Streptomyces Fumigatiscleroticus VIT-SP4 For Drug Delivery and

Saudi Journal of Biological Sciences xxx (xxxx) xxx
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Saudi Journal of Biological Sciences

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Original article
Characterisation of pectin and optimization of pectinase enzyme from

novel Streptomyces fumigatiscleroticus VIT-SP4 for drug delivery and
concrete crack-healing applications: An eco-friendly approach
Praveen Kumar Govindaraji, Suneetha Vuppu ⇑
Department of Biotechnology, School of Biosciences and Technology, VIT, Vellore, India
a r t i c l e i n f o a b s t r a c t
Article history: Pectinases are enzymes which are widely distributed in microbes that are present in pectin enriched
Received 6 January 2020 sites. The agro-industrial residues can be utilized in the industrial scale for low-cost and efficient pecti-
Revised 19 July 2020 nase production in an eco-friendly approach. This study employs low-cost substrates (i.e. culinary fruit
Accepted 20 July 2020
peels) for maximum pectinase production from novel Streptomyces fumigatiscleroticus VIT-SP4. The
Available online xxxx
extraction and characterization of pectin from different fruit peels were investigated and pectinase activ-
ity was analyzed. The orange pectin gave maximum pectinase activity of about 45.93 (U/mL). Further, sta-
Keywords:
tistical optimization of process parameters was studied by using Taguchi method showed optimum
Fruit pectin
Pectinase
values of pH-6, temperature 35 °C, orange pectin% 2.5, incubation time- 48 h and RPM- 200 rpm
Statistical optimization and pectinase activity was found to be 98.65 (U/mL). The response surface methodology (RSM) was used
Drug delivery for the optimization of media components which revealed that starch 1.17%, yeast extract-2%, and
Crack-healing orange pectin% 0.75% produces maximum pectinase of about 170.05 (U/mL). The drug-delivery study
showed drug release was not observed at initial pH 3 after 4 h. The immediate drug release was noted at
pH 6 caused due to disintegration of pectin by the pectinase activity. The self-healing of cracks by spray
culture technique was investigated. The crack healing was observed up to 0.50 mm wide after 12 days.
This confirms the ability of actinomycete spores to survive and they react to form calcite complex directly
helps in crack healing process. This low-cost microbial pectinase can be used in drug delivery and con-
crete crack-healing applications sectors in future.
Ó 2020 Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction require an eco-friendly approach. Citrus waste residues accumu-

