Optimizing the process of extraction of a polysaccharide from Zizyphus

mauritiana fruit pulp.

This report is for the partial fulfilment of Bachelor of Technology degree in the
Department of Chemical Engineering, National Institute of Technology
Durgapur

Submitted by:

GAUTAM MISHRA (21CH8053)

Under the guidance of:

Dr. Bimal Das

Assistant Professor

Department of Chemical Engineering

National Institute of Technology Durgapur
 National Institute of Technology Durgapur
Department of Chemical Engineering

CERTIFICATE

This is to certify that, the project thesis entitled Optimizing the process of extraction of a
polysaccharide from Zizyphus mauritiana fruit pulp submitted by GAUTAM MISHRA
(21CH8053) to the Department of Chemical Engineering, National Institute of Technology,
Durgapur, in partial fulfilment of the requirement for the degree of Bachelor of Technology
in Chemical Engineering is a bonafide record work carried out by him in the session 2024-
2025 under my supervision and guidance.

Date:

Dr. Bimal Das

Assistant Professor
Department of Chemical Engineering
National Institute of Technology Durgapur
 National Institute of Technology Durgapur
Department of Chemical Engineering

Certificate of Approval

The foregoing thesis is hereby approved as a creditable study of a technological subject,

carried out and presented in a manner satisfactory to warrant its acceptance as a prerequisite

to the bachelor’s degree for which it has been submitted.

It is to be understood that by this approval the undersigned do not necessarily endorse or

approve any statement made, opinion expressed or conclusion drawn therein, but approve this

thesis only for the purpose for which it has been submitted.

BOARD OF EXAMINERS

1.

2.

3.
 ACKNOWLEDGEMENT
At the outset we would like to extend our sincere gratitude to the authority of National

Institute of Technology, Durgapur for providing us the state-of-the-art facilities and ambience

atmosphere necessary to carry out our project work.

We like to express our sincere gratitude and deep regards to Dr Bimal Das, Assistant

Professor, Department of Chemical Engineering, National Institute of Technology

Durgapur for his sincere guidance and positive motivation throughout the course of our

assigned research work which inspired us to find the exact path of doing the research work.

We consider ourselves fortunate for having got the opportunity to work on such an interesting

topic.

We owe our profound gratitude to Dr Sayantan Sarkar, Research Scholar for his constant

assistance, sustained enthusiasm, creative suggestion and exemplary guidance towards the

completion of the project.

We are also very much thankful to our respected faculty members and Non-teaching staff of

Chemical Engineering Department, NIT Durgapur for their immense support during the four

years that we have spent at this institute. This acknowledgement would remain incomplete if

we fail to remember our parents who always give us whole hearted support and

encouragement to complete our thesis work. It would not be out of place to mention here that

their silent presence behind all our endeavour enable us to complete the project work

assigned to us.

Last but not the least we extend our sincere thanks to all our batch mate who provided great

support and encouragement during the course of our assigned project work.

GAUTAM MISHRA (21CH8053)

 DECLARATION

We hereby declare that the thesis entitled Optimizing the process of extraction of a
polysaccharide from Zizyphus mauritiana fruit pulp submitted in the in Department of
Chemical Engineering of National Institute of Technology Durgapur, is our own work and
that, to the best of our knowledge and belief, it contains no material previously published or
written by another person nor material which has been accepted for award of any other
degree or diploma of the university or other institute of higher learning, except where due
acknowledgement has been made in the text.

Gautam Mishra
B.Tech
Department of Chemical Engineering
Roll No: 21CH8053
 Contents

S. No. Content Page No.

1 Certificate 1

2 Certificate of Approval 2

3 Acknowledgement 3

4 Declaration 4

5 Contents 5

6 List of figures, Tables and Nomenclature 6-7

7 Abstract 8

8 Introduction 9

9 Materials 9

10 Objectives 10

11 Methodology 10

12 Isolation of Polysaccharide 11

13 Optimization of the Yield value 12

14 Result and Discussion 12- 17

15 Conclusion 17

16 Future Scope 18

17 References 19

List of Figures
Figure No. Figure Caption Page No.

