معاونت پژوهش و فناوری
به نام خدا
منشور اخلاق پژوهش
با یاری از خداوند سبحان و اعتقاد به این که عالم محضر خداست و همواره ناظربر اعمال انسان و به منظور پاس داشت مقام بلند دانش و پژوهش و نظر به اهمیت
جایگاه دانشگاه در اعتلای فرهنگ و تمدن بشری ، ما دانشجویان و اعضاء هیات علمی واحدهای دانشگاه آزاد اسلامی متعهد میگردیم اصول زیر را در انجام
فعالیت های پژوهشی مد نظر قرار داده و از آن تخطی نکنیم:
1-اصل برائت : التزام به برائت جویی از هرگونه رفتار غیر حرفهای و اعلام موضع نسبت به کسانی که حوزه علم و پژوهش را به شائبههای غیر علمی میآلایند.
2- اصل رعایت انصاف و امانت : تعهد به اجتناب از هرگونه جانب داری غیر علمی و حفاظت از اموال ، تجهیزات و منابع در اختیار.
3- اصل ترویج : تعهد به رواج دانش و اشاعه نتایج تحقیقات و انتقال آن به همکاران علمی و دانشجویان به غیر از مواردی که منع قانونی دارد .
4- اصل احترام : تعهد به رعایت حریمها و حرمتها در انجام تحقیقات و رعایت جانب نقد و خودداری از هرگونه حرمت شکنی.
5-اصل رعایت حقوق : التزام به رعایت کامل حقوق پژوهشگران و پژوهیدگان ( انسان ، حیوان و نبات ) و سایر صاحبان حق .
6- اصل رازداری: تعهد به صیانت از اسرار و اطلاعات محرمانه افراد ، سازمانها و کشور و کلیه افراد و نهادهای مرتبط با تحقیق.
7- اصل حقیقت جویی : تلاش در راستای پی جویی حقیقت و وفاداری به آن و دوری از هرگونه پنهان سازی حقیقت.
8-اصل مالکیت مادی و معنوی : تعهد به رعایت کامل حقوق مادی و معنوی دانشگاه و کلیه همکاران پژوهش.
9- اصل منافع ملی : تعهد به رعایت مصالح ملی و در نظر داشتن پیشبرد و توسعه کشور در کلیه مراحل پژوهش .

Islamic Azad University
Damghan Branch
Faculty of Agriculture

A Thesis Submitted in Partial Fulfillment of the Requirments For
the Degree of M.sc(Ph.D)in Food scienc and technology

Title
Investigation on Rheological Behaviour of Dually Modified Cassava Starch//-Carrageenan as Gelatin Alternative in Pharmaceutical Hard Capsules
Supervisor:
Abdorreza Mohammadi Nafchi, PhD
by:
Maliheh Saeidi
November 2013
ACKNOWLEDGEMENT

I would like to express my gratitude and thank to my father for their endless love, support and patience.
I also would like to express my deep gratitude and thanks to my supervisor Dr Abdorreza Mohammadi Nafchi for his professional guidance, moral support and encouragement during the experimental work, detailed comments and editing throughout the writing process of this thesis.
I am also greatly indebted to my friends and technicians of laboratories in Azad University, Damghan branch for their help, support and friendship.
Special thanks also go to Professor Abd Karim Alias from Food Science Malaysia for his special supports.

TABLE OF CONTENTS
Acknowledgementii
Table of Contentsiii
List of Tablesvi
List of Figuresvii
Abstract1
Chapter 1: Introduction2
1.1 Background3
1.2 Rational of study5
1.3 Objectives of the study5
1.4 Research Flowchart6
Chapter 2: Literature Review8
2.1 PHARMACEUTICAL CAPSULES9
2.1.1 Pharmaceutical hard capsules10
2.1.2 Manufacture of gelatin capsules11
2.1.3 Properties of gelatin capsules15
2.1.4 Alternatives to Gelatin17
2.2. POLYSACCHARIDES STUDY20
2.2.1 Starch20
2.2.1.1 Composition and primary structure of starch21
2.2.1.2 Morphology and ultra-structure of starch grains24
2.2.1.3 Semi-crystalline structure of starch grains27
2.2.1.4 Thermal transitions30
2.2.1.5 Starch modification35
2.2.1.6 Cassava41
2.2.2 Carrageenan53
2.2.2.1 Chemical Structure53
2.2.2.2 Conformation of κ-carrageenan54
2.2.2.3 Gelation of κ-carrageenan60
2.2.2.4 Thermoreversibility of gels and rheological properties61
2.3 POLYSACCHARIDE MIXTURES65
2.3.1 Phase Behavior65
2.3.2 Thermodynamic Incompatibility66
2.3.3 Gels based on mixtures polysaccharides68
2.3.3.1 Rheological properties69
2.3.3.2 Rheology of blends of starch70
Chapter 3: Materials and Methods72
3.1 Materials73
3.1.1 Gelatin73
3.1.2 κ-carrageenan73
3.1.3 Acid hydrolyzed hydroxypropylated cassava starch73
3.2 Methods74
3.2.1 Preparation of solutions74
3.2.1.1 Gelatin solutions74
3.2.1.2 Starch and κ-carrageenan solutions74
3.2.2 Rheological properties77
3.2.2.1 Flow properties77
3.2.2.2 Viscoelastic properties78
Chapter 4: Results and Discussions79
4.1 Rheological behavior of gelatin80
4.1.1 Gelatin solution at 50 °C80
4.1.2 Sol-gel transitions82
4.1.3 Viscoelastic properties of gelatin gels at 20 °C86
4.2 Rheological behavior of starch-κ-carrageenan blends90
4.2.1 Rheological behavior at 50 °C90
4.2.1.1 Dually modified cassava starch (HHSS)90
4.2.1.2 κ-carrageenan95
4.2.1.3 Dually modified cassava starch/κ-carrageenan blends96
4.2.2 Rheological behavior in sol-gel transitions (from 50 °C to 20 °C)102
4.2.2.1 Influence of κ-carrageenan content104
4.2.2.2 Influence of the different extents of starch hydrolysis106
4.2.3 Rheological properties of gels at 20 °C107
4.2.3.1 κ-Carrageenan gels107
4.2.3.2 Composite gels108
Chapter 5: Discussion and Conclusion113
5.1 Synergy and gel state114
5.1.1 Dually modified cassava starch and κ-carrageenan114
5.1.2 Mixtures115
5.2 Comparison with gelatin120
5.2.1 Solution properties120
5.2.2 Jellification121
5.3 Conclusion and recommendation for future research123
References126

