Ltd., Shanghai, China). In an
inverted position, the plates were incubated at 37 ◦C for 24 h [40]. Staphylococcus aureus
colonies on Baird Parker agar are characterized by round shape, being slimy/smooth,
convex with a diameter of 2 to 3 mm, with a greyish to black colour, and having a clear
halo that develops around the edge of the colony [41]. Colonies with typical black shiny
and narrow white margins, surrounded by a clear zone extending into the opaque medium,
were counted and expressed as log cfu mL−1. Finally, the yeast and mould count of
the chickpea samples was determined by spread plating 0.1 mL of appropriate dilutions
aseptically on Rose Bengal chloramphenicol agar (RBCA) supplemented with 100 mg/L
chloramphenicol and incubated at 25 ◦C for 5 days [40,42]. The colonies with pink colour
were counted and expressed as log cfu mL−1.
2.6. Sensory Profile
2.6.1. Colour Determination
The colour of the chickpea beverage samples was measured using a CR-400 model
colourimeter with a xenon lamp as a light source. The angle was 45◦, and viewing was
carried out at 0◦. After standardization, the colour values L* representing light to dark,
a* representing redness to greenness, and b* representing yellowness to blueness were
measured. The total colour changes (∆E), chroma, browning index, and hue angle (Ho)
were calculated using Equations (7)–(12) [43]. The hue parameter describes what people
think when they describe the colour green, red, and yellow, among others, while chroma
represents the intensity or purity of the hue. The browning index refers to food products’
enzymatic and non-enzymatic browning, which can significantly impact their flavour,
colour, and texture [44]. √
Total colour change (∆E) = (L∗ − L∗)2 + (a∗o − a∗o )
2 + (b∗ − b∗ 2o ) (7)
where “o” denotes the colour values of raw√chickpeas with no treatment.
Chroma = (a∗)2 + (b∗)2 (8)
X − 0.31
Browning index = 100 × (9)
0.17
X = (a∗ + 1.75L∗)(6.645L∗ + a∗ − 3.012b∗) (10)
Hue angle (Ho) = tan − 1(b∗a∗), (when a > 0) (11)
Hue angle (Ho) = 180 + tan − 1(b∗a∗), (when a < 0) (12)
2.6.2. eNose Analysis
The UAF’s effect on the beverage’s aroma was examined using an electronic nose and
the method outlined by Ref. [45]. The PEN3 electronic nose apparatus, which the German
company Airsense Analytics created, was used in this investigation. Two positioned pumps,
one of which is utilized to monitor the sample gas compounds being sucked through the
sensor array, and an array of ten partly selective metal-oxide semiconductors (MOS) gas
sensors, are its main components. Table 1 lists the characteristics of these sensors made of
metal-oxide-semiconductor (MOS) gas sensors that are partially selective.
Fermentation 2023, 9, 495 8 of 24
Table 1. Gas sensors of the PEN3 electronic nose system and their features.
Sensor Number Sensor Name Main Applications
S1 W1C Aroma component 1
S2 W5S Broadrange (nitrogen oxide)
S3 W3C Aromatic component (ammonia)
S4 W6S Hydrogen
S5 W5C Aromatic hydrocarbons
S6 W1S Methane
S7 W1W Organic sulfide
S8 W2S Alcohols, aldehydes, and ketones
S9 W2W Aromatic sulfide
S10 W3S Alkanes
A 5 mL chickpea milk sample was added to a 20 mL transparent vessel, sealed with
plastic wrap, after which the sampling needle connected to Teflon was inserted. The needle
sucked in the volatile compounds in the headspace at a 300 mL/min injection flow rate
while a Luer Lock needle connected to a charcoal filter maintained constant air pressure in
the sampling vessel. The measurement and flush time were 120 and 140 s, respectively. The
stable response signs of each sensor were extracted for analysis.
2.6.3. Consumer Sensory Analysis Tests
Consumer selection for the sensory evaluation of the chickpea products was con-
ducted using the method reported by Ref. [45] with slight modification. The selection
criterion was the nonexistence of allergic reactions to soy products and the consumption of
fermented chickpeas. The consumers were divided into three separate sessions. Each of
the three consumer sessions had 35 different participants. Throughout the test, 60% of the
participants were female, and 40% were male. All the consumers that participated in the
study were chickpea products consumers and were in Jiangsu University (Zhenjiang city,
Jiangsu Province, China). The participants in the sensory analysis completed an informed
consent form before data collection after approval of the study protocol by the School of
Food and Biological Engineering, Jiangsu University.
The test was conducted at the School of Food and Biological Engineering, Jiangsu
University, in a food laboratory that had fluorescent lights with a uniform level of lighting,
was well ventilated (good ventilation), quiet, comfortable, and had controlled conditions
to avoid distractions. All the conditions were equal for all tests. The room temperature
was 24 ± 1 ◦C. A 9-point hedonic scale was used for the sensory evaluation of the chickpea
products, and the participants were instructed in a brief orientation session about the
evaluation. Each chickpea product was assigned a random 3-digit code and randomly
presented to the participants to avoid a first-order effect [46–48].
Next, 25 mL of chickpea samples were served in a monodic format to the participants
in transparent (colourless) plastic cups coded with 3 random digits at room temperature
(24 ± 1 ◦C). Each participant was given a glass of water (and crackers, biscuits) to rinse their
mouth (clear taste buds) before and after each sample tasting, and a total of three samples
were evaluated. The attributes evaluated were the appearance, colour, texture, flavor,
aroma, taste, mouthfeel, consistency, and overall acceptability of the chickpea samples.
The scores of 1 = dislike extremely, 2 = dislike, 3 = dislike moderately, 4 = dislike slightly,
5 = neither like nor dislike, 6 = like slightly, 7 = like moderately, 8 = like, and 9 = like
extremely were used by the participants after each sample tasting to assign a numerical
value to individual attributes.
2.6.4. Statistical Analysis
The samples were analyzed in triplicate, and the results are presented as means
standard deviations. A multiple T-test was used to compare paired variables, and Tukeys’
test assessed the mean difference at p < 0.05.
Fermentation 2023, 9, 495 9 of 24
3. Results
3.1. Proximate Analysis of Treated and Untreated Chickpea Beverages
A common technique for figuring out the nutritional makeup of food products is
proximate analysis, which measures the amounts of macronutrients such as proteins, carbs,
and fats, as well as micronutrients such as vitamins and minerals [36]. According to
research by Ref. [32], fermentation and ultrasound-assisted fermentation are beneficial
for increasing the proximate content of a variety of food products, including fermented
milk, soybeans, and grape pomace. Although not many studies particularly focus on
the effect of these techniques on the proximate content of chickpea-based beverages, the
advantages of these methods could make them useful for improving the nutritional value
of these drinks as well. The proximate contents of the chickpea beverages (Table 2) did not
show significant changes. However, the protein content reduced significantly between the
various samples with values of 0.71, 0.69, and 0.67 for UCB, FCB, and UAFCB, respectively.
The carbohydrate content also saw a significant increase between the unprocessed and
processed beverages with values of 7.85 ± 0.20, 12.95 ± 0.32, and 13.45 ± 1.45 for UCB,
FCB, and UAFCB, respectively.
Table 2. Comparison of the various proximate contents of chickpea beverages.
Proximate Content UCB FCB UAFCB
Moisture 85.16 ± 2.50 a 80.63 ± 0.55 a 81.12 ± 2.60 a
Solids 14.84 ± 2.49 a 19.37 ± 0.55 a 18.88 ± 2.60 a
Ash 0.32 ± 0.08 a 0.38 ± 0.15 a 0.39 ± 0.19 a
Proteins 0.71 ± 0.01 a 0.69 ± 0.01 b 0.67 ± 0.005 c
Fats 6 ± 1.00 a 5.67 ± 2.08 a 4.33 ± 1.53 a
Carbohydrates 7.85 ± 0.20 a 12.95 ± 0.32 b 13.45 ± 1.45 b
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; and UAFCB-ultrasound-assisted
fermented chickpea beverages. Results are presented as mean ± standard deviation. Values with different
superscripts were significantly different at p < 0.05.
Complex carbohydrates and proteins can be broken down into simpler forms during
fermentation, improving their availability for absorption and utilization by the body [49],
contrary to the observations made in this study. The crude carbohydrate content after
fermentation and UAF increased significantly (Table 2). To further understand this, it is
important to note that the solid contents of the chickpea beverages also increased due to the
presence of the microorganisms used for the fermentation, thus affecting the carbohydrate
content. It is safe to say the carbohydrate content obtained from the treated samples is not
accurate. Similarly, Refs. [19,50] found that the application of ultrasonic waves causes a
more even distribution of microorganisms throughout the fermentation medium, which
affects the action of the microorganisms on the product.
