desizing of cotton by extracted aloe gel extracted from aloe vera plant by Biniyam Nur

THESIS REPORT 2021 CHAPTER ONE 1. DESIZING OF COTTON USING ALOE GEL EXTRACTED FROM ALOE VERA PLANT 1.1 Introduction Aloe Vera has a long history of providing a myriad of health benefits and being one of the herbal remedies most frequently used throughout the world [1]. It’s known as a tropical or subtropical plant with a turgid green leaves joined at the stem in a rosette pattern. These leaves can be separated in to three layers which are the inner gel, the yellow sap known as latex, and the outer thick layer, called the rind [2]. Currently, aloe vera has become one of the important raw materials in the food industry, since it represents an emerging source of bioactive components. The potential use of aloe vera gel in the food industry is mainly focused on the development of functional food due to its beneficial properties in treating constipation, coughs, diabetes, headaches, arthritis and immune system deficiency [2]. However, in the past few years, researchers have found another usage of aloe vera gel where it can be used in textile industries specially, in the wet processing. De-sizing is the part of wet processing that includes in the pre-treatment of textile materials before finishing. Desizing is the process of removing the starch molecules found in the textile fabrics, in which the size applied to the warp yarn before weaving is removed to facilitate the penetration of dyes and chemicals in the subsequent wet-processing operation. Almost all woven fabric contains size and about 65% of the cotton used for textile is made into woven fabric. Most cotton and synthetic fiber staple yarns are sized with various types of natural or synthetic polymers. Sizing is desirable for maintaining high processing speed and can prevent processing damage during weaving that could subsequently lead to a non-uniform fabric for dyeing and finishing operations. For cotton fabrics, the film former is usually starch and modified starch. All starches are by their nature either water-insoluble or only sparingly soluble. Chemically starches are composed of amylose and amylopectin. De-sizing of cotton fabric can be accomplished by physical, chemical or combined of the two.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 The most commonly used method for cotton is enzymatic de-sizing. Acid steeping and oxidative de-sizing (the chemicals used for removal of the size) are risky process and may result in degradation of cotton cellulose while rot steeping, hot caustic soda treatment and washing detergents are less efficient for the removal of the starch sizes. Enzymatic de-sizing is the classical de-sizing process of degrading starch size on cotton fabric using enzymes. Enzymes are complex organic, soluble bio-catalysts chemical reaction in biological processes. Enzymes are quite specific in their action on a particular substance. A small quantity of enzymes is able to decompose a large quantity of the substance it acts upon. Enzymes are usually named by the kind of substance degraded in the reaction it catalyzes. Amylases are the enzyme that hydrolyses and reduce the molecular weight of amylose and amylopectin molecule in the starch, rendering it water-soluble enough to be washed off the fabric. Aloe vera is a perennial, tropical, drought-resistant plant belonging to the liliaceae family which, turgid green leaves joined to the stem in a rosette pattern. It is a stem less or very short stemmed succulent plant growing to 60-100 cm tall, spreading by offsets. Aloe leaves consist of a thick epidermis (skin) covered by a cuticle surrounding the mesophyll that includes chlorenchyma cells and thinner walled cells that form the parenchyma (filet). The mesophyll cells contain a transparent mucilaginous jelly called Aloe vera gel [1]. The gel consists primarily of water (>98%) and polysaccharides such as pectins, cellulose, hemicellulose, glucomannan, acemannan, and mannose derivate. The aloe plant consists of 95% water, with an average 4.5 ph. The remaining solid material contains over 75 different ingredients including vitamin, minerals, enzymes, sugars, anthroquinone or phenolic compounds, lignin, saponins, sterols and so on. Aloe vera also contains potentially act constituents, such as vitamin, enzymes, minerals, sugars, lignin, saponins and amino acids Some of the most important enzymes in aloe vera are Peroxidase, Aliases, Catalase, Lipase, Cellulase, Carboxypeptidase, Amylase and Alkaline Phosphatase. These enzymes have active centers, which are the points where substrate molecule can join later the substrate molecules are converted into the product. It is well documented that plants are plants are abundant source of Enzymes specially Amylase [4]. But determining the characteristics (activities and stability studies required) is an essential process that determines the efficiency of ability of the enzymes to perform their intended action. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 It is important to note that biotechnology is not just concerned with biology, but it is truly interdisciplinary subject involving the integration of natural and engineering sciences. Now familiar with the application of modern biotechnology in medicine and agriculture: so-called red and green biotechnology. There is less general awareness of the white variety: the use of biotechnology for industrial applications. These are all examples of biotechnology in action, a sector that driving force of interdisciplinary application. The use of aloe vera products often involves some type of processing such as heating, dehydration, and grinding. Unfortunately, as a result of improper processing procedures, many aloe products contain only very little or virtually no active ingredients, so that there is an imperative need to develop better methods of preservation, which would increase the shelf life of aloe vera gel simultaneously maintaining its high quality. As S Senthlinathan (2012) published in a case study several enzyme biochemical catalysts, such as amylase and lipase, can digestion by breaking down fats and sugars. One important enzyme, a carboxypeptidase, inactivates bradykinins and produces an anti-inflammatory effect. During the inflammatory process bradykinins produces pain associated with vasodilation and, therefore, its hydrolysis reduces these two components and produces an analgesic effect. Aloe vera is advantageous plant in harness the potential of biotechnology application in textile pretreatment due to the presence of catalyst chemicals, there are all examples of biotechnology in action, a sector that is constantly growing and expanding into other industrial sectors, a true driving force of interdisciplinary application. The current trend deals with the potential of aloe vera gel extracted from aloe vera plant as a biotechnology and bio-polymer in textile industries specially in de-sizing of cotton fabrics.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 1.2 Statement of the Problem There are a lot of drawbacks in chemical transformation processes used in various industries from a commercial and environmental point of view. Nowadays, highly alkaline chemicals are used in the textile materials to enhance the dye ability of the fabric, results in destruction of fiber structure. Intensive rinsing and more acid is needed for reutilization of the fiber, which enlarge the volume of effluent. These chemicals also attack the cellulose leading to heavy strength loss and weight loss in the fabric. Furthermore, the hazardous chemicals result in increase in pollutant in waste water. Non-specific reactions may result in poor product yield. High temperature and pressure are required to complete reactions leads to high energy cost and may enlarge the volume of effluent, harsh or hazardous chemical processes involving high temperature, pressure and alkalinity requires high capital investment, specially designed equipment and control system. Unwanted by-product may occur difficult or costly for dispose. The net result is low quality control and polluted environment with high usage of energy, time, chemical and water. In many cases, these drawbacks can be virtually eliminated by using enzymes. However, the use of enzymes in the textile wet processing has added a new line research and likely eco-friendly substance to give a good solution to the problem of highly toxic chemicals causing environmental. So de-sizing of cotton using aloe gel is the promise solution to minimize conventional de-sizing method.

1.3 Limitation  After extraction process is done the researchers were unable to continue for the desizing processes quickly thus the stability of the gel can have an effect on the process.  The shortage of soxhlet extraction instrument in the lab make the researchers to conduct the extraction process with different method (centrifuge) that influence on the prepared literature as well as the time.  Shortage of auxiliary chemicals and dyes was the main limitation of all factors that reduce the quality and ability of the process to satisfy its objective.  Lack of shelf life information of the dye paste might highly affect the depth of the color of the treated samples.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 1.4 Objectives 1.4.1 General objective  To de-size cotton fabric by using extracted aloe gel from aloe vera plant. 1.4.2 Specific objective  To extract aloe gel from aloe vera plant  To de-size cotton fabrics by aloe Vera gel.  To compare de-sizing of cotton using conventional methods and new de-sizing methods  To analysis dye ability of aloe gel treated cotton fabric.

