LWT - Food Science and Technology 139 (2021) 110744

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LWT
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Kinetics of growth, plantaricin and lactic acid production in whey permeate

based medium by probiotic Lactobacillus plantarum CRA52
Abhay Sharma , Sandipan Mukherjee , Subbi Rami Reddy Tadi , Aiyagari Ramesh **,
Senthilkumar Sivaprakasam *
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

A R T I C L E I N F O A B S T R A C T

Keywords: In the present study, the prospect of alternate carbon and nitrogen sources was ascertained for plantaricin (PL)
Kinetic modelling and lactic acid (LA) production from the probiotic lactic acid bacteria Lactobacillus plantarum CRA52. The
Whey permeate alternate carbon sources used in the present study were whey permeate (WP) and palmyra palm sugar (PJ), and
Plantaricin
the alternate nitrogen source was whey protein hydrolysate (WPH). Experimental use of WP as carbon source and
Lactic acid
Lactobacillus plantarum
WPH as nitrogen source led to enhanced production of LA and PL, with the yield being 17.69 gL-1 and 462.32
AUmL−1 respectively. WP and WPH concentration ratio of 2:1 was found to be optimal for the production of PL
and LA. Kinetic modeling revealed that Aiba substrate inhibition model exhibited best fit for the experimental
data. On performing a medium based process economic analysis, WP and WPH-based medium were economical
for the production of LA. The present investigation validates the potential of WP-WPH based medium for
heightened economical production of PL and LA. It is envisaged that this study will lend fundamental insights in
order to leverage the innate nutritional potential of raw products such as PJ and WP for economical production of
industrially relevant products.

production of bacteriocins (Ghaffar et al., 2014). The bacteriocins find

1. Introduction
Lactic acid bacteria (LAB) are key microbes with a significant role in
the food and fermentation industry (Yépez et al., 2017). Among the LAB
strains, Lactobacillus plantarum has particularly wide application in the
food industry for enhancement of texture, essence and preservation; and
in the healthcare sector for bacteriocin synthesis and lactic acid pro-
duction (Brizuela et al., 2019; da Silva Sabo, Vitolo, González, & de
Souza Oliveira, 2014).
LA is a multi-functional organic acid, with applications in industries
ranging from food and fermentation to textile and leather (Guilherme,
Silveira, Fontes, Rodrigues, & Fernandes, 2012).The estimated global LA
market is forecasted to reach 8.77 billion USD by 2025 (Grand view
research, Inc., USA, LA Market Analysis Report 2019). In the last decade,
there has been a significant down turn in the chemical synthesis of LA,
which is attributed to rising cost of raw materials and associated envi-
ronmental concerns. These concerns have mandated a shift to microbial
fermentation, focused on using LAB with the added benefit of concurrent
application in food preservation and in health care; their action against
pathogenic Listeria monocytogenes and Clostridium difficile is especially
noteworthy (Cotter, Ross, & Hill, 2013; Mokoena, 2017). Unfortunately,
fermentative production of LA entails a prohibitive 30–68 % of overall
production expenditure debited to cost of conventional media (Guil-
herme et al., 2012).Therefore though a range of refined substrates have
been explored to date, the high cost factor remains unresolved (based on
current price values adopted from Alibaba.com) and ranges from 4000
to 340 $/MT (Yeast extract 4000, Glucose 420–600, Lactose 480, Su-
crose and Glycerol 340 $/MT). Consequently, the cost effectiveness of
unrefined, low cost renewable agricultural waste, such as recycled paper
sludge, sugar cane bagasse, corn stover, including Corn steep liquor at
600 $/MT, have been explored (Ghaffar et al., 2014). In the latter, the
treatment of precursors is arduous and expensive, limiting the
cost-effectiveness and hindering their utilization on a large scale. Thus,
it is vital to identify cheap substrates for the co-production of LA and
bacteriocins. Feasible alternative substrates are whey permeate (250
$/MT), whey protein concentrate (40 $/MT), and PJ (275 $/MT),

* Corresponding author.
** Corresponding author.
E-mail addresses: abhay.sharma@iitg.ac.in (A. Sharma), m.sandipan@iitg.ac.in (S. Mukherjee), t.subbi@iitg.ac.in (S.R. Reddy Tadi), aramesh@iitg.ac.in
(A. Ramesh), senthilkumar@iitg.ac.in, sjsenthil@gmail.com (S. Sivaprakasam).

https://doi.org/10.1016/j.lwt.2020.110744
Received 25 June 2020; Received in revised form 25 November 2020; Accepted 9 December 2020
Available online 11 December 2020
0023-6438/© 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Sharma et al. LWT 139 (2021) 110744