Furthermore, proteins and carbs, for example, can be released more readily during
ultrasound treatment, encouraging their breakdown and increasing their bioavailability [25].
Using ultrasonic waves can shorten fermentation times while increasing cell development
and product yield. Ref. [2] in a study discovered that during fermentation, the quantities of
some critical amino acids for human health, including lysine and tryptophan, increased.
This accounts for the slight decrease in crude protein contents observed after treatments.
This observation can be applied to the other proximate contents.
3.2. Effect of Processing on Structural Qualities of Ultrasound-Assisted Fermentation on
Chickpea Beverages
3.2.1. Influence of Ultrasonication on Structural Integrity via AFM
The atomic force microscope (AFM) visualizes conformational changes in structures of
samples that cannot be viewed with other visual-enhancing methods and displays image
surfaces of biological structures in their natural environment and oligomeric forms. This
analysis compared the topographic and nanostructural changes in UCB to those in UAFCB.
Both samples’ structures were distinct when viewed with an atomic force microscope, with
Fermentation 2023, 9, x FOR PEER REVIEW 10 of 25 
 
3.2. Effect of Processing on Structural Qualities of Ultrasound-Assisted Fermentation on Chickpea 
Beverages 
3.2.1. Influence of Ultrasonication on Structural Integrity via AFM 
The atomic force microscope (AFM) visualizes conformational changes in structures 
of samples that cannot be viewed with other visual-enhancing methods and displays 
Fermentation 2023, 9, 495 image surfaces of biological structures in their natural environment and oligo1m0 oefr2i4c 
forms. This analysis compared the topographic and nanostructural changes in UCB to 
those in UAFCB. Both samples’ structures were distinct when viewed with an atomic 
force microscope, with varying distributions and sizes of aggregates present. The ultra-
vsoaurynidn gtrdeaisttmriebnutt ifounrtshaenr daisdiezdes ino fthage gerveegna tdeisstprirbeusetinotn.  Tohf ethuel tpraarstoiculnesd ptrreeasetmnte dnut efu trot htheer 
abirdeeadkdinowthne oefv beonnddiss tprribeusetniotn ino fththe esapmarptliecl. eTshper etosepnotlodguiceatl oretphreebserneataktdioonw onf otfheb oUnCdBs  
pshreoswenedt  innatrhroewsa amnpdl eev. eTnhlye dtoisptorilbougtiecdal argegprreegsaentetsa,t wiointho of nthlye aU fCewB  sehspoewceiadllyn asrirgonwificaanndt 
esvtreuncltyudreisst, rmibuakteindga gthgere sgtartuecst,uwreit hhaovnely aa sfmewooetshp-elickiael lvyieswig.n Iifinc aa nrtesvtireuwct ubrye sR, emf.a [k5i1n]g, tthhee 
saturuthcotursr erehvaevaeleads mthoato tthh-el ipkleavnite lwiq.uInidas roerv sieuwspbenysRioenf.s[ 5h1a]d,  tah esmauotohtohr sanredv heaolmedogthenateothues 
psularnfatcleiq wuiidths oocrcsaussiopnenals iboings ahgagdreagasmteso,o wthhaicnhd ish oinm loingee nweiothu sthseu rofbace with occasional bigaggregates, which is in line with the observation made in this studyse. rTvhaetiaount mhoardseo ipni ntheids 
tshtuatdyth. eThper easuetnhcoerso foppirnoetdei nthsaitn ththe epbreesveenrcaeg eofp portoetnetiinasl liyn ctohnet briebvuetreadget opothteenftoiarlmlya ctioonn-
otrfibagugterdeg taot eths.e formation of aggregates. 
IInn ccoonnttrraasstt,, tthhee UUAAFFCCBB eexxhhiibbiitteedd aa mmoorree hheetteerrooggeenneeoouuss ssuurrffaaccee wwiitthh llaarrggeerr aaggggrree--
ggaatteess aanndd aa hhiigghheerr ddeeggrreeee ooff ssuurrffaaccee rroouugghhnneessss dduuee ttoo tthhee ddisisppeerrssiioonn ooff ppaarrttiicclleess.. TThhee 
ccoommbbiinneedd aacctitoionnsso of fu lutrlatrsaosnoincitch ethraepraypayn danfder mfeernmteantitoant,iownh, iwchheicnhc oeunrcaoguertahgeeb trheea kbdroewakn-
odfocwonm pofle cxopmroptleeixn spraontdeipnos laynsadc cphoalryisdaecschinatroidseims pinletor msimolepcleurl ems,oalreecurelseps,o anrseib rleesfpoorntshiebslee 
afoltre rthateisoen asl[t5e2ra].tiAonccso [r5d2i]n. gAtcocothrdeirnegs utolt sth(Fe irgeusruelt2s) ,(Fuilgtruarseo 2n)i,c uatlitoranssoinginciafitcioann tsliyginmifipcaacntteldy 
tihmepCacBt,ewd itthheU CABF,C wB’isths trUuActFuCraBl’sm satkreuuctpudraisl pmlaaykineugpa sduisppelraiyoirnfge ram esunpteedricohr icfkerpmeaenbteevd- 
ecrhaigckep. UeaA bFehvaersatghee. pUoAteFn thiaalst othbee paoutesenftuiallt etcoh bneiq au eufsoerfuiml tpercohvniinqguteh efopr hiymspicroocvhienmg itchael 
cphhayrsaicctoecrhisetmicsicaanl dchnaurtarcitteiorinsatilcvsa alnude  onfubtreivtieornaagle vsamluaed oeff rboemvecrahgiceksp meaasd.e from chickpeas. 
 
Figure 22.. AAttoommicicf oforcrecem micricorsocsocpoypiym iamgeasgsehs oswhoinwgitnhge tdhieff edrieffnecreesnincetsh eint otphoel otogpyoolfotghye uofn ftehrem uenfterd-
cmheicnktpedea cbheicvkepraega eb(eAve)raangde t(hAe) ualntrda tshoeu nudlt-rasssoiustned-faesrsmisetendte fderbmevenerteadge b(eBv)e.rage (B). 
33..22..22.. IInnflfluueennccee ooff UUllttrraassoonniiccaattiioonn oonn SSttrruuccttuurraall IInntteeggrriittyy vviiaa SSEEMM 
UUllttrraassoonniiccaattiioonn aanndd ffeerrmmeennttaattiioonn aarree ttwwoo ffoooodd pprroocceessssiinngg tteecchhnnoollooggiieess tthhaatt wweerree 
sshhoowwnn ttoo cchhaannggee tthhee mmiiccrroossttrruuccttuurree ooff ffoooodd pprroodduuccttss.. TThheessee cchhaannggeess aaffffeecctt tthhee pprrooppeerrttiieess 
ooff tthhee pprroodduucctt aass aa wwhhoollee.. UUltlrtraassoouunndd, ,fofor rininstsatnancec,e w, wasa ksnkonwown ntot cohcahnagneg teheth setrsutcrutucrteu roef 
opfroptreointesi,n tsh,etrheebrye bmyamkiankgi nthgetmhe emaseiears iteor dtoissdoilsvseo lavneda nbedttbeer tatet reamtuelmsifuylisnifgy i[n5g3].[ 5I3t ]w. aIst  
wshaoswshno twhant tuhlatrtauslotnraicsaotnioicna taiolsno ahlesolphs eblpresabkr edaokwdno cwonmcpolmexp cleaxrbcoahrybdorhaytdesr,a tseusc,hs uacsh oalis-
ogloigsaocscahccahriadreidse, sim, imprporvoivnign gthteh etetxetxutruer eanandd ddigigeestsitbibiliiltiyty oof ftthhee pprroodduucctt aanndd tthhee sshheellff lliiffee ooff 
ffoooodd pprroodduuccttss [[5544]].. TThheessee cchhaarraacctteerriissttiiccss ooff uullttrraassoonniicc ttrreeaattmmeennttss aarree ssiimmiillaarr ttoo pprroodduuccttss 
tthhaatt uunnddeerrwweennttf efermrmeenntatatitoionn. .S cSacnanninnignge leelcetcrotrnomn imcriocsrcoospcoyp(yS E(MSE)Mw)a ws uasse udsteodi dtoe nidtiefynttihfye  
mthoer pmhoorlpoghioclaolgcihcaarl acchtearriasctitcesriosftiUcsA oFfC BU.AAFcCoBm. pAa rcisoomnpoafrtihseonim oafg ethseo fimSEaMgebse otwf eSeEnMU CbeB-,
FtwCeBe,na nUdCtBh, eFUCBA,F aCnBd sthheo wUeAdFCthBa tshthoewperdo tcheasst etdhes apmropcelesssewde sraemmpolerse wpoerroe umsocroem ppoaroreuds 
to the unprocessed samples, with the UAFCB sample having a more porous and rough
surface (Figure 3). The compact nature observed in the UCB due to the bonds present was
cleaved during fermentation. Ultrasonication further cleaved the bonds, making the various
 components of the sample free and available to be utilized by the LAB. The micrograph of
the unfermented sample, as seen in Figure 3A, revealed a smooth continuous surface with
no visible fractures and a few tiny pores on the surface, as described by [7]. The structure
also showed visible continuous sheet-like structures that indicate the presence of starch
and protein structures, similar to the observations made by [55]. A visible degradation of
the cell wall is seen, changing it to a more loosened, irregular, rough surface with pits of the
Fermentation 2023, 9, x FOR PEER REVIEW 11 of 25 
 
compared to the unprocessed samples, with the UAFCB sample having a more porous 
and rough surface (Figure 3). The compact nature observed in the UCB due to the bonds 
present was cleaved during fermentation. Ultrasonication further cleaved the bonds, 
making the various components of the sample free and available to be utilized by the 
LAB. The micrograph of the unfermented sample, as seen in Figure 3A, revealed a 
smooth continuous surface with no visible fractures and a few tiny pores on the surface, 
as described by [7]. The structure also showed visible continuous sheet-like structures 
that indicate the presence of starch and protein structures, similar to the observations 
made by [55]. A visible degradation of the cell wall is seen, changing it to a more loos-
ened, irregular, rough surface with pits of the fermented chickpea sample in Figure 3B, 
which is also characterized by the increase in the sizes of the pores present with visible 
structural differences. The particles of the fermented chickpea sample characteristically 
showed a heterogenous and rough surface depicted by some cavitations due to the 
breakdown of cell structures by Lacticaseibacillus paracasei S2601-8 during fermentation. 