1.5 Significance of the Project Textile industries are one of the most environmental polluted factories due to the chemical reaction and the chemicals used during wet-processing. The process of de-sizing of cotton fabrics also contributes to increase the pollution and might cause cotton degradation. Rot steeping, acid steeping and oxidative de-sizing are one of the method used for desizing of cotton fabric, but they (the chemicals used for removal of the size) are risky processes and may result in degradation of cotton cellulose. In rot steeping, the fermentation is not controlled thus cross infections might occur like mildew that degrade the cellulose of the fiber, the weight in loss in acid steeping is higher than rot steeping because dilute of mineral acid and acid producing salt like ZnCl2 and AlCl2 which produce a hydrocellulose that cause tendering of fibers. And also when sodium bromite in the oxidative de-sizing method is used the concentration of bromite should be restricted to ensure the cellulose is kept to an eligible proportion otherwise degradation will occur that result low quality control and poor product yield. This is not only the problem but also, the removal for the chemical after carryout process has an extremely high effect on the environment. As the main objective of de-sizing process is to improve the dye ability and to make the fabric suitable for further processes, de-sizing should be controlled and done effectively by considering the method of process that should be conducted, and to innovate a biopolymer catalysts that are compatible with the textile processes to minimize the number of chemicals used while processing. So, in this project the researchers’ surveys and find aloe vera plant are applicable in textile pretreatment process especially for de-sizing of cotton fabric due to the presence of compatible enzymes present in the leaves of the plant to hydrolyze the starch. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 CHAPTER TWO 2. LITERATURE REVIEW 2.1 Introduction Aloe vera is a plant with high therapeutic properties of which thousands of useful compounds are derived. Among different species of Aloe in the world, Aloe barbadensis has considerable properties. The gel obtained from this plant has a high viscosity, which its level depends on environmental conditions and the age of plant. The high gel viscosity causes problems in related industries. The chemical composition of plant constituents depends on geographical distribution, soil quality, water availability, solar radiation and temperature. Water availability is important, since the physiological role of leaf gel is to retain water. The amount of water in the gel fluctuates between 98.5% and 99.5% of fresh weight, while more than 60% of the remaining gel consists of polysaccharides [1-3]. About 99% of gel of this plant has composed of water and the remaining 1% contains compounds such as polysaccharides, anthroquinone, chromones, vitamins, amino acids and proteins. The vascular bundle if the leave portion is adherent to the rind, whereas the remainder of the vascular bundle protrudes in to the mesophyll layer (Danhof 1987). The mesophyll can be differentiated into chlorenchyma cells and thinner-walled parenchyma cells. The parenchyma (filet and pulp), which is the major part of the leaf by volume, contains a clear mucilaginous gel known as Aloe vera gel [8]. Aloe vera gel is fresh and colorless and has placed in the internal part of the plant while the latex is a yellow liquid and is placed in the vessel elements adjacent to shell of the plant. The latex is laxative, due to the presence hydroxyl anthracene compounds like anthroquinone [1-2] To process the leaf, its products are separated into gel and cortex. The latex is in the cortex and corresponds to the bitter yellow juice of the green part of the leaf produced in the leaf epidermis and spiny portion of the leaves. The bitter juice contains hydroxyanthracene derivatives (15-40%). Aloe gel is the colorless mucilage within the inner part of the fresh leaves. Aloe vera gel is commercially presented as a powder. The gel of this plant is used externally, for example, wound healing, burn shooting, etc., and dermal diseases and also applied for internal use such as treatment of coughing, constipation, peptic ulcer, diabetes, headache and immunodeficiency [3-4] The use of aloe vera products often involves some type of processing such as heating, dehydration, and grinding. Unfortunately, as a result of improper processing procedures, many De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 aloe products contain only very little or virtually no active ingredients, so that there is an imperative need to develop better methods of preservation, which would increase the shelf life of aloe vera gel simultaneously maintaining its high quality

Fig 2.1 Aloe Gel from Aloe Vera Plant The gel consists primarily of water (>98%) and polysaccharides such as pectins, cellulose, hemicellulose, glucomannan, acemannan, and mannose derivate. Chemical pre-treatment may be broadly defined as a procedure mainly concerned with the removal of natural as well as added impurities (figure 2.2) in fabric (Cotton) to a level necessary for good whiteness and absorbency by utilizing minimum time, energy and chemical as well as water. The ice breaker for chemical pre-treatment is commonly de-sizing, which is used to degrade the size materials with different methods as shown in the figure below. Aloe vera is considered to be the most biologically active of the aloe species (world health organization 1999). More than 75 potentially active constituents have been identified in the plant.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021

Fig 2.2 Impurities of Cotton Fiber and Desizing Method of Size Components from Cotton The composition of aloe vera extract differs according to the plant variety, climatic and seasonal variations and the age of the plant [3]. The commercial production process of aloe vera products typically involves crushing, grinding, or pressing of the whole aloe vera leaf to produce juice, followed by various steps of filtration, and stabilization to achieve the desired extract. This method provides ease of processing and higher efficiency in the recovery of the solids [18], but it can result in a product that contains little or no active ingredients. A further issue with the commercial production process is that during the commercial extraction of aloe vera gel, it is impossible to prevent the contamination by leaf exudates [3]. The adulteration of aloe vera products using fillers such as maltodextrin, glucose, glycerin, and malic acid represents a major concern for the aloe vera market [17]. As such misrepresentation in the industry the international aloe science council develops a certification program that validates the quality and quantity of aloe vera in approved commercial products.

2.2 Enzymes Enzyme is a Greek word “Enzymos” meaning “in the cell” or “forms the cell”. They are protein substances made up of more than 250 amino acids. Enzymes are high molecular weight protein secreted by living organisms capable of catalyzing the chemical reaction of biological process. Based on the medium for their preparation they are classified as bacterial, pancreatic (blood, lever etc.) and malt (germinated barely) etc. their major function are fails on hydrolysis, oxidation, coagulation and decomposition. Grouped under the following groups:  Oxidoreductases – Oxidation, reduction reaction  Transferases – Transfer of functional group

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021  Hydrolases – Hydrolysis reaction (Mostly used in textiles)  Lyases – Addition to double bond or its reverse  Isomerases – Isomerization  Ligases – Formation of bonds with ATP cleavages Beside full length enzymes there are various advantages of truncated enzymes (from any of the termini of proteins) for production of robust and stable enzymes such as proteases, cellulases, amylases, pectinase, etc. Truncation can lead to enhance stability by altering structural conformations, improved activity due to increased affinity with substrate, more enzyme expression limit proteolysis in the expression host, etc. Other reports of truncated enzymes even if in the other processes compare the efficiency of full-length with the truncated alpha amylase for their better production, enhanced stability and activities for employing them in various applications. Determination and activity of enzymes can be analyzed by Bradford assay using bovine serum albumin as standard for determination and activity of enzymes estimated according to the method of Rick and Stegbauer for alpha Amylase [15]. Enzymatic De-sizing method is the common method for removing the starch molecules from the fabric. Amylases are important enzymes employed in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents of starch. Besides their use in starch saccaharification, they also find potential application in a number of industrial processes such as in food, baking, brewing, detergent, textile and paper industries. With the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into many other fields, such as clinical, medical and analytical chemistry [5-6]. In 1894 Emil Fischer provided the lock-and-key model assuming that the active site was a perfect fit for a specific substrate and that once the substrate binded to the enzyme no further modification was necessary or irreversible depending on the enzyme. In 1958 Daniel Koshland suggested a modification to the lock and key model. Instead of flexible structures, the active site is continually reshaped by interaction with the substrate as the substances interact with the enzyme. As a result, substrate does not simply bind to a rigid active site; the amino acid chains which make up the active site are molded into precise position that enable the enzyme to perform its catalytic function. In some case such as, glycosidase, the substrate molecules also change shape slightly as it enters the active site. The active site De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 continues to change until the substrate is completely bound, at which point the final shape and charge is determined. The product is usually unstable in the active site due to steric hindrances that force it to be released and return the enzyme to its initial unbound state. Enzymes can originate from animal or vegetable sources. Around 1900, the German Diamalt Co. of Munich introduced Devastator, the commercial desizing compound, to the trade. Around 1912 enzymes extracted from slaughter house wastes became available. Generally, these contained four enzymes two of which reacted primarily with albumin and casein, one decomposed and reacted with starch. In 1919 bacterial diastase became available under the trade name Rapidase (Wallenstein & Co.), which rapidly liquefied starch to dextrin. The main enzymatic development within starch desizing has so far been the introduction of amylase products (Diastafor) optimized to different temperature ranges. Malt amylases fall into two categories and are named the (alpha) and (beta) species which are found to be present in the ratio of 1.5 to 1.6. a-amylases are capable of hydrolyzing starch molecules at random present in the sizing preparation, transform starch to dextrin’s, breaking them down to soluble sugars thus helping in eventual desizing. α-amylase attacks straight chains, cleaves the units and produces maltose, so that molecular chain of starch is shortened gradually. When an (α -amylase is applied to a starch solution, it is found that viscosity of the solution decreases rapidly, but for β-amylase the viscosity drops slowly. Thus it is clear that the proportion of (α and β-amylases in a desizing mixture determines the period (time) of effective desizing. It is also clear that the molecular structure of cotton is unaffected by amylases. The alteration or destruction of the natural morphology of an enzyme which causes a decrease in enzyme activity and stability is due to undesirable extraction conditions. Therefore, it is crucial to optimize the extraction process to produce enzymes with high activity and stability. Some of the most important enzymes in aloe vera are peroxidase, alliase, catalase, lipase, cellulase, carboxypeptidase, amylase and alkaline phosphates. These enzymes have active center, which are the points where substrate molecule can join. Just as a particular key fits into a lock, a particular substrate molecule fits into the active site of the enzyme. The substrate forms a complex with the enzyme. Later the substrate molecule is converted into the product and the enzyme itself is regenerated as shown in figure 2.4.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 The removal of hydrophobic part of the sizes (the lubricants) is often especially problematic. These are not removed during desizing, but are expected to be stabilized or emulsified in the alkaline scouring. The total material present in the cotton fiber is up to 20% of the fiber weight including that of 4-12% natural impurities. In the process of desizing, not only sizing agents, but also some natural impurities are eliminated from fibers. About 75% of the sizing agents used throughout the world today consists of starch and its derivatives because of its low cost. Chemically starch is composed of amylose and amylopectin. Amylose molecule is in the form of helix with six glucose units per turn (Fig. 2). The low molecular weight of amylose is water soluble straight chain polysaccharides of glucose whereas amylopectin (70-80%) being water insoluble is difficult to remove from cotton due to its higher molecular weight and branches chain.

