TECHNOLOGY AND EDUCATIONMINISTRY OF EDUCATION AND TRAININGHO CHI MINH CITY UNIVERSITY OF GRADUATION THESIS FOOD TECHNOLOGYINVESTIGATE EXTRACTION CONDITIONS AND CHARACTERIZATION OF PECTIN
INTRODUCTION
Problem
Vietnam's agricultural sector accounts for a high proportion of the economy In addition to rice output, which is always among the world's largest exporters, Vietnam also develops fruit tree cultivation in large quantities and with a variety of types As one of the fruit trees with high economic value, durian is increasingly popular and is widely consumed not only in the domestic market but also for export Durian is a genus of plants in the mallow family, currently there are more than 30 identified species, of which about 9 species have edible fruits, Durio Zibethinus is the most common species on the market The distribution area of durian is in Southeast Asian countries, including Vietnam In addition to directly using the fruit flesh, durian is also processed into many dishes such as ice cream, candy, and jam
The durian rind typically accounts for over half of the total fruit weight, presenting a green to yellowish-brown, thick, semiwoody composition adorned with sharply pointed pyramidal thorns The excessive disposal of durian rinds during the durian season raises environmental concerns The rind of durian are highly cellulose-containing, according to research done in Thailand on using durian peels to make particleboard with lower heat conductivity [1] Particleboard, also known as chipboard in the UK, Australia, and some other countries, is an engineered wood product made by pressing and extruding wood particles- such as wood chips, sawdust, or shavings from sawmills- with a synthetic resin or other appropriate binder
A variety of polysaccharides, including cellulose, hemicelluloses, lignin, and pectin, make up the majority of the plant cells in durian [2] This agricultural waste might also be used to make a low-cost sorbent that would remove acid dye from aqueous solutions by acting as a gelling agent, tablet disintegrator, and binder[3] The water-soluble polysaccharide fraction from the rind of durian trees that has a high pectin content is particularly interesting [4]
Research objective
- The process of extracting pectin from durian rind
- Optimization parameters of extraction conditions.
Object and scope of the research
- Research Object: Pectin extracted from durian rind
- Scope of the study: This study was carried out on a laboratory scale
Research content
- Research the process of extracting pectin from durian rind, and optimization parameters of extraction conditions
- Study the properties of pectin: ash and moisture content, total anhydrouronic acid content, degree of esterification, morphology properties
Scientific and practical significance
- Provide complete pectin extraction process This serves as a premise for further studies
- Research the factors affecting the extraction process in order to improve the efficiency and quality
- Enhance the value and widen the range of applications of durian rind
LITERATURE REVIEW
Introduction about pectin
Pectin is a powder that ranges in color from white to light brown and is commonly made from apple pomace and citrus peel Although pectin was once only accessible as a liquid extract, its use as a dried powder has increased since it is easier to handle, store, and transport As a water-soluble fiber that is used in a variety of food products, including ice cream, jam, yogurt drinks, and fruity milk drinks Pectins are also included as nutritional supplements because of their possible health benefits Multiple research studies have documented the advantageous impacts of a diet rich in fiber on various areas By modifying the gut microbiota, recent research has also investigated the effects of pectin on allergic sensitization, which has been shown to have positive benefits of pectin supplementation on allergies [5]
2.1.1 Structure and chemical compositions of pectin
The molecular structure of pectin is derived from pectic acid, which itself is a polymer of D- galacturonic acid linked together by α 1-4-glycoside bonds The length of the polygalacturonic acid chain can vary from a few units to hundreds of galacturonic acid units The molecular weight of pectin isolated from different fruit sources varies widely, depending on the number of galacturonic acid molecules, and typically falls within the range of 10,000 to 100,000[6] In terms of glucid substances, when comparing molecular length, pectin has a higher molecular weight than starch but lower than cellulose For instance, in apple sources, pectin is obtained with a molecular weight of 25,000-35,000, while pectin from oranges can have a molecular weight of up to 50,000 [7] The pectin content of 1% in the
5 solution has high viscosity When 60% sugar is added and the pH is adjusted to a range of 3.1-3.4, coagulation of the product occurs
Commercial pectin does not have a definite molecular weight but depends on the raw material, extraction method, and type of product The pectin unit molecule is D-galacturonic acid Additionally, there are some neutral sugars such as rhamnose, galactose, arabinose, and some other sugars in smaller amounts Carboxyl groups (-COOH) can exist freely or in the form of ester bonds with methanol, phenolic acid or in the form of salts of Na + , K + , NH4 +, etc In amidized pectins, a portion of the carboxyl radicals in the pectin molecule is amidized to form an amide group (-CO=NH2).[8]
Three major types make up the structure of pectin: rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HGA)[9] Furthermore, due to their similar main chains to HGA, xylogalacturonan (XGA) and apiogalacturonan (AGA) are frequently regarded as pectins [10] Within the primary and mid cell walls, all three forms of polysaccharides are believed to form a pectin network through covalent bonds Enzymes on the cell wall control the structure of this network [11]
HGA is the most common form of pectin About 100 GalA units make up HGA, a linear homopolymer composed of a-(1-4)-D-GalA linkages Additionally, some of the carboxyl groups are esterified with methanol or acetyl groups[9, 12] HGA is also known as the smooth region due to its linear structure [13]