Nomenclature n, m Dimensionless numbers

S* Inhibitory substrate concentration (gL−1)
t Fermentation time (h) SSE Sum of square error (dimensionless)
vx Volumetric biomass growth rate (gL−1h−1) EY Economic yield ($ of LA $ of nutrient−1)
X0 Initial biomass concentrations (gL−1) EP Economic productivity ($ of LA $ of nutrient−1t−1)
X Biomass concentration (gL−1) R2 Correlation coefficient (dimensionless)
Xm Maximum biomass concentration (gL−1) df Degrees of freedom
S Substrate concentration (gL−1) Ks Monod saturation constant (gL−1)
S0 Initial substrate concentration (gL−1) KI Inhibition constant (gL−1)
YX/S Biomass yield on substrate consumed (gg−1) ms Maintenance coefficient (gg−1h−1)
rs Volumetric substrate consumption rate (gg−1h−1)
PL Plantaricin concentration (AU mL−1) Greek Letters
rPL (vPL) Volumetric plantaricin production rate (AU mL−1h−1) μ Specific growth rate (h−1)
PLm Maximum plantaricin concentration (AU.mL−1) μmax Maximum specific growth rate (h−1)
LA Lactic acid concentration (gL−1) λx Log period of biomass (h)
rLA (vLA) Volumetric lactic acid production rate (gL−1h−1) λPL Log period of plantaricin (h)
LAm Maximum lactic acid concentration (gL−1) λLA Log period of lactic acid (h)
YP/S Product yield on substrate consumed (gg−1)

sourced from the dairy and cottage industries.
PJ is a rough brown sugar made in southern India from the extract of
Palmyra palm trees. It is rich in vitamins and minerals which are
required for the growth of fastidious microorganisms like LAB. Further,
it has been tested as a sole carbon source in different fermentation
processes (Rohit, Jyoti, Subbi, Naresh, & Senthilkumar, 2018). Whey is a
major by-product of the dairy industry and the major constituents are
lactose (5.0 % w/v), protein (1.0 % w/v), fat (0.4 % w/v) and mineral
salts. Ultrafiltration of whey yields two fractions: (a) whey permeate
fraction (WP), rich in lactose and retentate; and (b) whey protein
concentrate fraction (WPC), replete with casein (Amado, Vázquez,
Pastrana, & Teixeira, 2016). Individual supplements of WP and hydro-
lyzed WPC have facilitated microbial production of bio-chemicals such
as D-LA (Tadi, Arun, Limaye, & Sivaprakasam, 2017) and ascorbic acid
(Yang et al., 2017). The co-utilization of lactose and protein fractions of
whey in the D-LA fermentative production has also been reported
(Prasad, Srikanth, Limaye, & Sivaprakasam, 2014).
A clear perception of the critical process parameters of growth pat-
terns and product formation are vital prerequisites of economical in-
dustrial scale production of LA and bacteriocin. Consequently, kinetic
modelling becomes a vital tool for optimizing design and control pa-
rameters, media reformulation, amending operational specifications and
development of large-scale fermentation process for the target metab-
olite. In this regard, it would be pertinent to study the influence of initial
substrate concentration on co-production of bacteriocin and LA from
pure sugars and renewable sources, as very few studies have detailed
these factors. Based on the aforementioned rationale, in the present
study, whey protein concentrate (WPC) and whey permeate (WP) or
palmyra plam sugar (PJ) were probed as alternative nitrogen and carbon
sources, respectively. This study primarily focuses on formulation of an
economically viable media for the production of LA and PL using
Lactobacillus plantarumCRA52. In addition, the development of a kinetic
model to explain the biomass growth, PL and LA production using the
formulated medium has been attempted.
2. Materials and methods
2.1. Bacterial strains and reagents
Lactobacillus plantarum CRA52 strain used in the present investiga-
tion was isolated previously from salt-fermented cucumber and char-
acterized for its bacteriocinogenic and probiotic traits (Mukherjee &
Ramesh, 2015; Mukherjee, Singh, Adhikari, & Ramesh, 2013; Singh,
Mukherjee, Adhikari, & Ramesh, 2012). L. plantarum CRA52 was grown
and maintained as stock cultures as described in a previous investigation
(Singh & Ramesh, 2009). Listeria monocytogenes Scott A was used as an
indicator organism for determination of PL activity. Powdered whey
permeate (WP) and whey protein concentrate (WPC) was obtained from
M/s Nutrimed Healthcare Limited, New Delhi, India. Palm sugar (PJ)
was procured from cottage industries, Chennai, India. MRS media, su-
crose and lactose, Anthrone reagent, dinitrosalicylic acid (DNS) were
procured from M/s Himedia, Mumbai. 5 (and 6) carboxyfluorescein
diacetate succinimidyl ester (cFDA-SE), alcalase 2.4L were obtained
from M/s Sigma-Aldrich Chemicals, St. Louis, USA.
2.2. Preparation of growth media
2.2.1. Enzymatic hydrolysis of WPC
WPC was hydrolyzed using alcalase 2.4L according to the method
reported previously (Doucet, Otter, Gauthier, & Foegeding, 2003).
2.2.2. Growth and production of LA and PL by L. plantarum CRA52 on
alternate growth medium
L. plantarum CRA52 was grown in MRS, WP and PJ-based media. The
composition of MRS, WP and PJ based media is provided in Table A
(Supplementary Information). Prior to examining the influence of salts
on the growth and PL production, the media was formulated without
salts present in the commercial MRS medium. All the experiments were
performed in 250 ml conical flasks at static conditions, and the cultures
were incubated at 37 ◦ C.
2.2.3. Optimization of the concentration of WPH (nitrogen source) and WP
(carbon source)
The nitrogen source in the selected medium was optimized using one
factor at a time (OFAT) method. Nitrogen source concentration was
varied as follows: 0.5, 1.0, 3.0, 5.0, 10, 15, 20, 25 and 30 gL−1, while
concentrations of all other medium components were kept constant.
Similarly, for carbon source optimization, the carbon source concen-
tration was varied as follows: 6.94, 15.03, 22.56, 41.06, 62.5, 95.31,
126.25, 159.25, 203.25 gL−1. In order to optimize the concentration of
carbon to be used, the optimized nitrogen source concentration from the
experiment was used. The concentrations of all other medium compo-
nents were kept constant. In all of the experiments, the initial pH of the
medium prior to autoclaving was maintained at 7.0. All the experiments
were performed in 250 ml conical flasks at static conditions, and the
cultures were incubated at 37 ◦ C.