LIST OF TABLES
Table 2. 1: Properties and applications of modified starches.35
Table 2. 2: Performance of starch slurry dewatering by a conventional centrifuge from a typical cassava starch factory.51
Table 3.1: Compositions of the starch- κ-carrageenan solution76
Table 4.1: Changes in viscosity of gelatin as a function of concentration. Experiments were performed at 50 °C81
Table 4.2: Gelation temperatures, TGEL and melting temperature TM (G’= G”) during cooling from 50 to 25 °C and heating from 25 to 50 °C. The rate of heating or cooling was 1°C/min. Frequency: 1 rad/s. Strain amplitude: 1%.86
Table 4.3: Viscosity of κ-carrageenan in different concentrations95
Table 4. 4: Gelling temperatures (TGEL) and melting temperatures (TM) of κ-carrageenan alone and the mixture HHSS12-κ-carrageenan determined from cooling and heating ramps at 1 °C/min and 1 rad/s.104
Table 4.5: Storage and loss moduli G’ and G” of κ-carrageenan alone and HHSS12-κC0.5 mixture determined from temperature ramps during cooling and heating at 1 °C/min by rheological measurements. Frequency: 1 rad/s111
LIST OF FIGURES
Figure 1.1: Research flowchart7
Figure 2. 1: Formation of hard gelatin capsules by dip molding12
Figure 2. 2: Position fingers dipping during passage through the drying ovens13
Figure 2. 3: Steps removing (a) trimming (b), and assembly of capsules (c).14
Figure 2. 4: Water content at equilibrium of pharmaceutical hard empty gelatin capsules in relationship with the mechanical behavior. The capsules are stored at different relative humidities for two weeks at 20 ° C.16
Figure 2. 5: Isothermal sorption-desorption capsules hard gelatin and HPMC at equilibrium at 25°C.19
Figure 2. 6: Test for fragility of the capsules: the percentage of broken capsules according to their water content. a: resistance to pressure with capsules filled with corn starch. b: impact resistance with empty capsules.19
Figure 2. 7: Structure of amylose22
Figure 2. 8: Structure of amylopectin23
Figure 2. 9: Grains of different starches observed in scanning electron microscopy SEM (magnification × 280)24
Figure 2. 10: The different levels of grain starch25
Figure 2. 11: Organization of starch grains in “blocklets”27
Figure 2. 12: X-ray diffraction diagram for crystalline starch type A, B and C.28
Figure 2. 13: Crystallinity of potato starch: influence of water content on the resolution of the diffraction pattern of X-rays29
Figure 2. 14: Crystalline arrangement of double helices of amylose type A and B30
Figure 2. 15: Variation of classical transitions of the potato starch as a function of water content33
Figure 2. 16: Hydroxypropylation reaction38
Figure 2. 17: Mass balance of cassava starch manufacturing process in a starch factory with a decanter.47
Figure 2. 18: Mass balance of cassava starch manufacturing process in a starch factory without a decanter.48
Figure 2. 19: Starch granules trapped in discharged pulp of cassava starch process.49
Figure 2. 17: Ideal repeating units of λ-carrageenan (a) (R = H or SO3-), and (b) for ι- carrageenan (R1 = R2 = SO3-) and κ- carrageenan (R1 = H ; R2 = SO3-).54
Figure 2. 18: Percentage of order of κ-carrageenan solution by polarimetry (0) and conductivity measurements (F)55
Figure 2. 19: Change in transition temperature Tm at cooling κ-carrageenan based on the total concentration of CT different monovalent cations (1) Rb+, (2) Cs+, (3) K+ ,(4) NH4+, (7) N(CH3)4+ (8) Na+, (9) Li+ and divalent cations (5-6) Ba2+, Ca2+, Sr2+, Mg2+, Zn2+, Co2+57
Figure 2. 20: Phase diagram of κ-carrageenan representing the variation of transition temperature on cooling and heating according to the total concentration of potassium (Rochas, 1982; Rochas & Rinaudo, 1980).59
Figure 2. 21: κ -Carrageenan gelation model, cation to promote gelation. (Morris et al., 1980)60
Figure 2. 22: Variations of G’ and G” as a function of temperature for a concentration of 1% κ-carrageenan, Frequency 1 Hz, Tg: temperature of gelation, Tm: melting temperature. Cooling G’ (■), G” (•). Heating G’ (□), G” (◊). (Fernandes, Gonçalves & Doublier, 1992).63
Figure 2. 23: Kinetics of evolution of κ-carrageenan at a concentration of 1%. Temperature is 25 ° C. Frequency 1Hz. G’ (■), G” (•).64
Figure 2. 24: Phase diagram at 25 °C mixture of waxy hydroxypropyl starch/κ-carrageenan.67
Figure 3.1: Phase diagram of κ-carrageenan representing the variation of transition temperature on cooling and heating according to the total concentration of potassium75
Figure 4.1: Newtonian behavior of gelatin at 50 °C and 20% concentration.80
Figure 4.2: Mechanical spectrum of 25% gelatin solution. G’: filled symbols, G”: empty symbols. Experiments were performed at 50 °C, strain amplitude was 1%82
Figure 4.3: Storage and loss moduli Gg, G, for a 25% gelatin sample during a cooling ramp. Temperature was ramped from 50 to 20 °C at 1°C/min. Frequency: 1 rad/s. Strain amplitude: 1%84
Figure 4.4: Storage and loss moduli GF, G, as a function of temperature during a heating ramp of a 25% gelatin sample. Temperature was ramped from 25 °C to 50 °C at 1 °C/min. Frequency: 1 rad/s. Strain amplitude: 1%85
Figure 4.5: Mechanical spectrum of 25% gelatin. G’: filled symbols, G”: empty symbols. The temperature was 20 °C. Strain amplitude: 1%.87
Figure 4.6: Changes in modulus G’ and G” as a function of time for a 27% gelatin gel. Measurement temperature was 20 ° C. Frequency: 1 rad / s. Strain amplitude: 1%.88