RG-I accounts for about 20-35% of the structural form of pectin A primary chain with a molecular weight of roughly 100 kDa, comprising repeated disaccharides [-α -D-GalA-1,2- a-L-Rha-1-4-]n, makes up the structure of RG-I The expression of the RG-I construct depends on the growth of the species and the number of sugar residues and branched oligosaccharides attached to the main chain of RG-1[14] The side chains contain galactose and arabinose residues attached at C-4 of the rhamnose residues These side chains consist of monosaccharides or combined chains of arabinan, galactan, or arabinogalactan RG-I is known as a hairy region because of its branching structure[13]
RG-II is the pectin with the most complex structure, accounting for 10% of pectin It has a low molecular weight of 5-10 kDa GalA (HGA-like) residues with a minimum of eight α - (1-4)-D-GalA links form the main chain of RG-II's structure RG-II has more branches than HGA Additionally, the branch chains contain 12 different sugars linked to C-2 and C-3[9]
Pectin is a charged hydrocolloid, which means that variations in pH and the kind and concentration of cations present in the solution can affect it These features allow it to be separated into two categories: acid gel and calcium gel, which are derived from HMP and LMP, respectively
Under pH ranges of 3.0 to 5.0 and soluble solid concentrations of 10% to 80%, LMP gels in the absence of Ca2+ ions [15]
The gelation of LMP occurs as a result of a process in which two carboxyl groups from separate pectin chains come into contact at the junction region, forming ionic connections through calcium bridges At the junction, the galacturonic acid monomers in the chains are linked together through electrostatic and ionic bonds of the carboxyl groups [16]
Figure 2.3 The gelation mechanism of LMP
The gelation process is facilitated by the free electron pairs of the oxygen atoms bound between the monomer units, in the ring, and in the hydroxyl groups, as illustrated in figure 2.3[16]
HMP forms a gel under pH conditions below 3.6 When there are solutes present and the concentration of sucrose is more than 55% by weight
Oakenfull provides a description of the HMP gelation mechanism Polymer chains are cross- linked when segments of two or more pectin molecules expand to form junctional areas These areas are sustained by a mixture of hydrogen bonds and hydrophobic contacts of methyl-ester groups between pectin molecules The degree of hydrophobic interactions and hydrogen bonding also affects the stability of HMP gels[17]
Classification based on the solution: [18]
• Water-soluble pectin: It also known as free pectin, is composed of galacturonic acid radicals Some of these radicals contain (-COOH) groups and are predominantly present in the cell fluid
• Insoluble pectin: It is found in the cell wall, existing in the form of pectin combined with arabinan and is not soluble in water
Classification based on the degree of methylation:
• High Methoxyl Pectin (HMP): DE > 50% (typically 55–75%) It is usually used to produce high-calorie, high-sugar foods Traditional jams and jellies ( high sugar content) are a typical application for it, not recommended for diabetics This type of pectin may increase the viscosity of the product To form coagulation, it is necessary to have pH 3,1 - 3,4 and sugar concentration above 60%.[19]
• Low Methoxyl Pectin (LMP): DE 1 The precise value is determined by the design's desired features as well as the number of components involved
Figure 2.7 Comparison of the Three Types of Central Composite Designs
The features of the three types of central composite designs are central composite circumscribed (CCC), central composite inscribed (CCI), and central composite face centered (CCF) Figure 2.7 depicts three different forms of central composite designs for two elements It is worth noting that the CCC investigates the greatest process space, whereas the CCI investigates the smallest process space The CCC and CCI are both rotatable, while
17 the CCF is not The design points of the CCC design describe a circle enclosed by the factorial square The CCC design points depict a sphere around the factorial cube for three factors
In this research, the choice of Central Composite Rotatable Design (CCRD) stems from its ability to anticipate responses using a limited set of experimental data This design is particularly advantageous as it allows for the variation of all parameters within a specified range [40, 41]
Furthermore, the flexibility provided by CCRD enhances the construction of more robust models, particularly in scenarios where experimental errors may influence certain experiments [42] Constructing statistical models can prove valuable for predicting and comprehending the outcomes of various experimental factors The key strength of RSM with Central Composite Rotatable Design (RSM-CCRD) lies in its ability to optimize multiple operational variables concurrently through a limited number of experiments, resulting in time and labor efficiency
Figure 2.8 Central composite rotatable design (CCRD)
Summary of previous studies on durian rind
Durian is a tropical fruit, one of the important fruit trees, and has high nutritional value in Southeast Asia and around the world Because durian peels are frequently discarded as rubbish Thus, it would be highly advantageous to develop it into a value-added product like pectin Therefore, in 2019, Hasem et al researched the extraction and partial characterization of durian rind pectin using the conventional acid extraction method and described the properties of pectin in terms of yield, water activity, moisture, and ash content Pectin was extracted with HCl, pH 2.5 at 85°C, for 60 minutes The results showed that the pectin yield in durian peel was 73.67%, moisture was 11.53%, ash content was 4.67%, and water activity was 0.452 Furthermore, this study demonstrates that pectin extraction was successful and offers possible environmental and economic advantages for industrial pectin extraction[43]