Refs. [7,56] opined that there was a degradation of starch and protein components of the 
cell walls by the LAB, which accounts for the availability of organic acids, phenolic con-
tent, and free amino acids among other nutraceuticals, bioactive, and nutritional com-
ponents of the beverages by facilitating the diffusion of nutrients, gases, and metabolic 
Fermentation 2023, 9, 495 products and improved digestibility of nutrients. These changes indicate the bio1lo1 gofic2a4l 
and chemical transformations that occur during fermentation, which were shown to im-
prove the nutritional quality, flavour, and shelf-life of legume-based products. 
fermeTnhteed UchAicFkCpBe asasammpplele pinarFtiicgluesr ed3iBsp, wlayheicdh ais malosorec hroauragchte, rpizoerdoubsy, tahnedin lcoroesaes esiunrftahcee 
swizietsho efvtehnelyp odriesstrpibreusteendt pwairtthicvleissi balneds tervuecntu lraarlgderif fcearveintacetiso.nTsh tehpanar tthicel efseromf tehnetefder msaemnpteldes 
c(hFiicgkupreea 3sBa)m, spimleilcahr atroa tchteer disetisccarlilpytisohno owf ewdhaeyh eptreortoeginens ooubssearnvdedr obuyg Rhesf.u [r5f7a]c.e Tdheep ainctaeldy-
bsyiss foomunedc atvhiatta tthioen csadvuiteattioonth pehberneoamkdeonwa,n wohficcehl lpsrtorduuctcuerde sa bsypoLnagctei ceaffseeicbta, cciollnutsrpibauratceads etio 
St2h6e0 U1-A8 FdBu’rsi nlogofseernmeden sttartuiocntu.rRe e(fFsi.g[u7r,5e6 3]Co)p. iTnhederteh watatsh aenre iwncarseaased eingr tahdea taimonouonf ts toafr crhe-
adnudcpinrgo tseuingacro mprpeosennent tbseocaf uthsee cthelel pwhaelnlsobmyetnhoenL cAaBu,swedh cicohvaalcecnotu, nntosnf-ocrotvhaeleanvta, ihlaybdilriotygeonf, 
oarngdan dicisauclifidds,ep choennnoelcicticoonnst ebnett,waenednf trheee ammoilneocualceisd sofa mthoen cgelolt whearlln tuot rbarceeaukt,i cianlcsr,ebaisoiancgti vthee,  
acnodnnceuntrtritaitoinoanl coof mlapcotince natcsido.f tBhoenbde vberreaagkeisngby cfoaucilldit ainticnrgeathse dthifefu bsiionavoafilnaubtirliietyn tso,fg pasoelys,-
apnhdenmoeltsa. bTohleic bpreroakdaugcets oaf nthdei mbopnrdosv oedf tdhieg cehsitcibkiplietay coefllnsu rtersiuenlttesd.  Tinh semseacllhear npgaersticinledsi cwaitteh 
tahne binioclroegaisceadl asnudrfacchee marieca latnradn ismfoprmroavteidon bsitohaavtaoilcacbuilritdyu orifn ngufterrimenetns tfaotiro tnh,ew fhericmhewnteirneg 
smhoicwronotrogiamnpisrmovse [5th8e]. nutritional quality, flavour, and shelf-life of legume-based products.
 
FFigiguurree 33. . SSccaannnniinngg eelleeccttrroonn iimmaaggeess sshhoowwiinngg tthhee ddiiffffeerreenncceess iinn tthhee mmiiccrroossttrruuccttuurree ooff tthhee uunnffeerr--
mmeennteteddc chhicickkppeeaab beevveerraaggee( A(A),),t htheef eferrmmeennteteddb beevveerraaggee( B(B),),a annddt htheeu ultlrtraassoouunndd-a-assssisisteteddf eferrmmeenntetedd 
beverage (C). 
beverage (C).
The UAFCB sample particles displayed a more rough, porous, and loose surface
with evenly distributed particles and even larger cavitations than the fermented samples
 (Figure 3B), similar to the description of whey proteins observed by Ref. [57]. The anal-
ysis found that the cavitation phenomena, which produced a sponge effect, contributed
to the UAFB’s loosened structure (Figure 3C). There was an increase in the amount of
reducing sugar present because the phenomenon caused covalent, non-covalent, hydrogen,
and disulfide connections between the molecules of the cell wall to break, increasing the
concentration of lactic acid. Bond breaking could increase the bioavailability of polyphe-
nols. The breakage of the bonds of the chickpea cells resulted in smaller particles with
an increased surface area and improved bioavailability of nutrients for the fermenting
microorganisms [58].
3.2.3. Influence of Ultrasonication on Structural Integrity via FTIR
The positions of the chemical structure of chickpea beverages shown as peaks of
lignin, hemicellulose, and cellulose examined the structural changes of the UCB, FCB,
and UAFCB were observed with FTIR (Figure 4). Minor increases in peak values were
observed in the C-I and C-Br stretch and the carbonyl (C=O) stretch after each processing
technique. However, the C≡C terminal alkyne (monosubstituted) stretch and the aromatic
and amines stretch observed a decrease in the peak values. The peak ranges observed were
within the iodo C-I, C≡C terminal alkyne, carbonyl (C=O), and NH2 stretches with varying
peak values. Chickpea fermentation results in beverages high in protein, soluble fibre,
Fermentation 2023, 9, x FOR PEER REVIEW 13 of 25 
 
Fermentation 2023, 9, 495 12 of 24
ids, peptides, proteins, alkaloids, DNA, and RNA because of amine (NH2) groups [7,63]. 
aAnmdibnioo aacctiidvse, cpoemptpidoense,n atns dco pmroptaeriends taoreu nkfneorwmne nttoe dbeb epvreesraegnet si,nm laarkgien gcothnecemntarpaptieoanlsin ign 
tcohihcekaplethas-m [6in6]d. eTdhdesrein ckhearsn,gaecsc oinr dpinrogtetion Rsetrfsu.c[t5u9r,e6s0 ]c.anIn scilgundiifincganutlltyra ismonpiaccatt itohne mbeavkeers-
faegrem’se notveedraclhl isctkrupcetaurdarli ninktsegmriotrye, estnahbalencainngd iutsn isftoarbmilitbyy acnhda nfguinncgtiothneals ptrruocptuerrteieosf. Athce-
pcorordteiinngs taon tdhem fiankdininggist oebatsaieinrefdor, cthhieckppaertai ccloensttaoinssp sriegandifiocuatn, tt haemreobuyntasl toefr icnagrbtohheyodvreartaelsl, 
cphreomteiinca, lasntdru lciptuidres ionf athdedditrioinnk to[6 s1e]v. eFrTaIlR fusnpcetciotrnoaslc gorpoyuipssa, spuocwh earsf ualmainnael,y ctaicrbalomxyel,t haondd 
thhyadtrcoaxnyli.n Pvreostteigina tweams orelelactuelda rtos ttrhuec taumreinceh garnogueps, iwn hfoeoredass yfasttteym asci[d6s2 ]a.nIdt  ccaanrbroehvyedarlatthees 
pwreersee nrceelaotefda ltcoo hthoel  acnadrbhoyxdyrl ogxryolugpr.o Tuhpes ,haylkdarnoexyaln gdraolukpe nise cgoronunpecst,endit rtoo gbeont-ho xliypigdrso uapnsd, 
scuarlfbuorhoyxdyrgartoeus.p s, aryl groups, and aliphatic iodo groups in legumes [63].