lated as unfavorable waste which are perceived as valueless but
In recent times the market value of pectinases was increased after degradation by pectinolytic actinomycetes results in produc-
and it acquires the highest position among commercially used tion of by-products like cellulose, fibers, pectin that can be utilized
enzymes in the industries and it is estimated to be 41.4$ billion for various industrial processes (Kaur, 2017). Many industries dis-
in future (Garg et al., 2016; El Enshasy et al., 2018). As, enzymes card agro-industrial residues as municipal solid wastes which can
are applied enormously in different fields and decreasing its probe utilized for enzyme production (Marzo et al., 2019) and they are
duction cost becomes the main problem and it has to be focused. dumped by industries contain high amounts of carbon, nitrogen
Most of the fruit dump yard wastes lead to hazardous pollution and other minerals and these wastes may be used as a substrate
and their disposal is alarming in less developed countries that for pectinase production (Mehmood et al., 2019). The citrus peels
and seeds of about 34 million tons were dumped in food process-
ing industries and accumulation of them causes serious environ-
⇑ Corresponding author. mental problems. According to the industrial point of view, the
E-mail address: [email protected] (S. Vuppu). production cost of enzymes is mainly caused due to the cost of
Peer review under responsibility of King Saud University. the medium and it has to be minimized. Hence, citrus peels which
are discarded as wastes that can be valorized as an alternate
substrate for pectinase production (Viayaraghavan et al., 2019).
The Taguchi method has been employed for the prediction of the
Production and hosting by Elsevier important contribution of variables that are designed and
https://doi.org/10.1016/j.sjbs.2020.07.024
1319-562X/Ó 2020 Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: P. K. Govindaraji and S. Vuppu, Characterisation of pectin and optimization of pectinase enzyme from novel Streptomyces fumi-
gatiscleroticus VIT-SP4 for drug delivery and concrete crack-healing applications: An eco-friendly approach, Saudi Journal of Biological Sciences, https://doi.
org/10.1016/j.sjbs.2020.07.024
2 P.K. Govindaraji, S. Vuppu / Saudi Journal of Biological Sciences xxx (xxxx) xxx
optimum conditions of each variable by running experiments on 2.2.2. Fourier Transform Infrared Spectroscopy (FTIR) characterization
flask experiments (John et al., 2019). Valorization of industrial of fruit pectin
wastes and their utilization by statistical analysis may be the pos- The samples were blended with potassium bromide (FT-IR
itive approach to minimize the production cost of enzyme. grade) and characterized by FT-IR (Perkin Elmer-Spectrum RX1
Response surface methodology (secondary mathematical model) spectrometer). The spectra were scanned wavenumber range
which is an integration of statistical and mathematical methodolo- between 4000 and 500 cm1.
gies that are significant for their modelling and problem scrutiny.
This model is predominantly used for optimizing the components 2.2.3. In vitro analysis of fruit peel pectin after pectinase activity
of media required for microbial derived pectinase and it has been The pectin samples were dried in the oven at 105 °C to evaluate
immensely used for the optimization of biochemical and chemical the moisture content present in it. Ash, proteins, lipids and crude
reactions like enzyme yield (Barbosa et al., 2010), optimization of fibers were analyzed according to AOAC methods (Feldsine et al.,
media (Kunamneni and Singh, 2005), constraints for enzyme 2002).
hydrolysis (Shieh and Lai, 2000), setting up of criteria for food pro-
cessing (Ozer et al., 2004). Pectinase enzyme has been reported for 2.3. Inoculum preparation for pectinase production
the drug-release studies for colon treatment by the degradation of
pectin-coated tablets and it also used for pulsatile drug delivery The potential pectinolytic isolate Streptomyces fumigatiscleroti-
studies (Zhu et al., 2019; Liu et al., 2012). In recent scenario, there cus VIT-SP4 (Genbank accession no. KM875468) was selected for
is an interest in the microbial self-healing process in case of crack the study (Kumar and Suneetha, 2017). The isolate VIT-SP4 was
repair of concrete due to its advantage like long-lasting, eco- inoculated in production (CSPY-ME) media contains (K2HPO4 0.5,
friendly and efficiency (Wang et al., 2016). Microbial-induced cal- casein 3.0, starch 10.0, peptone 1.0, yeast extract 1.0, pectin 1.0,
cium carbonate precipitation (MICP) plays an important role in malt extract 10.0 g L-1) where commercial pectin was replaced
bacterial healing of concrete cracks (Zhang et al., 2016). The cal- with extracted fruit pectins. The media were incubated at
cium precipitation depends on the spore concentration of the bac- 28 ± 2 °C in a shaker at 100g for 72 h. incubation, then the suspen-
teria (Achal and Mukherjee, 2015). On the other hand, some spores sion of potential isolate (1 mL) were pooled, centrifuged at
grown as vegetative cells because they cannot form germinated 10,000 rpm for 10 min at 4 °C and analyzed for pectinase assay
spores. There is a research lacuna on the statistical optimization (Panda et al., 1999). The isolate Streptomyces lydicus MTCC 7505
of pectinase production from Streptomyces sp. by valorizing fruit was taken as control for all experiments.
pectin.
Thus, our current study focuses on extraction, morphological 2.4. Taguchi methodology: Optimization of process variables for
and chemical characterization of pectins from different fruit peels. maximum pectinase yield
The extracted pectin was utilized for the statistical optimization of
pectinase from novel strain Streptomyces fumigatiscleroticus VIT- The statistical optimization of pectinase was carried out by the
SP4 by Taguchi orthogonal array method and Response Surface Taguchi orthogonal array methodology by Design expert software
Methodology (RSM). The yielded pectinase was utilized for con- version 7.0. Five main factors namely temperature, pH, Incubation
trolled drug delivery study and potential isolate VIT-SP4 were time, pectin (%), RPM were considered. The experiment’s size was
investigated for concrete- crack healing process. designed by arrays with 25 experimental trails. The experiments
were investigated by flask experiments for different trails which
involve the 72 h-old culture of Streptomyces fumigatiscleroticus
VIT-SP4 in production media (100 mL) with different parameters
2. Materials and methods based on experimental design and it was pectinase activity was
assayed for each trial. The results of the experiments were ana-
2.1. Extraction and selection of economically viable pectin lyzed by design expert software for individual and interaction of
the factors, optimal conditions for maximum pectinase production
The fruit peels like mosambi (Citrus limetta) – [MO], pomegra- by Streptomyces strain. The optimized conditions were validated by
nate (Punica granatum) [PO], Orange (Citrus aurantium) - [OR], the software. The signal to noise (S/N) ratio was calculated.
mango (Mangifera indica) - [MA] and commercial pectin (control)
were selected for the study. The pectin was extracted followed 2.5. Central Composite design (CCD): Optimization of media
by the protocol. (Liew et al., 2014). components for pectinase yield by RSM
The percentage production of fruit peel pectin was determined
by below formulae: The effect of media components (critical variables) was opti-
mized for maximal pectinase production from Streptomyces fumi-
gatiscleroticus VIT-SP4 was carried out by Central Composite
ProductobtainedðgÞ
Pectin yieldð%Þ ¼ 100 Design (CCD). The full factorial design was investigated with inde-
5goffruitpeelpowder pendent factors (3) namely starch, yeast extract, pectin% substrate
levels (5) and replicates (3) to obtain second-order three-
dimensional response surface over the factors and levels using
Design Expert Version 11.0.0 software shown in supplementary
2.2. Characterization of pectin information Table 3. Each variable was analyzed with 5 various
levels and it results in 20 experimental runs with 6-axial and 6-
2.2.1. Scanning Electron Microscopy (SEM) characterization of fruit centre points (Yu and Xu, 2018).
pectin The analysis of variance (ANOVA) was assessed by determining
The morphological features of pectin were visualized by the statistical analysis of the model and the significance of the
Scanning Electron Microscope (SEM) (ZEISS EVO 18 RESEARCH) model was determined. The F-value, variance’s measure was deter-
provided with retro-disperse electrons operated inside an Oxford mined by RSM and it was represented as R2 value. The quadratic
X-ray probe under low vacuum conditions at the pressure at model was performed and the 2-Dcontour plot and 3D- response
different magnifications at 1, 3 and 5 kV respectively. surface curves were generated to determine variable’s effect for
org/10.1016/j.sjbs.2020.07.024
P.K. Govindaraji, S. Vuppu / Saudi Journal of Biological Sciences xxx (xxxx) xxx 3
maximum pectinase production by using Design Expert 11.0. Soft- 3. Results