In 2009, Wong Weng Wai et al used the response surface approach to improve the pectin extraction process from durian rind and study the impact of temperature, pH, and heating duration on the degree and performance of esterification (DE) Based on the dry weight of the durian rind, the yield and DE of the extracted pectin were found to range from 2.27% to 9.35% and 47.66% to 68.6%, respectively 85 degrees Celsius, 4 or 1 hour, and a pH of 2 or 2.5 were the ideal parameters for obtaining maximum yield and DE [44] Prakash Maran conducted research using the Box-Behnken response surface methodology to investigate and optimize water extraction conditions for maximizing pectin extraction from durian peel The studied parameters Using the Box-Behnken response surface methodology, Prakash Maran studied and optimized water extraction conditions to maximize pectin extraction from durian rind These conditions included solid-liquid ratios ranging from 1:5 to 1:15 g/ml, pH 2 to 3, extraction times spanning from 20 to 60 min, and extraction temperatures varying from 75 to 95°C The optimal results were determined to be obtained with the following parameters: a pH of 2.8, an extraction time of 43 minutes, an extraction temperature of 86°C, and a ratio of 1:10 g/ml.[45]
In 2023, Sze Hui Jong et al conducted research on the impact of acid type and concentration on durian rind pectin yield, AUA, and DE They utilized five different concentrations (0.0001, 0.001, 0.01, 0.1, and 1.0 M) of different acids, including H2SO4, HCl, HNO3,
C6H8O7, tartaric, and acetic As a result, the concentration of acid increased (0.1, 1.0 M), and
19 the yields of pectin and DE dropped The homogalacturonan content of durian rind pectin is high, as indicated by the FTIR spectra Given the response yield of 9.13 % and purity (AUA
= 66.72%), 0.001M H2SO4 holds promise as an effective extractant for the commercial extraction of LMP (DE = 20.11%) from durian rind LMP has the potential to be used as a stabilizer and thickener for low-calorie meals and beverages[46]
MATERIALS AND METHODS
Materials, chemicals, and equipment
Durian rinds were gathered from fruit shops in Thu Duc District, Ho Chi Minh City, and transported to the laboratory for testing The durian rinds were examined for flaws such as browning, rotten areas, mold growth, holes caused by pests, and wet, watery rinds
Ethanol 95%, Ethanol 75% - are purchased at Bach Khoa Trading Service Co., Ltd, address
354 Ly Thuong Kiet, Ward 15, District 11, Ho Chi Minh City
Hydrochloric acid 37% (HCl), Sodium hydroxide (NaOH), and Sulphuric acid 95 – 98% (H2SO4)- are sourced from China
- Thermostatic Shaking Water Bath (Korea)
- Magnetic Stirrer (phoenix RSM-03010K, Germany)
- Texture Profile Analysis system (Brookfield's CT3, America)
- 2- and 4-digit analytical balance (Sartorius, Germany)
Beakers (500 mL, 50mL, 1000mL, 250mL), glass rods, thermometers, pipets (1mL,5L, 10mL), rubber squeeze bulb, micropipette (20-200àL), pipette tips, sieve (ỉ 0.6–1.5 mm), stainless steel plastic gloves, graduated cylinder (10mL,25mL, 100mL), volumetric flasks (100mL, 1000mL), Erlenmeyer flasks (250mL), burette (25mL), crucibles, petri dishes ,tongs, cuvettes, filter paper, separatory funnel, mortar, and pestle.
Research scheme
Figure 3.2 Diagram of experimental research
• Moisture and ash content of optimum pectin
• Intrinsic viscosity and viscosity- average molecular weight
Manufacturing
Figure 3.3 Flowchart diagram for durian rind drying
Purpose: Washing helps clean contaminants, and cutting prepares the durian rind for the drying stage
The durian rinds were examined for flaws such as browning, rotten areas, mold growth, holes caused by pests, and wet, watery rinds The durian shell is composed of three unique structural layers: the exocarp (an exterior layer covered in spines), the mesocarp (a thick white layer behind the spines), and the exocarp (a thin white layer in touch with the outer layers) [47] Pectin extraction only uses the white (mesocarp and exocarp) portion of the durian rinds because it contains a higher concentration of pectin After eliminating the durian thorns and sharp, the white layers were washed, Then, they were sliced into small pieces (2×
Purpose: Drying helps remove water from the rinds, prevents the growth of mold and bacteria, and prolongs storage time
The durian rind was dried at 60 °C in a convection oven until the moisture of durian rinds were kept from 10 to12%
Purpose: Grinding helps to reduce the surface area of raw materials, preparing them for the extraction process
The dried rind was then crushed by a grinder and sieved using a 150 mesh sieve Durian rind powder was stored at room temperature in ziplock bags for further investigation
1 × 95% EtOH Adjusted to pH 6 by NaOH 0.01N
Figure 3.4 Flowchart diagram for alcohol insoluble preparation
Purpose: Eliminate from the materials any free monosaccharides, colors, and contaminants
Proceed: Take 10g of durian powder and put it into 250ml erlen with 112.5 ml of 75% ethanol solution (EtOH) and 7.5ml of 5% HCl, and 30ml of distilled water according to the S:L ratio of 1:15 (g/mL) After that, the sample was immersed in a thermostatic shaker and continuously agitated for six hours at ambient temperature
Purpose: to obtain durian powder after being washed with alcohol
Proceed: Filter the sample through the vacuum filtration system
Purpose: Washing second time with alcohol helps to remove any remaining contaminants
Proceed: The powder sample rinsed with 75% EtOH three to four times until the filtrate was colorless After that, 95% EtOH was used to wash the rind powder
Note: In order to ensure adequate acid elimination during washing, NaOH 0.01N was added to the sample to bring its pH to 6
Purpose: to obtain an alcohol insoluble residue also known as AIR
Proceed: Filter the sample through the vacuum filtration system
Purpose: Drying helps remove water, prevents the growth of mold and bacteria, and prolongs storage time
Proceed: AIR was dried overnight at 60 °C in a convection oven Then, stored in a desiccator at ambient temperature
Add 95% EtOH, stand for 1 hour
Figure 3.5 Flowchart diagram for pectin extraction process