Fig 2.3 Molecular Structure of Starch Amylose and Amylopectin In many cases efficiency of one type of size is inadequate and they must therefore be combined with other sizing agents to accommodate good adhesion, film flex- ability, viscosity, and compatibility with salts in hard water, abrasion resistance, washing out properties and possibilities of removal of the size and recycling the size from the effluent. By using synthetic polymers not only the quality of weaving is improved, but also reduce the amount of sizing additive, thus reducing the pollution and improvement in handle. Solvent soluble sizes are, however, a development for the future. Further, the easily removable sizing materials are preferred as the milder de-sizing and scouring treatments are required for synthetic fibers. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021

Fig 2.4 Lock and Key Model of Enzyme Specificity The process continues until the enzyme is poisoned by a chemical bogie or inactivated by extremes of temperature, pH or by other negative conditions in the processing environment as shown in figure below.

Fig 2.5 Active Site of Enzyme Blocked by Poison Molecule Several enzyme biochemical catalysts, such as amylase and lipase, can digestion by breaking down fats and sugars. One important enzyme, a carboxypeptidase, inactivates bradykinins and produces an anti-inflammatory effect. During the inflammatory process bradykinins produces pain associated with vasodilation and, therefore, its hydrolysis reduces these two components and produces an analgesic effect [9].

2.3 Optimization procedure 2.3.1 Determination of Enzyme Activity Assay and Protein Concentration The enzyme activity was determined through different chemical process reaction mixtures. The reaction mixture consist of 500 µL crude enzyme, 500 µL of 0.1% soluble starch prepared in 0.1 M sodium acetate buffer at pH of 5.0. The reaction mixture incubates at 70°C for 30 min. subsequently, adding 1000µL of DNS to the reaction mixture and heating it at 100°C for 5min; the mixture going to be cooled in tape water. After that the released reducing sugar will determined by spectrophotometry (BioMate-3, Thermo Scientific, Alpha Numerix, Woodfield Dr. Webster, NY,USA) at ranging from 500-540nm using maltose as a standard reducing De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 sugar. One unit of enzyme activity is defined as the amount of enzyme that produces 1µmol of maltose per minute under the enzyme activity conditions applied. The protein concentration determined by the Bradford [21] method using BSA as a standard 2.3.2 Determination of Specific Activity Enzyme activity and protein concentration are two important parameters that determine the specific activity of enzyme. Thus, all variables such as time, ratio, buffer pH, and temperature that affect the activity of enzyme could also have an effect on the specific activity of the enzyme. Differences in protein concentration create differences between the specific activity and activity of an enzyme. The protein concentration is determined by the Bradford, and to evaluate the extraction process of the enzyme the following formula used to measure the specific activity [21]. Specific activity (U/mg) = Total activity (U) …….Equation (1) Total Protein (mg) 2.3.3 Determination of Temperature Stability The thermo-stability of enzymes is determined by incubating 500µL of the extracted enzyme in 500µL of 50 mM acetate buffer (pH 5.0) at various temperatures for processing time (30 minutes). The residual enzymatic activity was then measured using n amylolitic enzyme activity assay [20]. Small change in the active site of enzyme results in loss of stability. Temperature is one of the important parameters that can cause enzyme denaturation by enhancing molecular vibration, which is responsible for the breaking of intermolecular bond. 2.3.4 Determination of pH Stability The effect of pH on enzyme stability is determined by incubating the reaction mixture at various pH levels using the buffers of: 0.1 M glycine-HCl (2.0 - 3.5), 0.1M sodium acetate (pH 4.0 - 5.5); 0.1M sodium phosphate (pH 6.0 – 7.5); 0.1 M Tris-HCl (pH 8.0 – 9.0); 0.1 M glycine –NaOH (pH 9.5 – 10). After the reactions the extracted enzyme pH stability is tested by pre-incubating at different pH level in the mixed buffer for 30 minutes at room temperature prior to assay [22]; the remaining enzyme activity is then determined using standard assay.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 2.4 Response Surface analysis and Optimization Response surface analysis is used to determine the regression coefficients and statistical significance of the model as well as fit the regression model to the data required to achieve on overall optimum region for all response variables studied. The optimum enzymatic extraction condition is predicted according to the following equation: Y= β0 + β1X1 + β2X2 + β3X3 + β4X4 +β11X1+ β22X2 + β33X3 + β44X4 + β12X1X2 + Β13X1X3 + … and so on according to the number of sample taken. Or Y=

∑

……..Equation 2

Where is the constant coefficient; βi, βij, βij are the coefficients for linear quadratic, and the interaction terms, respectively; xi, and x_j are the independent variables; and e is the error. The resultant matimatical model helps to optimize and determine the level of factors that provide the maximum value for the response variable. The graphical optimization procedure expressed as three-dimensional; (3D) response surface plots. Overlaid plots drawn to visualize the significant (P <0.05) interaction effects of the enzyme extraction variables on the enzymatic properties of the enzyme extracted from the species.

Fig 2.6 Response Surface for Degree of Hydrolysis

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 2.5 Mechanism of Enzymatic Hydrolysis of Proteins Enzymatic hydrolysis of a protein, which consists of the enzyme-catalyzed cleavage of the peptide bonds that structure the protein chains, consumes one molecule of water in a nucleophile attack for each broken link. This results in the release of amine and terminal carboxyl groups with the dependence of the protonation state on the pH of the medium [46]. In the hydrolysis of an amide bond at an alkaline pH, the following steps are followed: -CHR’-CO-NH-CHR”-+H2O

-CHR’-COOH+NH2-CHR”

The terminal carboxyl group is completely dissociated: -CHR-COOH+NH2-CHR”

-CHR’-COO+NH3-CHR”

The added base neutralizes the proteins: NH3-CHR”+OH

NH2CHR”+H2O

2.6 Methods of Hydrolysis Control The degree of hydrolysis (DH) is commonly used to quantify the Progress of the reaction, which is defined as the ratio between the number of peptide bonds released and the number of bonds present in the native protein. In general, the methods for the determination of DH are based on the following: 1) Estimation of soluble nitrogen 2) Determination of released α-amino groups 3) Evaluation of the protein released in the hydrolysis, and 4) Decrease in the cryoscopy Point. [21] DH = Number of peptide bonds hydrolyzed …….Equation 3 Total number of peptide bonds Among the methods frequently used for calculation of DH is the determination of soluble nitrogen after precipitation with tricloacet acid (TCA), trinitrobenzenesulfonic acid (TNBS), and orthophenylaldehyde (OPA), which react with free α-amino groups. There is also titration with formaldehyde, osmomertic and pH-stat titration, which is the most used approach for enzymatic hydrolysis due to its speed and simplicity [21]. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 Generally to carry out the protein hydrolysis reactions, it is necessary to define the appropriate enzyme-substrate ratio, temperature and pH conditions and the time required by the final DH as determinants on the reaction rate. The pH-stat method allows for obtaining a large amount of information over time, so it is possible to estimate the progress of the reaction in fractions of time to expand the analysis landscape and substantially reduce the amount of effort and resource needed for the study. Through the use of this method, it has been established, for example, that the concentration of available hydrolysable bonds is a factor controlling of available hydrolysis time control the rate if hydrolysis, while the hydrolysis time controls the molecular weight of the peptide fractions and the type of hydro lysates in aloe vera proteins. In addition it has been possible to analyze and improve the controlled recovery of peptide to analyze and improve the controlled recovery of peptide with biological applications [19]. The enzymatic hydrolysis reaction is highly complex due to I. II.