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A. Sharma et al. LWT 139 (2021) 110744

Table 1 HPLC (Shimadzu, Kyoto, Japan) based method (Kwon, Yoo, Lee, Chang,
Logistic model based estimated kinetic parameters corresponding to biomass & Chang, 2001).
growth, substrate consumption and product formation on different media.
Kinetic Medium 2.3.3. Estimation of plantaricin
parameter
MRS LAC WP SUR PJ
To determine the concentration of plantaricin in the sample, a zone
Biomass
formation
of inhibition and a fluorescence-based method were pursued (Singh
et al., 2012).
Xm (gL−1) 2.20 2.66 2.25 2.8 1.81
vx (gL−1h−1) 0.35 0.24 0.13 0.16 0.14
λx (h) 3.42 1.91 2.87 1.94 1.44 2.4. Kinetic modelling
R2 0.99 0.99 0.98 0.98 0.97
MSE 0.01 0.01 0.01 0.02 0.01 2.4.1. Microbial growth kinetics
p-value 8.03 × 4.43 × 3.02 × 1.91 × 2.24 ×
Biomass growth can be explained by the modified logistic model
10−9 10−8 10−7 10−7 10−7
F (df = 8, α = 1004.97 614.61 352.44 402.98 384.50 previously reported to describe the growth of Lactococcus lactis and
0.05) Pediococcus acidi lactici for the production of bacteriocins nisin and
pediocin respectively (Vázquez & Murado, 2008). It is described by the
Sugar following equation (1).
consumption
S0 (gL−1) 19.79 19.52 20.06 19.55 20.12 Xm
YX/S (gg−1) 0.11 0.14 0.10 0.14 0.13 X= [ ] (1)
mS (gg−1h−1) 0.33 0.36 0.36 0.26 0.35 1 + exp 2 + 4v
Xm
x
(λx − t)
R2 0.99 0.99 0.97 0.99 0.97
MSE 0.7 0.36 1.85 0.81 0.87
p-value 4.64 × 4.24 × 1.02 × 6.43 × 1.27 × where, Xm is the maximum biomass (gL-1); λx, (h) and the maximum
10−8 10−9 10−6 10−8 10−6 biomass growth rate vx (gL-1h-1).
F (df = 8, α = 606.40 1207.64 247.40 551.84 231.56
0.05)
2.4.2. Substrate utilization kinetics
Plantaricin
production Substrate utilization in a batch fermentation process was explained
PLm (AU mL−1) 1060.22 323.16 543.48 552.50 1007.58 using following expression (2):
vPL (AU 50.24 40.94 31.58 57.34 36.42
mL−1h−1)
⎡ ⎤
λPL (h) 15.1 6.56 7.91 12.32 11.8 1 ⎢ Xm ⎥
R2 0.95 0.99 0.98 0.98 0.95 S = S0 − ⎢ [ ] − X0 ⎥
MSE 1294.65 128.31 613.226 520.51 994.56
Y X/S ⎣ ⎦
1 + exp 2 + 4v
Xm
x
(λx − t)
p-value 6.56 × 3.82 × 3.03 × 3.67 × 3.76 ×
⎛ ⎞
10−5 10−9 10−7 10−8 10−6
F (df = 8, α = 70.92 1244.44 352.26 648.90 168.27 ⎡ ⎜ X4vx t ⎟ ⎤
0.05) X0 ⎝e m − 1⎠ + X m
( ) ⎢ ⎥
ms X m 2 ⎢ ⎥
Lactic acid − ln⎢ ⎥ (2)
4vx ⎣ Xm ⎦
production
LAm (gL−1) 16.40 21.01 17.69 14.54 13.62
vLA (gL−1h−1) 2.68 1.3 1.68 1.48 1.61
λLA (h) 4.76 9.57 8.56 7.16 8.08 where, S0 is initial substrate concentration (gL−1), Where, ms is the
R2 0.99 0.98 0.99 0.97 0.99 maintenance coefficient (gg−1h−1), YP/S and YX/S are product and
MSE 0.25 0.72 0.56 0.99 0.09
biomass yield coefficients (gg−1) respectively.
p-value 1.41 × 1.25 × 2.48 × 5.39 × 1.76 ×
10−9 10−7 10−8 10−7 10−10
F (df = 8, α = 1656.43 455.33 726.82 297.88 3006.81 2.4.3. Product formation kinetics
0.05)