Figure 4.7: Changes in G’ as function of gelatin concentration. Data obtained after 6 h of time sweep measurement at 20 °C. Frequency: 1 rad/s. Strain amplitude: 1%.89
Figure 4.8: Flow curves of hydrolyzed hydroxypropylated cassava starch dispersions at a concentration of 25% (g/g): HHSS6 (●), HHSS12 (■), HHSS18 (o), HHSS24 (/). Measurements were performed at 50 °C91
Figure 4.9: Flow curves for dually modified cassava starch (HHSS12) dispersions at a concentration of 25% (g/g). Measurement was performed at 50 °C92
Figure 4.10: Flow curves of dispersions of hydroxypropyl cassava starch HHSS12 at concentrations of 20% (■), 23% (●) and 25% (▲). Temperature was 50°C93
Figure 4.11: Mechanical spectra of different dually modified cassava starches at concentrations of 25%: a) HHSS6, b) HHSS12, c) HHSS18, d) HHSS24. G’: filled symbols, G”: empty symbols. Measurement temperature was 50 °C and strain amplitude was 1%94
Figure 4.12: Newtonian behavior of κ-carrageenan in the concentration range of 0.25% to 1% at 50 °C96
Figure 4.13: Flow curves of the mixture HHSS12-κC0.5 (•), 20%HHSS12 and 0.5% κ-carrageenan, κC0, 5 (×), and starch dispersions HHSS12 20% (□), 23% (○) and 25% (Δ). The temperature was 50 °C97
Figure 4. 14: Flow curve of the HHSS12-κC0.5. Shear rate up 0 to 100 s-1 empty symbols, and down 100 to 0 s-1 filled symbols98
Figure 4.15: Flow curves of mixtures of 25% starch HHSS12 with κ-carrageenan at different concentrations. Measurements were taken at 50 °C99
Figure 4.16: Flow curves for 0.5% κ-carrageenan and mixtures of 25% dually modified cassava starches/κC0.5. Measurement temperature was 50 °C.100
Figure 4.17: Mechanical spectrum of κC0.5 (solid lines ■, □), HHSS12 (solid lines ●, ○), and the mixture κC0.5-HHSS12 (■, □). Concentration of HHSS12 alone was 25% and in combination total concentration was 25%. G’: filled symbols, G”: empty symbols. Measurement temperature: 50 ° C. Strain amplitude: 1%101
Figure 4.18: Variation of viscoelastic modulus G’ and G” as a function of temperature for κC0.5 and for the mixture of κC0.5 and HHSS12. a) Cooling from 50 °C to 20 °C. b) Heating from 20 °C to 50 °C. Heating/cooling rate: 1 °C/min. Frequency: 1 rad/s. Strain amplitude: 1%103
Figure 4.19: Variations of modulus G’ and G” as a function of temperature during cooling from 50 °C to 20 °C for 25% HHSS24 alone and in combination with κ-carrageenan. G”: filled symbols; G’: empty symbols. Cooling rate: 1 °C/min. Frequency: 1 rad/s. Strain amplitude: 1%105
Figure 4.20: Variations of modulus G’ and G” as a function of temperature during cooling from 50 °C to 20 °C for 1% κ-carrageenan and 25% starch mixtures. G’: empty symbols; G”: filled symbols. Cooling rate: 1 °C/min. Frequency: 1 rad/s. Strain amplitude: 1%106
Figure 4.21: Variations of modulus G’ and G” as a function of temperature during heating from 20 °C to 60 °C for 1% κ-carrageenan and 25% starch mixtures. G’: empty symbols; G”: filled symbols. Cooling rate: 1 °C/min. Frequency: 1 rad/s. Strain amplitude: 1%107
Figure 4.22: Mechanical spectra of κC1 (■, □), κC0.75 (●, ○) and κC0.5 (▲, Δ). G’: filled symbols, G”: empty symbols. Temperature: 20 ° C. Strain amplitude: 1%.108
Figure 4. 23: Mechanical spectrum of κC0.5 (●, ○), 25% HHSS12 (dashed line with ▲, Δ) and the mixture of κC0.5-HHSS12 (■, □) at 20°C. G’: filled symbols, G”: empty symbols. Strain amplitude: 0.1% for mixtures and 1% for constituents.109
Figure 4.24: Mechanical spectrum of mixtures HHSS12-κC1(▲, Δ), HHSS12-κC0.5 (dashed line with ●, ○) and HHSS12-κC0.25 (■, □) at 20 °C. G’: filled symbols, G”: empty symbols. Strain amplitude: 0.1%110
ABSTRACT
With the goal of finding an alternative to gelatin in the processing of pharmaceutical capsules, the effects of k-carrageenans on dually modified cassava starch were investigated. While film forming and mechanical properties are important in all pharmaceutical capsules, solubility at high solid concentration and thermo-reversibility are important factors for hard capsule processing. Casava starches were modified first by hydrochloric acid (0.14 N for 6, 12, 18, and 24 h at 50 °C) and secondly by propylene oxide (10, 20, and 30% of solid for 24 h at 40°C).
To improve the gel setting property of the dually modified starch, dually modified cassava starches were combined with -carrageenan (0.25, 0.5, 0.75, and, 1%). The concentration of the K+ ion in the composite mixture was adjusted appropriately to achieve the same sol-gel transition temperature. The rheological properties of the mixtures were measured and compared, with gelatin as the reference material. The solution viscosity, sol-gel transition, and mechanical properties of the films made from the mixtures at 50 °C were comparable to those of gelatin. The viscoelastic moduli (G’ and G”) for the gel mixtures were lower than those of gelatin. The composite gels had temperatures of gelation similar to that of gelatin. Both viscosity in solution and stiffness in gels could be adjusted using high levels of κ-carrageenan and was relatively independent of the molecular weight of the starch. These results illustrate that dually modified cassava starch in combination with -carrageenan has properties similar to those of gelatin, thus these starches can be used in dip-molding processes, such as those used to make pharmaceutical hard capsules.