The alcohol insoluble residue of durian rinds powder samples were extracted in a thermostatic bath running at 90 rpm using various mass over volume ratio (S:L) of concentrations of acid (1:20 to 1:60 g/mL) at mutiple temperature values (75 to 95°C) for varied times (30 to 270 min) For every extraction, the constant acid concentration H2SO4
0.001M After extraction, cool the solution with cold water to complete the extraction process
Purpose: to separate the viscous solutions
Proceed: Centrifuge at 3500 rpm (50 × g) for a duration of 20 min
Purpose: To create gel-like precipitation
Proceed: In order to aid in the precipitation process, the supernatant was precipitated using 95% EtOH in a 1:3 ratio This mixture was then left undisturbed at room temperature for a duration of 1 hour to facilitate the precipitation process
Proceed: Centrifugation was used for 15 minutes at 3500 rpm (50 × g) to collect the gel precipitate
Purpose: Pigments and contaminants are removed from the sample by alcohol washing
Proceed: With a magnetic stirrer, the resultant precipitate was rinsed three times, twice with 75% EtOH then once with 95% EtOH for ten minutes each time
Purpose: to extract the alcohol from the gel-like precipitation
Proceed: Centrifugation was used to collect the gel precipitate for 15 minutes at 3500 rpm (50 × g)
Purpose: Drying helps remove water, prevents the growth of mold and bacteria, and prolongs storage time
Proceed: Durian pectin was dried for 15 hours at 50°C in a convection oven
Purpose: Grinding helps to reduce the surface area of pectin
Proceed: The pectin was then ground using a grinder and sieved through a 150-mesh sieve The pectin was stored at room temperature in ziplock bags.
Analytical method
3.4.1 Moisture content of durian rind and pectin powder
Procedure: In an oven set to 105 °C, dry the petri dish to constant weigh (remember to label the lid and plate) Then let it cool in a desiccator before weighing it using an analytical balance that has a 0.0001g accuracy After that, 2g of the sample was put to the petri dish and dried at 105°C until achieve a constant weight After drying, place petri dish and sample into a desiccator to cool it, then use an analytical balance to weigh it, repeat the experiment three times and analyze the data The moisture content of sample was calculated as a equation (1)
% MC: moisture content (%) m2: Weigh of sample and petri dish before drying (g) m1: Weigh of sample and petri dish after drying (g) m: Weigh of initial sample (g)
3.4.2 Ash content of durian rind and pectin powder
Procedure: The porcelain crucible should be heated in a kiln between 550 and 600 °C to a constant weight, cooled in a desiccator, and weighed it by using analytical balance with an accuracy of 0.0001g Then, place 2g of the sample in a porcelain crucible, and accurately weigh each one with an analytical balance as described above Finally, calcined at 550–
600°C, heat the sample in the kiln until it becomes white (approximately 6 to 7 hours), then cool it in a desiccator and weigh
Repeat the experiment three times and analyze the data The ash content of sample was calculated as a equation (2)
G1: Weigh of sample and porcelain crucible before drying (g)
G2: Weigh of sample and porcelain crucible after drying (g)
Procedure: 1 gram of AIR was extracted at various S/L ratios (1/20 – 1/60 g/mL), at multiple temperature values (75 – 95 °C), and varying time intervals (30 - 270 min) in a thermostatic bath, agitated at a speed of 90 revolutions per minute A consistent 0.001M H2SO4 was used for all extraction procedures Pectin was dried in a convection oven for 15 hours at 50°C after extraction The quantification of pectin yield percentage was determined through the application of Equation 3, and the results were denoted as the expressed yield (%)
The AUA of pectin in the extraction solution was determined following the method of Khamsucharit, P., et al [48] Dissolve 0.01g of pectin with 0.1 ml of 95% ethanol, 0.02g of NaCl, and 1-2 drops of phenolphthalein Subsequently, 2 mL of distilled water was added The mixture was titrated with a 0.1 N NaOH solution to obtain the volume y (ml) Then, 0.5 ml of 0.25 N NaOH solution was added, and the mixture was stirred at room temperature for
30 minutes Afterward, 0.5 ml of 0.25 N HCl solution was added and shaken until the pink color disappeared The mixture was titrated with a 0.1 N NaOH solution, and the volume z (ml) was obtained The AUA was determined using the following equation 4:
Molecular unit of AUA (1 unit) = 176 g z = mL of 0.1N NaOH from equivalent weight determination y = mL of NaOH 0.1N from methoxyl content determination w = weight of pectin sample
The DE and chemical structure of pectin was analyzed by the Fourier Transform Infrared (FT-IR) spectroscopy method[49] Prior to measuring DE, distilled water was used to dissolve the pectin sample, which weighed roughly 10 mg In order to guarantee that all Carboxylate ions are a kind of non-esterified carboxylic groups The pectin solution's pH was subsequently increased to pH 6 using 0.01N NaOH due to the pKa of 3.38 of polygalacturonic acid After that, the pectin solution was dried for at least 15 hours at 60 °C in a vacuum oven with silica gel present
Using an FT/IR 4700, 16 scans were performed in attenuated total reflection (ATR) mode, covering a wavenumber range of 400 to 4000 cm −1 at a resolution of 2 cm −1 Using equation (5), the pectin's DE was determined [50] Using software, the peak areas for C=O peak from
1740 to 1720 cm −1 and the COO − peak from 1630 to 1600 cm −1 were determined [51]
C=O: Area of esterified carboxy group
COO - : Area of non-esterified carboxyl
Figure 3.6 FT-IR spectrum of pectin
3.4.6 Intrinsic viscosity and viscosity- average molecular weight
An Oswald viscometer was used to determine the pectin solution's apparent viscosity This measurement method measures the time required for a specified amount of liquid to flow through a capillary of known diameter and length, which is then compared to the time required for the liquid to have a known viscosity (usually distilled water) The limit of viscosity decline as concentration approaches zero is called intrinsic viscosity Finding the viscosity reduction at zero solute concentration and extrapolating it is the primary technique for calculating intrinsic viscosity[ 60]