All the cultures were grown in static condition at 37 ◦ C. Modified logistic models previously used to explain the LA and hy-
aluronic acid production (Amado et al., 2016) were adopted to model
2.3. Analytical methods the dynamic changes in PL and LA using equations (3) and (4):
PLm
All the experiments were repeated independently at least three times PL = [ ] (3)
and the data was represented as mean with standard deviation. 1 + exp 2 + 4vPL
PLm
(λPL − t)

2.3.1. Estimation of bacterial biomass LAm

LA = [ ] (4)
Bacterial biomass concentration was estimated spectrophotometri-
1 + exp 2 + 4vLA
(λLA − t)
cally at 600 nm using a microplate reader (Tecan,Grödig, Austria). LAm

Subsequently, measured Optical density (OD) values were transformed

into Dry Cell Weight (DCW) values by using estimated correlation be-
tween OD and DCW; where,1.0 OD600 = 0.55 DCW gL−1.
2.3.2. Estimation of glucose, sucrose, lactose, and lactic acid
Substrate analysis was performed by two independent methods.
Since the non-reducing sugar sucrose is the principal component for
palm sugar, total sugar present in the PJ was estimated using anthrone
method (Morris, 1948). For estimation of lactose present in WP, DNS
method was used (Miller, 1959). Glucose and LA were estimated using
where PLm and LAm are the maximum product concentrations of PL
(AUL−1) and LA (gL−1); vPL and vLA are the maximum PL (AUL−1h−1)
and LA (gL−1 h−1) production rates, and λPL and λLA are the lag phase of
PL and LA production (h).
2.5. Kinetic parameters estimation
Kinetic parameters were estimated by minimizing the sum of squared
errors (SSE) between models predicted and experimental data using

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A. Sharma et al. LWT 139 (2021) 110744

Fig. 1. Effect of different nutritive media on bacterial biomass, substrate utilization and product formation by L. plantarum CRA52. Experimental (scattered points)
and model simulated (continuous lines) dynamic profiles of biomass growth (●), substrate consumption (■), lactic acid (▴), and plantaricin (▾) production on (A)
glucose-, (B) sucrose-, (C) lactose-, (D) PJ-, (E) WP- based modified-MRS medium and (F) Zone of inhibition assay on target bacteria, L. monocytogenes Scott A (1–8
numbers represents samples collected at different time intervals).

nonlinear least square method. All the kinetic parameters estimation
and model fitting procedures were carried out using Microsoft Excel
solver tool (Rohit et al., 2018).
n ficient (R2) and Sum of squared error (SSE) were used to verify the
∑ ( )2
SSE = Zsim − Zexp
i=1
where, Zsim is the simulated data and Zexp is the experimental data.
3. Results and discussion
3.1. Growth of L. plantarum CRA52 on MRS and modified MRS media
The ability of L. plantarum to co-produce lactic acid (LA) and plan-
taricin (PL) in large amounts renders the organism as a virtual industrial
microbial factory. However, the commercial prospects of L. plantarum
are hindered by the economic costs of media components. Therefore, the
present investigation reports on the formulation, production and
viability of cost-effective medium components for production of LA and
PL. MRS medium was modified with WP, PJ (carbon source) and WPH
(nitrogen source) to assess growth of L. plantarum CRA52, LA and PL
production. Modified logistic model (Equations (4), (8) and (9)) was
utilized to explain the dynamic behavior of L. plantarum CRA52 growth,
PL and LA production under uncontrolled conditions. Correlation coef-
(5) goodness of the fit of the logistic model to the experimental data.
Fisher’s F and p-values are used to track the significance and consistency
of the kinetic model (Table 1). Since F-value is a ratio between the
regression variation and residual variation, the higher the F-value and
lower the p-value are considered as significant (Germec, Cheng, Karhan,
Demirci, & Turhan, 2019). Dynamic profiles of both simulated and
experimental data are depicted in Fig. 1.R2 value was observed to be
more than 0.9 for all carbon sources, implying that the Modified Logistic
model renders a better explanation of the kinetic parameters of
L. plantarum CRA52. This fact is also supported by the concurrence be-
tween the simulated values and the experimental values of the kinetic
parameters. Model estimated kinetic parameter values are presented in
Table 1. Lactose and sucrose-based medium resulted in high biomass
titre of 2.66 gL-1 and 2.8 gL-1, respectively. Biomass obtained with WP
based medium (2.25 gL-1) was comparable to control MRS medium (2.2
gL-1). In MRS and WP, a longer lag phases for growth resulted (3.42 h

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A. Sharma et al. LWT 139 (2021) 110744

Fig. 2. Effect of minimal salts on L. plantarum CRA52 measured through (A) biomass, (B) substrate utilization, (C) production of lactic acid, (D) bacteriocin. The
different media utilized are: MRS medium (●), PJ-based medium (■) and WP-based medium (▴).