The varied and often unknown nature of the substrates, The formation of new substrates for hydrolysis as the reaction progresses,

III.

Inactivation of the enzyme over time [20]

IV.

Effects associated with the reactivity of the linkages related to the enzyme used as well as the accessibility if specific reaction site, and

Sometimes reaction occurs in heterogeneous system [21], which mainly occurs when muscular fractions and insoluble fat portions are hydrolyzed, as is the case for hydrolysis of some aquaculture product and by products.

Textile chemical processing today, particularly the pre-treatment processes require a highly sophisticated technology and engineering to achieve the well-known concepts of “Right first time, Right every time and Right on time" processing and production. Chemical pre-treatment may be broadly defined as a procedure mainly concerned with the removal of natural as well as added impurities in fabric to a level necessary for good whiteness and absorbency by utilizing minimum time, energy and chemical as well as water. DH includes the concentration of the intact protein and the released peptides. In this approach, Marquez and Fernandez [22] and González-Tello et al., developed the structure of a model that considers the effects of inhibition by substrate and product as well as the contribution of

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 enzymatic deactivation phenomena simultaneously through the experimental analysis of kinetics of enzymatic hydrolysis of protein based on MM equation. d(DH) = α EXP [-b(DH)] ……….Equation (4) dt Thus, the models of the general form of the equation (1) can be used successfully in the study of protein hydrolysis reactions. This equation models the DH as a function of the reaction time (t) as well as an independent variables along with two kinetic constants (a,b) (see equation 4). In this model a & b can have different expressions depending on the proposed reaction mechanism, which defines the kinetic parameters of the system. The model successfully describes the typical shape of protein hydrolysis curve over time and was adjusted to the experimental data with high precision, combining kinetic bases, simplicity and predictability for many applications [20]. In this project the researchers aimed to provide a sophisticated technology to enhance the dye uptake of the fabric (Cotton), by contributing an enzyme found in the aloe vera plant (carboxypeptidase, amylase) for the de-sizing agent biocatalyst extracted from Aloe vera plant (aloe gel).

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 CHAPTER THREE 3. MATERIALS AND METHODS Extracting Aloe Gel from the Aloe Vera Plant

De-Sizing of Cotton Fabric by Aloe Vera Gel

Comparing Conventional De-Sizing and New De-Sizing Method

Analyzing the Dye Ability of Aloe Gel Treated Cotton Fabric Fig 3.1 Work Flow of Project Methodology

3.1 Materials Required 3.1.1 Plant Material The herb, Aloe Vera plant were gathered in Ethiopia, Region of Addis Ababa. Specimens were delivered to the school of Textile, apparel and fashion design and plants were identified by the Acadamic advisors Mr. Melese and Mr. Khalid as Aloe vera plant. Samples were brought in the chemical lab within 3-4 hour for rapid preparation of gel in order to save the bioactive from losing their effectiveness; the leaves used for extractions measured between 60 and 80 cm in length and were taken around 2-3year old plants. Either soxhlet or centrifuge extraction method were selected according to the quality of the gel.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.1.2 Fabric Sample Collection  Grey Fabric Weight (g) = 5.4

 Weave structure - Plain weave (1/1)

 Ends/cm - 42

 Dimension of the samples - 10.5 X 8 (cm)

 Picks/cm - 32

 Material – 100% Cotton Grey Fabric

3.1.3 Equipment  Extraction Equipment Sharp knife for cutting the aloe leaf and filleting the leaf, Mechanical stirrer for crushing the aloe gel in to liquid, distillation flask for separating the gel from the liquid, Centrifuge Model 800B, Vacuum pump for filtrating the extracted gel, Plastic bottle for Storing the gel and Refrigerator for storing the extracted gel.  Processing Equipment: Digital weight balance for weighing the fabric samples and chemicals, digital temperature sensor, Litmus paper for pH controller, timer watch for timing the process, essential laboratory instruments (Beakers, Stoves, Stirrers, gloves, etc...).  Testing Equipment: Dropper, Digital weight Balance, Spray rate tester, magnification glass,

3.1.4 Chemicals used for the Project 3.1.4.1 Chemicals used for Extraction of Aloe Vera  Ethanol used for aloe vera extraction, Citric acid for maintaining the pH and improving the flavor of Aloe Vera gel and Charcoal 3.1.4.2 Chemicals used for Pretreatment and dyeing of the Fabric A, Desizing Chemicals  Hydrochloric acid (HCL), Sodium Chloride (NaCl), Extracted Aloe gel and Wetting agent (Anionic & Non-ionic). B, Combined Treatment Chemicals (Scouring + Bleaching)  Caustic Soda (NaOH), Hydrogen peroxide (H2O2), Silicate (Si), Trisodium Phosphate (TSP) De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 C, Testing Chemicals  Iodine for testing the presence of starch, distilled water for absorbency test, D, Dyeing Chemicals  Direct dyes in the form of paste, Sodium Hydroxide (NaCl), Sodium Carbonate (Na2CO3),

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.2 Methods 3.2.1 General Procedure for Extraction of Aloe Vera Plant

Aloe vera leaf

Fresh water

Sun Light

Washing

Dirt Materials

Cutting

Tips and butts

Treatment the Leave

Fileting

Rind and Exudate

5°C temperature Ethanol

Homogenizing

4000 rpm speed 30 minutes

Separation process

Pulp (Unwanted materials)

4000 RPM Time 30 minutes

Extraction

Temperature 5°C

Crude Gel Charcoal

Purification

Anthroquinone

Vacuum Filtration Extracted Aloe

Optical density: 0.218(abs) Viscosity 0.675 Refractive index: 1.33550 TSS content: 0.93 (Brix)

Storage at 4°C in air tight bottle

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.2.1 Procedures for Separation and Extraction of Aloe Vera Leaves 3.2.1.1 Preparation and separation of aloe vera Leaves 1) Washing with fresh water to remove the dirt, 2) Trimming the tip and butts of the leave, 3) Pre-treat the aloe leaf, 4) Filleting the rind and exudates. (Remove the rinds), 5) Addition of Ethanol according to volume of the gel Grinding the filleted aloe gel by mechanical stirrer, 6) Separation of the gel from the liquid using Distillation flask, 3.2.1.2 Extraction of aloe gel by centrifugation process 1) Gel extraction by centrifuge (5°C, 4000 rpm for 30min), 2) Crude gel of approximate 100 ml obtained, 3) Purification of the gel by adding Activated Charcoal of 0.1 g/100ml crude gel, 4) Filtration continues using Vacuum Filtration , 5) Obtain a pure gel with the following properties (Chandegara, 2005 ), -

Viscosities 0.675 (stocks)

Refractive index: 1.33550

Optical density: 0.218 (abs)

TSS content: 0.93 (brix)

6) Finally store the pure gel at 4°C in air tight bottle.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.2.2 Preparation and Extraction of Aloe gel 1

Washing and Cleaning

Fig 3.2 Washing and Cleaning of Aloe Vera Leave Cleaning (washing the leave by tape water and scrub brushes), this will remove the dirt and dust particles that are found in the skin of the leaves. The leaves were cleaned with cleaning brush. 2

Cutting the Leave in to Three Section

Fig 3.3 Cutting the Top, Bottom and Rind Part of the Leave Cutting 1 inch of the base (white part), the tapering point of the leaf top (2-3 inch) and the short sharp spines located along the leaf margins are removed by a sharp knife. Cutting process is done carefully in order to separate the green leaves from other browning colors found in the plant. The process is done carefully in order to avoid contamination with other substances.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3