ware (Wahab et al., 2018).
The shake flask experiments was performed to evaluate the pre- 3.1. Characterization and utilization of pectin for pectinase production
dictive values of media components namely starch, yeast extract,
and pectin%. The media components were taken in different con- This study deals with the valorization of fruit pectin for the pro-
centrations based on the design. For this experiment, inoculums duction and statistical optimization of pectinase from potential
of the potential isolate were inoculated into 100 mL of media in isolate Streptomyces fumigatiscleroticus VIT-SP4 (Genbank acces-
250 mL conical flask and it was incubated at 30 °C in a shaker with sion no. KM875468). The pectin was extracted from different fruit
100 rpm for 120 h. The physical parameters were optimized by full peels and pectin yield was calculated by using the above formulae
factorial design before the experiment. (Liew et al., 2014). The pectin yield was more in orange peel and it
was estimated to be 42.3% respectively shown in Supplementary
information (Table 1). The morphological characterization of pec-
tin was analyzed by SEM (Scanning Electron Microscopy). The
2.6. Pectinase effect on a pectin-coated tablet in controlled drug-
results revealed that mosambi pectin shows porous structure with
delivery system
hard and cervices; pomegranate pectin shows rough and uneven
structures with pores; orange pectin shows heterogeneous struc-
The pectin-based drug delivery system was carried out in this
ture with pores, mango pectin shows cuticles on cuticular wax
study. Indomethacin, an anti-inflammatory drug (NSAID) was
ridges shown in (Fig. 1). The surface property of pectin was exam-
selected as a model drug. The Ca-pectinate tablets were prepared
ined to understand pectin structures, its types namely protopectin
with extracted orange pectin and the purified enzyme from Strep-
or pectinic acid present in fruit peels and also structural changes in
tomyces fumigatiscleroticus VIT-SP4 was chosen. The enzyme is
pectin caused by acid extraction method. Pectinase acts on differ-
purified and molecular weight was found to be 45 kDa. The purity
ent pectin by various mechanisms based on its pectin structures
of the enzyme was confirmed by HPLC prior to the experiment. The
and this study helps for detailed study about the mechanism of
experiment was carried out for the observation of drug release at
pectinase on different pectins.
different pH in CSPYME media and time intervals (Maestrelli
The interpretation of the FT-IR result was based on the corre-
et al., 2008).
sponding peaks respective to the functional groups present in the
fruit pectins. The spectra showed that peaks at 3331.07 cm1 of
Mosambi [MO], 3323.35 cm1 of pomegranate [PO],
2.7. Crack healing of concrete specimen by Streptomyces 3334.92 cm1 of orange [OR], 3342.64 cm1 of mango [MA] and
fumigatiscleroticus VIT-SP4 3556.74 cm1 of commercial pectin. The peaks at 2916.37 cm1
of [MO], 2922.16 cm1 of [PO], 2978.09 cm1 of [OR],
2.7.1. Culture preparation and sporulation kinetics 2916.37 cm1 of [MA] and 2937.59 cm1 of commercial pectin.
The prepared inoculum of isolate S.fumigatiscleroticus VIT-SP4 The peaks 1728.22 cm1 of [MO], 1722.43 cm1 of [PO],
were added to the sterilized CSPY-ME media and then it was incu- 1728.22 cm1 of [OR], 2848.86 cm1 of [MA] and 2937.59 cm1
bated for 72 h under agitation 100 rpm at 28 ± 2 °C for maximum of pectin (commercial). The characteristic peak at 1633.71 cm1
pectinase production. The endospores of S.fumigatiscleroticus VIT- of [MO], 1614.42 cm-1of [PO], 1627.92 cm-1of [OR], 1625.99 cm1
SP4 (72 h-old culture) was inoculated in CSPY-ME media were of [MA] 1629.85 cm1 of commercial pectin. The peaks
applied to cement concrete varying in pH to determine the surviv- 1238.30 cm1 of [MO], 1016.49 cm1 of [PO], 1055.06 cm-1of
ability of organisms in adverse conditions. [OR], 1165.00 cm-1of [MA],1012.63 cm1 , 1066.64 cm1,1103.28 -
cm1, 1008.77 cm1, 1031.92 cm1. The FTIR spectral peaks of fruit
pectins were compared with commercial pectin shown in (Fig. 2).
The peaks correspond to the functional groups confirm the pres-
2.7.2. Cement specimen preparation and survivability of
ence of pectic acid and other functional groups similar to that of
actinomycetes spores
pectin. The in-vitro analysis of fruit peels was characterized by
The actinomycete spores were introduced into cement speci-
using the AOAC method for the evaluation of proximate composi-
men in addition to water for determination of spore viability. The
tion present in fruit peels (AOAC, 1995). The results showed the
survivability of spores was studied for a period of 1 to 90 days.
amount of lipid, ash, carbohydrate, and fiber present in the fruit
Each specimen was pulverized by sterilized mortars to escalate
peels shown in Supplementary information (Table 2). The potential
the extraction of spores. Then, it was serial-diluted in sterilized
pectinolytic isolate Streptomyces fumigatiscleroticus VIT-SP4 was
Tris-HCl buffer. The concrete cylinders were prepared for a partic-
inoculated in the production media (CSPYME) media in which
ular size of about (75 75 mm). The cracks were made on the con-
commercial pectin was replaced with fruit pectin. After incubation,
crete cylinders by the compression machine. Then, culture was
the supernatant was taken for pectinase assay. The Orange pectin
sprayed after the spore formation and observed for the crack heal-
gave maximum pectinase activity of about 45.93 (U/mL) shown
ing process shown in Supplementary information (Fig. 1).
in (Fig. 3). So, orange pectin will be valorized for further optimiza-
tion studies for pectinase production by Streptomyces fumigatiscle-
roticus VIT-SP4.
2.7.3. Ultrasonic treatment on concrete cracks
The ultrasonic waves (Steinkamp ultrasonic tester type BP III) of
frequency range of 400–600 kHz were passed through hardened 3.2. Optimization of process parameters for maximum production of
concrete cylinders of 75 75 mm with a crack of 0.50 mm with pectinase by Taguchi method
10 mm depth on an open fissure in the concrete and observed for
maximum transmission time. The instrument is connected to the The results for the optimization of process parameters by Tagu-
machine which measures the time of a wave to passes from one chi method was shown in (Table 1). The results revealed that the
sensor to another sensor. The sensors were placed at the upper statistics for the Taguchi model were analyzed by F-value for
end of a concrete specimen on the cracks and it was measured ANOVA and results were shown in (Table 2). The F-value is 63.08
(Van Tittelboom et al., 2010). for the ANOVA model recommends it is the signature model for
org/10.1016/j.sjbs.2020.07.024
Fig. 1. Morphological study of culinary fruit pectins by SEM (Zeiss EVO 18) analysis at different magnifications (a) and (b) Mosambi (Citrus limetta) (c) and (d) Pomegranate
(Punica granatum); (e) and (f) Orange (Citrus aurantium); (g) and (h) Mango (Mangifera indica).
pectinase production. The chance is only 0.05% that F-value is large and RPM- 200 shown in (Fig. 4). The maximum pectinase activity
occur due to noise. The p-value of the model is<0.0500 indicates produced by Streptomyces fumigatiscleroticus VIT-SP4 under these
variables in the model are significant. In Taguchi model, all the optimum conditions was found to be 98.65 (U/mL).
model terms including A, B, C, D, and E are significant. The R2 value
should be more than 0.75 suggests the model’s fitness. In this 3.3. Media components optimization: Response surface methodology
Taguchi model, the R2 value is 0.9968. So, the model can analyze (RSM)