1 Changes in color with maturity and ripening of Indian jujube 10

fruits.
2 Design Summary for central composite design (CCD) 13

3 Experimental design matrix with factor and yield 14

4 Regression equation for yield 15

5 Model summary statistics 15

6 Statistical model analysis 16

7 Contour plot for yield vs temperature, ratio and pareto chart 16

of the standardized effects (response yields, α = 0.5)

Table No. Table Caption Page No.

1 Extraction condition and 11

yield of polysaccharide from
ziziphus mauritiana.
2 Effect of pulp-to-water ratio
and temperature on 12
polysaccharide yield.
List of Table

Nomenclature
FTIR : Fourier Transform Infrared Spectroscopy

DSC : Differential Scanning Calorimetry

HPLC : High Performance Liquid Chromatography

GC-MS : Gas Chromatography-Mass Spectrometry

HPMC : Hydroxypropyl Methylcellulose

NMR : Nuclear Magnetic Resonance

RSM : Response Surface Methodology

Abstract
The increasing demand for novel drug carriers has driven the exploration of new biopolymers
in pharmaceutical research. Among these, polysaccharides stand out due to their abundance
in nature and their biocompatible and biodegradable properties as well as in industries they
are generally regarded as safe. This study aims to extract and characterize a novel
polysaccharide suitable for drug delivery. The polysaccharide will be extracted from Ziziphus
mauritiana and characterized for compatibility using FTIR, thermal behaviour using DSC,
and structural analysis using HPLC, GC-MS, and NMR. Propranolol hydrochloride will serve
as a model drug, and the efficacy of the Z. mauritiana polysaccharide in tablet formulation as
a binder and release retardant will be evaluated. Results will be compared with those of starch
and HPMC K 15M, standard binders, and release retardants, respectively. The binding effect
of Z. mauritiana-derived polysaccharide will be compared with that of starch, and the release
retardant efficacy will be compared with that of HPMC K 15M at similar concentrations.
Depending on the drug release data, the release mechanism will be established. Accelerated
Stability studies will be conducted for six months to determine the stability of the selected
formulations. Through this study, we aim to elucidate the biocompatible and biodegradable
characteristics of Z. mauritiana polysaccharides, while also unveiling their diverse
phytopharmaceutical properties. Consequently, this research paves the way for the
development of a multifacility, industry-grade adjuvant tailored for pharmaceutical
applications. Polysaccharides play diverse roles in biological processes and offer immense
potential in healthcare, food, and cosmetic industries due to their therapeutic benefits and low
toxicity. In this study, a water-soluble polysaccharide was extracted from the fruit pulp of
Ziziphus mauritiana (commonly known as jujube or ber) using water as the extraction
solvent, followed by ethanol precipitation. The extraction process was optimized by varying
two key parameters: the ratio of water to plant material (ranging from 1:3 to 1:9) and the
extraction temperature (30°C to 75°C). Employing a central composite design, the percentage
yield of polysaccharide was found to vary with these parameters. The optimal extraction
conditions were determined to be a 1:5 ratio of jujube pulp to water and an extraction
temperature of 60°C, yielding the highest polysaccharide content of 9.4% ± 0.21. After the
RSM study the R-sq value is 99.97% and R-sq(adj) is 99.94% and R-sq(pred) is 93.96% and
the F-value is large compared to P-value and P-value.

Keywords: Ziziphus mauritiana fruit pulp, polysaccharide, extraction, optimization,

phytopharmaceutical properties.
1. Introduction

In recent years, increasing attention is being placed on polysaccharides extracted from plants
and microbial sources by biochemical and nutritional researchers, due to their various
potential biological activities (especially anti-oxidant, immunostimulatory, anti-oxidative and
anti-tumor effects) as well as their adjuvant properties (delayed releasing property, binding
property, sustained release property, emulsifying property) that could be applied to
pharmaceutical, healthcare foods or medicine. The polysaccharide obtained from plant
sources are generally regarded as safe. They are chemical complex biopolymers which have
attracted scientists all over the world for years, due to their higher degree of biocompatibility,
renewability, and biodegradability. Different functional groups can be observed in the
structure of polysaccharides from different sources and they display different
physico-chemical properties. These properties make them suitable for different food and
pharmaceutical applications, especially in drug delivery systems. Most of the polysaccharides
are preferred over their synthetic counterparts due to being inexpensive, abundant
availability, and non-toxic. This study deals with a water-soluble polysaccharide isolated
from the fruit pulp of Ziziphus mauritiana. Z. mauritiana, sometimes called Indian jujube or
Ber, belongs to the Rhamnaceae family. It is a small tree or large shrub that can grow up to
26 feet tall with a canopy diameter of 30 feet with oblong-elliptic leaves. It has been used
traditionally as a functional food for many centuries. In this study, a polysaccharide isolated
from Z. mauritiana fruit pulp in different conditions and the parameters were put through
RSM study to optimize the best yield conditions.