CHAPTER 1: INTRODUCTION

1.1 Background
The capsule is one of the formulations of the oldest pharmaceutical in history, known especially from the ancient Egyptians. In Europe, it was not until the nineteenth century that the first gelatin pharmaceutical capsule with the patent of Mr. Dublanc pharmacist and his student Mr. Mothes. Over the years, this invention has been so successful that the production of capsules has grown rapidly in many countries. This has led to many improvements including the invention of hard gelatin capsules in 1846 by Mr. Lehuby (Podczeck & Jones, 2004).
The development of pharmaceutical capsules, used for therapeutic purposes, originates in the keen interest shown by the numerous researches in pharmacology. This has greatly expanded the range of possible formulations using pharmaceutical capsules. Today, pharmaceutical capsules are mainly based on animal gelatin from porcine or bovine. Gelatin is an animal protein that is a traditional ingredient in many fields, including food. Gelation properties at temperatures close to room temperature and formation of homogeneous films, potable, gelatin as a choice for the manufacturing of pharmaceutical capsules.
However, the use of animal gelatin in the food and pharmaceutical industry is governed by regulations becoming more stringent. The precautionary principal applied, for example, the risk of transmission by animal gelatin; the bovine spongiform encephalopathy (BSE) has questioned its use. Even if today the rules on the origin of the gelatin are very strict and that gelatin is no longer a risk to health, development of alternative products of interest to pharmaceutical and food industries. The sources from which gelatin can also be problematic for ethical or religious populations. Many people around the world do not consume products made from pork (vegetarians, Hebrews, and Muslims) or beef base (vegetarian Hindus). It is therefore that the replacement of gelatin with other texturing agents of non-animal origin has been much research in recent years.
The most important properties that potable gelatin as capsule forming material are heat sealability of films for soft capsule processing and solubility in high concentration, film formability and thermo-reversibility for making hard capsules.
Starch as a plant based material is one of possible alternative for gelatin due to cost and accessibility. Native starches can form films, but the films have not heat sealability, also starches are non soluble biopolymer, and form non-reversible gels. So changes or supporting the structure likely improve the starch property to consider as gelatin replacement in some cases.
The proposed system is a mixture of starch and T-carrageenan. Starch would give the mixture of film-forming properties and solubility in aqueous and carrageenan bring its ability to gel. The selected starch has focused on the use of such modification(s) on starch that able it to dissolve at temperatures below 100 °C and form stable solutions at high concentrations (≈ 20-30%). The botanical origin of the cassava starch is due to its proper amylose content, which improves mechanical properties of films and availability of this starch in Southeast Asia. The gelling agent has been studied was κ-carrageenan/K+ for its ability to form thermo reversible gels and easily adjustable thermo-physical transition temperatures. The film-forming mixtures were prepared by casting method.
The main objective of this research project is to replace the gelatin with a composite cassava (tapioca) starch film for manufacturing of pharmaceutical capsules especially hard capsules. The idea for hard capsule processing is to develop a new system whose characteristics in the solution and solid state would be closer to existing formulations. The constraints imposed industrial development concentrated formulations (25-30%) prepared at temperatures below 100 °C capable of forming a gel by physical cooling and forming a film after drying.