Procedure: To determine viscosity, 5 ml of distilled water was injected into the capillary viscometer to determine the flow rate by starting the clock to measure time Next, pectin solution is mixed at a different concentration of 0.02 – 0.01 (g/l), the mixture is stirred well Then, 5 ml of pectin solution was injected into the capillary viscometer to determine the flow rate After each sample was taken, the viscometer was rinsed with distilled water at least 3 times Repeat the measurement 3-4 times for each sample and take the average value of the time Equation 6 was used to determine the pectin solution's relative viscosity [52] By
33 extending the Martin, Kraemer, Huggins curve to zero concentration between 0.002 and 0.01 (g/l), the intrinsic viscosity , was calculated [61] Three equations are frequently used to calculate [g] of food : Kraemer (9), Huggins (8), and Martin(10) Equation 11 illustrates the calculation of the viscosity-average molecular weight (Mv) using the Mark-Houwink- Sakurada equation.[53]
: is relative viscosity, ղ is the viscosity of pectin solution (Pa.s)
: is the viscosity of distilled water (Pa.s), t: is the time taken by pectin solution to flow in the viscometer (s), ts: is the time taken by solvent to flow in the viscometer (s) sp: is a spec ic viscosity
C: is the concentration of pectin solution (g/L), and KM is Martin's constant kh: is Huggins 's constant, kk: is Kraemer 's constant α & k: constants for temperature and specific solute-solvent system k = 2.34 ×10 −5 and α = 0.8224 [53]
Utilizing a scanning electron microscope, morphological study was performed on the powdered form of pectin DP was sieved using 150 mesh screens before examination Using
34 double-sided carbon tape adhesive, the pectin powder was attached to circular metal stubs These were then coated with a layer of gold using a sputter-coating technique SEM pictures were obtained at magnifications of × 30 and × 200 [54]
Experiment design
3.5.1 Experimental design of pectin extraction optimization
We used a CCRD-based experimental design to maximize extraction conditions In order to determine the effects of pectin extraction parameters on yield (Y1, %), DE (Y2, %), and AUA (Y3,%) of pectin This study looked at three different ranges: X1:80–90 O C; X2: 1:30– 1:50 g/mL; and X3:90–210 min Statistical software (Design Expert) was used to create a five-level, three-factor CCRD in 17 trial runs An extended range beyond the required values of each variable was covered by the experimental design, which comprised of eight factorial points (coded -1 and +1), six axial points (coded -2 and +2), and three center points (coded 0) To calculate the experimental error, three center point replications were carried out [55]
To mitigate the influence of uncertain variability from unrelated causes on response outcomes, experiments were conducted in a random order[56]
Table 3.1 Factor settings for CCRD model for three factors
Each experiment was conducted in triplicate, and the results were expressed as the mean ±
SD Design Expert software was utilized for experimental design, response prediction, and results analysis To determine the regression coefficients (β), the experimental data were fitted to the generalized second-order polynomial model (Eq 12) in the response surface analysis
In which, y: represents the anticipated reaction
36 and : are the independent variables ε: is the error
The coefficient of determination (R 2 ) was used to evaluate how well the model fit the data ANOVA was used to determine the variations among the independent variables, with a p- value of less than 0.05 being used as the threshold for statistical significance [57] Design Expert was used to generate three-dimensional response surface graphs The desirability function (D) was ultilized with an range of 0 ≤ D ≤ 1, using numerical optimization to optimize the pectin extraction parameters based on the desired results For this optimization procedure, the Design Expert program used a numerical optimization technique Attaining an AUA content of at least 65% and optimizing pectin output were the goals
By comparing experimental data with theoretically anticipated data, the anticipated response values and extraction parameters were verified in order to evaluate the dependability of the RSM model Responses were assessed in triplicate using the suggested extraction settings For every response, the percentage of difference between the experimental and anticipated values was determined, with different fewer than 10% being preferred [58]
3.5.2 Solve multi-objective optimization problem
The multi-objective optimization problem is formulated based on the regression equation determined through the design of experiments method, describing the dependence of product recovery efficiency and AUA content on the factors of temperature (°C), time (min), and the ratio of raw material to solvent (g/mL) The Design Expert software is utilized to model the experiments using the response surface method (RSM), analyze the experimental results based on the model, and solve the multi-objective optimization problem
Finally, a validation experiment is conducted to verify pectin recovery under the optimal extraction conditions regarding temperature, time, and the ratio of raw material to solvent (g/mL) The yield, AUA and DE are determined, followed by a comparison and evaluation of the predicted results to assess the accuracy of the model (Validation Experiment)
RESULTS AND DISCUSSIONS
Moisture and ash content of durian rind
Based on the results of the raw materials obtained in table 4.1, the moisture content of the durian rinds was 7.72 ± 0.22% These results are less than 12% With low moisture content, the raw materials will have the ability to diffuse molecules, easily extract the compounds, and at the same time convenient to preserve the raw materials for a long time Ash content reached values of 0.853 ± 0.15% for durian rinds This facilitates the extraction of polysaccharide content