 
Figure 4.. FTIR analysis showing the difffferences in chemical structures of the unfermented chickpea 
beeveerraagee,, tthee ffeerrmeentteed beeveerraagee,, aand tthee ullttrraassound--aassssiisstteed ffeerrmeentteed beeveerraagee.. 
3.3. HAigccho-PrderifnogrmtoanRce fLsi.q[u6i3d, 6C4h],roFmTIaRtogrerasuphltys (uHsPuLaClly) Ahnaavleyspise aks observed at the aliphatic
i3o.3d.o1.c Oomp
−1
rgaonuicn Adcsi,dasn CdoCn-tIeannt dofC U-Bltrrasstoreutnchd-bAestswiseteend 5C0h0icaknpde6a0 B0ecvmerag.eIsn this study, the
peak observed for the unfermented sample recorded at 571.86 cm−1 significantly shifted to
589.9T9hcem a−m1 ofourntth oeff eorrmgaennitce dacsiadm pproled.uScimedi lianr lyc,htihckepaeliap-bhaastiecdi obdeovepreaagkeso bcasenr vbeed afffoerctthede  
UbyA FfeCrBmwenatsartieocno rdanedd after6m05e.8n6tactmio−n1 ,awsshisitcehdi sbsyi gunlitfircaasnoutlnydd.i fTfehreesnet ftreocmhntihqeuUesC mB iagnhdt tihne-
FcrCeBa.seT htheep rpersoednucectoiof na paenadk rinelethaesea loipf hoartgicanbirco macoid-iso,d iomcpormovpionugn dthses tfirenticshheindd picraotdesuctht’es 
pserensseonryce qoufailoitdieins eaanndd nburtorimtiiodneacl owmoprtohu. nBdasctaesrinau ctariutsioen tahlec bormeapkodnoewntns ooff cchaircbkopheyadsr[a6t5e]s. 
Adugariinng, t hfeermFTeInRtastpioecnt,r aprsoadmupcliensgs hmoewtaebdoclhitaens,g ienscilnudthinegfi nogrgerapnriicn at criedgsi osnuochf  2a1s1 1la.6c4ticc mac−i1d, 
2a1n0d4 .a1c2etcimc a−c1i,da [n4d9]2. 1M32o.r5e6ovcmer−, 1thfeo rretlheeasael koyf nbeoruengdio onrsgoanf itch eacUidAs FdCuBri,nFgC fBe,rmanedntUatCioBn, 
rceasnp becet aivcehliye.veAdc bcoyr tdhien gacttoivRiteyf .of[ 6m4]i,crtohbeiaCl ≡enCzytemrmesi,n walhaiclkhy enneha(mncoens otshueb bsitoitauvtaeidla)bfialiltlys  
wofi tthhienseth aec2id1s0 0a–n2d1 4th0ec mac−id1itrya nlegvee, lism opfl ythine gfitnhiasht ethde ppreoadkuoctb s[7e0rv].e Tdhies ctrriepalteiobno nodf ocragrabnoinc 
caocmidps oduunrdinsg.  Afesrimgnenifitactainotna cltaenr aitmiopnroinveth tehpe enauktrloitcioatniaoln vfaolruteh eofC c≡hCickteprema ibneavl earlkagyense aiss 
owbeslel ravse dthienirt hflisavstourd aynadf tceorntsreisattemnceyn,t ,mcoanksinisgte tnhtew fiinthalt hperoradnugcet pmroorpeo sdeedlebcyta[b6l4e] .and ap-
pealiMnge aonvwerhaillle ,[t3h6e,7c1h]a. rTahctee roirsgtiacnriecg icoonntfeonr tt hoef ttyhpei cparloacbessosrepdt iobnevoefrpagheesn oinliccrceoamsepdo uconmds-
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pcoarntciceunltarraltyioonr goafn picrocpomanpooicu nadcisd,  irnecclourddiendg kweatso nthese, halidgehheystd, eess,peescteiarlsl,ya nind tchaer bFoCxBy,l sw, gitahv ea 
ovfafluabe soofr 5p.t5i6o nmbga/mndLs, fboeltlwoweeend 1b8y0 l0acatnicd a7c5id0, cwmh−il1e [c5i6tr,i6c4 a].ciIdn cloinnetewnti trhedthuicse, dth derapsetaickaslloyf. 
1636.C27hcicmkp−e1,a1s 6a3r6e.3 1reccmor−d1e,da ntdo 1c6o3n6t.3a8inc mso−m1 efo or rUgCanBi,cF CacBid, as.n dAUccAorFdCinBgf atlol  wRitehf.i n[7th2]e, 
acbhoicvkeprean vgaer.ieIttiwesa hsanvoet evdartyhiantgu nltartausroanlliyca oticocnurcraingc hoargnagneicF CacBid’s cliopnitdenantsd. Icna robrodhery dtor ainte-
corenatesne tth[1e9 f]o. Trmhiastoiobns earnvdat rioenlealsseo oafl iogrngsawniicth ahciidgsh ifna tbtyevaecridag, seus cdhearisvleindo flreoicma ncdhiocklepiceasc,i dit, 
aisn fdeathsiebpleh teon torleiactc cohnitceknpteoafs cuhsiicnkgp uealtsra[6s6o]u.nTdh.e Tmhios daipfipcraotiaocnhs winopuleda kwpoorksi twioenlls doubes etorv tehde 
cpoouslsdibrielistuyl tthfraotm thteh eualtsryamsomunetdr itcraelasttmreetcnht ionfg cvhiibcrkaptieoans odfahmemagieces lltuhleoirs ecaenlld wcaellsu,l omsaekdiunge  
tohetmhe mtroeraet mvuelnnteprarbolcee stose tsheu taiclitziveidtya onfd mciocuroldbifaul retnhzeyrminefls uaenndc entchoeufreargminegn tehde rcehliecakspe eoaf 
beverage’s structural integrity and overall quality.
 
Fermentation 2023, 9, 495 13 of 24
Additionally, the wide range between 3500 and 3000 cm−1 characterizes aromatic
compounds and amines found in amino acids, peptides, proteins, alkaloids, DNA, and
RNA because of amino (NH2) groups [7,63]. The peaks obtained are similar to what [67]
described. Ref. [68] found that applying ultrasonication to a sample can cause changes in
the amide I and amide II regions of FTIR spectra, which are related to protein secondary
structures. According to [69], ultrasonication could induce changes in protein conforma-
tions, leading to higher protein unfolding. This alteration can be observed in the peak
values of 3289.14 cm−1 in the UAFCB, being different from the UCB, which recorded a
peak value of 3315.35 cm−1 and the FCB, which also recorded a peak value of 3309.57 cm−1
(Figure 4). The measured peak values fell between the broad range of 3500 and 3000 cm−1,
which is typical of aromatic chemicals and also amines that were identified in amino
acids, peptides, proteins, alkaloids, DNA, and RNA because of amine (NH2) groups [7,63].
Amino acids, peptides, and proteins are known to be present in large concentrations in
chickpeas [66]. These changes in protein structures can significantly impact the beverage’s
overall structural integrity, enhancing its stability and functional properties. According
to the findings obtained, chickpea contains significant amounts of carbohydrates, protein,
and lipids in addition to several functional groups, such as amine, carboxyl, and hydroxyl.
Protein was related to the amine group, whereas fatty acids and carbohydrates were related
to the carboxyl group. The hydroxyl group is connected to both lipids and carbohydrates.
3.3. High-Performance Liquid Chromatography (HPLC) Analysis
3.3.1. Organic Acids Content of Ultrasound-Assisted Chickpea Beverages
The amount of organic acid produced in chickpea-based beverages can be affected by
fermentation and fermentation assisted by ultrasound. These techniques might increase
the production and release of organic acids, improving the finished product’s sensory
qualities and nutritional worth. Bacteria cause the breakdown of carbohydrates during
fermentation, producing metabolites, including organic acids such as lactic acid and acetic
acid [49]. Moreover, the release of bound organic acids during fermentation can be achieved
by the activity of microbial enzymes, which enhances the bioavailability of these acids
and the acidity levels of the finished product [70]. The creation of organic acids during
fermentation can improve the nutritional value of chickpea beverages as well as their flavor
and consistency, making the final product more delectable and appealing overall [36,71].