2. Materials

Z. mauritiana fruits were obtained from the local markets of Barasat and authenticated in
Botanical Survey of India. Ethanol used was of 99% pure provided by Oxford Fine Chem
LLP. All the other solvents and chemical used was of analytical grade. The entire
experiments were performed in distilled water.
 Fig. 1 Changes in colour with maturity and ripening of Indian jujube fruits.

3. Objective

The primary objective of this experiment is to isolate a water-soluble polysaccharide from

the fruit pulp of Ziziphus mauritiana and optimize the extraction conditions to achieve
the best yield. This optimization was carried out using Response Surface Methodology
(RSM).

4. Methodology

1. Material Collection and Preparation:

 Ziziphus mauritiana fruits were sourced from local markets and

authenticated by the Botanical Survey of India.
 Ethanol and other chemicals used were of analytical grade, with distilled
water being the solvent for experiments.

2. Extraction Process:

 The fruit pulp was sieved through a #100 mesh and cooked in distilled
water with pulp-to-water ratios ranging from 1:3 to 1:9.
 Cooking was performed for 6 hours at temperatures between 30–75°C.
 The cooked mixture was cooled, chilled at 4°C overnight, and filtered
through a muslin cloth.

3. Centrifugation and Concentration:

 The filtrate was centrifuged at 3500 rpm for 20 minutes to remove debris.
  The supernatant was concentrated to one-fifth of its volume.

4. Precipitation:

 The concentrated supernatant was precipitated in 95% ethanol in a 1:5

(v/v) ratio.
 The precipitate was separated by centrifugation at 3500 rpm for 2
minutes.

5. Dialysis:

 The precipitate was dialyzed using a cellulose membrane to remove

low-molecular-weight materials after 12 hours of storage at 4°C.

6. Yield Optimization:

 Polysaccharide yield was evaluated at various temperatures (30°C,

45°C, 60°C, 75°C) and pulp-to-water ratios (1:3 to 1:9).
 Yield percentages varied from 5.6% to 9.4%, with maximum yield
observed at a ratio of 1:5 and a temperature of 60°C.

Sl. No Ratio of Amount of Amount of Temperature % of Yeild

jujube pulp jujube pulp water (℃)
and water (gm) (ml)
(During
extraction)
1 1:3 – 1:9 200 600-1400 30-75 5.6 ± 0.23-
9.4 ± 0.21

Table 1: Extraction Conditions and Yield of Polysaccharide from Ziziphus mauritiana

(This table describes the pulp-to-water ratios, amounts used, temperatures, and yield
percentages.)

Isolation of polysaccharide
Soft pulp of Z. mauritiana was sieved through #100 mesh and cooked in distilled water in a
pulp: water ratios between 1:3 to 1:9 for 6hr at a range of 30-75 ºC. The cooked pulp in
solvent is cooled to room temperature and After chilling at 4 ºC overnight, the dispersion was
filtered through a muslin towel. The filtered dispersion was centrifuged for 20 min at 3500
rpm (R-8C, Remi, India). The residual debris was removed, and the supernatant was
concentrated to one-fifth of its original volume. The concentrated supernatants were
precipitated in 95% ethanol at a ratio of 1:5 (v/v). The precipitate was separated by
centrifugation (3500 rpm for 2 min) and dialysed using cellulose membrane (Hi media 110
M.W. 12-14 kDa) to remove low molecular weight material after 12 hours of storage at 4 °C.