the response is correct. The (S/N) ratio for this model was deter-
mined by ‘Adeq precision’. The ratio should be greater than 4 to The experiments were carried out based on the optimized val-
confirm the model is the desired model. In this, the ratio is ues of media components and results were shown in (Table 3).
26.9112 implies an adequate signal. This model can be utilized to The statistical results of the model was determined by F-Value
direct the design space. The point in the parity plot has two co- for the analysis of variance (ANOVA) and results are shown in
ordinates namely (x and y) where indicates actual value and y (Table 4). The F-value for the ANOVA model of 97.03 suggests that
indicates predicted value. The line equation (y = x) was considered the model is significant. There is only a 0.01% chance that Fisher’s
to be a reference. When both experimental and predicted values value (F-value) this large may occur due to noise. The p-value of
are similar, the points lie on the same line (Fig. 6 (a). In our model, the model terms is less than 0.0500 implies terms are significant.
experimental and predicted values lay on the same straight line. In this study, A, B, C, AB, AC, A2, B2, C2 are the most significant
This confirms the model’s fitness. model terms. In case, there are more insignificant model terms,
The results revealed that optimum conditions for maximum the model reduction can be helpful to improve the model.
pectinase activity from isolate VIT-SP4 were found to be pH-6, The R2 value should be greater than 0.75 implies the fitness of
temperature- 35 °C, orange pectin%- 2.5, incubation time- 48 h the model. In this model, the Correlation coefficient (R2 value) is
org/10.1016/j.sjbs.2020.07.024
Table 1
Experimental design for optimization of process parameters by Taguchi method.
Run Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Pectinase activity Pectinase activity
A:pH B: Temp C:Pectin% D: Incubation time E: RPM (Actual value) (Predicted value)
3 1 5 30 2 48 150 92.35 92.02
16 2 8 20 2.5 36 250 89.23 88.90
11 3 7 20 2 96 100 78.89 79.47
24 4 9 35 2 36 50 95.23 95.07
6 5 6 20 1.5 48 200 96.75 96.59
14 6 7 35 1 48 250 87.54 87.54
21 7 9 20 3 72 150 94.52 94.52
1 8 5 20 1 24 50 94.06 93.96
23 9 9 30 1.5 24 250 94.31 94.89
7 10 6 25 2 72 250 93.57 93.47
9 11 6 35 3 24 100 97.54 97.21
12 12 7 25 2.5 24 150 80.23 80.07
18 13 8 30 1 72 100 88.01 87.85
4 14 5 35 2.5 72 200 97.36 97.94
17 15 8 25 3 48 50 87.59 88.17
2 16 5 25 1.5 36 100 90.25 90.25
25 17 9 40 2.5 48 100 97.23 97.13
13 18 7 30 3 36 200 85.56 85.46
10 19 6 40 1 36 150 96.23 96.81
20 20 8 40 2 24 200 86.34 86.34
22 21 9 25 1 96 200 95.25 94.92
8 22 6 35 2.5 48 200 98.65 98.65
19 23 8 35 1.5 96 150 87.38 87.28
5 24 5 40 3 96 250 97.89 97.73
15 25 7 40 1.5 72 50 84.56 84.23
0.9887. The ‘Predicted R2 value’ is 0.9320 is a reasonable agree- P-value is 0.0413 which is less than 0.05 confirms the interaction
ment with ‘Adjusted R2 Value’ is 0.9785 (i.e.) difference is less than between starch and yeast extract showed an increase in the pecti-
0.2. So, the model can predict the response is correct. The signal to nase production. Found to be significant. The response surface
noise ratio for the model was calculated by ‘Adeq Precision’. This graph (Fig. 5 (b)) shows the particular concentration and interac-
value should be greater than 4 to confirm the model is the desired tion between starch (A) and orange pectin% (C). The yeast extract
model. In this model, the ratio is 28.116 indicates an adequate sig- (B) was kept as constant for higher pectinase production. When
nal. This model can be utilized to route the design space shown in starch and orange pectin % concentrations increase, the pectinase
(Table 5). activity was also increased. At, the very higher concentration of
The Coefficient of variation (CV) implies a degree of precision orange pectin% mild decrease in the pectinase activity was
which is utilized for the comparison of experimental values. The observed. The p-value is 0.0001 which is less than 0.05 confirms
model’s final equation in terms of coded factors was the interaction between starch and orange pectin% was found to
be significant. The response surface graph (Fig. 5 (c)) shows the
interaction between yeast extract (B) and orange pectin% (C). The
Pectinase activity ¼ 158:95 þ 11:59A þ 4:91B þ 6:57C 3:11A
starch (A) was kept as constant for maximum production of pecti-
B þ 7:89 A C 0:6025B C 3:39A2 nase. The results showed that a higher concentration of yeast
extract decreases the pectinase production. When, orange pectin
20:00 B2 16:91 C2 Where; A
% concentration increases, there is a gradual increase in pectinase
¼ Starch; B ¼ Yeast extract; C production. The p-value is 0.6599 which is greater than 0.05 con-
¼ Orange pectin% firms the interaction between yeast extract and orange pectin%
influences less for the maximum pectinase production. Found to
The ‘Lack of Fit F-value’ shows 1.57 denotes lack of Fit is be insignificant. The contour plot (Fig. 5 (d)) represents a 2D-plot
insignificant following pure error is shown in (Table 6). There is showed the contour curves are oval which confirms interaction
a 31.62% chance that ‘Lack of Fit F-value’ was larger which obtains between starch (A) and yeast extract (B) found to be is significant
due to noise (i.e. Non-significant lack of fit is considered as good). for pectinase production. The contour plot (Fig. 5 (e)) represents
When both experimental and predicted values are similar, the the 2D-plot of interaction between Starch (A) and Orange pectin%
points lie on the same line (Fig. 6(b). In our model, experimental (C) for pectinase production. The oval shape of the contour curve
and predicted values lay on the same straight line. This confirms indicates more interaction between factors starch (A) and orange
the model’s fitness. The Response Surface Methodology (RSM) pectin% (C) which confirms it is significant. The contour plot
and 3-D graphical representation of the regression equation were (Fig. 5 (f)) shows a 2D-plot of interaction between yeast extract
generated by the model. These graphs were utilized to determine (B) and orange pectin% (C) for pectinase production. The contour
the factors which were significant for maximal pectinase produc- curve is a circular shape confirms the interaction between factors
tion. Based on the combinations between the factors three (B) and (C) are less interactive for pectinase production. The opti-
response graphs were generated by the software. mum concentrations were predicted for the media components
The response surface graph (Fig. 5 (a)) implies the concentration by Design-Expert software (Ver.11.0) were Starch 1.17%, Yeast
and interaction between starch (A) and Yeast extract (B) for max- extract-2%, orange pectin%-0.75%. Based on the predicted concen-
imum pectinase production. The results showed that when starch trations of the media components, the predicted value for maxi-
and yeast extract concentrations increase, there is a gradual mum pectinase activity was found to be 170.05 U/mL. To
increase in pectinase production. At high concentration of yeast investigate the optimum concentrations of media components
extract leads to a decrease in the pectinase production. The generated by the model experiments were carried out in shake
org/10.1016/j.sjbs.2020.07.024
Fig. 2. Fourier Transform Infrared Spectroscopy analysis of (a) Mosambi (Citrus limetta) (b) Pomegranate (Punica granatum) (c) Orange (Citrus aurantium) (d) Mango
(Mangifera indica) (e) Commercial pectin.
Table 2
Analysis of variance of factors and summary of statistics for taguchi model.
Source Sum of squares Df Mean square F-Value p-value