The organic content of the processed beverages increased compared to the unprocessed
ones. However, the organic acid concentrations obtained after ultrasonic treatment were
significantly lower than that of the fermented sample. The concentration of propanoic acid
recorded was the highest, especially in the FCB, with a value of 5.56 mg/mL, followed by
lactic acid, while citric acid content reduced drastically.
Chickpeas are recorded to contain some organic acids. According to Ref. [72], chickpea
varieties have varying naturally occurring organic acid contents. In order to increase the
formation and release of organic acids in beverages derived from chickpeas, it is feasible to
treat chickpeas using ultrasound. This approach would work well due to the possibility
that the ultrasound treatment of chickpeas damages their cell walls, making them more
vulnerable to the activity of microbial enzymes and encouraging the release of intracellular
substances such as organic acids [25]. Additionally, the capacity of ultrasonic treatment to
speed up the fermentation process leads to a higher output of organic acids in a shorter
amount of time [73]. Ultrasound-assisted fermentation is beneficial for increasing the
organic acid content of several food products, including fermented milk and kefir [74].
However, compared to the outcomes achieved when the beverages were just fermented, the
use of UAF resulted in a sizable drop in the organic content discovered in this experiment
(Table 3).
Fermentation 2023, 9, 495 14 of 24
Table 3. Organic acid content of chickpea beverage samples after fermentations.
Target Concentration (mg/mL)
Organic Acid
UCB FCB UAFCB
Malic acid 0.53 ± 3 × 10−5 a 0.93 ± 2 × 10−5 b 0.56 ± 2 × 10−5 c
Lactic Acid 0.71 ± 2 × 10−5 a 1.62 ± 1 × 10−5 b 1.01 ± 3 × 10 −4 c
Acetic Acid 0.09 ± 3 × 10−5 a 0.36 ± 2 × 10−5 b 0.21 ± 4 × 10−5 c
Citric acid 0.15 ± 1 × 10−5 a 0.05 ± 1 × 10−5 b 0.03 ± 2 × 10−5 c
Propanoic acid 1.79 ± 3 × 10−5 a 5.56 ± 2 × 10−5 b 3.50 ± 0.00344 c
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; and UAFCB-ultrasound-assisted
fermented chickpea beverages. Results are presented as mean ± standard deviation. Values with different
superscripts were significantly different at p < 0.05.
Even though the generation of lactic acid was the main focus of this work, other
organic acids were identified in the samples. Except for citric acid, most of the organic acid
levels in chickpea beverages increased during fermentation. This finding is comparable
to that of Ref. [38], who discovered that fermentation reduced the amount of citric acid in
chickpea beverages. Using ultrasound during fermentation resulted in an extra reduction
in citric acid content. The naturally occurring organic acid noted to be the most abundant
was propanoic acid, a short-chain fatty acid. According to Ref. [75], Lactiplantibacillus
paracasei may ferment substrates such as lactose and other sugars to produce propanoic
acid. In this process, lactose is broken down into lactic acid, which is subsequently changed
into propanoic acid by the action of certain enzymes generated by the bacterium. This
process further explains the reduced lactic acid concentration observed compared to the
propanoic acid concentrations. In contrast, acetic acid was discovered to have the lowest
concentration of naturally occurring organic acids, as seen in the UCB sample (Table 3),
though it increased after fermentation. The significant drop in organic acid levels observed
after UAF was potentially caused by damage to microbial cells, which are crucial for
generating enzymes required for forming organic acids.
3.3.2. Phytochemicals of Ultrasound-Assisted Chickpea Beverages
Phytochemicals, also known as phytonutrients, are naturally occurring compounds
that may be found in plants and are known to give various advantages to human health [76],
with factors including the genotype, the conditions of production, and the processing
processes used. This study analyzed and compared the effect of ultrasound-assisted
fermentation of chickpea beverages to unfermented beverages. According to the findings
presented in Table 4, the total polyphenol derivatives were high, with concentrations
ranging from 0.1040 ± 003 to 35.60 ± 05 µg/mL. In contrast, the phytosterol content
reduced significantly in the processed samples. There are approximately seven different
polyphenols, with phenolic acids being the most common.
The manner of product processing could affect the finished product’s phytochemical
profile. Fermentation is one type of processing that might change the amount of polyphe-
nols present in chickpeas. According to Ref. [77], certain microorganisms, such as lactic
acid bacteria, can create enzymes that hydrolyze bound forms of polyphenols during
the fermentation process. This enhances the polyphenols’ bioavailability as well as their
antioxidant activity. Additionally, particular fermentation conditions might encourage the
creation of additional phenolic compounds, which can increase the health-promoting char-
acteristics of fermented chickpea products. This interpretation may be seen quite plainly in
the results obtained in Table 4. Some phytochemicals, including chlorogenic acid, biochanin
A, p-coumaric acid, cinnamic acid, rosmarinic acid, quinic acid, salvianolic acid, and el-
lagic acid, had significantly higher concentrations after fermentation. The breakdown of
plant cell walls and the release of cell-bound compounds during fermentation by enzymes
produced by Lacticaseibacillus paracasei could account for these increases in phytochemical
concentrations. This makes it easier for these chemicals to be released and used. In contrast,
others, such as stigmasterol and β-sitosterol, had significantly lower concentrations.
Fermentation 2023, 9, 495 15 of 24
Table 4. Phytochemical content of chickpea beverage samples.
Target Concentration (µg/mL)
Phytochemical
UCB FCB UAFCB
Chlorogenic acid 7.44 ± 0.04 a 10.3 ± 0.05 b 10.20 ± 0.05 b
Biochanin A 0.10 ± 0.00 a 0.205 ± 0.00 b 0.21 ± 0.00 b
p-coumaric acid 1.44 ± 0.02 a 7.63 ± 0.05 b 5.15 ± 0.03c
Phenol, 4-ethenyl-2,6-dimethoxy 0.29 ± 0.00 a 0.26 ± 0.00 b 0.33 ±0.00 c
Cinnamic acid 3.33 ± 0.03 a 5.07 ± 0.04 b 4.69 ± 0.04 c
Rosmarinic Acid 5.58 ± 0.04 a 8.7 ± 0.05 b 13.20 ±0.05 c
Stigmasterol 11.70 ± 0.05 a 4.67 ± 0.04 b 2.41 ± 0.03 c
β-sitosterol 20.30 ± 0.05 a 14.7 ± 0.05 b 9.31 ± 0.04 c
Quinic acid 0.80 ± 0.01 a 10.70 ± 0.01 b 14.10 ± 0.05 c
Salicylic acid 0.69 ± 0.01 a 0.70 ± 0.01 a 0.68 ± 0.01 a
Caffeic acid 0.86 ± 0.01 a 0.84 ± 0.01 a 0.85 ± 0.01 a
Tanshinone IIA 0.11 ± 0.01 a 0.11 ± 0.00 a 0.11 ± 0.00 a
Protocatechuic acid 0.60 ± 0.01 a 0.52 ± 0.01 b 0.63 ± 0.004 c
Epicatechin gallate 0.44 ± 0.01 a 0.43 ± 0.01 a 0.44 ± 0.01 a
Salvianolic acid 0.76 ± 0.01 a 0.78 ± 0.01 b 0.80 ± 0.005 c
Glycyrrhetinic acid 0.11 ± 0.00 a 0.11 ± 0.00 a 0.11 ± 0.01 a
Ellagic acid 0.90 ± 0.01 a 1.00 ± 0.00 b 1.29 ± 0.02 c
Total phytochemical content 55.41 66.71 64.51
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; and UAFCB-ultrasound-assisted
fermented chickpea beverages. Results are presented as mean ± standard deviation. Values with different
superscripts were significantly different at p < 0.05.
According to Ref. [19], ultrasound-assisted fermentation is emerging as a potentially
useful method for boosting the levels of bioactive chemicals in various foods, including
chickpeas. Again, according to [78,79], phytochemicals bound to their polysaccharide
subunits are less easily released. However, with ultrasonication, the chemical bond dissoci-
ation effect enhances phenolic acids bond cleavage into their aglycon derivatives, releasing
the phenolic compound into the samples as observed; consequently, ultrasound-assisted
fermentation can lead to a higher release of bound polyphenol [80]. According to previous
research, adding fermentation to ultrasound treatment significantly increases the total
polyphenolic content (TPC) and antioxidant activity of various food products, including
soybean and grape pomace [81]. The current investigation supports this finding. Compared
to the samples that were not fermented, the phytochemical content of the UAF samples
showed a considerable rise.