Table 4.1 Moisture and ash content in durian rind
Values in the table represent mean ± standard deviation (SD)
Results of experimental design
To determine the optimal conditions for achieving high efficiency in DE and AUA output,
17 experiments, as arranged in table 4.2, were conducted under various extraction conditions
Table 4.2 Experimental results of objective responses
The data represents the mean ± standard deviation (SD) The means with different letters in the same column indicate a statistically significant difference (p 0.05; the larger the pvalue, the better the model fits
After evaluating the regression equation, the response surface models were built according to the regression equation having form as equation that had been found, then analyze the experimental results and solve optimization problems The ANOVA outcomes for the DE
39 model, characterized by lower values of R 2 , predicted R 2 , and adjusted R 2 along with a p- value that is greater than 0.05, suggest that the linear model's prediction is not statistically significant
Table 4.3 ANOVA for linear model Y 2 (DE)
Source Sum of squares df Mean
Square Coefficient F value p value Comments
The presented data pertains to a linear regression analysis, particularly within the framework of DE model The overarching model, designated as linear, fails to achieve statistical significance, as evidenced by its elevated p-value of 0.0888 This outcome implies a lack of robust fit between the model and the observed data Respective p-values, all exceeding the conventional threshold of 0.05 Consequently, their inclusion in the model does not contribute significantly to explaining the variability in the response variable The Lack of Fit test further underscores the inadequacy of the model in capturing the underlying patterns within the data Specifically, this test indicates a significant lack of fit, suggesting that the model inadequately represents the true relationship between the predictors and the response variable
Table 4.4 ANOVA for quadratic model Y 2 (DE)
Source Sum of squares df Mean
Square Coefficient F- value p-value Comments
The results in table 4.4 show that the results of the experiment did not fit with models of regression, indicating that the extraction conditions using 0.001M H2SO4 at pH 2.39 had no effect on the DE Some studies on melon and banana peel have shown notable variations in
DE as a result of temperature-pH and time-pH interactions [59] [60] Our investigation found that the impact of temperature and time on DE (18.56 – 22.18%) was insignificant when using a lower acid concentration (0.001M H2SO4) This implies that the DE may occur at a slower rate when subjected to variations in temperature and time
Moreover, an alkaline environment was found to exhibit a more noticeable pectin de- esterification process in comparison to an acidic This is supported by the fact that the acid demethylation rate is considerably lower (less than 0.01%) than the alkaline demethylation rate, even when employing similar temperature and chemical concentration conditions[61]
Effect of extraction parameters on pectin yield
Table 4.5 displays that the yield linear model exhibited a higher coefficient of predicted R 2 and a p-value that is less than 0.0001 in comparison to the other models Therefore, the linear model was chosen to match the data given from experiment of yield
Table 4.5 Summary model response of yield
Lack of Fit p- value Adjusted R²
The yield of pectin extracted from durian rind differed from 6.75 to 11.28% based on the concentration of 0.001M H2SO4 The results of pectin yield generated from the ANOVA for the linear model are shown in Table 4.6 With remarkably low p-values, all independent variables: time, temperature, and S:L ratio affected pectin yield during acid extraction noticeably Moreover, positive coefficients for duration, S:L ratio, and extraction temperature indicated beneficial effects on yield The extent of the effect on pectin yield is shown by the magnitude of the coefficient values
Table 4.6 ANOVA for linear model Y 1 (Yield)
The equation using coded factors defines the functional expression that captures the relation between the dependent variable (yield) and the independent variables (temperature, S:L ratio, and time)
In this case, the coded independent variables are A, B, and C (temperature, S:L, and time, respectively)
Source Sum of squares df Mean Square
Lack of fit 8 11 0.7271 10.74 0.0882 Not significant
The perturbation plot seen in figure 4.1 indicates temperature had a greater effect on the yield of pectin extraction than the other two parameters combined
Figure 4.2 further illustrates that the pectin yield increased as the extraction temperature rose The highest pectin yield was achieved at the maximum temperature (95 °C), in conjunction with the central levels of solid-to-liquid ratio (S:L) (1:40 g/mL) and extraction time (150 min)
The process of extracting pectin follows the principles of mass transfer, encompassing three main steps [62]
Step 1: The protopectin in the raw materials is hydrolyzed
Step 2: The generated pectin is dissolved in the extraction solvent until saturation is achieved
Step 3: The solubilized pectin's degradation
Insoluble protopectin hydrolysis within the cell wall of plants as well as the subsequent solubilization of the pectin in the acidic solvent are both influenced by temperature, which is a critical factor in pectin extraction [62] The efficiency of insoluble protopectin hydrolysis appears to be diminished at lower acid hydrolysis temperatures, resulting in a lower pectin output [63]
Moreover, a rise in temperature promoted solvent penetration into plant tissues and softened plant tissue, which made it easier for pectic polysaccharides to move out of the cell wall into the extraction liquid A higher rate of pectin extraction was facilitated by this improvement in the coefficient of extraction and the diffusion rate of pectin from the solid matrix to the solvent [45] Furthermore, by supplying an adequate energy to the break bonds of materials at high temperatures-a frequent event where solubility often increases with temperature-the dissolving reaction was triggered [45]