1.2 Rational of study
The main objective of this research was to replace gelatin with a composite casava starch for the manufacture of pharmaceutical capsules. The methods proposed for hard capsule processing involve creating a starch that has characteristics in the solution and solid states that are similar to those of existing gelatin-based formulations. Any gelatin alternative material would have to meet these criteria: solid concentration of 25–30% w/v, preparation temperature below 100 °C, ability to form a gel by physical cooling, and ability to form a film after drying.
The system proposed herein is a mixture of cassava starch and carrageenan. The starch would provide the film-forming property and solubility in an aqueous environment and the carrageenan is expected to enhance the gelling property and improve the durability of the system. Thus, the modification of the starch was designed so that it can dissolve at temperatures below 100 °C and form stable solutions (in terms of rheological properties and functionality) at high concentrations (20–30%).

1.3 Objectives of the study
The objectives of this study are:
1. Investigation on effects of carrageenan on dually modified cassava starch flow properties.
2. Investigation on effects of carrageenan on dually modified cassava starch sol-gel transition.
3. Investigation on effects of carrageenan on dually modified cassava starch gel properties.

1.4 Research Flowchart
This research was designed as the following research flowchart.
Figure 1.1: Research flowchart
CHAPTER 2: LITERATURE REVIEW
2.1 PHARMACEUTICAL CAPSULES
The capsules, also called caps (from the Latin word meaning capsulatus container), are solid preparations consisting of a hard or soft shell, shape and capacity variables, usually containing a unit dose of active ingredient. The contents of capsules can be solid, liquid or pasty. The soft capsules mean capsule consists of a single party whose shape can be cylindrical, spherical, ovoid, etc. It usually contains a plasticizer which gives the properties of flexibility where the adjective that means. The hard capsules are made up of two parts (head and body) of cylindrical shape with a diameter slightly different for their engagement. The hard capsules contain very little plasticizer which makes them rigid.
The first patent on the use of gelatin capsules for therapeutic use has been filed in 1834 by MM. J.G.A. Dublanc and fob Moths. Many attempts were undertaken to mask the unpleasant taste of certain drugs in vogue at that time (turpentine, Copaiba). The solution that met the most success was the invention of a cap based on a gelatin film containing the drug.
MM. Dublanc and Moths then develop a process for manufacturing capsule; which is to dip a brass cylindrical object in an aqueous solution of gelatin flavored and sweetened, then remove and place it vertically for drying. After evaporation of water, gelatin is in the form of a solid film covering the walls of the cylinder, forming a capsule. The capsules, which can be regarded as soft capsules are then filled and sealed with a drop of gelatin solution. Moths has only continued to work on improving the process of development and the use of gelatin capsules. The incredible success of his capsules in the following years led to their use worldwide. For more details, you can refer to a book on history of pharmaceutical capsules, their method of manufacture and their characteristic (Podczeck & Jones, 2004). We limit ourselves in the following sections describe the characteristics of pharmaceutical hard capsules, because main part of this thesis focused on hard capsules.
2.1.1 Pharmaceutical hard capsules
In the mid-nineteenth century, the remarkable growth of Mothes pharmaceutical soft capsules, leads the development of many alternative procedures. In 1846, slightly more than ten years after the invention of the first capsule-based gelatin, MJC Lehuby published a patent under the heading “My drug envelopes”. It is the first to suggest a capsule consisting of two parts which are produced by dipping the fingers of casting metal in a gelatin solution and then drying them. The capsules are cylindrical and consist of two half cylinder with a diameter slightly different for easy assembly. It is important to note that this process has been improved by the inventor over the years, was originally intended to produce hard capsules based on cassava starch, and then based on a mixture of carrageenan and gelatin then called lichen capsules. However, these formulations have been abandoned due to their fragility in comparison with gelatin.
Unlike the early success experienced by the Mothes soft gelatin capsules, development of hard capsules has been delayed by technical difficulties posed by the manufacture of the head and body of the capsule. It was not until the early twentieth century to emerge in the U.S. the first industrial production of hard capsules made from gelatin. From 1931, the Parke, Davis & Co., managed to develop a machine capable at the same time to produce the head and body of the capsule and assembling them. The production of hard capsules is still based on this process. Some minor changes have been made over the years, mainly to automate and optimize the various stages of production. The largest producers of pharmaceutical capsules are now Capsugel (USA) and Shionogi Qualicaps (Japan) companies.

