Applied Microbiology and Biotechnology (2023) 107:1189–1204

https://doi.org/10.1007/s00253-023-12372-z

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Optimization and cholesterol‑lowering activity of exopolysaccharide

from Lactiplantibacillus paraplantarum NCCP 962
Asma Afreen1 · Zaheer Ahmed1 · Nauman Khalid2 · Ifra Ferheen3 · Iftikhar Ahmed4

Received: 28 October 2022 / Revised: 28 December 2022 / Accepted: 4 January 2023 / Published online: 21 January 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Exopolysaccharides (EPSs) are biological polymers with unique structural features have gained particular interest in the
fields of food, chemistry and medicine, and food industry. EPS from the food-grade lactic acid bacteria (LAB) can be used
as a natural food additives to commercial ones in the processing and development of functional foods and nutraceuticals.
The current study was aimed to explore the EPS-producing LAB from the dahi; to optimize the fermentation conditions
through Plackett–Burman (PB) and response surface methodology (RSM); and to study its physicochemical, rheological,
functional attributes, and cholesterol-lowering activity. Lactiplantibacillus paraplantarum NCCP 962 was isolated among
the 08 strains screened at the initial stage. The PB design screened out four independent factors that had a significant posi-
tive effect, i.e., lactose, yeast extract, ­CaCl2, and tryptone, while the remaining seven had a non-significant effect. The RSM
exhibited lactose, yeast extract, and ­CaCl2, significantly contributing to EPS yield. The maximum EPS yield (0.910 g/L) was
obtained at 6.57% lactose, 0.047% yeast extract, 0.59% C ­ aCl2, and 1.37% tryptone. The R2 value above 97% explains the
higher variability and depicts the model’s validity. The resulted EPS was a heteropolysaccharide in nature with mannose,
glucose, and galactose monosaccharides. FTIR spectrum reflected the presence of functional groups, i.e., O–H, C–H, C = O,
C–O–H, and ­CH2. SEM revealed a porous and rough morphology of EPS, also found to be thermally stable and negligible
weight loss, i.e., 14.0% at 257 °C and 35.4% at 292.9 °C was observed in the 1st and 2nd phases, respectively. Rheological
attributes revealed that strain NCCP 962 had high viscosity by increasing the EPS concentration, low pH, and temperature
with respectable water holding, oil capacities, foaming abilities, and stability. NCCP 962 EPS possessed up to 46.4% reduc-
tion in cholesterol concentration in the supernatant. Conclusively, these results suggested that strain NCCP 962 can be used
in food processing applications and other medical fields.
Key points
• The fermentation conditions affect EPS yield from L. paraplantarum and significantly increased yield to 0.910 g/L.
• The EPS was heteropolysaccharide in nature and thermally stable with amorphous morphology.
• Good cholesterol-lowering potential with the best rheological, emulsifying, and foaming capacities.

Keywords Exopolysacchride · Lactiplantibacillus paraplantarum · Response surface methodology · Rheology ·

Cholesterol-lowering activity

3
* Zaheer Ahmed Department of Biosciences, and Veterinary Medicine,
[email protected] University of Camerino, 62032 Camerino, Italy
4
1 National Culture Collection of Pakistan (NCCP), Bioresource
Department of Nutritional Sciences and Environmental
Conservation Institute (BCI), PGRI Building, National
Design, Research Complex, Allama Iqbal Open University,
Agricultural Research Centre (NARC), Park Road,
Islamabad, Pakistan
Islamabad 45500, Pakistan
2
Department of Food Science and Technology, School
of Food and Agricultural Sciences, University
of Management and Technology, Lahore 54000, Pakistan

13
Vol.:(0123456789)
1190 Applied Microbiology and Biotechnology (2023) 107:1189–1204

Introduction interactions of the factors could not be assessed. So, this

is being done through appropriate statistical designs such
Since the last few decades, the exploration of new bioac- as central composite design (CCD) and response surface
tive polymers with effective potential and low toxicity are methodology (RSM) in recent years to optimize the condi-
in demand. Among those, exopolysaccharides (EPSs) are tions of a targeted response (Wang et al. 2019a).
important as high molecular weight polymers produced by Hence, the isolation of LAB strains has been in practice
microbes in the extracellular space (Jiang and Yang 2018; for a long, and still, research is going on just because of their
Noureen et al. 2019; Rajoka et al. 2022). The EPS produced medicinal and industrial value. In the sub-continent, dahi is
by lactic acid bacteria (LAB) have diversified attributes one of the famous fermented dairy products of milk and is
such as safeguarding probiotic bacteria from toxic metals, consumed almost daily by a large number of the populace
surface adhesion ability, drug, and micronutrient delivery regardless of age (Hassan et al. 2020). It possesses a sig-
and biofilm formation (Limoli et al. 2015). Microbial EPS nificant source of beneficial bacteria and nutritional value
also displayed their role in the food industry as an emul- as well. It is evident from the research that dahi contains
sion stabilizer, suspension capability, rheological agent, distinctive microbiome of LAB like Lactobacillus rham-
and significant biocompatibility (Jiang and Yang 2018). In nosus, Lactobacillus bulgaricus, Lactobacillus casei, and
addition, EPS has been reported for several health benefits Streptococcus thermophilus, and these beneficial microbes
such as it was depicted as an anti-tumor, immunomodula- are added in the commercially prepared probiotic dahi too
tory, anti-HIV, cholesterol, and triglyceride-lowering agent (Aziz et al. 2020). However, L. plantarum is seldom isolated
(Ayadi et al. 2016; Caggianiello et al. 2016; Górska et al. from dahi as other species studied; thus, its contribution to
2017). Various LAB strains and their derived EPS have the EPS yield, characterization, and cholesterol-lowering abil-
capability to reduce blood cholesterol levels via either cho- ity is barely determined. The hypothesis behind this study
lesterol absorption, microbial transformation, binding to was that the dahi is a good habitat for the isolation of EPS-
the bacterial cell wall, and enzymatic de-conjugation of bile producing bacteria with novel functional attributes. There-
salts (Park et al. 2018). The gel formation and rheological fore, the current study was aimed to screen out the probiotic
attributes of EPS as well as its tendency to sequester bile LAB from the dahi; isolated EPS-producing LAB, laterally
acid conjugates may responsible for the decrease in choles- identified as L. plantarum. Then, the medium composition
terol absorption by enhancing the bile acid excretion and was optimized through PB design and RSM for maximum
promoting compensatory oxidation of cholesterol in the liver EPS yield. Additionally, the physicochemical, rheological,
(Gawande et al. 2021). functional attributes and cholesterol-lowering effect of the
Most of the Lactobacillus plantarum strains reported produced EPS were also studied in detail.
in the literature are considered probiotic and present a
poly-functional role in meat, dairy products, vegetables
and lactic beverage’s processing and preservation, as Materials and methods
well as other enterohepatic fermentation and medicinal
formulations (Lee et al. 2016; Ołdak et al. 2017; Russo Sampling, isolation, and identification
et al. 2017). Principally, the EPS-producing Lactobacillus of EPS‑producing LAB
plantarum strains exhibited functional and physiochemical
attributes of healthy foods (Al-Dhaheri et al. 2017; Jiang Dahi sample was collected from the local market and stored
and Yang 2018). For any microbial production, optimi- in the laboratory at 4 °C for further analysis. The sample was
zation is an essential bioprocess, especially for commer- homogenized, serially diluted, and inoculated on MRS/M-17
cial-scale procedures. The yield of the EPS produced by (Oxoid-UK) agar supplemented with 2% lactose and min-
the LAB differs among various species as it depends on gled in distilled water to make a volume of up to 100 mL.
many factors like composition, culture conditions of the The pH was adjusted to 5.2 and placed under an anaerobic
medium, carbon and nitrogen sources, pH, temperature, incubator at 37 °C for 3–4 days. Initially, LAB was screened
and the incubation period (Adesulu-Dahunsi et al. 2018). out based on the ropy, sticky, and mucoid behavior of the
Studies indicated that this species exhibited excellent colonies on agar medium. Then, it was confirmed with the
properties (Kumar et al. 2022; Wang et al. 2019b), but its help of Gram staining, microscopy, biochemical, and car-
low yield rigorously delayed further study and applica- bohydrate fermentation. Finally selected EPS-producing
tion at the commercial scale. Previously one single-factor strain was reactivated by two spikes in MRS broth from a
optimization, i.e., changing one factor at a time and keep 10 μL/mL inoculum and incubated at 37 °C for 3–4 days.
the other factors constant, was used for bioprocess opti- Again, this reactivated culture (10 μL/mL inoculum) was
mization. However, in this method, the effect between the transferred into vials containing 500 mL MRS broth and