Once more, the concentration of phytosterols found was dramatically reduced. The
phytosterol stigmasterol dropped from 11.70 ± 05 µg/mL in the UCB to 4.670 ± 04 and
2.410 ± 03 µg/mL in the FCB and UAFCB, respectively. At the same time, the level of
β-sitosterol dropped from 20.30 ± 05 µg/mL to 9.31333 µg/mL in the UCB and UAFCB,
respectively. A similar observation was made by Ref. [17]. They concluded that the decline
in phytosterol content might be attributable to the dehydration or oxidation that occurs
in an oxygen-rich environment when substrates are subjected to heat or ultraviolet light.
Subsequently, the exposure of the samples to prolonged heat as an essential parameter in
the fermentation process of the heat produced due to the ultrasound treatment significantly
impacted the phytosterol contents. This was found to be the case when the samples were
exposed to heat for a longer period of time. Table 4 and Figure 5 show that salicylic
acid, caffeic acid, tanshinone IIA, epicatechin gallate, and glycyrrhetinic acid were not
substantially different from one another in any of the samples (p > 0.05). According
to Ref. [82], the fluctuations in pH during the fermentation process may cause these
polyphenolic chemicals without causing any substantial alteration.
Fermentation 2023, 9, x FOR PEER REVIEW 16 of 25 
 
Rosmarinic Acid 5.58 ± 0.04 a 8.7 ± 0.05 b 13.20 ±0.05 c 
Stigmasterol 11.70 ± 0.05 a 4.67 ± 0.04 b 2.41 ± 0.03 c 
β-sitosterol 20.30 ± 0.05 a 14.7 ± 0.05 b 9.31 ± 0.04 c 
Quinic acid 0.80 ± 0.01 a 10.70 ± 0.01 b 14.10 ± 0.05 c 
Salicylic acid 0.69 ± 0.01 a 0.70 ± 0.01 a 0.68 ± 0.01 a 
Caffeic acid 0.86 ± 0.01 a 0.84 ± 0.01 a 0.85 ± 0.01 a 
Tanshinone IIA 0.11 ± 0.01 a 0.11 ± 0.00 a 0.11 ± 0.00 a 
Protocatechuic acid 0.60 ± 0.01 a 0.52 ± 0.01 b 0.63 ± 0.004 c 
Epicatechin gallate 0.44 ± 0.01 a 0.43 ± 0.01 a 0.44 ± 0.01 a 
Salvianolic acid 0.76 ± 0.01 a 0.78 ± 0.01 b 0.80 ± 0.005 c 
Glycyrrhetinic acid 0.11 ± 0.00 a 0.11 ± 0.00 a 0.11 ± 0.01 a 
Ellagic acid 0.90 ± 0.01 a 1.00 ± 0.00 b 1.29 ± 0.02 c 
Total phytochemical content 55.41 66.71 64.51 
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; and 
Fermentation 2023, 9, 495 UAFCB-ultrasound-assisted fermented chickpea beverages. Results are presented as m1e6aonf  24± 
standard deviation. Values with different superscripts were significantly different at p < 0.05. 
 
Figure 55.. Muullttiivvaarriiaatteeh heeaatmtmaapps hsohwowinigngth tehve avrayrinyginpgh pyhtoycthoecmheimcailccaol nctoenttesnotfs tohfe tvhaer ivoaursiosuams spalmes-.
ples. 
The sample that was fermented had the highest overall phytochemical content, whereas
t3h.4e. sMamicrpolbeisalt hEantuwmeerreatnioont fermented had the lowest. It is reasonable to conclude that fer-
mentAat ifoonowd apsraodsuigcnt’isfi cpahnytscioconctrhiebmutiocralt,o cthheemriisceali,n atnhde obbiosaecrvtievde pcohmytpocohsietmioinc ails csoingtneinfit-.
Icnansptliyte inoflf utheins,ctehde bayp pitlsi cmatiicornoboifouloltgraicsaolu qnudahliatyd [a8s3i]g. nInifi acdandtitiimonp,a ac tfoonodth perpohdyutcotc’sh emmicircoal-
cboiomlopgoisciatilo qnuoaflitthye ssiagmnipfilceasnatslyc oimmppaarcetsd htoowth elosnagm ipt lceatnh abtew parsenseortvterdea [t8ed4].. TThheed miffaeirne ndcee-
bteertmwieneanntths eofF CthBe arinsdk UofA mFiCcrBobiniatle irnmfescotifotnh eint obteavleprahgyetso cmheamdei cfarol mco nhtiegnht-rwesaisstnaontcev eerny-
lvairrgoenemitehnetra.l bacteria are the circumstances of the chickpeas in their raw state and their 
3h.a4n. dMliincrgo,b iparloEcneussminerga,t iaonnd storage [85]. Yeast and mesophilic bacterial growth can be 
impacted by the water activity of raw materials and end products [86]. Creating a con-
sumaAblfeo ogdoopdro tdhuact ti’ss ps hysicochemical, chemical, and bioactive composition is significantlinfluenced by its microbeicouloreg iacnadl q sutaabliltey o[8v3e]r. tIinmaed ddeitpioenn,das fcoroitdicparlolyd uonct ’tshme micriocrboioblioogloicgai
y
l-
qcaula qliutyalsitigyn oiffi cthaen tclhyicimkppeaac tbsehvoerwagloesn gpriot dcauncebde. pInr elsigehrvt eodf [t8h4e] .pTrehveiomuas,i ncodnestiedremr itnhaant tist 
owfatsh eessriesnktioalf tmo iecvraolbuiaatlei nthfeec btieovneriangbe’esv meriacgroesbimolaodgeicafrl oqmuahliitgyh d-ruees itsot atnhcee heignhv imroonismtuerne- 
tcaolnbteanctte, reiaxaamreinthinegc itrhceu mprsotadnuccetss’o fmtihcerocbhiioclkopgeicaasl incotmhepiorsriatiwons tfaotceuasnedd tohne irmheasnodplhinilgic, 
pbraoccteersisai,n Sgt,aapnhdylosctoocrcaugse a[u8r5e]u. sY, eyaesatsat,n adnmd emsoopuhldil.i cHboawcteevreiarl, gthroerwe thwecraen nboe idmetpeacctetedd mbiy-
tchroeowrgaatenrisamctsi veixtcyeopft rfoawr mmeasoteprhiaillisc abnadcteenrida. products [86]. Creating a consumable good
that is secure and stable over time depends critically on the microbiological quality of
the chickpea beverages produced. In light of the previous, consider that it was essen-
 tial to evaluate the beverage’s microbiological quality due to the high moisture content,
examining the products’ microbiological composition focused on mesophilic bacteria,
Staphylococcus aureus, yeast, and mould. However, there were no detected microorganisms
except for mesophilic bacteria.
Table 5 provides a summary of the data that were gathered. There was no
Staphylococcus aureus present or any indication of yeast or mould growth. They were
potentially preoccupied with making the drinks, which could be one explanation for their
absence. The obtained chickpea milk was heated in a water bath at 60 ◦C for 30 min as a
means to obtain a sterile product. Staphylococcus aureus cannot endure high temperatures
for an extended period of time, as opined by [87]. Additionally, it is well known that yeast
cannot thrive in hot environments [88]. The aerobic mesophilic bacteria count is typically
used to determine the degree of food freshness, estimate product stability or shelf life, and
evaluate the microbiological quality of foods [89,90].
Fermentation 2023, 9, 495 17 of 24
Table 5. Microbial quality of processed chickpea beverages.
Organism UCB (log cfu mL−1) FCB × 104 (log cfu mL−1) UAFCB × 104 (log cfu mL−1)
Mesophilic bacteria n.d 2.26 ± 0.03 2.41 ± 0.02
Staphylococcus aureus n.d n.d n.d
Yeast and mould n.d n.d n.d
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; and UAFCB-ultrasound-assisted
fermented chickpea beverages; n.d- not detected. Results are presented as mean ± standard deviation.
On the other hand, mesophilic bacteria were found potentially because Lacticaseibacil-
lus paracasei S2601-8 was present in the treated beverages. The mesophilic bacteria cell
counts of 2.26 ± 0.03 and 2.41 ± 0.02 log cfu mL−1 for FCB and UAFCB, respectively, are
lower than the recommended daily minimum consumption level [91]. The absence of any
signs of microbiological contamination could be because meticulous attention was paid to
maintaining a high degree of hygienic standards during the processing step.
3.5. Sensory Profile
3.5.1. Effect of Processing on Appearance and Colour UAF on Chickpea Beverages
Given that colour is the first feature consumers consider when selecting whether to
accept a product, colour is a crucial quality index that influences perception, preference,
and purchasing power [48,92]. Chickpea beverages’ quality can be primarily judged
by their colour, significantly impacting how well consumers welcome their products.