2.1.2 Manufacture of gelatin capsules
The manufacturing process of a hard capsule uses a very old process that is casting by dipping. The parameters of molding capsules are based on characteristics specific to gelatin. Each step of the production of capsules should be carefully controlled to ensure continuous operation of production machines at very high speed. The materials used are gelatin, colorants, preservatives, and surfactants. The various stages of manufacture of hard capsules are placed in solution, casting by dipping also called dipping or dip-molding, drying, and assembly.
Dissolution. Gelatin powder form, is dissolved in deionized water at a concentration of 30-40% is then heated to about 60 °C. The mixture is homogenized under vacuum to limit the presence of air bubbles may be trapped in the viscous solution of gelatin and then create appearance defects on the capsule. At this temperature, the gelatin solution can undergo hydrolysis reactions over time, can significantly alter its physical properties such as viscosity in solution or gel strength. For this reason, the production cycle of the capsules must be planned and monitored rigorously. The gelatin is then transferred into tanks regulated to about 55 °C lower volume that will feed into each production line. In those tanks, additives such as dyes, preservatives, and surfactants will be added. Preliminary measurements of viscosity are generally made directly into the tank using a rotational viscometer. Both concentration and viscosity solution are very important parameters because it determines the thickness and mass of the capsule.
Capsule formation. The gelatin solution is placed in a tank with overflow at a temperature of about 55 °C. At this temperature, continuous evaporation takes place so requiring control the viscosity and readjusts if necessary. The concentration of the solution was kept between 25% and 30%. The container used is fitted in the center of a rectangular plate below which is a Archimedes screw. This device allows one hand to obtain a constant height of bath and, secondly, to homogenize the solution with a constant motion of the liquid. It also helps to filter the gelatin solution and thus eliminate any residues. The fingers of casting steel (coated with a thin layer of lubricant: soy lecithin based solution) with a temperature of about 25 °C, are dipped in a gelatin bath with a temperature of about 50-55 °C (Figure 2.1). The gelatin solution gelled instantly on the surface of the fingers. The fingers are then withdrawn slowly from the solution to ensure uniform thickness. The gel helps to set the gelatin over the finger. The viscosity of the solution determines the amount of material removed and then the final thickness of the capsule. Different velocity profiles fingers molding are also programmed to control the thickness. To avoid the formation of drops or streams of material formed during the ascent, fingers molding undergo several rotations to even thickness of material on the surface of the capsule. A gentle stream of cold air is then blown to permanently set the surface of gelatin film. The molding fingers have a diameter greater for the upper (head) which fits into the bottom of the capsule (body).

Figure 2. 1: Formation of hard gelatin capsules by dip molding

Drying of capsules. The gelatin films are formed successively in the past
drying tunnels, inside which the temperature and relative humidity (RH) are,
controlled very precisely (Figure 2-2). The temperature is between 22 and 28 ° C
relative humidity between 38% and 43% depending on the drying compartment. The
temperature cannot be too high because of the thermo reversibility gelatin gels.

Figure 2. 2: Position fingers dipping during passage through the drying ovens

The drying cycle takes about 30 to 40 minutes. The drying conditions are adjusted to
obtain a slow drying rate at the beginning of the cycle, then reaching a peak
mid-cycle and then decreasing at the end of the cycle. These velocity profiles are needed to
make uniform drying. Indeed, if the drying rate is too high, a “hard skin”
material can be formed on the surface so acts as the insulating film and inside the capsule that remains in gel form. When the capsules out of the drying tunnel, they are not totally
dried. They still contain a residual amount of water between 15% and 18%. (Standard water content at equilibrium is 13-16% under standard temperature and relative humidity: 25 °C, 60% RH). Water is an integral part of the capsules and plays a role of plasticizer.
The higher the water content leads the higher the ductility and flexibility of gelatin film. Instead, for low water contents (<10%), they become fragile and brittle. The capsules are then extracted from the furnace with a water content of 15-18% for them to resist mechanical steps of removing and trimming.

Removing, trimming, assembly. The solid gelatin films are removed from the molds using
metal jaws around the casting fingers, which allow release capsules
molding fingers (Figure 2.3 (a)). The capsules formed are deliberately too long because
thickness defects usually appear at the base of the head and body. A trimming step
can remove the defective part and thus get a good capsule dimension (Figure 2.3 (b)). The scrap which represents 20% of the capsule mass will be reused later. Indeed, these pieces of film will be dissolved in order to be recycled in the production chain. The head and body of the capsule are then assembled and formed the capsule is stored at 25 ° C and 40% RH to complete the drying (Figure 2.3 (c)).
abc
Figure 2. 3: Steps removing (a) trimming (b), and assembly of capsules (c).