Pre-conditioning the Aloe leaves

Fig 3.4 Pre-conditioning of the Aloe Vera The aloe vera leaves are pre conditioned by sun drying make it suitable for extraction of gel that gives 93.42% gel extraction efficiency. The sun drying, yielded highest crude gel recovery and lesser residual time without aloin contain as compared to fresh leaves (Anonymous, 2008c). 4

Filleting Operation

Fig 3.5 Filleting Process and Filleted gel The filleting (skinning) of the leave is critical process that is used to separate the gel from the rind (cuticle). Hand-filleting method has been used to remove the gel from the rind by using sharp knife, keeping anthroquinone level low, but in this process most of the gel is left on the working table. This process is done carefully to avoid the decomposition of the biological activity; the filleting operation is completed within 36 h of harvesting the leaves [10]. After filleting 200ml of crude gel were obtained thus 100ml of ethanol were added with the gel.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 5

Homogenizing/Grinding

Fig 3.6 Mechanical Stirring of the Gel In this method the whole leaf after filleting operation the colorless gel was ground in a mechanical stirrer of 10,000 rpm and centrifuged for 20 minutes in order to remove the fibers. The method used to extract gel polysaccharides by alcohol precipitation has been conducted by adding 100 ml of ethanol to the aloe gel and stirred for 20 minutes by mechanical stirrer. 6

Separation of the liquid gel

Fig 3.7 Separation of Liquid Gel from Unwanted Materials After stirring (grinding) process is completed the alcohol-aloe gel mixture is then mixed in the Distillation flask and left to stand for 4hr. The clear supernatant that forms is decanted or siphoned off, without disturbing the precipitate in the beaker and the gel with the alcohol (ethanol) mixture changes its color due to long storage (24 hr). De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 7

Extraction by Centrifuge Method

Fig 3.8 Extraction of the Liquid Gel with Centrifuge The gel in the beaker is collected in the small tubes (7) of the centrifuge with 5°C, 5000 rpm for 30 minutes, and obtains a crude gel. The crude gel is being pressed with a pressure and the liquid gel was obtained in the beaker. Citric acid was also added prior to purification in order to reduce the browning reaction, to improve the flavor of Aloe vera gel juice and to stabilize the juice [12, 13, 14, and 15]. 8

Purification and Stabilization

Fig 3.9 Purifying the gel and removing of the Cellular Material Citric acid was added prior to purification in order to reduce the browning reaction, to improve the flavor of Aloe vera gel juice and to stabilize the juice (0.1 g/ 100ml) [12, 13, 14, and 15]. The pH value of the gel is then 5 which is a weak acid.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 9

Filtering the Crude gel

Fig 3.10 Vacuum Filtration of the Liquid Gel

The filtration process is conducted by using a vacuum pump. The pump goes through the tube to the beaker hole, the gel is filtered with the filtration paper in the same beaker, and by using an air pressure, the vacuum pump pull the aloe gel under the filtration paper without the presence of oxygen in the filtrated gel. The filtration paper takes 6hr. 10. Storage

Fig 3.11 Pure Gel Stored in Bottle After filtration was done the pure gel juice is of 100ml were obtained and poured in the bottle that is covered with aluminum foil to prevent light that reduce the ability of bioactivity of the gel. Finally the bottle was stored by freeze dried at 4°C. 11. Storage Stability The storage stability is one of the most important parameter to be considered in enzyme extraction procedure. The temperature did not show any significant effect on the storage De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 stability of enzyme after two weeks, a favorable characteristic did not exhibited by the enzyme in this study. In addition the sample extracted at pH 5.0 demonstrated the highest storage stability, which suggests that the active site of the enzyme is stable at this pH and could properly interact with the substrate after the storage time. The enzyme mixing time for 20 min was more stable than long or shorter period of extraction. This indicates that the tertiary structure of the enzyme was not damaged during this mixing period.

3.3 Processing Design of Wet Processing Preparation of samples

Optimization of Process Variables

Desizing of Samples

Comparing the Desized Samples

Scouring + Bleaching the Samples

Testing the Pretreated Samples

Dyeing the Treated Sample

Analyzing the Dye ability of Samples Fig 3.12 Processing Design of Wet-processing De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.3.1 Procedures for Desizing 1

Take sample of grey fabric for each desizing method according to the material liquor ratio (MLR) provided.

Dry the sample and weight the dried sample.

Make 65X65 mm size on each of the sample for shrinkage test.

Prepare the desizing liquor according to the respective recipes for the each method.

Cheek the solution PH.

The fabric sample for enzymatic desizing method is washed in hot water (80-95°C) to gelatinize the starch.

Put the fabric sample in the respective desizing liquor, and carry out desizing according to time and temperature specified by the condition for the respective desizing.

Carry out after treatment – rinse with hot water (70 - 90°C) and give a cold wash

Dry – and weigh each sample.

10 Finally level the de-sized fabric samples

3.4 Standardization of desizing parameter 3.4.1 Optimizing the Enzyme Concentration Temperature and pH Recommended process parameter for the aloe gel enzyme were not available for desizing, but pH 5 – 7.5, temperature 50 - 60°C and 5 – 20 % of enzyme concentration were used for degrading the starch. To achieve optimum recipe of aloe gel for desizing of a given fabric, the researcher conduct the research by keeping the MLR constant but varied the process variables according to the range of recipe given for the recipe. Finally, the best result is selected based on the testing result of samples property (Test result) and selecting the best sample for the project.  Concentration of aloe gel using the following % of concentration to determine the optimal Conc. = 3%, 4%, 5%, 6% and 7%.  Temperature using the following temperatures to determine the suitable process Temp = 40°C, 50°C, 60°C & 70°C  pH using the following stabilizes to determine the best process pH = 5 - 6, 6 - 7 & 7 - 8 De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 The concentration of aloe gel is determined based on the processing of conventional Biolase enzyme of enzymatic desizing method. The aloe gel contain enzymes that is used for degrading the starch, thus

concentration of enzymatic desizing method is the source for

conducting the present experiment. The concentration of enzymatic desizing is performed in a concentration of 3 – 7% according to material liquor ratio (1:20). And also, the temperature and pH highly affect the enzymatic activities. Lower and higher temperature with pH is selected according to the concentrations. Researchers used 12 samples with different concentration, temperature and pH for determining the optimum recipe of aloe gel for the process of desizing. To find the effect of above mentioned factors the researchers have carried out the desizing process by the following ways. 1. Determining by varying concentration and making the temperature and pH constant. 2. Determining by varying temperature and making the concentration and pH constant. 3. Determining pH by making by making the concentration and temperature constant.

Fig 3.13 Determination of Optimal Concentration, pH and Temperature

3.5 Desizing Activities of Conventional type and New Method Enzymatic Process By assigning A for the Acid Desizing Sample B for the Aloe gel Enzyme and C for the Oxidative desizing samples then the process can be compared. 3.5.1 Conventional Desizing using Acid (A) Table 3.1 Recipe for Acid Desizing of Cotton Fabric Chemical used

Concentration (% o.w.f)

MLR: 1:20 PH: 1 - 2

0.5 – 1

HCL Wetting agent

0.3[optional]

Temperature: 30 - 60°C Time: 2 hr

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.5.2 Conventional Desizing using Oxidative (C) Table 3.2 Recipe for Oxidative Desizing of Cotton Fabric Chemical used

Concentration (% o.w.f) MLR: 1:20

Hydrogen peroxide

2-4

Caustic soda

3-4

PH: 8 - 9

Sodium silicate

2-3

Temperature: 90°C

Magnesium sulphate

0.05

Wetting agent

0.5

Time: 10-15 min

3.5.3 Enzymatic Desizing (B) Table 3.3 Recipe for Enzymatic Desizing of Cotton Fabric Chemical used

Concentration %(o.w.f)

MLR: 1:20

Enzyme (Biolase)

(5-20)