Model 762.58 20 38.13 63.08 0.0005 Significant
A-pH 644.85 4 161.21 266.70 <0.0001
B-Temp 42.05 4 10.5 17.39 0.0085
C-Pectin% 42.56 4 10.64 17.60 0.0084
D- Incubation time 38.45 4 9.61 15.88 0.0078
E- RPM 24.66 4 6.16 10.20 0.0225
Residual 2.42 4 0.6045
Cor total 765.00 24
Std. dev 0.7775 R2 0.9968
Mean 91.46 Adjusted R2 0.9810
C.V.% 0.8501 Predicted R2 0.8765
Adeq precision 26.9112
flask experiments. The experimental actual value for maximal isolate was found to be 0.75%. The pectin (0.25%) utilized for max-
pectinase activity was revealed to be 168.84 U/mL which is in good imum production of pectinase from Streptomyces sp. (Kuhad et al.,
agreement with the predicted value. 2004). The orange pectin% utilized by isolate VIT-SP4 is compara-
The positive interaction between starch and yeast extract for ble to previous reports. The interaction between orange pectin%
pectinase production from isolate VIT-SP4 was observed. But, less and starch showed positive interaction for maximum pectinase
interaction was observed between yeast extract and orange pec- production. The interaction between orange pectin% and yeast
tin% for the production of pectinase from isolate VIT-SP4. The opti- extract showed less interaction for pectinase production by Strep-
mal orange pectin concentration for the potential pectinolytic tomyces fumigatiscleroticus VIT-SP4.
org/10.1016/j.sjbs.2020.07.024
Table 3
Experimental design for the optimization of variables for pectinase production.
Std. order Run Starch (g/L) Yeast extract (g/L) Pectin % (g/L) Actual Value PredictedValue
1 1 0.5 1 0.5 96.00 99.76
10 2 1.17045 2 0.75 170.05 168.84
5 3 0.5 1 1 100.15 98.33
2 4 1 1 0.5 115.33 113.38
12 5 0.75 3.68179 0.75 110.25 110.63
7 6 0.5 3 1 110.00 113.16
11 7 0.75 0.318207 0.75 96.21 94.12
9 8 0.329552 2 0.75 130.36 129.87
16 9 0.75 2 0.75 160.25 158.95
19 10 0.75 2 0.75 160.25 158.95
14 11 0.75 2 1.17045 123.54 122.18
13 12 0.75 2 0.329552 100.42 100.08
8 13 1 3 1 148.45 145.89
20 14 0.75 2 0.75 152.13 158.95
3 15 0.5 3 0.5 120.30 117.01
4 16 1 3 0.5 115.15 118.18
6 17 1 1 1 139.00 143.50
17 18 0.75 2 0.75 160.25 158.95
18 19 0.75 2 0.75 160.25 158.95
15 20 0.75 2 0.75 160.25 158.95
it helps in self-healing of cracks. The crack healing was observed