One important aspect that affects the pectin extraction yield is the solid to liquid ratio, especially when the pectin is solubilizing into the extractant [62]
Figure 4.2 One-factor plot illustrating the effect of temperture on pectin yield
From figure 4.3, an increase in ratio of solid over liquid was shown to provide a higher yield of extracted pectin, showing patterns akin to those of extraction of pectin from pistachio hulls and the outer layers of passion fruit [57, 64] The chosen extracting solvent, 0.001M
H2SO4 in this instance, can be likened to a "container" for collecting soluble pectin through mass transfer from the solid matrix to the acidic solvent As a result, a larger total pectin yield may be achieved by increasing the solvent volume since the extracting solvent can hold more extracted pectin Additionally, the elevated S:L ratio caused differences in concentration between the inside of the cell and the outside solvent, which amplified the solid matrix to solvent mass transfer from the solid matrix to the solvent and increased the pectin quantity [45, 57] The extraction solvent's pectin concentration increased as the process went on, increasing the viscosity of the solvent
After pectin reached a saturation point, the rate of mass transfer gradually reduced and finally stopped [62] However, a persistent rise in the S:L ratio could cause a negative effect since it could cause the extracted pectin to hydrolyze as a result of the solution being diluted excessively [62, 65]
Figure 4.3 One-factor plot illustrating the effect of S:L ratio on pectin yield
Extractive efficiency and fluid selectivity can both be impacted by the length of the extraction process [45]
As depicted in figure 4.4, the yield of pectin demonstrated an increase with the extension of the extraction duration An extended extraction time allowed the solvent to seep into the AIR and help the protopectin containing in the wall of plant cell hydrolyze A larger output of pectin was produced as a result of the longer time, which improved the mass transfer of the plant product’s soluble pectin into the solvent Consequently, an extended extraction time was conducive to the recovery of pectin However, it is crucial to note that prolonged extraction, especially at extreme temperatures, and excessive duration may lead to pectin degradation, as observed in studies on durian rind pectin extraction [45]
Additionally, the relationship between the components involved in extraction, either S:L and temperature, time and temperature, or time and S:L, promoted pectin formation, according to the 3D response surface graphs and countor in figure 4.5a–f
Figure 4.4 One-factor plot illustrating the effect of time on pectin yield
Figure 4.5 The 3D response surface graphs and countor demonstrate the relationship of extraction parameter
Figure 4.5 (a) and (b) indicate that an increase in both the solid-liquid (S:L) ratio and temperature contributes to an elevation in pectin yield Specifically, a higher S:L ratio is noted to enhance yield by providing a larger contact surface between the plant material and the solvent This promotes swelling of the material, facilitates cell disruption, and enhances the solubilization of pectin Additionally, prolonged exposure to an acidic extractant at a higher temperature is associated with an increase in pectin output
Figure 4.5 (c) and (d) show the duration of the extraction process has a pronounced impact on the production of pectin According to the theory, when the acidic extractant is in contact with the raw material for an extended period, especially at higher temperatures, it tends to enhance the overall yield of pectin The extended exposure allows for more efficient extraction of pectin from the raw materials, contributing to an increased output of this important substance.[66] The rationale behind the observed phenomenon lies in the nature of pectin, which is a carbohydrate polymer composed of (1, 4) linked galacturonic acid units The requirement for time during the extraction process stems from the need to soften the structure of pectin before effective extraction can take place In the initial stages of extraction, the increase in pectin yield with time is attributed to the fact that longer durations provide ample opportunities for the structural softening of pectin, allowing for greater reaction time and subsequently enhancing the overall yield.[67]
Figure 4.5 (e) and (f) illustrate that the increase in pectin yield is directly associated with an augmentation in both the solid-liquid ratio and the duration of extraction This phenomenon can be elucidated by the extended time frame, which provides an increased number of opportunities for reactions between the solvent and the constituents involved The prolonged contact period between the extraction medium and plant materials is instrumental in facilitating a more efficient mass transfer, allowing solid particles to migrate into the solution[45, 57] Furthermore, the higher solid-liquid ratio plays a pivotal role in enhancing the overall yield This is attributed to the augmented contact surface area between the plant material and the solvent, facilitating a more comprehensive interaction The increased ratio also contributes to the swelling of the plant material, promoting cell disruption This disruption, in turn, facilitates the solubilization of pectin, as the cellular structure becomes more amenable to the extraction process In essence, both the extended sonication time and higher solid-liquid ratio synergistically create favorable conditions for maximizing pectin yield through improved mass transfer and enhanced solubilization
Figure 4.6 Predicted and Actual values of Yield
The results depicted in the figure 4.6 show a high level of agreement between the predicted and experimental values The data points are closely clustered along the diagonal line, indicating a strong correlation and minimal dispersion There are no scattered points, underscoring the accuracy of the predicted values, which closely align with the actual experimental results.