Table 2
Kinetic parameters from bacterial growth in different media.
Maximum Specific Maximum lactic Lactic acid yield Volumetric lactic Volumetric Maximum Plantaricin yield
Media biomass growth acid produced, on substrate, acid productivity, plantaricin plantaricin on substrate, YPL/
produced, Xm rate, μ (h−1) LAm (gL−1) YLA/S (gg−1) rLA (gL−1 h−1) productivity, rPL produced, PLm S (AU g )
−1

(gL−1) (AUmL−1h−1) (AUmL−1)

Control 2.15 ± 0.18 0.24 ± 0.02 16.62 ± 0.25 0.88 ± 0.02 0.96 ± 0.03 16.52 ± 2.15 434.64 ± 51.6 21.12 ± 1.58
(MRS)
PJΔS 0.98 ± 0.06 0.08 ± 0.03 3.04 ± 0.21 0.84 ± 0.01 0.12 ± 0.02 0.54 ± 0.57 44.88 ± 13.6 3.87 ± 3.1
WPΔS 0.54 ± 0.04 0.05 ± 2.18 ± 0.13 0.57 ± 0.02 0.09 ± 0.01 0.09 ± 0.42 38.4 ± 10.08 0.65 ± 2.9
0.001

Product yield (YP/S, gg−1) was calculated as a ratio of product produced (g or AU) to substrate consumed (g). Volumetric productivity (rp, gL−1h−1) was calculated as a
ratio of concentration of product produced (gL−1) to fermentation time (h). All the cultures were grown in static condition at 37 ◦ C.

and 2.87 h, respectively), whereas with lactose, sucrose and PJ-based
medium exhibited a shorter growth lag phase. Maximum LA titre was
observed in WP-based medium (21.01 gL−1), followed by lactose-based
media, whereas PJ- and sucrose-based media displayed lower quantities
of LA. Interestingly, lactose-, sucrose-, WP-, and PJ-based medium led to
a longer lag phase for LA production (>6 h) as compared to
glucose-based medium (4.76 h). In case of plantaricin production,
growth of L. plantarum CRA52 in glucose-, sucrose- and PJ
based-medium yielded higher titers of bacteriocin as compared to
lactose-based media. High bacteriocin titre in glucose- and
sucrose-based medium has been reported for the different strains of
L. plantarum producing bacteriocin bacST8KF (Powell, Witthuhn,
Todorov, & Dicks, 2007) and PL ST31(S. Todorov, Gotcheva, Dousset,
Onno, & Ivanova, 2000). Further, lactose- and WP-based medium
resulted in relatively reduced lag phase for PL production compared to
glucose-, sucrose- and PJ-based medium (Fig. 1). This was a clear indi-
cation that certain components of these media supported the synthesis of
bacteriocins, most plausibly by increment of biomass. Further, in all the
cases high PL production was observed as the L. plantarum CRA52
entered the stationary phase. This could be due to the fact that bacte-
riocins are secondary metabolites, produced in increasing quantities in
the stationary phase. Similar results were reported for the bacteriocins
produced by L. plantarum ST8KF(Powell et al., 2007), L. plantarum
ST202Ch (Engelhardt, Szakmár, Kiskó, Mohácsi-Farkas, & Reichart,
2018). It is noteworthy, that these results are contrary to previously
reported higher yield in lactose – based media for L. plantarum (S. D.
Todorov, van Reenen, & Dicks, 2004). The advantage of WP- and
PJ-based medium over MRS medium for production of LA and PL can be
ascribed to the use of hydrolyzed whey protein as nitrogen source, as
compared to yeast extract, beef extract and peptone in MRS. Since whey
protein is widely available and economically viable, the production cost
of LA and PL is anticipated to be significantly reduced. Based on the
aforementioned rationale and the fact that WP-based media elicited
maximum LA and PL, whey protein (WP) was selected as carbon source
and used for all further experiments.

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A. Sharma et al. LWT 139 (2021) 110744

Table 3
Effect of initial nitrogen substrate concentration on plantaricin and lactic acid production.
Initial nitrogen substrate concentration(gL−1)

Kinetic parameters 0.5 1.0 3.0 5.0 10 15 20 25 30

Maximum biomass produced, 0.81 ± 0.05 1.16 ± 0.05 1.59 ± 0.08 1.58 ± 0.05 1.74 ± 0.06 1.82 ± 0.12 1.92 ± 0.18 1.95 ± 0.08 1.98 ± 0.11
Xm (gL−1)
Specific growth rate, μ (h−1) 0.13 ± 0.02 0.16 ± 0.01 0.15 ± 0.02 0.18 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.15 ± 0.02 0.14 ± 0.01 0.14 ± 0.01
Biomass yield on substrate, YX/S 0.07 ± 0.01 0.08 ± 0.01 0.09 ± 0.02 0.08 ± 0.02 0.09 ± 0.01 0.10 ± 0.01 0.10 ± 0.02 0.09 ± 0.01 0.11 ± 0.02
(gg−1)
Residual Lactose (gL−1) 5.37 ± 0.21 3.26 ± 0.83 0.80 ± 0.51 0.71 ± 0.16 0.40 ± 0.37 0.54 ± 0.34 0.30 ± 0.15 0.59 ± 0.22 0.49 ± 26
Maximum LA produced, 0.93 ± 0.24 11.84 ± 14.29 ± 14.92 ± 17.26 ± 17.59 ± 0.29 17.74 ± 17.59 ± 17.56 ±
LAm(gL−1) 0.15 0.18 0.11 0.43 0.63 0.31 0.49
Volumetric LA productivity, rLA 0.45 ± 0.05 0.57 ± 0.02 0.68 ± 0.05 0.76 ± 0.04 0.87 ± 0.06 0.95 ± 0.04 0.97 ± 0.05 1.02 ± 0.05 1.03 ±
(gL−1h−1) 0.0.9
LA yield on substrate, YLA/S (gg−1) 0.67 ± 0.03 0.77 ± 0.02 0.75 ± 0.02 0.78 ± 0.01 0.87 ± 0.02 0.85 ± 0.01 0.84 ± 0.02 0.88 ± 0.02 0.88 ± 0.01
Maximum PL produced, PLm(AU 299.49 ± 318.39 ± 356.72 ± 400.75 ± 412.66 ± 407.66 ± 421.94 ± 437.43 ± 434.67 ±
mL−1) 18.1 21.7 19.6 23.1 43.6 30.52 35.1 25.4 32.3
Volumetric PL productivity rPL 15.15 ± 16.10 ± 18.03 ± 19.93 ± 1.7 20.69 ± 22.03 ± 2.34 20.73 ± 19.04 ± 21.79 ±
(AU mL−1h−1) 1.37 1.16 1.54 2.11 1.42 3.51 2.66
PL yield on substrate YPL/S (AU 23.31 ± 20.96 ± 19.18 ± 20.24 ± 19.34 ± 21.64 ± 1.82 20.97 ± 20.56 ± 20.42 ±
g−1) 2.25 0.93 1.58 1.34 1.66 1.53 2.19 2.00