13
Applied Microbiology and Biotechnology (2023) 107:1189–1204 1191

incubated under the same conditions for EPS production, precipitated protein was removed as per the centrifugation
purification, and quantification. conditions described earlier. The pH of the resultant super-
natant was increased to 8.5 by adding 6 M NaOH drop by
Identification of the main EPS‑producing LAB strain drop and again centrifuged at 11,000 rpm for 15 min. Again,
by 16S rRNA gene sequencing and phylogenetic the pH of the resultant supernatant was lowered to neutral by
analysis adding 1 mL of 10% TCA and purified exopolysaccharide
was gathered by adding chilled ethanol in an equal volume.
Finally, high EPS-yielding strain NCCP 962 (strain deposit Furthermore, the resultant pellet was liquefied in deionized
No from NCCP) was selected for species identification, ­H2O and dialyzed (molecular weight cutoff of 12,000 to
briefly, 16S rRNA gene was amplified using primers (9F, 14,000 Da) for 48 h against distilled water with four times
5′-GAG​TTT​GAT​CCT​GGC​TCA​G-3′ and 1510R, 5′-GGC​ of water per day and finally lyophilized. The total yield of
TAC​CTT​GTT​ACGA-3′) and Premix Taq™ DNA Polymer- EPS was determined by the phenol–sulfuric method, and
ase (Ex Taq™ Version 2.0, Takara Cat# RR003A). PCR protein fractions were estimated by the Bradford method
product was purified and sequenced using 16S rRNA gene (Wang et al. 2019b).
sequencing primers (27F, 5′-AGA​ GTT ​ T GATCMTGG​
CTC​AG-3′ and 1492R, 5′-TAC​GGY ​TAC​CTT​GTT​ACG​ Optimization of EPS yield by Lactiplantibacillus
ACTT-3′) at Macrogen, Korea (https:// ​d na. ​ m acro ​ gen.​ paraplantarum NCCP 962
com/). The resulting sequence fragments of both prim-
ers were quality checked and assembled using BioEdit. A Identification of significant factors through PB design
total of 1431 bp were obtained as a consensus sequence of
strain NCCP 962, which was submitted to NCBI (accession A Plackett–Burman (PB) statistical design was used to ini-
number LC712922). Identification was performed using the tially select the most significant independent variables that
EzBioCloud server (www.​ezbio​cloud.​net). Similarity with influenced the EPS production through the Design Expert
closely related validly named species was calculated and the 11.0 version (Stat Ease Inc., Minneapolis, USA). Eleven
ClustalX software package was used to perform multiple (11) factors based on the medium’s composition and culture
alignments of the 16S rRNA gene sequences data. Phylo- conditions were chosen. The analysis was done at three lev-
genetic analysis was performed using MEGA 5.05 software els, i.e., low, medium, and high, denoted by − 1, 0, and + 1,
and the tree was reconstructed using the neighbor-joining respectively, for each factor as depicted in Table 1 (part a).
(NJ) method with Kimura’s two-parameter model (Rajoka The design matrix of medium composition is presented in
et al. 2022). Table S1. The 95% probability level was set to have a sig-
nificant effect of strain NCCP 962 on EPS yield and hence
Acid and bile tolerance utilized for optimization of EPS through RSM.

The main EPS-producing strain was assessed by its growth Central composite rotatory design (CCRD)
at higher concentration of bile and low pH (Li et al. 2015).
After screening the significant factors from the PB design,
Extraction and purification of EPS the central composite rotatory design (four level-four fac-
tors) was applied to optimize independent variables further:
The highest yielded strain NCCP 962 was cultured in MRS lactose, yeast extract, C
­ aCl2, and tryptone. The levels of
(Oxoid) broth at 37 °C for 3–4 days under anaerobic condi- independent variables were presented in Table 1 (part b),
tions. After fermentation, the EPS was extracted and puri- and the yield of the EPS (g/L) was recorded as the response
fied as per the procedure given in the literature with some based on a triplicate experimental mean. Finally, a second-
modifications (Yang et al. 2019). Briefly, the obtained turbid order polynomial model denoted multiple regression of the
culture was heated at 80 °C for 10 min to destroy enzymes responses. The results were presented in 3-dimensional and
capable of degradation of polymers. Then, centrifugation contour plots.
was done to detach the exopolysaccharide at 11,000 rpm
for 15 min at 4 °C. The resultant supernatant was precipi-
tated by adding chilled ethanol in a double volume and Physicochemical characterization of EPS
kept overnight at 4 °C. It was followed by centrifugation at
11,000 rpm for 15 min at 4 °C. To get rid of protein which The spectrum of FT-IR was assessed through a BIO-RAD
precipitated at lower pH, the resultant pellet was first dis- IR spectrometer (FTS3000, Bruker, Karlsruhe, Germany)
solved in deionized water and its pH was decreased to 4.5 by having a detector resolution of 1 ­cm−1 with a scanning
adding 1 mL of 10% TCA—trichloroacetic acid. Then, the range between 500 and 4000 ­cm−1. The monosaccharide

13
1192 Applied Microbiology and Biotechnology (2023) 107:1189–1204

Table 1  Levels of independent a. Levels of coded values for PB design b. Levels of coded values for CCRD design
variables used for PB and
CCRD. design Variable (%) Levels Variable (%) Levels
−1 0 +1 −α −1 0 +1 +α

Glucose 5.0 7.5 10.0 Lactose 2.5 5.0 7.50 10.0 12.5
Fructose 5.0 7.5 10.0 Yeast extract 0.005 0.02 0.035 0.05 0.065
Lactose 5.0 7.5 10.0 CaCl2 0.15 0.30 0.45 0.60 0.75
Sucrose 5.0 7.5 10.0 Tryptone 0.30 0.70 1.10 1.50 1.9
Yeast extract 0.02 0.035 0.05
Beef extract 0.05 0.1 0.15
Tryptone 0.70 1.1 1.50
MgSO4 0.15 0.2 0.25
CaCl2 0.30 0.45 0.60
K2HPO4 0.30 0.45 0.60
NaH2PO4 0.15 0.2 0.25

composition of EPS was estimated by gas chromatogra- Functional properties of EPS

phy (GC-Agilent 7890, USA) method with the help of a
hydrogen flame ion detector (HP-5 column having dimen- The emulsion capacity (EC) and emulsion stability (ES)
sions of 30 m × 0.32 mm). Macroscopic morphology was were measured by using various concentrations of EPS,
done through scanning electron microscopy (SEM) (S-4800, i.e., 0.25, 0.5, and 1.0% (w/v); these were thoroughly mixed
Hitachi, Tokyo, Japan) (Wang et al. 2021). The analysis was into corn oil (6 mL) and centrifuged at 10,000 rpm for
done at various magnifications with an accelerating voltage 2 min. The capacity of emulsification (EC) of purified EPS
of 10 kV. ED’s examination was done using energy disper- was calculated as EC = ev × 100 , where ev represents the
tv
sive spectrum (INCA energy 250, Oxford, Japan). Thermo- emulsion volume and tv is the total volume. The emulsion
gram analysis (TGA) and differential scanning calorimetry stability (ES) of the purified EPS was calculated by heat-
(DSC) were done through Thermo gravimetric analyzer ing the emulsion at a temperature of 80 °C for 30 min in a
(STA449f3, Netzsch, Germany) under atmospheric pressure. water bath and then placed for centrifugation for 5 min at
The X-ray diffraction analysis was done through an X-ray 1300 rpm. ES was measured as ES = iev fev
× 100, where fev is
diffractometer (X'Pert PRO MPD, Panalytical, Netherlands) the finally prepared emulsion, and iev is the initial volume of
furnished with Ni-filtered CuKα radiation. The sample was the emulsion. Oil holding capacity (OHC) and water holding
measured at a scanning speed of 0.02°/s at 40 kV/30 mA and capacity (WHC) of purified EPS were evaluated according
2θ angle ranging from 10 to 40° (Zhao et al. 2021). to the method reported previously with slight modification
(Rashid et al. 2018). For foaming capacity (FC) and foam-
ing stability (FC), 2.5 g of the purified EPS were mixed in
Rheological properties and milk coagulation distilled water and beat for 2 min at 10,000 rpm speed in a
test homogenizer by the method given by Rashid et al. (2018).
The initial volume before and after beating was measured,
Viscosity was assessed by viscometer DV2T Brookfield and the increase in volume was assessed. FS was measured
USA through spindle 64 at a fixed time of 30 s at 37 °C. as the decrease in foam volume after 2 h time at 25 ± 2 °C
Freeze-dried EPS with different concentrations, i.e., 15, 30, by transferring the foam slowly in a 1000-mL graduated cyl-
and 45 mg/mL were examined at a shear rate ranging from inder and calculated as Volumeincrease(%) = V2−V1 V1
× 100,
0.3, 0.6, 1.5, 3, 6, 12, 30, to 60 rpm. The effect of pH, tem- where: V1 is the volume of the solution prior to mixing and
perature, and viscosity of 45 mg/mL NCCP 962 EPS solu- V2 is the after-mixing volume of the solution.
tion was also analyzed at a temperature range of 4 to 55 °C
and pH of 3.0, 6.0, 9.0, and 12.0 (ambient temperature of
25 °C). For milk coagulation, pre-cultivated 5% (v/v) of the Cholesterol‑lowering activity
NCCP 962 strain was added to 10% (w/v) sterilized skim
milk. Coagulation/solidification of the milk with the addi- The cholesterol-lowering ability of EPS was also studied
tion of sucrose up to 2% was checked after incubation for by the protocol (Soh et al. 2003). Briefly, 1 mL of reac-
48 h at 37 °C temperature. tion mixture comprised of EPS (0.1%) and cholesterol