Effect of extraction parameters on AUA
The AUA concentration must be at least 65% as a benchmark for determining the purity of the extracted pectin [68] A lower AUA value suggests the potential presence of elevated levels of proteins, starch, and sugars in the precipitated pectins [69] In our study, the AUA content of pectin varied between 48% and 77.15% under different extraction conditions, including varying temperatures, solid-liquid ratios (S:L), and time in a 0.001M H2SO4 solution The findings suggest that not all extracted pectin met the purity criterion of AUA content exceeding 65% y = 0.9371x + 0.5419 R² = 0.9371
When compared to other models, the quadratic model for AUA performed better in terms of lower p-values, adjusted R 2 , predicted R 2 Consequently, the quadratic models were chosen to correctly reflect the experimental data for AUA, permitting an in-depth study of the interaction between independent and dependent variables
Table 4.7 Summary model response of AUA
The ANOVA in table 4.8 outcomes revealed that the regression models pertaining to AUA in DP were extremely important, evidenced by a p-value less than 0.0001 and a substantial F-value The effectiveness of the models was underscored by the coefficient of determination (R²), which stood at 0.9770 for the quadratic AUA model This indicated that a remarkable 97.70% of the variability found in the experimental AUA data was explained by the models Additionally, the AUA quadratic model's appropriate precision value of 21.478 showed that the model produced satisfactory signals
Table 4.8 ANOVA for quadratic model Y 3 (AUA)
The equation (8) using coded factors defines the functional expression that captures the relation between the dependent variable (AUA) and the independent variables (temperature, S:L ratio, and time)
Source Sum of squares df Mean Square
Lack of fit 18.49 5 3.7 7.68 0.1192 Not significant Pure error 0.9632 2 0.4816
In this case, the coded independent variables are A, B, and C (temperature, S:L, and time, respectively)
Temperature was shown to have the greatest impact on the AUA percentage, as seen by the perturbation plot in figure 4.7
The one-factor plot for temperature shown in figure 4.8 indicates that a temperature of about
90 °C was required to achieve pure pectin (AUA > 65%) This implies that at a sufficiently high temperature of about 90 °C, there was a noticeable reduction in sugar side chains from pectin Furthermore, the longer extraction time allowed for more contact between the heated acid solvents and the pectin to be extracted, giving enough time for the side chain removal of sugars to occur As such, the pectin's purity was improved
Figure 4.8 One-factor plot illustrating the effect of temperture on AUA
In figure 4.9, there was a progressive decrease in the AUA percentage and a rise in S:L ratio The hydrolysis of the extracted pectin brought on by the solution's extreme dilution could be the source of this event Due to this, uronic acid partially degrades into smaller molecules that cannot precipitate.[62, 70]
Figure 4.9 One-factor plot illustrating the effect of S:L ratio on AUA
Figure 4.10 The relationships of the extraction parameters were displayed by 3D surface response graphs and countor
In both figure 4.10 a and b, it is evident that the AUA content of pectin rises with higher extraction temperatures and longer durations This observation aligns with findings from some studies conducted, which investigated extracting process of pectin from skins of apple and pea ,respectively [70, 71] It has been reported that the glycosidic bonds in the pectin chain are resistant to hydrolysis to different degrees, with the links between neutral sugars being the least resistant [72] Consequently, elevated temperatures and extended durations could facilitate the degradation of side chains composed of pectic neutral sugars [73]
Figure 4.10 c and d, when increasing S:L ratio and temperature, the AUA value also increased, it can explain by the higher ratios and temperatures can help in effective pectin extraction by creating more contact between the solid plant material and the extraction liquid, which can enhance extraction efficiency by promoting breakdown the cell wall facilitates the release of pectin and increases the overall quality and purity of the extracted pectin Figure 4.10 e and f demonstrate, the pectin AUA content was positively impacted by the relationship between the extracting time and S:L ratios There was an growth in pectin AUA content followed after increasing S:L and time, the similar trend can be observed when extracting pectin from peel of grape pomace and ponkan The lengthier extraction period might have accelerated the solubilization and pectin’s mass transfer of the input material [74]
Figure 4.11 Predicted and actual value of AUA y = 0.977x + 1.3933 R² = 0.977
Pred ic te d val ue
Based on figure 4.11, representing the normality of the data, which includes experimental values with three factors: extraction time, temperature, and S:L ratio, along with simulation results A robust relationship exists between the actual and predicted values The data points are close to the diagonal, which can mean that the actual results will be close to the results predicted value of the model.
Optimization of the experiment and validation of the model
After creating models for each reaction to optimize the purity and yield of pectin, the optimization process focused on excluding DE due to its negligible impact as indicated by the results The Design Expert software generated 54 solutions with a desirability value of 0.885 Among these, optimal pectin extraction conditions were identified, involving less severe extraction conditions compared to other solutions The ideal parameters included 0.001M H₂SO₄ concentration, a temperature of 90°C, S:L ratio of 1:50 (g/mL), and a duration of 210 minutes This configuration resulted in a predicted extraction efficiency of 11.085% and an AUA content of 71.832% (refer to table 4.9)
To confirm the suggested extraction conditions and the generated model's projected outcomes, experiments were conducted A rigorous triple experiment was conducted for validation in order to evaluate the dependability and consistency of the suggested extraction settings The purpose was to verify whether the model's predictions aligned with the actual experimental results, thereby confirming the robustness and accuracy of the established extraction conditions Table 4.9 displays the experimental efficiency and AUA values for the chosen extraction conditions, which were 11.25±0.15 % and 72.258±0.32 %, respectively, with differences of 1.489% and 0.59% The results show that there is no significant difference between the predicted and actual values This is reasonably desirable, with experimental values with different of less than 10% [58] Thus, it may be said that there is a good degree of agreement between the experimental and projected values
Table 4.9 The response of verified samples at the optimal extraction conditions (90 °C,
Factors Predicted value Experimental value Different (%)
In each column, values with different latters are statistically different (p