Product Yield (YPS, gg-1) was calculated as a ratio of product produced (g or AU) to substrate consumed (g). Volumetric productivity (rp, gL-1h-1) was calculated as a
ratio of concentration of product produced (gL-1) to fermentation time (h). All the cultures were grown in static condition at 37 ◦ C.

3.2. Effect of minimal salts on growth and product formation
Considering the crucial role played by salts in bacterial growth and
production of metabolite, it was considered pertinent to probe the
growth of L. plantarum CRA52 on WP and PJ-based medium devoid of
salts. Thus, PJΔS and WPΔS represented PJ- and WP-based medium
without externally added salts (MgSO4.7H2O and MnSO4.7H2O).The
primary observation recorded a drastic fall in growth, substrate con-
sumption and product formation in PJΔS and WPΔS (Fig. 2, Table 2).
This finding indicates that although PJ and WP are rich in salts, external
supplementation of salts is essential for growth and production of LA
and PL by L. plantarum CRA52. This can be ascribed to the critical role
played by metal ions such as Mg2+ and Mn2+ in glucose metabolism
(Abo-Amer, 2011). Manganese also plays an important role in oxidative
stress resistance in LAB, particularly in microbes such as L. plantarum
which lacks superoxide dismutase activity (Watanabe, van der Veen, &
Abee, 2012). Further, manganese also acts as a cofactor for lactate de-
hydrogenase, which is responsible for production of LA from pyruvic
acid. Both Mg2+ and Mn2+ play important roles in biosynthesis of lip-
oteichoic acid and peptidoglycan (Lew, Liong, & Gan, 2013). Taking
into consideration the important role of salts, in all further experiments
salts were added to WP- and PJ-based medium.
3.3. Effect of initial nitrogen substrate (WPH) concentration on
L. plantarum CRA52 growth, substrate consumption and product
formation
To achieve optimal biomass growth, substrate consumption and
product formation, the concentration of WPH as nitrogen source, was
optimized using One – factor-at –a time (OFAT) approach. The con-
centration of WPH was varied at 0.5, 1.0, 3.0, 5.0, 1.0, 15.0, 20.0, 25.0
and 30.0 gL-1 and the kinetic parameters were calculated from the
experimental data. The kinetic data revealed that at lower concentra-
tions, nitrogen proved to be a limiting factor (Table 3). A proportional
maximal increase in the biomass, Xm (gL−1); lactic acid, LAm (gL−1) and
plantaricin, PLm (AU mL−1) resulted with concomitant increase in WPH
concentrations, within the lower range (0.5, 1.0, 3.0 and 5.0 gL-1).
Further, the residual lactose concentration was found to be high at low
WPH concentration (0.5, 1.0, 3.0 and 5.0 gL-1), with complete sugar
utilization only at WPH concentrations of 10 gL-1 and above. This
finding indicates that WPH concentration is critical for consumption of
lactose by L. plantarum CRA52. Further, there was no appreciable
change in final biomass and product yields above 5 gL-1 concentration of
WPH. This could be due to the complex nature of WPH, which acts as the
nitrogen source. Complex organic nitrogen sources such as yeast extract,
beef extract etc do not affect the bacteriocin production even at high
concentration (Nguyen et al., 2012). Considering the aforementioned
findings, WPH concentration was fixed at 10 gL-1 for the further kinetic
modelling experiments.
3.4. Effect of initial carbon substrate concentration on
L. plantarumCRA52 growth, substrate consumption and product formation
OFAT approach was employed to study the effect the initial carbon
source (WP) concentration on growth of L. plantarum CRA52and pro-
duction of LA and PL. To achieve this, initial WP concentration was
varied and the kinetic parameters were determined. In order to deter-
mine the kinetic parameters, logistic model was utilized to fit the
experimental data and the simulated biomass, amount of product
formed and substrate consumption profiles were generated. The primary
observation suggested that biomass growth, substrate utilization and
product formation were dependent on the concentration of WP (Fig. 3).
It was observed that complete utilization of carbon source was observed
at a sugar concentration ranging from 6.94 to 22.56 gL-1, while residual
sugar was observed to increase beyond these concentrations. The
maximum biomass growth was found to increase when the WP con-
centration ranged from 6.94 to 22.56 gL-1. Further, at high substrate
concentrations, up to 22.56 gL−1, the production of PL and LA were
saturated (Fig. 3). This finding can perhaps be ascribed to the allosteric
inhibition in the biosynthetic pathway of LA, which leads to a saturation
beyond a certain carbon concentration. Concurrently, synthesis of
bacteriocin is primarily dependent on availability of substrate and
presence of lower pH in medium, both of which are accomplished at
high substrate concentrations. The logistic model-based estimated ki-
netic parameters are presented in Table 4. The kinetic pattern obtained
was reasonably explained by the model with the regression coefficient
value being greater than 0.9 at different initial WP concentrations,
displaying agreement in simulated and experimental profiles of sub-
strate consumption, cell growth, and LA production (Fig. 3).
To decipher the precise role of substrate, different substrate inhibi-
tion and substrate limiting kinetic models were examined and compared
in this study (Supplementary Information Table B). Even though
Andrews model (B), Han-Levenspiel model (C), Aiba model (D) and Non-
competitive inhibition model (F) gave same correlation coefficient
values of 0.99, Aiba model (D) resulted in relatively low RMSE value
(2.27 × 10−5). Hence, Aiba model is considered suitable for defining the
production of LA and PL (Fig. 4). The estimated kinetic parameter values
for the Aiba model are presented in Table 5, and are as follows:

6
A. Sharma et al. LWT 139 (2021) 110744

Fig. 3. Experimental (scattered points) and Logistic model based simulated (continuous lines) dynamic profiles of biomass growth (●), substrate consumption (■),
lactic acid (▴) and plantaricin (▾) production on medium with varying initial concentration of carbon substrate, (A) 6.94 gL-1, (B) 15.03 gL-1, (C) 22.56 gL-1, (D)
41.06 gL-1, (E) 62.5 gL-1, (F) 95.31 gL-1, (G) 126.25 gL-1, (H) 159.25 gL-1 and (I) 203.25 gL-1.

7
A. Sharma et al. LWT 139 (2021) 110744

Table 4
Logistic model based estimated kinetic parameters corresponding to biomass growth, substrate consumption and product formation at different whey permeate
concentrations.
Kinetic Initial carbon substrate concentration (gL−1)
Parameter
6.94 15.03 22.56 41.06 62.50 95.31 126.25 159.25 203.25

Biomass formation

Xm (gL−1) 1.62 1.98 1.96 2.01 2.01 2.08 2.09 2.12 2.09
vx (gL−1h−1) 0.10 0.14 0.13 0.14 0.14 0.14 0.13 0.14 0.12
λx (h) 2.00 3.78 3.65 4.03 3.31 3.20 3.72 3.56 3.75
R2 0.98 0.96 0.96 0.96 0.95 0.96 0.97 0.95 0.96
MSE 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02

Sugar consumption
S0 (gL−1) 6.94 15.03 22.56 41.06 62.50 95.31 126.25 159.25 203.25
YX/S (gg−1) 0.48 0.19 0.14 0.26 0.14 0.14 0.16 0.19 0.19
mS (gg−1h−1) 0.20 0.38 0.25 0.36 0.20 0.36 0.32 0.38 0.46
R2 0.96 0.97 0.98 0.88 0.94 0.92 0.92 0.84 0.86
MSE 0.34 1.41 1.65 10.59 5.95 9.29 8.53 21.3 14.58

Plantaricin production
PLm (AU mL−1) 307.99 444.70 457.43 463.60 500.35 492.61 489.04 502.39 495.45
vPL (AU mL−1h−1) 22.92 38.71 43.50 39.10 41.57 44.49 37.86 40.99 41.41
λPL (h) 5.58 5.54 6.19 5.59 5.81 6.01 5.52 5.79 5.82
R2 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
MSE 140.76 283.56 174.89 288.74 149.82 73.51 382.75 126.04 141.47

Lactic acid production

LAm (gL−1) 6.55 14.10 19.14 17.00 17.75 16.58 18.86 19.60 16.14
vLA (gL−1h−1) 0.75 4.35 4.14 3.99 2.11 2.65 3.16 2.79 5.01
λLA (h) 4.75 10.91 10.26 10.55 10.64 10.80 10.99 9.26 11.12
R2 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
MSE 0.12 0.07 0.33 0.35 0.24 0.19 0.51 0.25 0.40

All the cultures were grown in static condition at 37 ◦ C.