13
Applied Microbiology and Biotechnology (2023) 107:1189–1204 1193

(30 µg) was mingled at 25 °C for 20 min. Then the mix- Extraction and purification of EPS
ture was added to 50 µL hexadecyl trimethyl ammonium
bromide solution and centrifuged at 12,500 × g. The Strain NCCP 962 was inoculated on MRS medium at 37 °C
resultant supernatant was assessed for total choles- for 48 h under anaerobic conditions. The obtained EPS was
terol content by Zak’s method (Zak and Epstein 1961) extracted by precipitation with chilled ethanol, deprotein-
and calculated as cholesterol-lower ing activity ized, and lyophilized for further characterization. Under
(%) = Conc.of cholesterol in control−Conc. of cholesterol in test
Conc.of cholesterol in control
× 100. such initial conditions, the calculated EPS yield obtained
was 0.157 g/L in media supplemented with lactose.

Results Screening of factors affecting EPS production

through PB design
Screening and isolation of EPS‑producing LAB strain
Depending upon the results of the PB design, a fitted order
Based on the morphological characteristics, a total of 8
polynomial model representing the correlation between the
strains were screened out as LAB, keeping in view the
eleven independent variables and the EPS yield is as follows:
ropy, mucoid, or slimy behavior of the colonies, which
were further propagated on fresh agar as a pure culture. Y = 6.43 − 0.325X1 − 0.0083X 2 − 1.08X3 − 0.5083X 4 + 1.12X5
Only one strain NCCP 962 was selected as high EPS- (1)
+ 0.4917X 6 + 1.06X 7 + 0.1417X8 + 1.04X 9 − 0.3917X 10 − 0.2583X11
producing LAB, slightly whitish in color, circular, con-
vex with creamy consistency on MRS plates containing The linear regression coefficient was found to be
lactose supplementation (Fig. 1). The morphological and R2 = 0.9948, with the adjusted determination coefficient of
biochemical assessment of NCCP 962 showed that it was adj-R2 = 0.9760 (Table 3, part a), showing that the model
a long rod, gram-positive, and catalase negative. It could could well validate the variability of the data in the response
utilize D-sucrose, D-lactose, D-maltose, D-mannose, yield. PB design screened out four factors, i.e., lactose, yeast
D-fructose, D-galactose, and D-arabinose but was not extract, tryptone, and ­CaCl2 showed the p-values less than
able to utilize D-ribose, D-sorbitol, and D-xylose as a 0.001 indicating more influential factors on EPS yield and
carbon source. Strain NCCP 962 was identified as Lac- were further optimized through RSM. The comparison was
tiplantibacillus paraplantarum with 99.72% sequence done between the experimental values and average values to
similarity of the 16S rRNA gene. Phylogenetic analysis see the need for the steepest ascent testing (Table S2). The
also confirmed the relationship of strain NCCP 962 with values of equivariance and unequal variance significantly
L. paraplantarum (Fig. S1 and S2) at a high bootstrap differed among the average and experimental responses. The
value, indicating that strain NCCP 962 belongs to this central value in the PB design was closer to the maximum
species. This strain was deposited in the National Cul- response area; hence, the steepest ascent test could not be
ture Collection of Pakistan (NCCP), National Agricul- performed. The main effects observed during the initial
ture Research Center (NARC), Islamabad, Pakistan. The screening were dominant in a linear fashion. In terms of
NCCP 962 strain presented a better existence at the pH contribution, yeast extract was at the top with 23% followed
conditions of 2, 3, 4, and 7 and bile concentrations of by lactose 21.08%, tryptone 20.36%, C ­ aCl2 19.72%, sucrose
0.2% and 0.4%, which indicates the probiotic behavior 4.69%, beef extract 4.39%, ­K2HPO4 2.78%, glucose 1.92%,
of the strain. ­NaH2PO4 1.21%, ­MgSO4 0.364%, and fructose 0.012%.

Fig. 1  Morphological charac-

teristics of strain NCCP 962. a
growth on MRS agar, b gram
staining, and c microscopic
view

13
1194 Applied Microbiology and Biotechnology (2023) 107:1189–1204

Optimization of significant factors through CCRD and appropriate for predicting EPS yield response, as
proved by the F-value of 35.92 having a probability value
Significant four factors, i.e., lactose, yeast extract, C
­ aCl2, of < 0.0001. The regression coefficient R2 (0.9710) and
and tryptone, were further approached for RSM. The adjusted coefficient Adj-R2 (0.9440) indicate the highest
design matrix, along with the response EPS yield, was pre- acceptance of the model. Lack of fit (F-value = 2.72) and
sented in Table 2. Depending upon the results, a second- corresponding p-value of 0.1408 implicit the non-signif-
order polynomial equation was employed for interactive icant value compared to the pure error because of noise.
expression between independent and response variables So, the model equation fits well with the experimented
as described below: responses under any grouping of independent variables.
In addition, the significance of each variable and inter-
Y = 0.855 + 0.0533XA + 0.049XB + 0.124XC + 0.0025XD − 0.1087XA XB
action among each coefficient could be perceived in the
+ 0.0150XA XC − 0.0025XA XD + 0.0163XB XC + 0.0213XB XD + 0.0250XC XD ANOVA (Table 3, part b), which depicted p values less
− 0.0508XA2 − 0.0108XB2 − 0.0846XC2 − 0.0671XD2 than 0.05 of linear coefficients, interaction coefficients,
(2) and quadratic coefficients.
The analysis of the variance of the quadratic regres- Depending upon the generated model, three-dimensional
sion model is presented in Table 3 (part b) showed that and corresponding contour plots (Fig. 2), a wide range of
the achieved model was found to be highly significant EPS yield extending from 0.25 to 0.95 g/L was obtained,

Table 2  Actual and predicted Run Lactose (%) Yeast extract (%) CaCl2 (%) Tryptone (%) EPS yield (g/L)
values for EPS yield from
CCRD design Actual value Predicted value

1 12.5 0.035 0.45 1.1 0.75 0.7583

2 5 0.02 0.6 0.7 0.51 0.5146
3 7.5 0.035 0.45 1.1 0.87 0.8550
4 7.5 0.005 0.45 1.1 0.73 0.7133
5 10 0.05 0.6 0.7 0.72 0.7446
6 7.5 0.035 0.75 1.1 0.78 0.7650
7 7.5 0.035 0.45 1.1 0.89 0.8550
8 5 0.05 0.6 0.7 0.85 0.8204
9 10 0.02 0.6 1.5 0.94 0.8813
10 7.5 0.035 0.45 0.3 0.59 0.5817
11 10 0.02 0.6 0.7 0.85 0.8738
12 2.5 0.035 0.45 1.1 0.55 0.5450
13 5 0.05 0.6 1.5 0.95 0.9229
14 7.5 0.035 0.45 1.1 0.85 0.8550
15 5 0.05 0.3 0.7 0.58 0.6196
16 5 0.02 0.6 1.5 0.46 0.5321
17 10 0.05 0.3 1.5 0.51 0.4763
18 5 0.05 0.3 1.5 0.62 0.6221
19 10 0.05 0.3 0.7 0.53 0.4838
20 7.5 0.035 0.45 1.9 0.58 0.5917
21 7.5 0.065 0.45 1.1 0.89 0.9100
22 5 0.02 0.3 1.5 0.35 0.2963
23 7.5 0.035 0.45 1.1 0.85 0.8550
24 5 0.02 0.3 0.7 0.38 0.3788
25 7.5 0.035 0.45 1.1 0.8 0.8550
26 7.5 0.035 0.45 1.1 0.87 0.8550
27 7.5 0.035 0.15 1.1 0.25 0.2683
28 10 0.02 0.3 1.5 0.53 0.5854
29 10 0.05 0.6 1.5 0.81 0.8371
30 10 0.02 0.3 0.7 0.68 0.6779

13
 Table 3  ANOVA tables of EPS. (a) ANOVA for the PB design and (b) ANOVA for response surface quadratic model
a. ANOVA for the PB design b. ANOVA for response surface quadratic model
Source Sum of Squares df Mean square F-value p-value Source Sum of squares df Mean square F-value p-value