PH: 5 - 7.5

Sodium chloride

Nonionic wetting agent

1.5

Temperature: 50 – 60°C Time – 90 minutes

3.6 Testing the Desized Fabric Samples 3.6.1 Weight loss calculation (Wt. %) Weights lose percentage was calculated by comparing the weight of de-sized sample with the original grey cotton fabric sample or with the given formula; Weight loss % = Grey Fabric Weight – Desized Fabric Weight X 100 Grey Fabric Weight 3.6.2 Test for Starch (Iodine Test) Iodine test is used for starch detection. A drop of iodine solution placed on a test samples resulting in a characteristic of change in color.  Blue color, the starch is not removed.  Resulting of brown color, the starch has been modified (removed). De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021  Resulting in a colorless, on the specimen the starch is fully removed. 3.6.3 Absorbency/wettability Test (AATCC 22) The wettability of the samples is measured by the spray rate testing method. This method is applicable to any textile fabrics, which may or may not have been given a waterrepellent finish. It measures the resistance of fabric to wetting by water. After conducting the test the fabric should immediately compare with the rating chart and select the nearest level on the rating chart.  Rating the Absorbency of Fabrics

Fig 3.14 Standard Spray Test Rating Chart The wettability of fabrics is immediately compared with the standard spray rating chart and grading according to their nearest similarity. As shown in figure 3.17 the desized fabrics and gray fabric were tested and occupies different wetting surface area in the fabric. 3.7 Combined Pre-treatment 3.7.1 Procedure for Combined Pre-treatment (Scouring + Bleaching) 1. Prepare solution and desized fabric sample according to MLR 2. Measure the absorbency of desized fabric 3. Dry and weigh the sample to be treated 4. Mark 65 X 65 cm on the sample for shrinkage test 5. Place the sample on the holder 6. Set the processing temperature, time and gradient of the treatment 7. Place the solution into beaker and heat it 8. Place the sample into the solution (beaker) with holder and carry out combined treatment at fixed temperature for given time De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 9. Then rinse with hot and following with cold water 10. Dry and condition the sample before weighing 11. Level the treated fabric 12. Calculate weight loss and measure reflectance, absorbency, picks and ends density per centimeter of treated fabric 3.7.2 Combined Scouring and Bleaching of Desized Fabric Samples Table 3.4 Recipe for Combined Pre-treatment (Sample A, B & C) Chemical used

Concentration %(o.w.f) MLR: 1:40

Caustic soda

Hydrogen peroxide

PH: 11

Silicate

Temperature: 70 - 90°C

TSP

Time: 2 hr

3.7.3 Testing the Combined Treated Fabric Samples 3.7.3.1 Weight loss calculation (Wt. %) Weights lose percentage was calculated by comparing the weight of de-sized sample with the original grey cotton fabric sample or with the given formula; Weight loss % = Grey Fabric Weight – Desized Fabric Weight X 100 Grey Fabric Weight 3.7.3.2 Absorbency/wettability Test (AATCC 22) The wettability of the samples is measured by the spray rate testing method. This method is applicable to any textile fabrics, which may or may not have been given a waterrepellent finish. It measures the resistance of fabric to wetting by water. After conducting the test the fabric should immediately compare with the rating chart and select the nearest level on the rating chart (figure 3.18).

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 3.8 Dyeing with Direct dyes 3.8.1 Type of direct dyes used for the project  Class C: Temperature controllable dyes 3.8.2 Preparation of dye solution: The dye is dissolved by pasting it with minimum amount of water and soda ash, and then boiling water was added to the paste according to recipe and follow with constant stirring. 3.8.3 Dyeing Procedure The dye bath is set with required volume of concentration of dyes 0.5% soda ash and water to make the desired M: L ratio. Then the processes material is entered in the dye bath at 40°C and dyeing is carried out for 15 minutes. The required amount of salt is added in even interval of 15 minutes. The salt varies between 5 to 20% on weight of material for light to heavy shade. The temperature of dye bath is slowly raised to boil and is continued as this temperature for the period of 45 to 60 minutes and then it is cool for 15 to 20 minutes for better exhaustion. The samples are then removed from the dye solution, squeezed and dried and after treatment in solution of dye fixing agent after precise drying stage. 3.8.4 Recipe of Direct Dye Table 3.5 Recipe for Direct dyes Chemical used Direct Dye Stuff Soda Ash Common Salt

Concentration %(o.w.f) 5% 0.5-1%

MLR: 1:20 Temperature: 100°C Time: 15 – 60 min

----

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 CHAPTER FOUR 4. RESSULT AND DISCUSSION 4.1 Optimization the Enzyme Extracted from Aloe Vera 4.1.1 Effect of Concentration By varying the concentration of aloe gel and keeping the temperature, pH and MLR constant, the researchers observed that the best aloe gel can be obtained in the following condition:  MLR 1:20  pH: 5 – 7.5  Temperature: 50-60°C  Time: 1hr To observe the effect of concentration of enzymes in aloe gel, the researchers have been desized the samples with 3%, 4%, 5%, 6% and 7% of aloe gel concentration which results obtained are depicted in the following table: Table 4.1 Effect of Concentration of Enzymes in Aloe gel Desizing Result Properties

Concentration of aloe gel 3%

Fabric weight

5.197 g

5.185 g

5.117 g

5.120 g

5.156 g

5.175 g

5.23 g

Weight loss

3.759%

3.981%

5.241%

5.185%

4.519%

4.16%

3.14%

Color Change

Bluish

Brown

From this table, the lower the fabric weight the higher the starch is removed from the fabric. The result weight of the fabric weight loss % leads to identify the best process for determining the concentration. Thus, it can be concluding that “as the aloe gel concentrations increases, the weight loss increase significantly”. Increase in concentration of enzymes cause an increase in strength loss. Optimum percentage weight loss is obtained at near 5% concentration.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 6 5 4 3

fabric weight

weight loss%

1 0 3%

Fig 4.1 Effect of Concentration of aloe gel enzyme desizing on Fabric weight 4.1.2 Effect of Temperature Temperature affects the performance of enzymes found in the aloe gel. Each enzyme has an optimum temperature range, where enzyme activities gone maximum. Hence it is essential to determine the optimal temperature of aloe gel enzymes. Increase in temperature decrease the enzyme activity and the enzymes activity came to almost zero and deactivated at temperature of 70°C. Lower temperature shows reduction in reaction speed but doesn’t deactivate the enzyme. It is therefore possible to use lower temperature by longer cycle. To observe the effect of Temperature of enzymes in aloe gel, the researchers have been treated the samples with 45°C, 50°C, 55°C and 60°C temperature that results obtained are depicted in the following table.  MLR 1:20  pH: 5 – 7.5  Time: 1:30hr  Concentration: 5% Table 4.2 Effect of Temperature of Enzymes in aloe gel Desizing Result Properties

Weight loss (%)

Temperature 40°C

45°C

50°C

55°C

60°C

70°C

2.6481

3.2778

4.225

5.122

5.5741

4.305

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 The optimum result is obtained at 55-60°C temperature. Thus, the rate of reaction of the enzyme activated rapidly at this point.

weight loss % 6 5 4 3

weight loss %

1 0 40°C

45°C

50°C

55°C

60°C

70°C

Fig 4.2 Effect of Temperature of Aloe Gel Enzyme Desizing on Fabric Weight 4.1.3 Effect of pH pH is a critical factor affecting the efficiency of enzymes. To observe the effects of pH on aloe gel the researchers desized the samples with 5% concentration at various pH of 5-6, 67, 7-8 using citric acid as pH controller.  MLR 1:20  Temperature: 55 - 60°C  Concentration: 5%  Time: 1 hr Table 4.3 Effect of pH of Enzymes in Aloe gel desizing Result Properties

Weight loss (%)

pH Values 5

5.5

6.5

3.185

3.256

4.25

4.98

5.741

4.8

The researchers observed from the result indicated that pH 7-7.5 was more suitable for the enzyme activity. Either the increase or decrease in pH beyond the optimum value showed decline in the enzyme activity. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 pH / weight loss % 7 6 5 4 pH / weight loss %

2 1 0 5

5.5

6.5

7.5

Fig 4.3 Effect of pH of Aloe Gel Enzyme Desizing on Fabric Weight From the above experiments it was conclude that the enzymes are active at the period of 40 – 60 minutes and concentration of 5%, temperature of 55-60°C and pH of 7-7.5. Thus, the Standardization of the enzymes as follows: Table 4.4 Recipe for Enzymatic Aloe gel Desizing of Cotton Fabric Chemical used

Concentration %(o.w.f)

Enzyme ( Aloe gel)