Therefore, after being produced with fermentation and UAF, measurements of the colour
characteristics of the chickpea beverages were taken. The findings are presented in Table 6.
The results demonstrate that most colour indices did not exhibit significantly significant
variation. Notwithstanding, the processing technique made the processed beverages appear
more brown in colour compared to the unfermented beverages.
Table 6. Colour of chickpea beverage samples before and after processing.
Samples L* a* b* Chroma Hue Browning Index ∆E
UCB 31.35 ± 1.12 a 0.34 ± 0.37 a 5.98 ± 0.38 a 36.23 ± 4.13 a 86.61 ± 3.81 a −12.01 ± 0.54 a 0.05 ± 1.12 a
FCB 33.00 ± 1.22 ab −0.12 ± 0.26 a 5.66 ± 0.40 a 32.34 ± 4.61 a 91.09 ± 2.64 a −14.63 ± 0.06 b 0.05 ± 1.22 a
UAFCB 34.63 ± 0.71 bc −0.29 ± 0.03 a 6.15 ± 0.05 a 38.11 ± 0.68 a 91.09 ± 0.261 a −14.45 ± 0.33 b 0.05 ± 0.71 a
UCB-unfermented chickpea beverages; FCB-fermented chickpea beverages; UAFCB-ultrasound-assisted
fermented chickpea beverages; L*-lightness, a*-redness; and b*-yellowing. Results are presented as
mean ± standard deviation. Values with different superscripts were significantly different at p < 0.05.
An object’s ability to reflect or transmit light is described by the chromatic indicator
L*, an indicator of lightness value. It denotes the sample’s whiteness. The green colour
is represented by negative (−) a*, the red colour by positive (+) a*, the yellow colour by
positive (+) b*, and the blue colour by negative (−) b* [93]. Except for the L* value obtained
for UCB and UAFCB, there was no significant difference (p < 0.05) in the L* values of any
of the samples. The raw freshly prepared samples had a pale-yellow colour. Although
all samples contained yellow, the treated samples also contained green, as shown by their
negative (−) a* values. However, the three samples did not show a significant difference in
these colour differences. The use of heat during the production of the beverages potentially
caused a Maillard reaction, which would possibly result in the existence of the yellow
colour observed [94]. Ref. [13] claims that chickpea beverages have this effect, with a higher
level of browning observed after fermentation. According to Ref. [36], fermentation time
and temperature were important variables affecting the beverage’s final colour as a factor
significantly affecting Maillard reactions.
The browning index is an important metric since it determines the purity of the brown
colour produced during processing by enzymatic and non-enzymatic reactions [48]. The
non-enzymatic browning begins with a low energy order, eventually transitioning into
autocatalysis [94]. Compared to the FCB and the UAFCB, the UCB’s browning index
value was statistically (p < 0.05) different. Compared to UCB, FCB and UAFCB had higher
Fermentation 2023, 9, 495 18 of 24
browning indices, in line with the finding by Ref. [94] that cooking or processing foods
cause chemical processes that give them a brown colour. Additionally, the samples became
discoloured potentially due to fermentation and ultrasonic cavitation, which encouraged
the formation of free radicals due to the presence of minerals in chickpeas and the oxidation
of phenolic compounds, causing a series of changes that increased the browning index [95].
The type and amount of phenolic compounds in the chickpea substrates impact how much
of this enzymatic browning occurs [96].
Chroma values describe the intensity of the colour value and the saturation level [97].
Colour purity can be seen in chroma values. With no grey or white colour present, a
high chroma value indicates the presence of highly reflecting materials or fluorescent
surfaces [97]. Consequently, the more pure or bright the colour is, the higher the chroma
values are [48]. The chroma values found in this research varied from 32.0 to 38.2. No
significant (p < 0.05) difference was found since the values obtained were so close together.
However, the sample that underwent fermentation had the lowest chromatic index. A
previous investigation by [98] discovered that fermented food items treated with ultrasound
had a larger increase in chromaticity. The high chromaticity of the ultrasound-treated
sample is a sign of the purity of the colours in the sample. This may be accounted for by
the chickpeas’ cellular-bound yellow pigmentation’s sensitivity to cavitation, as well as the
effects of the processing methods’ osmotic and hydrodynamic properties [99].
Hue is a phrase used to describe colour perception in its most basic form, as the
dominant wavelength dictates. It refers to the degree of redness and yellowness, with 0
or 360 denoting red, 90 denoting yellow, 180 denoting green, and 270 denoting blue [93].
Chickpea beverages’ appearance is influenced by several elements, including colour, opti-
cal, and geometric properties. The hue angle was calculated to determine the colour of the
chickpea beverages. The chickpea beverages tested in this study had a yellow tint, accord-
ing to the hue angle values that ranged from 86.61 ± 3.81 to 92.69863 ± 0.26106. Enzymes
produced by bacteria during the fermentation process may impact the pigments and result
in the creation of metabolites, which in turn may impact the samples’ chroma [100]. Further-
more, the samples’ colour can be definitively attributed, in large part, to the fermentation
processing technology used to improve the chickpea beverage.
3.5.2. eNose Analysis
A radar chart of eNose sensor responses, which showed distinctive fragrance patterns
for each sample, was used to highlight the sensor array reactions to the volatile organic
compounds (VOCs) contained in UCB, FCB, and UAFCB. Similar scent print patterns
were seen in all three samples’ eNose sensor responses to VOCs (Figure 6). The sensitiv-
ity of the FCB and UAFCB were very similar, with a minute distinction of the nitrogen
oxide compound present. The volatile compounds in the UCB recorded the least volatile
compounds. According to Ref. [101], the sensors’ non-specificity enables them to identify
volatile compounds of both organic and inorganic origins. The three samples’ headspace
volatile compositions were not noticeably different based on the sensor array’s response to
VOCs, which produced scent prints with similar patterns and intensities.
Different reactions were seen from sensors S2 (primarily sensitive to nitrogen oxide),
S6 (primarily sensitive to methane), S7 (primarily sensitive to sulfides), and S9 (primarily
sensitive to aromatic sulfides). Although fermented and the UAF beverages had the
maximum and similar intensities in the sensors stated above, the UAFCB displayed a
minutely higher sensitivity at S2 (primarily sensitive to nitrogen oxide), further indicating
that the UAF improved the fermentation process even by a minute improvement [102].
This result implies that the optimized sample outperformed the others in the fragrance
analysis despite the composition of all samples being similar.
3.5.3. Consumer Acceptability Test
Between the processed chickpea beverages (fermented and ultrasound-treated) and
the control (unfermented), the analysis of variance revealed no appreciable variations
Fermentation 2023, 9, x FOR PEER REVIEW 19 of 25 
 
identify volatile compounds of both organic and inorganic origins. The three samples’ 
headspace volatile compositions were not noticeably different based on the sensor array’s 
response to VOCs, which produced scent prints with similar patterns and intensities. 
Fermentation 2023, 9, 495 Different reactions were seen from sensors S2 (primarily sensitive to nitrogen o1x9idofe2)4, 
S6 (primarily sensitive to methane), S7 (primarily sensitive to sulfides), and S9 (primarily 
sensitive to aromatic sulfides). Although fermented and the UAF beverages had the 
maximum and similar intensities in the sensors stated above, the UAFCB displayed a 
mininseuntesolyr yhicghhaerra csteenrsisittiivcsity(T aatb lSe2 7()p. rTimhearailcyc espetnasbitiilvitey toof neiatcrhogseanm opxliedbey), cfuonrtshuemr ienrds iwcaats-
ienvga ltuhaatte dthue sUinAgFi tismapccroepvetadb itlhitey fienrdmeexnt(aFtiigounr epr7o).ceTssh eevUenC Bbys aam mplienustheo wimepdroavhemighenert 
[a1c0c2e]p. tTahnicse riensdueltx iomfp7l.i0e3s ±tha1t. 8t0hef oorpatlilmcirzietedr isaamthpanle tohuetpperorfcoersmseedd stahme poltehse;r[s4 5in]  dtheefi nfread-
garcacnepceta abnlealayssias ndeascpcietpe ttahbei lciotymspcoosrietioofn loefs saltlh saanm7p0l%es, baenindgt hsiumsilsacro. res below 70% were
deemed undesirable.
 
FFiiggurree 66.. AAr ardaadrapr loptlootf goafs gsaens ssoernssoofrcsh iocfk pcheaicbkepveear abgeevsearmagpel essafmropmledsi fffreormen tdpirffoecreesnsitn pgrmoceethssoidnsg. 
methods. 
Fermentation 2023, 9, x FOR PEER REVIE
 Ta
Wbl e 7. Sensory characteristics of processed chickpea beverages based on a 9-point hedon2ic0 socfa l2e5. 