Model 65.67 11 5.97 52.65 0.0038** Significant Model 102.46 14 7.32 35.92 < 0.0001 Significant
A-Glucose 1.27 1 1.27 11.18 0.0443* A-Lactose 6.83 1 6.83 33.51 < 0.0001
B-Fructose 0.0008 1 0.0008 0.0073 0.9371 B-Yeast 5.80 1 5.80 28.48 < 0.0001
extract
C-Lactose 13.87 1 13.87 122.30 0.0016** C-CaCl2 37.00 1 37.00 181.63 < 0.0001
D-Sucrose 3.10 1 3.10 27.35 0.0136* D-Tryptone 0.0150 1 0.0150 0.0736 0.7898
Applied Microbiology and Biotechnology (2023) 107:1189–1204

E-Yeast 15.19 1 15.19 133.94 0.0014** AB 18.92 1 18.92 92.88 < 0.0001
extract
F-Beef 2.90 1 2.90 25.58 0.0149* AC 0.3600 1 0.3600 1.77 0.2036
extract
G-Tryptone 13.44 1 13.44 118.54 0.0017** AD 0.0100 1 0.0100 0.0491 0.8276
H-MgSO4 0.2408 1 0.2408 2.12 0.2411 BC 0.4225 1 0.4225 2.07 0.1704
J-CaCl2 13.02 1 13.02 114.83 0.0017** BD 0.7225 1 0.7225 3.55 0.0792
K-K2HPO4 1.84 1 1.84 16.23 0.0275* CD 1.00 1 1.00 4.91 0.0426
L-NaH2PO4 0.8008 1 0.8008 7.06 0.0765 ­A2 7.09 1 7.09 34.79 < 0.0001
Residual 0.3402 3 0.1134 ­B2 0.3219 1 0.3219 1.58 0.2280
Lack of fit 0.2802 1 0.2802 9.34 0.0925 Not significant ­C2 19.62 1 19.62 96.32 < 0.0001
Pure error 0.0600 2 0.0300 ­D2 12.34 1 12.34 60.59 < 0.0001
Cor total 66.01 14 Residual 3.06 15 0.2037
Lack of fit 2.58 10 0.2581 2.72 0.1408 Not significant
Pure error 0.4750 5 0.0950
Cor total 105.51 29
⁎⁎
Part a (ANOVA for the PB design): R2 = 0.9948; Adj-R2 = 0.9760; predicted R2 = 0.9145. ⁎p < 0.05. p < 0.01. Part b (ANOVA for response surface quadratic model): R2 = 0.9710; Adj-
R2 = 0.9440; Pre R2 = 0.8526. ⁎p < 0.05. ⁎⁎p < 0.01

13
1195
1196 Applied Microbiology and Biotechnology (2023) 107:1189–1204

13
 Applied Microbiology and Biotechnology (2023) 107:1189–1204 1197

◂Fig. 2  3-dimensional graphical presentation of RSM and their corre- increasing the concentration, i.e., 45 mg, it was observed to
sponding contour plots of NCCP 962 EPS yield be 4278 Pa.s at the same rpm (Fig. 5b). However, the viscos-
ity was decreased while increasing the shear rate. At pH 3.0,
whereas a ratio of maximum to minimum was attained to the viscosity was observed to be 4678 Pa.s, while increasing
be 2.8. It was pertinent to mention that a ratio of more than the hydrogen ion concentration, the viscosity decreases, i.e.,
ten usually shows that alteration was required. For ratios 997 Pa.s. at pH 12.0 (Fig. 5c). The isolated strain also could
less than 3, the transformation power has an effect, indi- ferment milk in a depending upon the concentration of EPS
cating that the experimentation results were adequate. The upon addition of sucrose up to 10% without syneresis.
model equation was validated by performing three experi-
ments under set conditions within the experimental range Functional attributes of EPS NCCP 962
(Table 4). The analyzed results indicated that the experimen-
tal responses were consistent with the predicted values and EPS produced by strain NCCP 962 possessed a respect-
satisfied the model equation. able WHC: 475 ± 4.32% (Table 4) that indicated EPS had
a permeable polymer chain with water-holding capacity
Physicochemical characteristics of EPS through hydrogen bonding; however, OHC was observed
to be 658.45 ± 1.65%. High levels of foaming abil-
The FTIR, GC, SEM, and XRD results of EPS were pre- ity (37.57 ± 0.23%) and foam stability were found to be
sented in Fig. 3. The FTIR spectrum of EPS depicted in 28.85 ± 0.74 as compared to other commercially available
Fig. 3a showed the presence of absorption peaks at 3270, hydrocolloids. Food-grade biopolymers are extensively
2923, 1653, 1418, 1343, 1268, 1148, 1103, 1006, 909, and used as an emulsifier to stabilize the system of oil/water
755 ­cm−1. The gas chromatograph of the EPS (Fig. 3b) emulsions. Furthermore, it is evident that by increasing the
showed the presence of mannose, glucose, and galactose. concentration of EPS, the EC increases i.e. 35.95 ± 0.58,
The XRD analysis (Fig. 3c) confirmed the amorphous nature 42.63 ± 0.65 and 55.14 ± 0.29 at concentrations levels of
of EPS. The TGA analysis (Fig. 3d) showed appreciable 0.25%, 0.5% and 1.0% w/v respectively. Similarly, emulsions
weight loss of 14.46, 35.14, and 28.20% at 257.12, 292.98, containing the replica of the same concentrations of EPS
and 597.02 °C, respectively. for ES also exhibited pronounced emulsion stability, which
increased by improving the EPS concentration.
Scanning electron microscopy (SEM) and energy
dispersive X‑ray spectroscopy (EDS) Cholesterol‑lowering capability

SEM and EDS are the dominant tools for assessing the mor- The present study showed that NCCP 962 could reduce the
phological structure of polysaccharide polymers. The macro- cholesterol concentration. The cholesterol concentration in
scopic morphology of NCCP 962 EPS polymer at 50.0 K × , the supernatant was reduced up to 46.4% by the addition
20.0 K × , 10.0 K × , 5.0 K × , and 1.0 K × was presented in of NCCP 962 EPS when presented in percentage (Fig S3).
Fig. 4a, which seemed to be porous and rough. EDS analysis Thus, incorporating such EPS in food preparations may help
(Fig. 4b) showed that the core particles in NCCP 962 EPS to reduce the increased blood cholesterol level to an extent.
were mainly carbon and showed the presence of monomer
units of sugar carbon rings and oxygen with a weight per-
centage of 53.11 and 44.72, respectively. Negligible quanti- Discussion
ties of Na 0.15%, Mg 0.09%, P 0.85%, Mn 0.04%, Ca 0.78%,
Fe 0.01%, K 0.01%, and Zn 0.23% were also found in NCCP Regardless of the invention of novel EPS, improvement
962 EPS, that may be bound with hydroxyl and carboxyl in the yield along with its production at low cost is chal-
group of monosaccharide. lenging for the commercialization of new EPS. RSM is one
of the widely used statistical way of optimizing fermenta-
Rheological properties of EPS and milk coagulation tion conditions that can enhance microbial EPS yield and
reduce production costs. In this study, RSM was employed
Figure 5 depicts the effect of various temperatures, con- to optimize the culture conditions of the NCCP 962 for EPS
centrations, and pH on the viscosity of NCCP 962 EPS. production. Firstly, LAB was isolated from the dahi sample
Figure 5a shows that viscosity decreased by increasing the purchased locally, then highest yielded EPS-producing LAB
temperature to 55 °C. At 25 °C, the viscosity was measured was selected for further study. Initially, lactose supplementa-
to be 4776 Pa.s, and at the same shear rate of 3.0 and 55 °C, tion was done in addition to other MRS agar composition
the viscosity was observed to be 3529 Pa.s. In addition, the to identify the EPS-producing LAB, where the calculated
viscosity of 15 mg EPS was 2654 Pa.s at rpm 0.3, while by EPS yield obtained was 0.157 g/L, found to be double times

13
1198 Applied Microbiology and Biotechnology (2023) 107:1189–1204

Table 4  Model No Lactose (%) Yeast extract (%) CaCl2 (%) Tryptone (%) EPS yield (g/L)
validation experiments
for Lactiplantibacillus Predicted Experimental
paraplantarum NCCP 962
1 6.568 0.047 0.587 1.371 0.938 0.910
2 7.234 0.044 0.518 1.406 0.905 0.894
3 6.735 0.045 0.507 1.497 0.880 0.814