Ratio of jujube %of Yield at %of Yield at %of Yield at %of Yield at
pulp and water 30°C 45°C 60°C 75°C
(During
Extraction)
1:3 5.6 ± 0.23 5.7 ± 0.22 7.6 ± 0.24 7.5 ± 0.25
1:5 6.4 ± 0.21 6.6 ± 0.26 9.4 ± 0.21 9.2 ± 0.22
1:7 6.3 ± 0.18 6.4 ± 0.19 8.3 ± 0.23 8.1 ± 0.27
1:9 5.8 ± 0.24 5.7 ± 0.25 7.8 ± 0.26 7.6 ± 0.23

Table 2: Effect of Pulp-to-Water Ratio and Temperature on Polysaccharide Yield"

(This table details the yield percentages for different temperatures and pulp-to-water ratios.)

Optimization of the Yield value

The extraction of polysaccharides from Ziziphus mauritiana fruit pulp was optimized using
Response Surface Methodology (RSM) implemented in Minitab software. This section
provides a detailed account of the methodology, results, and implications of using RSM for
yield optimization.

Introduction to Optimization Using RSM

Response Surface Methodology is a robust statistical technique used for process optimization.
It involves designing experiments, building models, and analysing the effects of several
variables to identify optimal conditions for the desired response—in this case, maximizing
the yield of polysaccharides. The central composite design (CCD), a common design under
RSM, was chosen due to its ability to evaluate linear, quadratic, and interaction effects of the
variables.

Experimental Design

The study focused on two critical parameters influencing polysaccharide extraction:

 Pulp-to-water ratio: Varied between 1:3 and 1:9.

 Extraction temperature: Ranged from 30°C to 75°C.

A central composite design (CCD) was applied, consisting of:

 Factorial points to evaluate variable interactions.

 Axial points to assess curvature effects.

 Center points for replication and estimation of pure error.

In total, the design included 13 experimental runs to systematically analyze the effects of
these variables.

Fig 2 Design Summary for Central Composite Design (CCD)

After the first experimental run the analysis will generate a table and the obtained yield value
are filled as input to the table before running the analysis.

Fig 3. Experimental Design Matrix with Factors and Yield

Data Collection and Response Calculation

After each experimental run, the percentage yield of polysaccharide was calculated. The yield
was determined by the mass of the polysaccharide obtained relative to the initial mass of the
fruit pulp. Yields varied between 5.6% ± 0.23 and 9.4% ± 0.21, with the highest yield
observed at a pulp-to-water ratio of 1:5 and an extraction temperature of 60°C.

Modelling and Analysis in Minitab

The experimental data were entered into Minitab to develop a quadratic regression model.
The general equation for the model is:
 Fig 4. Regression Equation for Yield

Statistical Evaluation

Minitab's analysis provided the following key metrics to assess the model's adequacy:

 R-squared (R²): 99.97%, indicating that the model explains 99.97% of the variability
in the response.

 Adjusted R-squared (R² adj): 99.94%, showing a minimal penalty for the number of
predictors.

 Predicted R-squared (R² pred): 93.96%, demonstrating the model's predictive

capability.

 F-value and p-value: A high F-value and a p-value < 0.05 confirmed the model's
significance.

The model's residuals were normally distributed and showed no evidence of

heteroscedasticity, confirming its reliability.

Fig 5 Model Summary Statistics

 Fig 6 Statistical Model Analysis

Visualization of Results

Contour and 3D Surface Plots were generated in Minitab to visualize the relationships
between the independent variables (temperature and pulp-to-water ratio) and the response
(yield).

 Contour Plots: These 2D plots displayed regions of high yield, helping identify the
optimal range of process parameters.

 3D Surface Plots: These plots provided a more comprehensive view of how the
interaction between the two variables influenced the yield.
Fig 7 Contour Plot of yield vs temperature, ratio and Pareto Chart of the Standardized Effects
(response is yield, α = 0.05)

Optimization and Validation

Using the model, Minitab predicted the optimal conditions for maximum polysaccharide
yield:

 Pulp-to-water ratio: 1:5

 Temperature: 60°C

Under these conditions, the predicted yield was 9.4%. Experimental validation under these
parameters confirmed the prediction, with an actual yield of 9.4% ± 0.21%.