up to 0.55 mm wide was recorded after the specimens were sub-
merged in water for 12 days (Fig. 8(e)). The ultrasonic measure-
ment test was carried out for both cracked and healed
specimens. The results showed that the transmission time of the
cracked specimen and healed concrete specimen were found to
be 31.05 µs and 30.07 µs respectively. After, crack repair waves
cannot pass through sealed crack leading to a decrease in the trans-
mission time. Bio-mineralized calcite showed maximum resistance
to dissolution compared to inorganic calcite formation.
4. Discussion
The study deals with characterization of pectin and optimiza-

tion of pectinase from novel Streptomyces fumigatiscleroticus VIT-
SP4 and its industrial applications. The pectin was extracted from
Fig. 3. Selection and conventional optimization of cheap substrates (fruit pectin) different fruit peels. The maximum pectin yield was obtained by
for maximum pectinase production. citric acid extraction method was of about 42.3% respectively and
it was higher comparable to the pectin yield of 19.24% (Prakash
Maran et al., 2013), 20.44 ± 0.64 (Guo et al., 2012), 5.27% (Yeoh
3.4. Action of pectinase from potential isolate VIT-SP4 to pectin-based et al., 2008) seen in earlier reports. The media which has pectin
pellet for controlled drug delivery system materials can ultimately increase the production of enzymes that
were reported earlier (Aguilar and Huitron, 1990). The characteri-
The controlled drug release parameter studied for continued zation of pectin was carried out by scanning electron microscope.
hours in CSPY-ME media with variation in different pH at regular The results revealed the morphological features of fruit pectins
intervals. The result revealed that at initial pH (3.0) after 2 h there were similar to earlier reports (Arias and Ramon-Laca, 2005;
is an absence of drug release. When pH was attained to pH 6 after Mafra et al., 2001; Pathak et al., 2017; Wang et al., 2016). The FTIR
the period of 6 h rapid drug release was observed. At pH 3, drug spectra revealed that peaks at 3300.00 cm1 correspond to the OH
release was absent and at pH 6, immediate drug release was groups due to the presence of alcohol and pectic components of
observed after 17 h. The maximum pectinase activity from Strepto- pectin molecule. The peaks at 2900.00 cm1 is CAH asymmetrical
myces fumigatiscleroticus VIT-SP4 was observed at pH 6 which stretching vibration due to aliphatic structures caused by the result
cause controlled drug release and it was shown in (Fig. 7). of different vibration modes in carbohydrates and lignin. The peaks
at 1700.00 cm1 correspond to carbonyl group (C@O) stretching of
3.5. Crack healing of concrete cement specimen by potential isolate methyl esterified COCH3 in pectin which has been regarded as low
methoxy pectin. The peaks at 1600.00 cm1 represents stretching
The self-healing of crack concrete blocks was investigated with vibration due to ionic carboxyl groups. The peaks 1200.00 cm1,
potential isolate VIT-SP4 by spray culture technique. The stained 1100.00 cm1, 1000.00 cm1 are caused by C@O and C@C vibration
spores were observed (Fig. 8 (a)). Sporulation % was shown in of glycosidic bonds and pyrenoid ring. The FTIR spectra range
(Fig. 8(b)). Detection of viable spores was resistant after 90 days around 800–1300 cm1 represents the ‘fingerprint’ region due to
was shown in (Fig. 8(c)) confirms the survivability of spores. The OH bending CAOAC stretching and CH3 plane deformation. Finally,
cracked concrete block was taken as control (Fig. 8 (d)). After IR spectra at 3300 cm1 confirm the presence of pectic acid in the
sporulation, the inoculum was sprayed on the cracks. The spores fruit peels (Chatjigakis et al., 1998; Silverstein and Bassler, 1962;
react with chemicals present in the concrete to form calcite and Pappas et al., 2004; Kumar and Chauhan, 2010; Cerna et al.,
org/10.1016/j.sjbs.2020.07.024
Fig. 4. Taguchi model: Main effect plots of process parameters (a) pH (b) Temperature (c) Orange Pectin% (d) Incubation time (e) RPM.
Table 4
Analysis of Variance (ANOVA) for all variables in RSM model.
Source Sum of Squares Df Mean square F-value p-value