5-20

Sodium chloride

Nonionic wetting agent

1.5

MLR: 1:20 PH: 7-7.5 Temperature: 55 - 60°C Time – 40 – 60min

4.4 Determination of Amount of Water and Chemicals based on Fabric Weight  Amount of water - is prepared according to Material Liquor Ratio 1:20 and Grey Fabric weight as follows; 1,000 gram Sized Fabric weight (5.4 gram)

= 20,000 ml of water =

Amount of water used for the solution = 108 ml

 Amount of chemicals - is prepared according to Fabric weight and given concentration by the following Formula; Required amount of Chemicals = Fabric Weight X Concentration of Chemical

100

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021  Amount of chemicals for acid desizing Hydrochloric Acid - 1%

Wetting Agent – 0.3%

= 5. 4 gram X 1

= 5. 4 gram X 0.3

100

= 0.054 gram of HCL

= 0.02 gram of Wetting agent

 Amount of chemicals for aloe gel desizing Aloe gel = 5%

Sodium Chloride – 5%

Non-ionic wetting agent – 1.5%

= 5.4 gram X 5

= 5. 4 gram X 1.5

100 = 0.3 gram of Aloe gel

100 = 0.3 gram of NaCl

100 = 0.1 gram of N.I.W.A

 Amount of chemicals for oxidative desizing Hydrogen peroxide – 3% = 5. 4 gram X 3 100 = 0.162 gram of N.I.W.A

Caustic soda – 2%

TSP – 2%

= 5. 4 gram X 2

= 5. 4 gram X 1.5

100 = 0.1 gram of N.I.W.A

Silicate – 2% = 5. 4 gram X 1.5 100

= 0.1 gram of N.I.W.A

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 4.5 Experimental Process of Desizing 4.5.1 Desizing of Samples

Fig 4.4 Desizing of Samples with Optimal Temperature and pH

Fig 4.5 After-treatment Process of Hot and Cold Water Wash of the Samples 4.5.2 Testing the Desized Fabric Samples 4.5.2.1 Weight loss calculation (Wt. %) Weights lose percentage was calculated by comparing the weight of de-sized sample with the original grey cotton fabric sample or with the given formula; Weight loss % = Grey Fabric Weight – Desized Fabric Weight X 100 Grey Fabric Weight

Fig 4.6 Weighing Balance for Desized Fabric samples The grey fabric sample was weighed 5.4 gram and the desized fabric sample with acid is weighed 5.058 (W1) gram, 5.1 gram (W2) and 5.117 gram (W2) for enzymatic aloe gel desizing. So, the weight loss % of the samples is calculated. Weight loss percentage for aloe De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 gel enzymatic desizing method is 5.24% 5.5% for oxidative method and 6.3% for acid desizing method. 4.5.2.2 Test for Starch (Iodine Test) Iodine test is used for starch detection. A drop of iodine solution placed on a test samples resulting in a characteristic of change in color.  Blue color, the starch is not removed.  Resulting of brown color, the starch has been modified (removed).  Resulting in a colorless, on the specimen the starch is fully removed.

Fig 4.7 Determination of the Removal of Starch with Iodine  Absorbency/wettability Test (AATCC 22) The wettability of the samples is measured by the spray rate testing method. This method is applicable to any textile fabrics, which may or may not have been given a waterrepellent finish. It measures the resistance of fabric to wetting by water. After conducting the test the fabric should immediately compare with the rating chart and select the nearest level on the rating chart.

Fig 4.8 Wettability of Gray and Aloe gel Desized Fabric by Spray Rate Tester

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021  Rating the Absorbency of Fabrics

Fig 4.9 Standard Spray Test Rating Chart The wettability of fabrics is immediately compared with the standard spray rating chart and grading according to their nearest similarity. As shown in figure the desized fabrics and gray fabric were tested and occupies different wetting surface area in the fabric. 4.6 Comparing and Assessing the Desized Samples Table 4.5 Comparing De-sized Fabric Samples S.N

Parameters

Grey Fabric

Aloe gel Desizing

Acid Desizing

Oxidative Desizing

Rot Stepping

GSM

EPC

PPC

Iodine

Blue

Brown

Weight loss

---

5.2%

6.3%

5.5%

5.3%

Time

---

35 minutes

1:30 hr

15 minutes

16 hr

Wettability

Repellant

Absorbent

Feeling test

Hard

More Softer

Soft

More Softer

Rough

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 7 6 5 4 Fabric weight 3

Weight loss

2 1 0 Aloe Gel

Acid Desizing

Oxidative Desizing

Rot Steeping

Fig 4.10 Comparison of Conventional desizing Method with the New Method of Aloe gel

4.7 Comparative Study on the Effect of Aloe gel enzymatic desizing Process After conducting the desizing process the desized fabrics and gray fabric were analyzed by their fabric density, feeling test, test for the presence of starch by iodine, weight loss % and wettability. The grey fabric sample was weighed 5.4 gram (W1) and the desized fabric sample with acid is weighed 5.058 gram, 5.117 gram (W2) enzymatic aloe gel desizing and 5.1 gram (W3) for oxidative method. So, the weight loss % of the samples is calculated. Weight loss percentage for oxidative method is 5.5%, aloe gel enzymatic desizing method is 5.2% and 6.3% for acid desizing method. And also the fabric density varies in each process because of the weight loss presentation in the sample, 4.7.1 Comparative Study on the Effect of Desizing on Fabric Weight Loss % Table 4.5 clearly indicated that the fabric treated in conventional pre-treatment process is much higher than in enzymatic desizing. Higher weight loss in traditional method maybe due to severity of treatment, which result in greater removal of natural and added impurities. There is also the possibility of degradation of cellulose because of harsh alkaline treatment. Enzymatic processing shows lower weight loss and this may be due to controlled treatment. And also there is too much difference between the weight loss percentages of the samples that indicate degradation of fibers in acid desizing is higher than aloe gel sample. This results shows the reason that there is high (%) weight loss but less removal of starch indicates fiber De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 degradation while the process of acid desizing, about approximately 1% difference on weight loss between acid and aloe gel desizing. 4.7.2 Comparative Study on the Effect of Desizing on Presence of Starch (Iodine Test) Fig 4.7 clearly shown that there is a brown color appears when the iodine was added on the fabrics this indicate that all the samples including the aloe gel enzymatic process remove the starches. As the above picture shows that the starch is removed in both conventional and Aloe gel desizing methods. But, the depth of brown color in the aloe gel desized fabric (sample B) is a lighter than acid desized fabric (sample A), this indicates that approximate full starch materials is removed in aloe gel enzymatic desizing method while the starch are being modified in acid desizing. 4.7.3 Comparative Study on the Effect of Desizing on Feeling Test of Samples As the researchers described on the weight loss study the weight loss % between the aloe gel enzyme and acid desizing samples can further confirmed by the ability of samples to regain water absorbency test and also during feeling test. As the researchers described in the above (table 4.5) the feeling or the sensitivity (physical appearance) of the fabric surface for acid desized sample is less soft as compared to aloe gel sample. The more softness of samples infers the more ability of fabrics to absorb water or to become wettable. 4.7.4 Comparative Study on the Effect of Desizing on Wettability of Samples The wettability test of samples for aloe gel desized sample shows a complete wetting of the entire face of the sample that is near to AATCC 50 (ISO 1 Grade) & grade of AATCC 50 (ISO 1 Grade) is near to the acid desized fabric and AATCC 100 (ISO 5 Grade) is given for the gray fabric. Therefore, aloe gel desized fabric has an excellent absorbency as same as acid desized fabric and grey fabric as well. The result of wettability of samples results approved that, the aloe gel desized fabric has good absorbency in other word the starch present in fabric has been fully removed by the enzymes this make the sample suitable for next processes of pretreatment (Scouring and bleaching process) 4.7.5 Comparative study on the effect of Desizing on Fabric Density The desized fabrics varies in EPC and PPC hen compared to grey fabric, thus the presence of change in density of fabrics is based on the impurities removed in the Cotton swatches. Increase in fabric density resulted because of much removal of impurities and vise verse. De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 350 300 250

time/min

200 temerature °C pH

150 100 50 0 Acid Desizing

Rot Stepping

Oxidative Desizing

Enzymatic Desizing (Aloe gel)

Fig 4.11 Comparison between Conventional and Biocatalyst Desizing Method

4.8 Analysis of Combined Pre-treatment process The comparison above shows that the aloe gel desizing can further treated for achieving the objective of pretreatment which is preparing the samples for dyeing.