Maximum specific growth rate (μmax)- 0.17 h−1; Monod’s saturation
constant (KS)- 3.95 gL−1and substrate inhibition constant (KI)- 741.36
gL-1. When the concentration of carbon source was increased, from 6.94
to 15.03 gL-1, μmax increased from 0.09 to 0.14 h−1. However, a constant
μmax value of 0.135 ± 0.05 was observed when the initial concentration
of the carbon source ranged from 15.56 to 203.25 gL-1. The observed
saturation of maximum biomass and formation of LA and PL could be
due to the inhibitory effect of LA. μmax of a similar magnitude was re-
ported for L. plantarum CIL6 strain grown on whey based medium
(Filannino et al., 2014). Further, high values of the predicted KI for all
the tested models signifies, that the L. plantarum CRA52 growth is only
inhibited at very high sugar concentration (Fig. 4 and Table 4). This was
further confirmed from the nearly constant log period for biomass (λx)
for the sugar concentration from 15.56 to 203.25 gL-1 (Table 4).
3.5. Medium based economic analysis of LA production
Techno-economic feasibility of LA fermentation was analyzed by
estimating economic yield (EY) and economic productivity (EP) (Bustos,
Moldes, Alonso, & Vázquez, 2004). EP and EY of LA production on
different carbon and nitrogen source-based media were calculated using
equations (6) and (7).
Y Further optimization of process parameters such as temperature, pH,
Economicyield, EY = ∑ I (6)
(CN)
Y enhanced production of lactic acid and plantaricin in large scale. It is
Economicproductivity, EP = ∑ I (7)
t (CN)
where, Y is product (LA) concentration gL−1, I is selling price of product
(LA) ($g−1), C is the cost associated with nutrient ($), N is the concen-
tration of the nutrient (g), and t is fermentation time, (h). The detailed
cost information of individual medium components and products (LA)
were taken from the online bulk chemical trading site (Alibaba.com) and
presented in Supplementary Information Table C. Since, information on
commercial cost of plantaricin is not available; corresponding EY and EP
calculations were not performed for the bacteriocin. It was observed that
medium formulated with glucose resulted in very low EY and EP (0.22 $
of LA $ of nutrient−1 and 0.01 $ of LA $ of nutrient−1h−1). However, WP
has yielded highest EY and EP (1.25$ of LA $ of nutrient−1 and 0.05 $ of
LA $ of nutrient−1h−1) in comparison to sucrose-, lactose- and PJ-based
medium (Fig. 5). This shows WP-based medium is economically viable
for the production of LA using L. plantarum CRA52.
4. Conclusions
Raw material cost is the primary limiting factor in the economic
production of lactic acid and plantaricin. Alternate media were suc-
cessfully formulated using PJ, WP and WPH from dairy industry as
carbon and nitrogen source in lieu of glucose present in MRS medium for
lactic acid and plantaricin production using probiotic L. plantarum
CRA52. Modified logistic model used in the present study was able to
explain the growth behavior of L. plantarum CRA 52 and formation of
lactic acid and plantaricin. Even though WP- and PJ-based medium are
rich in minerals, externally added salts were found to be essential for
growth of L. plantarum CRA52 and formation of lactic acid and plan-
taricin. Initial WP and WPH concentrations were optimized. L. plantarum
CRA 52 growth was not affected by high initial WP concentration.
agitation rate, dissolved oxygen and developing an effective process
strategy for high cell density cultivation would facilitate economic and
envisaged that the present study would serve as a blueprint for utiliza-
tion of ubiquitous resources from the cottage industry, thereby
benefitting the industrial sector and small scale bioprocess units.
Funding
A.R. received a research grant from the Department of Biotech-
nology, Govt of India [BT/PR13560/COE/34/44/2015].

8
A. Sharma et al. LWT 139 (2021) 110744

Fig. 4. Effect of initial WP concentration on L. plantarum CRA52 specific growth rate. (A) Monod model, (B) Andrew model, (C) Han-Levenspiel model, (D) Aiba
model, (E) Competitive inhibition model, (F) Non-competitive inhibition model.

Table 5
Logistic model based estimated kinetic parameters corresponding to biomass growth; substrate consumption and product formation at different whey permeate
concentrations.
Name of the Kinetic Model μmax (h−1) KS (gL−1) KI (gL−1) n m S* (gL−1) R2 RMSE

Monod model 0.14 1.92 0.95 8.93 × 10−5

Andrews model 0.16 3.62 849.24 0.99 2.58 × 10−5
Han-Levenspiel model 0.16 3.98 0.98 0.96 836.23 0.99 2.32 × 10−5
Aiba model 0.17 3.95 741.36 0.99 2.27 × 10−5
Competitive inhibition model 0.14 1.87 286.44 0.95 9.05 × 10−5
Non-competitive inhibition model 0.17 4.32 618.79 0.99 2.34 × 10−5

CRediT authorship contribution statement
Abhay Sharma: Investigation. Sandipan Mukherjee: Investigation,
Writing - review & editing. Subbi Rami Reddy Tadi: Investigation,
Conceptualization, Writing - original draft, Writing - review & editing.
Aiyagari Ramesh: Funding acquisition, Supervision, Writing - review &
editing. Senthilkumar Sivaprakasam: Project administration, Super-
vision, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no conflict of interest.

9
A. Sharma et al.
Fig. 5. (A) Economic yield and (B) Economic productivity of lactic acid produced by L. plantarum CRA52 on different carbon substrate-based modified MRS medium.
Acknowledgements
S.M. and S.R.T. thank IIT Guwahati for a research fellowship. Au-
thors extended their gratitude for the support rendered by the Depart-
ment of Biosciences and Bioengineering, IIT Guwahati.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2020.110744.
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