Model 12333.86 9 1370.43 97.03 <0.0001 Significant
A-starch 1833.28 1 1833.28 129.81 <0.0001 Significant
B- Yeast extract 392.02 1 329.02 23.30 0.0007 Significant
C- Pectin% 589.20 1 589.20 41.72 <0.0001 Significant
AB 77.38 1 77.38 5.48 0.0413 Significant
AC 498.02 1 498.02 35.26 0.0001 Significant
BC 2.90 1 2.90 0.2056 0.6599 Not significant
A2 165.72 1 165.72 1.73 0.0065
B2 5764.09 1 5764.09 408.13 <0.0001
C2 4118.77 1 4118.77 291.63 <0.0001
Residual 141.23 10 14.12
Lack of fit 86.29 5 17.26 1.57 0.3162 Not significant
Pure Error 54.95 5 10.99
Cor Total 12475.09 19
Table 5
Statistical Summary of model terms to determine suggested model for pectinase production.
Source Sequential p-value Lack of fit p-value Adjusted R2 Predicted R2

Linear 0.2503 <0.0001 0.0744 0.1643
2FI 0.8431 <0.0001 0.0714 0.8081
Quadratic <0.0001 0.3162 0.9785 0.9320 Suggested
Cubic 0.2289 0.4533 0.9842 0.8654 Aliased
2003; Gan et al., 2010). The in-vitro analysis of fruit peels after pared to other pectins which is higher compared to previous stud-
pectinase treatment was characterized by using the AOAC method ies (Kuo et al., 2019).
and proximate composition present in fruit peels were evaluated The optimization of pectinase was carried out by Taguchi
(AOAC, 1995). The potential isolate VIT-SP4 was inoculated in the orthogonal array method. The maximal pectinase production from
production (CSPYME) media with different fruit pectins. The result Streptomyces fumigatiscleroticus VIT-SP4 was observed at pH 6
revealed that orange pectin gave maximal pectinase activity com- which is similar to earlier study (Zhu et al., 2019) and it was com-
org/10.1016/j.sjbs.2020.07.024
Table 6
Statistical summary of lack of fit.
Source Sum of Squares Df Mean Square F-value P-value

Linear 9668.65 11 878.97 79.99 <0.0001
2FI 9090.35 8 1136.29 103.40 <0.0001
Quadratic 86.29 5 17.26 1.57 0.3162 Suggested
Cubic 7.26 1 7.26 0.6605 0.4533 Aliased
Pure Error 54.95 5 10.99
Fig. 5. Response surface method: 3D-response graph relationship between (a) starch and yeast extract (b) starch and orange pectin% (c) yeast extract and orange pectin %,
Counter plot for interaction between (d) starch and yeast extract (e) starch and orange pectin% (f) yeast extract and orange pectin%
parable to pectinase production from Streptomyces erumpens MTCC which is higher compared to the previous reports on bacteria (180
7317 at pH 7 (Kar and Ray, 2011). The optimal temperature for the RPM) (Pant et al., 2015) and fungi (150 RPM) (Dinarvand et al.,
maximum pectinase production by the potential isolate VIT-SP4 2017).
was found to be 35 °C which is the same compared to the soil tem- The media optimization was carried out by Response surface
perature of dumpsites which was about 35 to 40 °C as reported. methodology (CCD). The optimal starch concentration was
The optimal orange pectin% for maximum pectinase production observed at 1.17% for maximum pectinase production by Strepto-
from Streptomyces fumigatiscleroticus VIT-SP4 was found to be myces fumigatiscleroticus VIT-SP4. The statistical optimization of
2.5% which is nearly comparable to the pectin% used in the earlier pectinase production from Bacillus sp.Y1 by RSM and optimal
studies. The concentration extracted pectin (2%) in media was uti- starch concentration was found to be 3.8% (Guo et al., 2019).
lized for pectinase production by bacteria. The result concludes The interaction between starch and yeast extract, starch and
actinomycetes utilize maximum pectin concentration for pectinase orange pectin% showed positive interaction towards maximum
production (Sharma et al., 2019). The incubation time for maximal pectinase production by isolate VIT-SP4. Maximum pectinase pro-
pectinase produced by Streptomyces fumigatiscleroticus VIT-SP4 duction was observed at 2% yeast extract concentration by poten-
was observed at 48 h which is similar compared to previous stud- tial isolate VIT-SP4. The yeast extract (0.3%) for maximal
ies on Streptomyces lydicus (Jacob and Prema, 2006). The optimized pectinase production by B.licheniformis KIBGE IB-21 (Rehman
agitation speed for pectinase production was found to be 200 RPM et al., 2012). The high concentration of nitrogen source has been
org/10.1016/j.sjbs.2020.07.024
Fig. 6. Parity plot: (a) taguchi model (b) response surface methodology (The point in the parity plot has two co-ordinates namely (x and y) where x indicates actual value and
y indicates predicted value. The line equation (y = x) were considered to be reference. The model is significant all points lie on same straight line).
healing process, actinomycetes germinate spores and they get col-