Fig. 3  Physicochemical analysis of NCCP 962 EPS. a FT-IR analysis, b monomer composition, c XRD, and d TGA analysis of NCCP 962 EPS

higher than L. plantarum produced EPS in a semi-defined most significant factors that make the strain Leuconostoc
media 58.7 mg/L (Wang et al. 2015b) and 2.11 g/L EPS dextranicum NRRL B-1146 for more EPS production. Like-
produced before optimizing the conditions by strain SM1127 wise, concentrations of tryptone and sucrose also greatly
(Sun et al. 2020). Among the various sources of carbon ana- impacted EPS yield by Leu. mesenteroides DRP105 (Wang
lyzed earlier for EPS yield, lactose was seen better for high et al. 2019b).
EPS yield, followed by sucrose, fructose, and glucose (Petry The RSM regression model was found to be highly signif-
et al. 2000; Wang et al. 2017). icant (Xing et al. 2018), the highlighted factors were lactose,
After initial fermentation, further screening of significant ­CaCl2, and tryptone with a significant effect (p < 0.0001) on
factors was done through PB and RSM for maximum EPS EPS yield. When considering the individual effect, it could
production. PB design screened out four factors, i.e., lactose, be assessed that with the increase in the percentages of these
yeast extract, tryptone, and ­CaCl2, in the present study. How- significant factors, the EPS yield increased in return. The
ever, many other factors affect the EPS yield, i.e., medium contour plot of the relationship among factors clearly depicts
composition, fermentation strain, culture conditions, extrac- that the maximum range of EPS yield exists around the mid-
tion, and refinement methods (Wang et al. 2019b). The PB dle region. Among the three tested experiments, the highest
analysis identified peptone, yeast extract, and sucrose as the EPS yield was optimized with 6.568% lactose, 0.047% yeast

13
Applied Microbiology and Biotechnology (2023) 107:1189–1204 1199

Fig. 4  a SEM and b EDS analysis of NCCP 962 EPS

extract, 0.587% C­ aCl2, and 1.371% tryptone. As such, the obtained from L. plantarum NTMI05 and NTMI20, respec-
predicted value for EPS yield was 0.938 g/L, and the actual tively, by Imran et al. (2016). However, high molecular
experimental response was 0.910 g/L. Hence, concluded that weight EPS was recovered with a yield of 2.8 g/L by Lb.
these tested experiments can be selected as the best response plantarum BR2 (Sasikumar et al. 2017). In the 3-D surface
for optimization defined at the beginning of the study. Previ- plots, the combined effect of lactose and yeast extract was
ously, the maximum EPS yield was 0.956 g/L and 0.827 g/L quadratic. With the increase in both variables, the EPS yield

13
1200 Applied Microbiology and Biotechnology (2023) 107:1189–1204

Fig. 5  Effect of a temperature, b concentration, and c pH on the viscosity of NCCP 962 EPS solution

increased sharply right from the very start. The trend went of mannose, glucose, and galactose monosaccharides, and
up to the center, after which the sharp trend in the increase EPS with similar compositions have been reported to be pro-
of EPS yield showered down a bit. In the case of the effect duced by L. plantarum KF5; L. Plantarum WLPL04 (Liu
of lactose and ­CaCl2, the trend differed a bit; the overall et al. 2017); Lactobacillus plantarum BR2 (Sasikumar et al.
increase in the yield of EPS was a bit lower compared to the 2017) and L. plantarum HY (Wang et al. 2010). Moreover,
first case. However, contrary to the case of lactose and yeast L.plantarum 70,810 produced homopolysaccharide, galactan
extract, the trend continued up to the end. There were many in nature (Wang and Lü 2014); whereas L. plantarum 14FE
combinations for the moderate EPS yield; more importantly, produced β-glucan EPS (Saif and Sakr 2020); L. plantarum
few were lying in the region of high production. In the con- EPS HM47 produced glucogalactan based EPS (Nambiar
tour plot for the interaction of lactose and tryptone, these et al. 2018); thus indicated that L.plantarum either produced
two were different combinations for same EPS yield produc- homo or heteropolysaccharide depending upon the culture
tion and more importantly the high yield was observed in conditions, medium and strain dependent. XRD analysis
the center of the plot. The interaction between yeast extract of EPS from L. plantarum NCCP 962 revealed a broader
and tryptone has been shown positive for the EPS yield up peak at 30°, reflected the amorphous nature of an isolated
to the center, but after that the yield decreased, in which the EPS. Several researchers have reported EPS with amorphous
yeast extract played more role as compared to the tryptone. nature from L. plantarum S123, Bacillus licheniformis, and
So, overall, it could be concluded that almost all these sig- Bacillus subtilis (Saleem et al. 2021; Sathishkumar et al.
nificant variables had a considerable effect on the trend of 2021; Singh et al. 2011).
EPS yield. The thermal stability of biomaterial is a vital attribute
The physiological characteristics of EPS play a consider- that decides its fate to be used in food products produced
able role in any industry application; however, EPS produc- at elevated temperatures. TGA revealed that EPS NCCP
tion by L. plantarum strains is recognized as a strain-depend- 962 was quite stable at elevated temperatures. Only 14%
ent, relying on medium composition and culture conditions of the weight was lost initially at 30–257 °C, attributed
(Jiang and Yang 2018). The FT-IR spectrum showed a broad to free moisture evaporation (Vaziri et al. 2018; Wang
absorption peak at 3270 ­cm−1, which confirmed the pres- et al. 2010) and indirectly indicating high carboxylic
ence of O–H groups in sugar moieties of EPS (Park et al. group concentration in NCCP 962 EPS (Nambiar et al.
2022). A peak at 2923 showed the C-H stretching frequency, 2018). It's important to mention here the NCCP 962 EPS
and the absorption peaks at 1653, 1268 and 1148 indicated was quite stable, and negligible weight loss, i.e., 14% at
the presence of C = O, C–O–C, and C-O–H, respectively, 257 °C and 35.4% at 292.9 °C was observed in ­1 st and
which showed typical functionalities in EPS (Park et al. ­2nd phases, respectively (Fig. 3c), which was much lower
2022; Saleem et al. 2021). The asymmetric deformation of as compared to EPS from L. plantarum HM47, where
­CH2 was ascertained by the presence of a peak at 1418. The loss of 65% of its original weight was observed at 273 °C
polysaccharide nature of the sample was further confirmed (Nambiar et al. 2018). Owing to high stability at elevated
by the presence of an absorption peak at 1103 (Wang et al. temperatures, NCCP 962 EPS isolated in the current study
2010). The GC analysis revealed the heteropolymer nature is suitable for products such as baked products and tetra-
of polysaccharides by L. plantarum NCCP 962. It is evi- packed juices where the elevated temperature is involved
dent from Fig. 3b that exopolysaccharide was composed during food processing.