5. Results and Discussion

The extraction of polysaccharides from the fruit pulp of Ziziphus mauritiana was successfully
optimized using Response Surface Methodology (RSM). The two key parameters
investigated were the pulp-to-water ratio and the extraction temperature. The experimental
results showed that the polysaccharide yield varied from 5.6% ± 0.23% to 9.4% ± 0.21%,
depending on the extraction conditions. The maximum yield of 9.4% ± 0.21% was obtained
at a pulp-to-water ratio of 1:5 and an extraction temperature of 60°C. The statistical analysis
using the central composite design (CCD) and the quadratic regression model revealed that
both the pulp-to-water ratio and the extraction temperature had significant linear and
quadratic effects on the polysaccharide yield. The interaction between these two variables
was also found to be significant. The high R-squared values (R² = 99.97%, R²(adj) = 99.94%,
R²(pred) = 93.96%) and the low p-value (p < 0.05) indicated that the developed model could
accurately predict the polysaccharide yield under different extraction conditions. The normal
distribution of residuals and the lack of heteroscedasticity further confirmed the reliability of
the model. The contour and 3D surface plots helped visualize the relationships between the
process parameters and the polysaccharide yield. These plots clearly showed the optimal
region where the maximum yield could be achieved, corresponding to a pulp-to-water ratio of
1:5 and an extraction temperature of 60°C. The experimental validation under the optimal
conditions yielded 9.4% ± 0.21% polysaccharide, which was in excellent agreement with the
model's prediction. This confirms the suitability and accuracy of the RSM approach for the
optimization of polysaccharide extraction from Ziziphus mauritiana fruit pulp.

6. Conclusion

In this study, a water-soluble polysaccharide was successfully extracted from the fruit pulp of
Ziziphus mauritiana. The extraction process was optimized using Response Surface
Methodology, which identified the optimal conditions as a pulp-to-water ratio of 1:5 and an
extraction temperature of 60°C. Under these conditions, the maximum polysaccharide yield
was 9.4% ± 0.21%. The statistical analysis of the experimental data showed that both the
pulp-to-water ratio and the extraction temperature had significant linear and quadratic effects
on the polysaccharide yield. The interaction between these two variables was also found to be
significant. The high R-squared values, low p-value, and the agreement between the predicted
and experimental results confirmed the reliability and accuracy of the developed quadratic
regression model. The visualization of the results using contour and 3D surface plots further
supported the identification of the optimal extraction conditions.

7. Future Scope

The successful optimization of the polysaccharide extraction process from Ziziphus

mauritiana fruit pulp opens up several avenues for future research and applications. Detailed
characterization of the extracted polysaccharide, including its chemical structure, molecular
weight distribution, and physicochemical properties, will provide a comprehensive
understanding of its properties. Evaluating the polysaccharide's biocompatibility,
biodegradability, and potential pharmaceutical applications is crucial. This includes assessing
its efficacy as a binder and release retardant in tablet formulations, investigating its ability to
control drug release and modulate the release kinetics of model drugs, exploring its potential
as a drug carrier for targeted delivery or controlled release applications, and studying its
suitability for developing novel dosage forms like hydrogels, microspheres, or nanoparticles
for drug delivery. Additionally, evaluating the polysaccharide's other functional properties,
such as its antioxidant, antimicrobial, or immunomodulatory activities, could expand its
applications in the food, cosmetic, and healthcare industries.

8. References
1. Kao, T. H., & Chen, B. H. (2014). Functional components in Zizyphus with emphasis on polysaccharides.
Polysaccharides, 1–28. doi:10.1007/978-3-319-03751-6_15-1.
2. Ray, P., Chatterjee, S., & Saha, P. (2021). Screening of polysaccharides from fruit pulp of Ziziphus mauritiana L. and
Artocarpus heterophyllus L. as natural mucoadhesives. Future Journal of Pharmaceutical Sciences,
7(1). doi:10.1186/s43094-020-00164-5.
3. Sangeethapriya, M., & Siddhuraju, P. (2014). Health related functional characteristics and antioxidant potential of
mucilage (dietary fiber) from Zizyphus mauritiana fruits. Food Science and Human Wellness, 3(2), 79–
88. doi:10.1016/j.fshw.2014.05.003.
4. Jethara S, Patel M (2014) Pharmaceutical controlled release drug delivery systems: a patent overview. Aperito J Drug
Des Pharmacol 1:1–22. https:// doi.org/10.14437/AJDDP-1-107.