3.5.3. Consumer Acceptability Test 
Overall
SaBmeptwleeen thCe oplrooucressed Fclhaivcokuprea bevTeerxatguerse (fermMeonuttehdfe aenl d uClotnrassisotuenncdy-treAatcecde)p ta-nd 
ththe ea cccoenptrtaobl i(luitnyf eorfm ceernttaeidn) ,f etahteu arensa laymsiosn ogf  tvhaer idaniffceer reenvte parleodce nssoi nagp pterechcinaibqlue evsa. rAiaabltli ilosiatnyms -in 
psleesn sfeolrl belowUCyB chara
 cthteer ithr6.78 ±sti1c
ess h(oTladb l(e≥ 770)).  fTohr ea lal csceenpstoarbyil iqtyu aolift ieesa,c hre ssuamltipnlge  inby l ocwon ascucmepetrasn ce, .65 6.38 ± 1.57 6.68 ± 1.53 6.46 ± 1.68 6.38 ± 1.52 7.03 ± 1w.80as acecvoarlduFiaCntBegd t ou stihn6eg. 4 [i94ts5± ]a 1ch.c4ye1potath5b.e6ils8iits±y,  i2wn.1dh2eicxh ( 5Fs.9tiag7tu±erse1  t.79h1)a. tT ah6ne.0  8aUc±CceB2p. 0st5aabmilpi6tl.y3e8  ss±hcoo1rw.e7 e7odf  a76 0.h5%i4g ±hise1 rr. 7ea8-c-
gcaerdpUetadAn FacCesB  iancdceepx5 t.oa95bf l±7e..0 1A3.3 s6±i d1e.85 0f.1r o4fom±r  a2th.l1le 3c rgiete5n.re9ir5aa ±tl ha1ac.n7c7 etphtea5 bp.8ir6loit±cye 1so.s9fe 6tdh esa 5Um.9C5pB±le ss1;a. 7m[145p]l ed6,. e2tfi2hne± esd1e. 7na6-c-
socUreCypB tq-auubnalfelir tmaieses n atwende rcaheci cakellp ebtaebloeilvwietry ta hgseecs o;thFreCre Bso-hffe orlmledsessn. t etdhachni c7kp0e%a,b eavnedra gtehsu; asn dscUoAreFsC Bb-eulltorwaso u7n0d%-a swsisetreed 
dfeermemenetedd uchnicdkepseiarabebvleer.a ges. Results are presented as mean ± standard deviation.
Table 7. Sensory characteristicsC oofl oprrocessed chickpea bever aUgCeBs based on a 9-point hedonic scale. 
7.5  FCB
 UAFCB
Sample Colour Flavour Textu7r.e0  Mouthfeel Consistency 
Overall 
Acceptability 
UCB 6.78 ± 1.65 6.3O8v ±er a1l.l57 6.68 ± 16..55 3 6.46 ± 1.68 Flavor 6.38 ± 1.52 7.03 ± 1.80 
FCB 6.49 ± 1.41 ac5ce.6pt8a b±il i2ty.12 5.97 ± 16..90 1 6.08 ± 2.05 6.38 ± 1.77 6.54 ± 1.78 
UAFCB 5.95 ± 1.36 5.14 ± 2.13 5.95 ± 1.77 5.86 ± 1.96 5.95 ± 1.71 6.22 ± 1.76 
UCB-unfermented chickpea5 .5 beverages; FCB-fermented chickpea beverages; and 
UAFCB-ultrasound-assisted fe5r.0mented chickpea beverages. Results are presented as mean ± 
standard deviation.  
Table 7 compares the various processing methods by utilizing analysis of variance to 
shCoownsi stthenec aycceptance outcomes for the processed beverage samples. The acceptance index 
for each attribute was under 60%. Furthermore, thTeexrteu rwe ere no appreciable variations in 
 
Mouthfeel
 
Figure 7. Radar plot showing sensory scores of chickpea beverage samples.
Figure 7. Radar plot showing sensory scores of chickpea beverage samples. 
4. Conclusions 
This study compared the effects of ultrasound-assisted fermentation on chick-
pea-based beverages’ nutritive and sensory components. The results indicate that the 
nutritional value of chickpea-based beverages was boosted by both the traditional and 
ultrasound-assisted fermentation procedures. The presence of different organic acids in 
the beverage was attributed to the fermentation time and the metabolic pathways avail-
able to the Lacticaseibacillus paracasei S2601-8 strain. The application of ultrasound treat-
ment successfully broke down complicated molecules and released enzymes, making it 
easier for microorganisms to access nutrients, leading to better fermentation and ho-
mogenization. However, the microstructural analysis indicated that the processing tech-
niques applied to the beverages, especially the application of ultrasound, significantly 
impacted the beverage’s quality. Despite this, the appearance of processed beverages, 
such as colour, did not significantly affect consumers’ acceptance. On the other hand, the 
aroma significantly influenced general acceptance. Overall, the fermented and ultra-
sound-assisted fermented beverages resembled one another, with only a few notable 
variations in their properties, proving that ultrasound therapy had no different effects on 
the beverage’s outcome from traditional fermentation techniques. The development of 
this novel, wholesome, and appealing food product may significantly benefit from fur-
ther study to evaluate the economic viability of this technique and further evaluate the 
processing parameters. Special consideration can be given to better optimize the fer-
mentation time to prevent degradation in the nutritional contents of the product. 
Author Contributions: Conceptualization, N.A.N.J. and Y.M.; Methodology, N.A.N.J. and G.B.; 
Software, J.-N.E. and G.B.; Formal analysis, E.I.; Investigation, N.A.N.J., Y.L., G.B. and E.I.; Re-
sources, Y.M.; Data curation, N.A.N.J., S.Y.-S.S.A., J.-N.E., Y.L. and E.I.; Writing—original draft, 
N.A.N.J. and S.Y.-S.S.A.; Writing—review & editing, J.-N.E. and Y.M.; Supervision, Y.M.; Funding 
acquisition, Y.M. All authors have read and agreed to the published version of the manuscript. 
 
Fermentation 2023, 9, 495 20 of 24
Table 7 compares the various processing methods by utilizing analysis of variance to
show the acceptance outcomes for the processed beverage samples. The acceptance index
for each attribute was under 60%. Furthermore, there were no appreciable variations in the
acceptability of certain features among the different processing techniques. All samples fell
below the threshold (≥70) for all sensory qualities, resulting in low acceptance, according to
the [45] hypothesis, which states that an acceptability score of 70% is regarded as acceptable.
Aside from the general acceptability of the UCB sample, the sensory qualities were all
below the thresholds.
4. Conclusions
This study compared the effects of ultrasound-assisted fermentation on chickpea-based
beverages’ nutritive and sensory components. The results indicate that the nutritional value
of chickpea-based beverages was boosted by both the traditional and ultrasound-assisted
fermentation procedures. The presence of different organic acids in the beverage was
attributed to the fermentation time and the metabolic pathways available to the Lactica-
seibacillus paracasei S2601-8 strain. The application of ultrasound treatment successfully
broke down complicated molecules and released enzymes, making it easier for microorgan-
isms to access nutrients, leading to better fermentation and homogenization. However, the
microstructural analysis indicated that the processing techniques applied to the beverages,
especially the application of ultrasound, significantly impacted the beverage’s quality. De-
spite this, the appearance of processed beverages, such as colour, did not significantly affect
consumers’ acceptance. On the other hand, the aroma significantly influenced general ac-
ceptance. Overall, the fermented and ultrasound-assisted fermented beverages resembled
one another, with only a few notable variations in their properties, proving that ultrasound
therapy had no different effects on the beverage’s outcome from traditional fermentation
techniques. The development of this novel, wholesome, and appealing food product may
significantly benefit from further study to evaluate the economic viability of this technique
and further evaluate the processing parameters. Special consideration can be given to
better optimize the fermentation time to prevent degradation in the nutritional contents of
the product.
Author Contributions: Conceptualization, N.A.N.J. and Y.M.; Methodology, N.A.N.J. and G.B.;
Software, J.-N.E. and G.B.; Formal analysis, E.I.; Investigation, N.A.N.J., Y.L., G.B. and E.I.; Resources,
Y.M.; Data curation, N.A.N.J., S.Y.-S.S.A., J.-N.E., Y.L. and E.I.; Writing—original draft, N.A.N.J. and
S.Y.-S.S.A.; Writing—review & editing, J.-N.E. and Y.M.; Supervision, Y.M.; Funding acquisition, Y.M.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Zhenjiang Key Research and Development Program (Modern
Agriculture) grant number NY2020020 and the APC was funded by Nana Adwoa Nkuma Johnson.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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