Thus the aloe gel

enzymes are further treated to corroborate the ability of the sample on the dyeing. The researchers choose one of the samples of conventional desizing process (Acid desizing sample) in order to compare the dye-ability. By assuming the samples A for the Acid desized sample and B for the aloe gel the process continues for further analysis. 4.8.1 Determination of Amount of Water and Chemicals based on Fabric Weight  Amount of water - is prepared according to Material Liquor Ratio (1:40) and Fabric weight. Sample are treated in different solution because the weight of the samples varies that determine the amount of water and chemicals. By considering the sample that is desized by Acid is (Sample A) and Aloe gel (Sample B). If, 1,000 gram

= 40,000 ml of water

Sample A 5.058 gram = Amount of water used for the solution = 202 ml Sample B 5.117 gram = Amount of water used for the solution = 204 ml

 Amount of chemicals - is prepared according to Fabric weight and given concentration by the following Formula;

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 Amount of Chemicals = Fabric Weight X Concentration of Chemical 100  Amount of Chemicals required for Sample A  Caustic soda – 2%

 Hydrogen peroxide = 3%

 Silicate – 2%

 TSP – 2%

= 5.058 gram X 2

= 5.058 gram X 3

= 5.058 gram X 2

100

= 0.1 gram

= 0.16 gram

= 0.1 gram

100 = 0.1 gram

 Amount of Chemicals required for Sample B  Caustic soda – 2%

 Hydrogen peroxide = 3%

 Silicate – 2%

 TSP – 2%

= 5.117 gram X 2

= 5.117 gram X 3

= 5.117 gram X 2

100

= 0.1 gram

= 0.153 gram

= 0.1 gram

100 = 0.11 gram

4.8.2 Combined Treatment of the samples

Fig 4.12 Combined Treatments of the Desized Samples

Fig 4.13 After-treatment Process with Hot and Cold Water

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 4.8.3 Testing the Combined Treated Fabric Samples 4.8.3.1 Weight lose calculation (Wt. %) Comparing the weight of treated samples with the original grey cotton fabric sample and find out the weight lose percentage. Additionally the deference between conventional method and aloe gel were determined for further analysis. Wt. % = W1 – W2 X 100 W1

Fig 4.14 Weighing the Desized Fabrics The weight of sample A is 5.003 gram and 5.055 gram for sample B. These results indicate that there is a loss in weight in both fabrics and little deference between the results. The weight loss % for sample A is 7.351 and 6.7422 for sample B. 4.8.3.2 Wettability/Absorbency The wettability of the samples is measured by the spray rate testing method. After conducting the test the fabric should immediately compare with the rating chart and select the nearest level on the rating chart.

Fig 4.15 Wettability Testing of Aloe Treated Fabric De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 The wettability of fabrics is immediately compared with the standard spray rating chart and grading according to their nearest similarity. As shown in figure 4.12 the aloe gel desized fabric was tested and too much area of wetting surface in the fabric. The combined treated weight of sample A is 5.003 gram and 5.055 gram for sample B. These results indicate that there is a loss in weight in both fabrics and little deference between the results. When compared the fabric weight of combined treated fabric with desized fabric there is a loss in weight that indicate the removal of impurities from the fabric about 0.114 gram of impurities from aloe gel desized sample and 0.003 gram for Acid desized fabric. Wettability of the samples after combined process increase because of hydrophobic part of impurities removed during the treatment. Thus, the result for wettability test shows that there is a complete wettability of a fabric AATCC 0 (ISO 0 Grade: 0), this implies that almost all the impurities in the cotton are successfully removed which means the fabric is >95% ready for through for further process like dyeing and printing.

4.9 Comparing the Treated Fabric Sample Table 4.6 Comparing the Combined Pre-treated Fabric Samples Parameters

Sample A

Sample B

EPC

PPC

7.3519%

6.7422%

Excellent absorbency

GSM

Weight loss % Wettability

weight loss % 7.5 7 weight loss %

6.5 6 Sample A

Sample B

Fig 4.16 Comparison the weight of Conventional and Biocatalyst Desizing Method De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 4.10 Experimental Process of dyeing 4.10.1 Dyeing of the Fabrics with Direct Days

Fig 4.17 Dyeing of the Pre-Treated Samples with Direct dyes 4.10.2 Color fastness of sample to washing (EN ISO 15703 / IUF423) Fastness of color of sample to hand washing is the resistance to washing under mild domestic laundering in water. In washing samples, only change in color can occur in the sample, but color substances may bleed from it and may stain adjacent samples. The sample of dyed samples in contact with un dyed textiles, agitated in natural solution of a synthetic detergent for 30 minutes at 30°C, then the samples are rinsed and dried and the samples was compared with standard Grey Scales.

Fig 4.18 Grey Scale Card for Assessing the Color Fatness to Washing

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 4.10.2 Analysis on the color fastness to washing Dyeing is processed with direct dye but the inability to find the fixing agents highly affect the color fastness of the samples. No matter what the depth of the color shows highly in the aloe gel desized fabric. The color fastness of the dyed fabric treated with the enzymatic or AG desized fabric has been tested to washing fastness, other color fastness testing method is not conducted because of the shortage or inability of testing instruments, thus color fastness to washing has been conducted on the sample. The color fastness of the dyed samples as shown in figure 4.19 the depth of the color shows a better in the aloe gel treated fabric than the acid, this indicate that the aloe gel treated fabric losses too much impurities than the acid desized fabric. And the color fastness test result shown the fabric has low color fastness to washing this result is achieved because of the inability of fixing agents present in the sample, thus the fabric shows transference of colored material to the other fabric which is a staining effect in the 3rd wash as shown in the figure below.

Fig 4.19 Dyed Fabric Sample and Staining of Color in the Fabric during Washing Fastness

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 CHAPTER FIVE 5. CONCLUSION AND RECOMMENDATION 5.1 Conclusion The aloe gel treated fabric was exhibited high desizing efficiency. This is due to key – lock mechanism of enzymes present in aloe gel. When compared the desizing efficiency of synthetic enzyme and aloe gel enzyme (natural enzyme amylase) the weight loss is approximate that means the weight loss in synthetic enzyme desizing is 6.3 % and in aloe gel case 5.24% without degradation, so it has good desizing efficiency but aloe gel desizing have side effect of coloring salt. And also, during desizing the softness of the fabric is much better than the chemical treated fabrics in addition to the sensitivity of the fabric that implies the absorbency of the fabric; Aloe gel treated fabric has an extreme wettability than other fabrics thus indicate that there is a high removal of starches than the conventional methods. During other treatment and dyeing process aloe gel treated fabric shows a magnificent ability of dye up take as well as water, this indicate that the weight loss during desizing by hydrochloric acid there is a degradation of cotton that is why the fabric weight is higher than the AG treated fabric & it clearly proved during the dyeing process. The better depth of the color appears in AG treated sample than Acid desized sample. The natural and valuable enzyme from AG has a requires less temperature and time effectiveness than other chemicals that decrease energy cost and because of it processes in neutral atmosphere it decrease the pollution of water to the environment. Generally the researchers conclude that AG treated sample shows a great performance during pretreatment specially in desizing and also at the dyeing process. In addition their low costs make the AG enzymes to be used as a potential enzyme in different industries and biotechnological application. Future work may be focused on commercialization of the process  Future work will focused on studying the characteristics of Aloe Gel,  Utilization of mixed substrate in column extraction method of enzymes for effective practice in desizing process,  Cheek for enzyme stability for further application in textile industry,

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design

THESIS REPORT 2021 5.2 Recommendation Wet processing in textile industries cause adverse changes in the immediate environment and necessitated the development of enzyme based process as alternative to currently employed chemical processes. There effective production, extraction and application will greatly affect their role as an alternative process to combat environmentally hazardous chemicals employed in desizing process. Enzymatic based hydrolysis of starches from the cotton surface is accomplished successfully because of the ability and potential active enzymes found the plant of Aloe vera, in addition there is amylase, and cellulase and catalase which are the most used enzymes in textile wet processing of pre-treatment. Further work can be carried out in combined pre-treatment process (DSB), (DS) and (SB). And also, the enzymes can further be optimized and determined with best extraction method and can be applicable on textiles in the finishing of textiles (softening) and also as a thickener agent in dyeing processes.

De-Sizing Of Cotton Using Aloe Gel Extracted From Aloe Vera Plant School of textile apparel and fashion design