13
Applied Microbiology and Biotechnology (2023) 107:1189–1204 1201

As evident from SEM results depicted in Fig. 4a, the sur- present lactic acid strain could be suggested as a potential
face morphology of EPS isolated from Lactiplantibacillus candidate for the dairy industry.
paraplantarum NCCP 962 seemed to be porous and rough, For technological applications in the food industry, the
similar to EPS produced by L. plantarum GD2 (Öztürk study of WHC, OHC, foaming and emulsifying capabilities
et al. 2018), bulgaricus B3 (Yuksekdag and Aslim 2008), are important and have been explored by many research-
and L. delbrueckii ssp. strains (Sirin and Aslim 2020). Fur- ers in the recent past (Korcz and Varga 2021; Saleem et al.
thermore, EPS isolated from L. plantarum KF5 was porous 2021). NCCP 962 EPS possessed a respectable WHC and
and possessed a sheet-like structure (Wang et al. 2010), OHC, indicated this strain has a permeable polymer chain
whereas L. plantarum HM47 EPS had a web-like structure with the ability to hold water through hydrogen bond-
(Nambiar et al. 2018). The appearance of polysaccharides ing (Rajoka et al. 2020). Lactobacillus plantarum S123
or other biomaterials is related to their physical attributes showed less WHC, i.e., 326.6%, as compared to its OHC,
(Xu et al. 2019). Rough and porous surface morphology i.e., 995.3% (Saleem et al. 2021). Polysaccharides and pro-
depicted the suitability of NCCP 962 EPS for the delivery teins, recognized as food macromolecules, played a vital
of micronutrients, vitamins and minerals etc. ED’s analysis role in foam stabilization. Foam is a colloidal structure with
is being used for the structural evaluation of elements and an airy segment sustained in a continuous medium (Rajoka
minerals present in biopolymers. Singh et al. (2011) reported et al. 2020). Its development and stability mainly depend
the presence of O, C, Na, P, Cl, and S in EPS produced on surface-active ingredients' interfacial properties in food
by Bacillus licheniformis, present similarly in NCCP 962 preparation and foams are used to enhance the organoleptic
EPS and depicted a strong affinity for various elements and attributes like consistency, appearance, and texture of foods.
minerals; hence, suitable for microbial-mediated synthesis Table 4 reflected the findings of foaming ability and sta-
of nanoparticles. Usage of EPS from L. planatrum TA4 bility, indicated high levels of foaming ability compared to
has been reported for the synthesis of silver nanoparticles other commercially available hydrocolloids (Li et al. 2015).
which exhibited antimicrobial activity against gram-positive Lactobacillus sp.Ca6 isolated from cooked beef sausages
and gram-negative bacteria; along with antioxidant activity depicted a foaming capacity of 30.76% with 2.0% EPS con-
(Mohd Yusof et al. 2020). NCCP 962 EPS could be used to centration and foaming stability of 26.96% and 22.64% of
develop various kinds of nanoparticles for multiple applica- foam remained after 30 and 60 min, respectively (Trabelsi
tions in different fields. et al. 2018). The NCCP 962 EPS exhibited higher foaming
Rheological is one of the most important attributes in stability and predicts its usage in food systems that require
the development and consumer acceptance of several food leavening or textural properties (Table 5). It is reported that
products such as juices, yogurt, soups, etc. Viscosity of. the addition of EPS-1 by Lactococcus lactis and EPS-2 by
NCCP 962 EPS was assessed in terms of temperature, Streptococcus thermophilus significantly improved the rhe-
concentration, and pH. A decrease in the viscosity was ology and texture of dairy products (Deep et al. 2012). Food-
observed as temperature increased, and a similar pattern of grade biopolymers are extensively used as emulsifiers to sta-
EPS at increased temperature was reported for EPS by L. bilize the system of oil/water emulsions. Results of NCCP
plantarum YW11 (Ahmed et al. 2013) and L. kefiranofa- 962 indicated that by increasing the concentration of EPS,
ciens ZW3 (Wang et al. 2015b). This was attributed to low
intramolecular interaction and weaker polymer structure.
As far as the impact of EPS concentration was related, a Table 5  Functional attributes of NCCP 962 EPS. Mean values ± standard
pseudoplastic behavior was noted, owing to the breakdown deviation (n = 3)
of the structural unit by hydrodynamic forces (Wang et al. Functional attributes (%) NCCP 962 EPS
2014). Furthermore, an increase in viscosity was observed
Foaming ability 37.57 ± 0.23
at lower pH, and this behavior was similar to EPS produced
Foaming stability 28.85 ± 0.74
from other lactic acid bacteria previously (Ahmed et al.
Oil holding ability 658.45 ± 1.65
2013; Wang et al. 2015a). This action may be attributed to
Water holding ability 475.15 ± 4.32
changes in electrostatic repulsion as influenced by charge
Emulsion capability (conc. %)
variation in EPS (Saif and Sakr 2020). Increased viscosity
0.25 35.95 ± 0.58
of NCCP 962 at low hydrogen ion concentration suggested
0.5 42.63 ± 0.65
its suitability as a stabilizer or biothickner for fermented
1.0 55.14 ± 0.29
milk products and drinks having low pH. The isolated
Emulsion stability (conc. %)
strain could ferment milk without syneresis, attributed to
0.25 9.23 ± 0.98
connections between the protein content present in milk
0.5 11.56 ± 0.12
and LAB. The present study findings are consistent with
1.0 15.04 ± 0.13
the previous results reported by (Wang et al. 2019a); hence

13
1202 Applied Microbiology and Biotechnology (2023) 107:1189–1204

the emulsifying capacity and emulsifying stability increased. References

The L3 EPS resulted in varied levels of emulsion indices
with a gradual increase in both EC and ES with different Adesulu-Dahunsi A, Sanni A, Jeyaram K, Ojediran J, Ogunsakin
vegetable oils, hence aligned to our studied results (Wang A, Banwo K (2018) Extracellular polysaccharide from Weis-
sella confusa OF126: production, optimization, and characteri-
et al. 2019b). Furthermore, Lac. paracasei M7 EPS showed zation. Int J Biol Macromol 111:514–525. https://​doi.​org/​10.​
an emulsion index of 68.3% (Bhat and Bajaj 2019), whereas 7717/​peerj.​5326
L. plantarum DM5 EPS exhibited higher levels of emulsion Ahmed Z, Wang Y, Anjum N, Ahmad A, Khan ST (2013) Char-
stability (80.6%) with n-hexadecane (Das et al. 2014). acterization of exopolysaccharide produced by Lactobacillus
kefiranofaciens ZW3 isolated from Tibet kefir – Part II. Food
The present study showed that the cholesterol concentra- Hydrocoll 30(1):343–350. https://​doi.​org/​10.​1016/j.​foodh​yd.​
tion in the supernatant was reduced up to 46.4% by adding 2012.​06.​009
NCCP 962 EPS. Similarly, 47.5%, 45%, and 42.24% choles- Al-Dhaheri AS, Al-Hemeiri R, Kizhakkayil J, Al-Nabulsi A, Abush-
terol-lowering ability was observed by Lactobacillus plan- elaibi A, Shah NP, Ayyash M (2017) Health-promoting benefits of
low-fat akawi cheese made by exopolysaccharide-producing pro-
tarum BR2 (Sasikumar et al. 2017), LAB strain NBHK006 biotic Lactobacillus plantarum isolated from camel milk. J Dairy
(Tsai et al. 2014) and L. planatrum RJF4 respectively (Dilna Sci 100(10):7771–7779. https://​doi.​org/​10.​3168/​jds.​2017-​12761
et al. 2015). Thus, the addition of EPS with such potential Ayadi F, Bayer IS, Marras S, Athanassiou A (2016) Synthesis of water
in food product development may help to reduce increased dispersed nanoparticles from different polysaccharides and their
application in drug release. Carbohydr Polym 136:282–291.
blood cholesterol levels to some extent. Earlier research https://​doi.​org/​10.​1016/j.​carbp​ol.​2015.​09.​033
also indicated that polysaccharides that are acidic in nature, Aziz G, Zaidi A, Bakht U, Parveen N, Ahmed I, Haider Z, Muhammad
are able to reduce cholesterol efficiently as compared to T (2020) Microbial safety and probiotic potential of packaged
neutral polysaccharide. Furthermore, dextran-based EPS yogurt products in Pakistan. J Food Saf 40(1):e12741. https://​doi.​
org/​10.​1111/​jfs.​12741
being a neutral polymer, also failed to remove cholesterol. Bhat B, Bajaj BK (2019) Hypocholesterolemic potential and bioactivity
However, the exact mechanism of cholesterol-lowering is spectrum of an exopolysaccharide from a probiotic isolate Lacto-
still understudy, but studies found that probiotic strains like bacillus paracasei M7. Bioact Carbohydr Diet Fibre. 19:100191.
Sreptococcus thermophilis and Lactobacillus delbrueckii https://​doi.​org/​10.​1016/j.​bcdf.​2019.​100191
Caggianiello G, Kleerebezem M, Spano G (2016) Exopolysaccharides
subsp. bulgaricus could bind with free bile acids resulting produced by lactic acid bacteria: from health-promoting bene-
in increased cholesterol excretion after digestion. In return, fits to stress tolerance mechanisms. Appl Microbiol Biotechnol
new bile acids are synthesized by the cholesterol from the 100(9):3877–3886. https://​doi.​org/​10.​1007/​s00253-​016-​7471-2
liver and thereby decreasing the level of circulating choles- Das D, Baruah R, Goyal A (2014) A food additive with prebiotic prop-
erties of an α-d-glucan from Lactobacillus plantarum DM5. Int
terol (Dilna et al. 2015). J Biol Macromol 69:20–26. https://​doi.​org/​10.​1016/j.​ijbio​mac.​
2014.​05.​029
Supplementary Information The online version contains supplemen- Deep G, Hassan A, Metzger L (2012) Exopolysaccharides modify
tary material available at https://d​ oi.​org/​10.1​ 007/​s00253-​023-​12372-z. functional properties of whey protein concentrate. J Dairy Sci
95(11):6332–6338. https://​doi.​org/​10.​3168/​jds.​2012-​5649
Acknowledgements The authors would like to thanks all persons who Dilna SV, Surya H, Aswathy RG, Varsha KK, Sakthikumar DN, Pan-
provide technical support in this research. Special thanks to Prof. Dr. dey A, Nampoothiri KM (2015) Characterization of an exopoly-
Asif Ahmed for providing guidance related to RSM. Special thanks saccharide with potential health-benefit properties from a pro-
to Dr. Zaman Ashraf who provided technical support to conduct this biotic Lactobacillus plantarum RJF4. LWT - Food Sci Technol
research. 64(2):1179–1186. https://​doi.​org/​10.​1016/j.​lwt.​2015.​07.​040
Gawande K, Kolhekar M, Kumari M, Kapila S, Sharma P, Ali SA,
Author contribution AA, ZA and NK conceived and designed the Behare PV (2021) Lactic acid bacteria based purified exopolysac-
research. AA conducted the experiments. IF contributed in characteri- charide showed viscofying and hypercholesterolemic capabilites.
zation techniques. IA contributed in microbiological techniques. AA Food Hydrocoll. Health 1:100042. https://​doi.​org/​10.​1016/j.​fhfh.​
wrote the manuscripts. ZA and NK reviewed and edited the manuscript. 2021.​100042
All authors read and approved the final manuscript. Górska S, Hermanova P, Ciekot J, Schwarzer M, Srutkova D, Brzozo-
wska E, Kozakova H, Gamian A (2017) Chemical characterization
Funding Study was supported from Higher Education Commission of and immunomodulatory properties of polysaccharides isolated
Pakistan Project No. NRPU-8709. from probiotic Lactobacillus casei LOCK 0919. Glycobiology
27(3):275–277. https://​doi.​org/​10.​1093/​glycob/​cww107
Data availability The datasets generated during and/or analyzed dur- Hassan MU, Nayab H, Shafique F, Williamson MP, Almansouri TS,
ing the current study are available from the corresponding author on Asim N, Shafi N, Attacha S, Khalid M, Ali N (2020) Probiotic
reasonable request. properties of Lactobacillus helveticus and Lactobacillus plan-
tarum isolated from traditional Pakistani yoghurt. Biomed Res
Declarations Int 2020. https://​doi.​org/​10.​1155/​2020/​88891​98
Imran MYM, Reehana N, Jayaraj KA, Ahamed AAP, Dhanasekaran
Ethics approval This study does not contain any studies with human D, Thajuddin N, Alharbi NS, Muralitharan G (2016) Statistical
participants or animals performed by any of the authors. optimization of exopolysaccharide production by Lactobacillus
plantarum NTMI05 and NTMI20. Int J Biol Macromol 93:731–
Conflict of interest The authors declare no competing interests. 745. https://​doi.​org/​10.​1016/j.​ijbio​mac.​2016.​09.​007