The castings fingers are then cleaned and lubricated to facilitate the stripping
capsules are then transferred to the top of the chain of production for the next round. The
produced capsules are controlled so that there is no default in appearance on the surface. Moreover, the dimensions of the capsules are very precisely controlled. The capsules are then
sent to pharmaceutical companies that provide open capsules, their filling and final closure in filling machines operating at very high speed. Any default of appearance or wrong size may cause cycle arrest filling. The molding fingers allow current to obtain capsules
which both parties have many improvements to forms (ribs, grooves,
etc …) to ensure strength and kept at optimum storage and filling.

2.1.3 Properties of gelatin capsules
The hard gelatin capsules have water content between 13 and 16% (db). Depending on storage conditions, temperature and relative humidity, the water content
can be significantly altered. The sorption/desorption isotherm of gelatin were
been widely studied and show the influence of water activity on the water content of
hard gelatin capsules (Sobral & Habitante, 2001). For moisture content
below 10%, films based on gelatin become brittle. They are deformed and
tear at water contents above 18%. The change in water content causes
not only changes the physical properties of the capsules but also dimensional changes. For moisture contents between 13 and 16%, a 1% change in water content causes a dimensional change of 0.5%. The relationship between relative humidity during storage, moisture, and mechanical properties of hard gelatin capsules has been established and is shown in Figure 2.4 (Bond, Lees & Packington, 1970). For optimum performance, the capsules should be stored
in conditions of relative humidity between 35% and 55%. Conventional methods
to assess the fragility of the capsules are based on measurements of
impact resistance. The capsules can withstand the impact or rupture thereby
defines a criterion of fragility based on storage conditions. However this method
empirical remains limited to determine accurately the influence of formulation on
rigidity of the capsule. Compression and bending measurements on the capsules were
also been conducted to determine the mechanical properties of the capsules (Kuentz & Röthlisberger, 2002; Missaghi & Fassihi, 2006). These studies showed that the rigidity of gelatin capsules is changed over time, depending on the moisture and
also according to the excipient. Measurements of traction are also relevant to
evaluate the mechanical properties of films based on gelatin.

Figure 2. 4: Water content at equilibrium of pharmaceutical hard empty gelatin capsules in
relationship with the mechanical behavior. The capsules are stored at different relative humidities for two weeks at 20 ° C(Bond, Lees & Packington, 1970).

An important feature of gelatin which explains its use in
pharmaceutical capsules since their invention is its ability to dissolve in
aqueous media at a temperature close to that of the human body. The study of
disintegration of the capsules and their solubility in different biological environments recreating gastric conditions has been the subject of numerous studies (Podczeck & Jones, 2004). The gelatin capsules are insoluble at temperatures below 30 ° C. Microscopic techniques allowed to observe the rupture of the capsules and also view
heterogeneities on the thick wall of the capsule. It has been shown in a solution 0.1M
HCl as the capsule begins to wrinkle after 40s submersion. Finally, by
scanning electron microscopy (SEM), dissemination of biological environment within
the wall of the capsule could be visualized. After only 30 s, 75% of the surface of the capsule was achieved.

2.1.4 Alternatives to Gelatin
Gelatin is widely used, particularly in the pharmaceutical industry and
to be the material most suitable for the manufacture of capsules. It is soluble in water
at high levels around 60 °C, it forms gels with cooling temperature at temperatures close to room temperature, it offers excellent film-forming qualities and it dissolves easily at a temperature close human body. Fish gelatin capsules have been marketed recently by some
manufacturers (of Roxlor AquaCap ®, OceanCapsTM Capsugel). The gelatin is produced
from the skin of certain species of fish in warm water with a proline and hydroxyproline composition (amino acids play an important role in gelation)
similar to mammalian gelatin. These capsules have equivalent chemical and
physical properties to mammalian gelatin, facilitating their implementation in
standard machines. However the cost of these capsules is high.
The hydroxypropyl methylcellulose (HPMC) or hypromellose is modified cellulose polysaccharide-based which has been very popular when marketed as
vegetarian capsule in the field of herbal medicine. The first hypromellose capsules,
for the American market to satisfy consumer demand vegetarians have
been marketed by the company A. P. Scherer West Inc. (GS Technologies Inc., 1998;
(Grosswald, Andrew & Anderson, 2002; Inc, 1998). However, as the material offered in the mechanical properties lower than those of gelatin, the HPMC based capsules (Vegicaps ®) were twice
thick. Shionogi Qualicaps Japan Society has made a point of HPMC capsules in which carrageenan as a gelling agent is added in small quantities, (Yamamoto, Matsuura & Kazukiyo, 1998). These capsules (Quali-V®) have properties similar to gelatin. They have same size and fit into the filling machines used for gelatin capsules. Subsequently, many companies have also developed cellulose-based capsules: Capsugel Division of Pfizer Inc. (Vcaps ® capsules) Natural Capsules Ltd.(Cellulose capsules). The HPMC capsules offer film-forming properties comparable to those of gelatin. In addition, under identical storage conditions, the water content of HPMC (2-5%) is much lower than that of gelatin (13-15%) (Figure 2.5)
This allows using these capsules to contain hygroscopic substances.
Moreover, the capsules retain their properties despite low water content (Figure 2.6).

Figure 2. 5: Isothermal sorption-desorption capsules hard gelatin and HPMC at equilibrium at 25°C(Nagata, 2002).