onized at the time of harsh conditions in presence of H2O and
causes CaCO3 precipitation to provide compressive strength and
compatibility to the concrete. The self-healing of concrete by acti-
nomycetes could decrease material permeability. As, it acts as cat-
alyst it can be incorporated as mineral precursor to form CaCO3
precipitation which offer self-sufficient repair process. The self-
healing of concrete with other inorganic materials like epoxy resin,
silica fume and fly ash lead to various disadvantages like thermal
expansion coefficient of the concrete that causes environmental
and health problems. Since, microbes were involved in the process
it seemed to be very cost-effective and recyclable in production
compared to other inorganic materials.
Fig. 7. In-vitro controlled drug release by pectinase enzyme. 5. Conclusion
The study highlights the cost-free production and statistical

utilized by isolate VIT-SP4 for maximum pectinase production optimization of pectinase from novel pectinolytic isolate Strepto-
compared to bacterial isolates. myces fumigatiscleroticus VIT-SP4 (Genbank accession no:
The Controlled drug release study was carried out with pecti- KM875468) by utilizing fruit pectin. The Orange pectin gave max-
nase from isolate VIT-SP4 to pectin based pellet. The main cause imum pectinase activity of about 45.93 (U/mL) compared to other
for drug release is the disintegration of pectin by pectinase enzyme peels. The statistical optimization of pectinase from isolate VIT-SP4
at pH 6 whereas, at pH 3, there was no disintegration and it con- by Taguchi method produces pectinase activity of 98.65 U/mL. Fur-
firms the disintegration of pectin by pectinase which facilitate ther, optimization of media concentrations by response surface
the controlled drug release as seen in earlier reports (Butte et al., methodology (CCD) showed maximum pectinase activity of
2014). 170.05 U/mL. The results conclude a 1.7-fold increase in the pecti-
The self-healing of crack concrete blocks was investigated with nase activity after optimization. The yielded pectinase was used for
potential isolate VIT-SP4 by spray culture technique. The crack controlled drug delivery study and drug release was observed at
healing was observed up to 0.55 mm wide was recorded and the pH 6. Further, downstream processing of the pectinase can be car-
specimens were submerged in water for 12 days. Whereas, crack ried out. The production and characterization of pectin-coated
healing was recorded up to 0.46 mm wide of 100 days period and drug before and after pectinase treatment can be studied. The in-
which is comparable to previous studies (Wiktor and Jonkers, vivo and clinical studies can be done prior to commercialize
2011). The results confirm that self-healing of cracks as there pectin-coated tablets in the market as anti-inflammatory or
was a decrease in the transmission time of the healed concrete colon-specific drugs that require specific drug delivery system.
specimen compared to the cracked specimen (Van Tittelboom The potential isolate VIT-SP4 was utilized for concrete crack-
et al., 2010). healing process and self-healing of cracks was observed up to
Streptomyces are spore-forming actinomycetes that are resistant 0.50 mm wide after 12 days. Hence, large-scale production of this
to high pH and remained viable after casting the concrete for sev- cost-effective pectinase in industrial level can be implemented as
eral days and helps directly in self-healing system. In the crack it makes less production cost.
org/10.1016/j.sjbs.2020.07.024
Fig. 8. Spore formation and sporulation kinetics in Streptomyces fumigatiscleroticus VIT-SP4 (a) spore-staining of isolate (b) % sporulation determined with stained spores (c)
Survivability of Streptomyces fumigatiscleroticus VIT-SP4 spores in cement paste up to 90 days (d) shows cracks on the cement paste (control) (e) crack-healing of concrete
block by spores of S.fumigatiscleroticus VIT-SP4 (test).
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Streptomyces Fumigatiscleroticus VIT-SP4 For Drug Delivery and

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Saudi Journal of Biological Sciences xxx (xxxx) xxx

Contents lists available at ScienceDirect

Saudi Journal of Biological Sciences

Characterisation of pectin and optimization of pectinase enzyme from

1. Introduction require an eco-friendly approach. Citrus waste residues accumu-

maximum pectinase production by using Design Expert 11.0. Soft- 3. Results

Source Sum of squares Df Mean square F-Value p-value

it helps in self-healing of cracks. The crack healing was observed

The study deals with characterization of pectin and optimiza-

Source Sum of Squares Df Mean square F-value p-value

Source Sequential p-value Lack of fit p-value Adjusted R2 Predicted R2

Source Sum of Squares Df Mean Square F-value P-value

healing process, actinomycetes germinate spores and they get col-

Fig. 7. In-vitro controlled drug release by pectinase enzyme. 5. Conclusion

The study highlights the cost-free production and statistical

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