13
Applied Microbiology and Biotechnology (2023) 107:1189–1204 1203

Jiang Y, Yang Z (2018) A functional and genetic overview of exopoly- medium. Appl Environ Microbiol 66(8):3427–3431. https://​doi.​
saccharides produced by Lactobacillus plantarum. J Funct Foods org/​10.​1128/​aem.​66.8.​3427-​3431.​2000
47:229–240. https://​doi.​org/​10.​1016/j.​jff.​2018.​05.​060 Rajoka MSR, Mehwish HM, Zhang H, Ashraf M, Fang H, Zeng X,
Korcz E, Varga L (2021) Exopolysaccharides from lactic acid bacteria: Wu Y, Khurshid M, Zhao L, He Z (2020) Antibacterial and anti-
techno-functional application in the food industry. Trends Food oxidant activity of exopolysaccharide mediated silver nanopar-
Sci Technol 110(2021):375–384. https://​doi.​org/​10.​1016/j.​tifs.​ ticle synthesized by Lactobacillus brevis isolated from Chinese
2021.​02.​014 koumiss. Colloids Surf B Biointerfaces 186:110734. https://​doi.​
Kumar A, Joishy T, Das S, Kalita MC, Mukherjee AK, Khan MR org/​10.​1016/j.​colsu​r fb.​2019.​110734
(2022) A potential probiotic Lactobacillus plantarum JBC5 Rajoka MSR, Mehwish HM, Kitazawa H, Barba FJ, Berthelot L,
improves longevity and healthy aging by modulating antioxida- Umair M, Zhu Q, He Z, Zhao L (2022) Techno-functional prop-
tive, innate immunity and serotonin-signaling pathways in Cae- erties and immunomodulatory potential of exopolysaccharide
norhabditis elegans. Antioxidants 11(2):268. https://​doi.​org/​10.​ from Lactiplantibacillus plantarum MM89 isolated from human
3390/​antio​x1102​0268 breast milk. Food Chem. 377:131954. https://​doi.​org/​10.​1016/j.​
Lee S-B, Kim D-H, Park H-D (2016) Effects of protectant and rehydra- foodc​hem.​2021.​131954
tion conditions on the survival rate and malolactic fermentation Rashid F, Hussain S, Ahmed Z (2018) Extraction purification and
efficiency of freeze-dried Lactobacillus plantarum JH287. Appl characterization of galactomannan from fenugreek for indus-
Microbiol Biotechnol 100(18):7853–7863. https://​doi.​org/​10.​ trial utilization. Carbohydr Polym 180:88–95. https://​doi.​org/​
1007/​s00253-​016-​7509-5 10.​1016/j.​carbp​ol.​2017.​10.​025
Li W, Xia X, Tang W, Ji J, Rui X, Chen X, Jiang M, Zhou J, Zhang Q, Russo P, Arena MP, Fiocco D, Capozzi V, Drider D, Spano G (2017)
Dong M (2015) Structural characterization and anticancer activity Lactobacillus plantarum with broad antifungal activity: A
of cell-bound exopolysaccharide from Lactobacillus helveticus promising approach to increase safety and shelf-life of cereal-
MB2-1. J Agric Food Chem 63(13):3454–3463. https://​doi.​org/​ based products. Int J Food Microbiol 247:48–54. https://​doi.​org/​
10.​1021/​acs.​jafc.​5b010​86 10.​1016/j.​ijfoo​dmicro.​2016.​04.​027
Limoli DH, Jones CJ, Wozniak DJ (2015) Bacterial extracellular poly- Saif FAA, Sakr EA (2020) Characterization and bioactivities of
saccharides in biofilm formation and function. Microbiol Spectr exopolysaccharide produced from probiotic Lactobacillus plan-
3(3):3.3.29. https://d​ oi.o​ rg/1​ 0.1​ 128/m
​ icrob​ iolsp​ ec.M
​ B-0​ 011-2​ 014 tarum 47FE and Lactobacillus pentosus 68FE. Bioact Carbo-
Liu Z, Zhang Z, Qiu L, Zhang F, Xu X, Wei H, Tao X (2017) Charac- hydr Diet Fibre 24:100231. https://​doi.​org/​10.​1016/j.​bcdf.​2020.​
terization and bioactivities of the exopolysaccharide from a pro- 100231
biotic strain of Lactobacillus plantarum WLPL04. J Dairy Sci Saleem M, Malik S, Mehwish HM, Ali MW, Hussain N, Khurshid M,
100(9):6895–6905. https://​doi.​org/​10.​3168/​jds.​2016-​11944 Rajoka MSR, Chen Y (2021) Isolation and functional characteri-
Mohd Yusof H, Abdul Rahman NA, Mohamad R, Zaidan UH (2020) zation of exopolysaccharide produced by Lactobacillus plantarum
Microbial mediated synthesis of silver nanoparticles by Lactoba- S123 isolated from traditional Chinese cheese. Arch Microbio
cillus Plantarum TA4 and its antibacterial and antioxidant activ- 203(6):3061–3070. https://​doi.​org/​10.​1007/​s00203-​021-​02291-w
ity. Appl Sci 10(19):6973. https://​doi.​org/​10.​3390/​app10​196973 Sasikumar K, Vaikkath DK, Devendra L, Nampoothiri KM (2017) An
Nambiar RB, Sellamuthu PS, Perumal AB, Sadiku ER, Phiri G, Jayara- exopolysaccharide (EPS) from a Lactobacillus plantarum BR2
mudu J (2018) Characterization of an exopolysaccharide produced with potential benefits for making functional foods. Bioresour
by Lactobacillus plantarum HM47 isolated from human breast Techno 241:1152–1156. https://​doi.​org/​10.​1016/j.​biort​ech.​2017.​
milk. Process Biochem 73:15–22. https://​doi.​org/​10.​1016/j.​procb​ 05.​075
io.​2018.​07.​018 Sathishkumar R, Kannan R, Jinendiran S, Sivakumar N, Selvakumar G,
Noureen S, Riaz A, Arshad M, Arshad N (2019) In vitro selection and Shyamkumar R (2021) Production and characterization of exopol-
in vivo confirmation of the antioxidant ability of Lactobacillus ysaccharide from the sponge-associated Bacillus subtilis MKU
brevis MG000874. J Appl Microbiol 126(4):1221–1232. https://​ SERB2 and its in-vitro biological properties. Int J Biol Macromol
doi.​org/​10.​1111/​jam.​14189 166:1471–1479. https://​doi.​org/​10.​1016/j.​ijbio​mac.​2020.​11.​026
Ołdak A, Zielińska D, Rzepkowska A, Kołożyn-Krajewska D (2017) Singh RP, Shukla MK, Mishra A, Kumari P, Reddy C, Jha B (2011)
Comparison of antibacterial activity of Lactobacillus plantarum Isolation and characterization of exopolysaccharides from sea-
strains isolated from two different kinds of regional cheeses from weed associated bacteria Bacillus licheniformis. Carbohydr Polym
Poland: Oscypek and Korycinski cheese. Biomed Res Int 2017. 84(3):1019–1026. https://​doi.​org/​10.​1016/j.​carbp​ol.​2010.​12.​061
https://​doi.​org/​10.​1155/​2017/​68203​69 Sirin S, Aslim B (2020) Characterization of lactic acid bacteria derived
Öztürk M, Aydin Y, Kiliçsaymaz Z, Önal C, Ba N (2018) Molecu- exopolysaccharides for use as a defined neuroprotective agent
lar cloning, characterization, and comparison of four bile salt against amyloid beta1–42-induced apoptosis in SH-SY5Y cells.
hydrolase-related enzymes from Lactobacillus plantarum GD2 Sci Rep 10(1):1–18. https://​doi.o​ rg/1​ 0.​1038/s​ 41598-0​ 20-​65147-1
of human origin. Food Biotechnol 32(3):191–205. https://d​ oi.o​ rg/​ Soh H-S, Kim C-S, Lee S-P (2003) A new in vitro assay of cholesterol
10.​1080/​08905​436.​2018.​15079​11 adsorption by food and microbial polysaccharides. J Med Food
Park S, Kang J, Choi S, Park H, Hwang E, Kang Y, Kim A, Holzap- 6(3):225–230. https://​doi.​org/​10.​1089/​10966​20036​07166​43
fel W, Ji Y (2018) Cholesterol-lowering effect of Lactobacillus Sun ML, Zhao F, Zhang XK, Zhang XY, Zhang YZ, Song XY, Chen
rhamnosus BFE5264 and its influence on the gut microbiome and XL (2020) Improvement of the production of an Arctic bacte-
propionate level in a murine model. PLoS One 13(8):e0203150. rial exopolysaccharide with protective effect on human skin cells
https://​doi.​org/​10.​1371/​journ​al.​pone.​02031​50 against UV-induced oxidative stress. Appl Microbiol Biotechnol
Park S, Saravanakumar K, Sathiyaseelan A, Park S, Hu X, Wang MH 104(11):4863–4875. https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00253-0​ 20-1​ 0524-z
(2022) Cellular antioxidant properties of nontoxic exopolysaccha- Trabelsi I, Ktari N, Triki M, Bkhairia I, Slima SB, Aydi SS, Aydi
ride extracted from Lactobacillales (Weissella cibaria) isolated S, Abdeslam A, Salah RB (2018) Physicochemical, techno-func-
from Korean kimchi. LWT 154:112727. https://d​ oi.o​ rg/1​ 0.1​ 016/j.​ tional, and antioxidant properties of a novel bacterial exopolysac-
lwt.​2021.​112727 charide in cooked beef sausage. Int J Biol Macromol 111:11–18.
Petry S, Furlan S, Crepeau MJ, Cerning J, Desmazeaud M (2000) Fac- https://​doi.​org/​10.​1016/j.​ijbio​mac.​2017.​12.​127
tors affecting exocellular polysaccharide production by Lactoba- Tsai CC, Lin PP, Hsieh YM, Zhang ZY, Wu HC, Huang CC (2014)
cillus delbrueckii subsp. bulgaricus grown in a chemically defined Cholesterol-lowering potentials of lactic acid bacteria based on