Figure 2. 6: Test for fragility of the capsules: the percentage of broken capsules according to their water content. a: resistance to pressure with capsules filled with corn starch. b: impact resistance with empty capsules (Nagata, 2002).

The main disadvantage of HPMC capsules is their high cost compared to that of gelatin. This material also has the disadvantage of having a taste and odor. In recent years the growing number of patents dealing with substitutes gelatin for the manufacture of pharmaceutical hard capsules, including pullulan (Robert, Cadé, Xiongwei & Cole, 2005) and hydroxypropyl starch, shows how research in this area remain active (Basquin, Darasse, Despre & Messager, 2003; Paris & Viau, 2001). Recently, new capsule containing pullulan (NPCAP ®) have been marketed by the company Capsugel Division of Pfizer Inc(Robert, Cadé, Xiongwei & Cole, 2005).
Capsules of starch were manufactured by injection molding (Wittwer, Tomka, Bodenmann, Raible & Gillow, 1998). These capsules had a significant thickness and a different shape compared with capsules containing gelatin and require the use of specific equipment for filling. The manufacture of hydroxypropyl starch capsules has also been described (Christen & Cheng, 1977). Unfortunately, due to the absence of gelation of these solutions of starch, the soaking time is relatively long (20 s compared to 1 to 2 s for gelatin), hindering the marketing of such capsules. Currently, despite the large number of patents on starch, no commercial exploitation is done.

2.2. POLYSACCHARIDES STUDY
2.2.1 Starch
Starch is a polysaccharide of plant origin. This is the main carbohydrate reserve substance of most plants. It represents a significant mass fraction of agricultural commodities. It is found in the storage organs of plants such as cereals (30-80% dry matter), tubers (60-90%), and legumes (25-50%). Starch is the main source of energy for food and feed. It is a nutritional compound abundant, renewable, inexpensive, which is in foods multiple functions as a thickener, gelling agent, binder, sweeteners. Starch is also used in many non-food industrial: paper production, pharmaceuticals, cosmetics, textiles, etc. It has also become in recent years an interesting raw material for renewable plastics production and biodegradable and poses as a potential candidate for the manufacture of biofuels.

2.2.1.1 Composition and primary structure of starch
Characteristics of composition, morphology and ultra structure depending on the botanical origin of starch have been many literature reviews (Banks & Greenwood, 1975; Buléon, Bizot, Delage & Multno, 1982; Zobel, 1988). Starch is a polymer of glucose C6H10O5, consisting of two homopolymers of different primary structures: amylose, macromolecule almost linear, and amylopectin, heavily branched macromolecule. The amylose content varies according to the botanical origin of starch. It varies between 0% (waxy maize starch or waxy) and 70-80% (wrinkled pea starch and high amylose maize). These extreme values are obtained for the mutated genotypes, whereas the amylose content of wild species such as potato, wheat, smooth pea is between 18 and 35%. Starch consist entities granular semi-crystalline result of an organization of its two constituents. The starch also contains small amounts of non-carbohydrate constituents representing 0.1 to 2% according to botanical origin. These are mainly minor components of lipids, proteins and minerals located both on the surface of starch grains and inside.

2.2.1.1.1 Amylose
Amylose is a linear polymer composed of D-glucose units linked by bonds of type α (1,4) (Figure 2.7). The native amylose contains 500 to 6000 glucose units according to botanical origin, divided into several channels including the average degree of polymerization is about 500, corresponding to a average molecular weight Mw between 105 to 106 g/mol(Banks & Greenwood, 1975). Some chains can be branched amylose bonds by α (1,6) (Banks & Greenwood, 1975). However the number of these bonds is low and they seem to be frequently located near the reducing end (Takeda, Hizukuri, Takeda & Suzuki, 1987).

Figure 2. 7: Structure of amylose(Carvalho, 2008)

Amylose has the specificity of complex power hydrophobic molecules such as iodine, fatty acids, alcohols etc. Its conformation and binding mode enables it to adopt helical forms comprising 6 glucose units per turn, stabilized by intramolecular hydrogen bonds. The hydrophilic groups are directed outwards and the hydrophobic groups inward, forming a hydrophobic cavity which will be accommodated molecules complexed. The binding capacity of iodine is 20 mg to 100 mg of amylose is characterized by maximum wave length absorption (λmax) between 620 and 640 nm.
Amylose can be extracted from starch granules dispersed in water by complexation with some alcohols (eg. butanol) (Schoch, 1945). Amylose can also be synthesized in vitro by enzymatic (Ball, van de Wal & Visser, 1998; Pfannemüller, 1987; Potocki-Veronese et al., 2005).

2.2.1.1.2 Amylopectin
Unlike the linear chain amylose, amylopectin is a highly branched polymer consisting of hundreds of short chains of glucose units linked together mainly by connections α (1,4) and by 5 to 6% bonds α (1 , 6) responsible ramifications (Figure 2.8).

Figure 2. 8: Structure of amylopectin(Carvalho, 2008)

Amylopectin is the main constituent of most starches. The first structural models of amylopectin proposed a homogeneous organization ramifications, but it was later determined that amylopectin consisted of a set of clusters of short channels (S chains or A chain) DPW 15-20, linked by longer chains of DPW (L chains



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