13
1204 Applied Microbiology and Biotechnology (2023) 107:1189–1204

bile-salt hydrolase activity and effect of potent strains on choles- from Leuconostoc mesenteroides strain XR1 and its application
terol metabolism in vitro and in vivo. Sci. World J. https://d​ oi.o​ rg/​ in fermented milk. LWT 146:111449. https://​doi.​org/​10.​1016/j.​
10.​1155/​2014/​690752 lwt.​2021.​111449
Vaziri AS, Alemzadeh I, Vossoughi M, Khorasani AC (2018) Co- Xing H, Du R, Zhao F, Han Y, Xiao H, Zhou Z (2018) Optimization,
microencapsulation of Lactobacillus plantarum and DHA fatty chain conformation and characterization of exopolysaccharide
acid in alginate-pectin-gelatin biocomposites. Carbohydr Polym isolated from Leuconostoc mesenteroides DRP105. Int J Biol
199:266–275. https://​doi.​org/​10.​1016/j.​carbp​ol.​2018.​07.​002 Macromol 112:1208–1216. https://​doi.​org/​10.​1016/j.​ijbio​mac.​
Wang X, Lü X (2014) Characterization of pectic polysaccharides 2018.​02.​068
extracted from apple pomace by hot-compressed water. Carbo- Xu Y, Cui Y, Wang X, Yue F, Shan Y, Liu B, Zhou Y, Yi Y, Lü X
hydr Polym 102:174–184. https://​doi.​org/​10.​1016/j.​carbpo​ l.2​ 013.​ (2019) Purification, characterization and bioactivity of exopoly-
11.​012 saccharides produced by Lactobacillus plantarum KX041. Int J
Wang Y, Li C, Liu P, Ahmed Z, Xiao P, Bai X (2010) Physical char- Biol Macromol 128:480–492. https://​doi.​org/​10.​1016/j.​ijbio​mac.​
acterization of exopolysaccharide produced by Lactobacillus 2019.​01.​117
plantarum KF5 isolated from Tibet Kefir. Carbohydr Polym Yang Y, Feng F, Zhou Q, Zhao F, Du R, Zhou Z, Han Y (2019) Iso-
82(3):895–903. https://​doi.​org/​10.​1016/j.​carbp​ol.​2010.​06.​013 lation, purification, and characterization of exopolysaccharide
Wang K, Li W, Rui X, Chen X, Jiang M, Dong M (2014) Structural produced by Leuconostoc citreum N21 from dried milk cake.
characterization and bioactivity of released exopolysaccharides Trans Tianjin Univ 25(2):161–168. https://​doi.​org/​10.​1007/​
from Lactobacillus plantarum 70810. Int J Biol Macromol s12209-​018-​0143-9
63:133–139. https://​doi.​org/​10.​1016/j.​ijbio​mac.​2013.​10.​036 Yuksekdag ZN, Aslim B (2008) Influence of different carbon sources
Wang J, Dai S, Yao Y, Cheng P, Lin Y, Zhan X (2015a) Spirobiflu- on exopolysaccharide production by Lactobacillus delbrueckii
orene-based acceptors for polymer solar cells: Effect of isomers. subsp. bulgaricus (B3, G12) and Streptococcus thermophilus
Dyes Pigm 123:16–25. https://​doi.​org/​10.​1016/j.​dyepig.​2015.​07.​ (W22). Braz Arch Biol Technol 51:581–585. https://​doi.​org/​10.​
020 1590/​S1516-​89132​00800​03000​19
Wang J, Zhao X, Tian Z, Yang Y, Yang Z (2015b) Characterization Zak B, Epstein E (1961) A study of several color reactions for the
of an exopolysaccharide produced by Lactobacillus plantarum determination of cholesterol. Clin Chim Acta 6(1):72–78. https://​
YW11 isolated from Tibet Kefir. Carbohydr Polym 125:16–25. doi.​org/​10.​1016/​0009-​8981(61)​90038-9
https://​doi.​org/​10.​1016/j.​carbp​ol.​2015.​03.​003 Zhao D, Jiang J, Liu L, Wang S, Ping W, Ge J (2021) Characterization
Wang X, Zhang L, Wu J, Xu W, Wang X, Lü X (2017) Improvement of exopolysaccharides produced by Weissella confusa XG-3 and
of simultaneous determination of neutral monosaccharides and their potential biotechnological applications. Int J Biol Macromol
uronic acids by gas chromatography. Food Chem 220:198–207. 178:306–315. https://​doi.​org/​10.​1016/j.​ijbio​mac.​2021.​02.​182
https://​doi.​org/​10.​1016/j.​foodc​hem.​2016.​10.​008
Wang B, Song Q, Zhao F, Han Y, Zhou Z (2019a) Production optimiza- Publisher's note Springer Nature remains neutral with regard to
tion, partial characterization and properties of an exopolysaccha- jurisdictional claims in published maps and institutional affiliations.
ride from Lactobacillus sakei L3. Int J Biol Macromol 141:21–28.
https://​doi.​org/​10.​1016/j.​ijbio​mac.​2019.​08.​241 Springer Nature or its licensor (e.g. a society or other partner) holds
Wang B, Song Q, Zhao F, Zhang L, Han Y, Zhou Z (2019) Isolation exclusive rights to this article under a publishing agreement with the
and characterization of dextran produced by Lactobacillus sakei author(s) or other rightsholder(s); author self-archiving of the accepted
L3 from Hubei sausage. Carbohydr polym 223:115111. https://​ manuscript version of this article is solely governed by the terms of
doi.​org/​10.​1016/j.​carbp​ol.​2019b.​115111 such publishing agreement and applicable law.
Wang L, Gu Y, Zheng X, Zhang Y, Deng K, Wu T, Cheng H (2021)
Analysis of physicochemical properties of exopolysaccharide

13