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Biopolymer-Based
Formulations
Biomedical and Food Applications
Edited by
Kunal Pal
Indranil Banerjee
Preetam Sarkar
Doman Kim
Win-Ping Deng
Navneet Kumar Dubey
Kaustav Majumder
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Contributors
Saumya Agarwal
Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India
Jasim Ahmed
Food and Nutrition Program, Environment and Life Sciences Research Center, Kuwait Institute
for Scientific Research, Safat, Kuwait
B. Amulyasai
Department of Biotechnology and Medical Engineering, National Institute of Technology,
Rourkela, Odisha, India
C. Anandharamakrishnan
Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing
Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur,
India
Arfat Anis
Department of Chemical Engineering, King Saud University, Riyadh, Saudi Arabia
Muhammad Arshad
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB,
Canada
Indranil Banerjee
Manav Bandhu Bera
Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and
Technology, Longowal, Sangrur, Punjab, India
Rakesh Bhaskar
Pravin Bhattarai
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing,
Haidian, China
Nisarani Bishoyi
Department of Chemistry, NIT Rourkela, Rourkela, Odisha, India
Prasanta Kumar Biswas
Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata,
West Bengal, India
Ragini G Bodade
Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India
Anand G Bodade
Department of Transfusion Medicine, Seth G S Medical College and KEM Hospital, Mumbai,
Maharashtra, India
xxi
xxii Contributors
Subhadeep Bose
Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata,
West Bengal, India
Muhammed Yusuf Ça glar
Istanbul Sabahattin Zaim University, Faculty of Engineering and Natural Sciences, Department
_
of Food Engineering, Istanbul, Turkey
Mustafa Çavuş
Igdır University, Engineering Faculty, Department of Food Engineering, Igdır, Turkey
Jianshe Chen
School of Food Science, University of Idaho, Moscow, ID, United States
Harish Kumar Chopra
Department of Chemistry, Sant Longowal Institute of Engineering and Technology, Longowal,
Sangrur, Punjab, India
Donghwa Chung
Food Technology Major, Graduate School of International Agricultural Technology, Institute of
Green Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon,
Republic of Korea
Geta David
Gh. Asachi Technical University of Iasi, Iasi, Romania
Raj Deb
Mehmet Demirci
_
Win-Ping Deng
School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; Stem
Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan;
Graduate Institute of Basic Science, Fu Jen Catholic University, New Taipei City, Taiwan
Chanda Vilas Dhumal
Navneet Kumar Dubey
Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
Rajni Dubey
Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan
Sayantani Dutta
India
Contributors xxiii
Rimpi Foujdar
Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and
Technology, Longowal, Sangrur, Punjab, India
Advaita Ganguly
Comprehensive Tissue Centre, UAH Transplant Services, Alberta Health Services, Edmonton,
AB, Canada; Health Sciences Education and Research Commons, University of Alberta,
Edmonton, AB, Canada
Ann Mary George
Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of
Higher Education, Manipal, Karnataka, India
Mukesh Kumar Gupta
Sadaf Hameed
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing,
Haidian, China
Thi Thanh Hanh Nguyen
The Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul
National University, Pyeongchang-gun, Gangwon-do, Republic of Korea; Department of
International Agricultural Technology & Institute of Green BioScience and Technology, Seoul
National University, Pyeongchang, Gangwon-do, Republic of Korea
Md Saquib Hasnain
Department of Pharmacy, Shri Venkateshwara University, Amroha, Uttar Pradesh, India
Monjurul Hoque
Margaret O. Ilomuanya
Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,
University of Lagos, Surulere, Lagos, Nigeria
John Jeslin
Department of Biotechnology, St. Joseph’s College of Engineering, Chennai, Tamil Nadu, India
Juhui Jin
Graduate School of International Agricultural Technology, Seoul National University,
Pyeongchang-gun, Gangwon-do, Republic of Korea
Padmaja Kar
Department of Chemistry, NIT Rourkela, Rourkela, Odisha, India
Hyo Jin Kim
Pyeongchang, Gwangwon-do, Republic of Korea; Institutes of Green Bio Science and
Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea
xxiv Contributors
Doman Kim
Department of International Agricultural Technology & Institutes of Green BioScience and
Technology, Seoul National University, Pyeongchang, Gangwon-do, Republic of Korea; The
Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National
University, Pyeongchang-gun, Gangwon-do, Republic of Korea; Graduate School of
International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-
do, Republic of Korea
Sanjeev Kumar
Department of Biotechnology, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan,
Himachal Pradesh, India
Chi-Ching Lee
_
Timothy Lee Turner
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern
University, Chicago, IL, United States
L. Mahalakshmi
Computational Modeling and Nanoscale Processing Unit, Indian Institute of Food Processing
Tamil Nadu, India
Tanushree Maity
Defence Research and Development Organization, DRDO Bhawan, Rajaji Marg, New Delhi,
India
Samrendra Maji
SRM Research Institute, SRM Institute of Science and Technology, Kanchipuram, Tamil Nadu,
India
Kaustav Majumder
Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE,
United States
M. Maria Leena
Computational Modeling and Nanoscale Processing Unit, Indian Institute of Food Processing
Tamil Nadu, India
Nupur Mohapatra
Jeyan A. Moses
India
Contributors xxv
Soma Mukherjee
Department of Veterinary Medicine School, Mississippi State University, Mississippi State, MS,
United States
Amit Kumar Nayak
Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj,
Odisha, India
Suraj Kumar Nayak
Bhagyashree Padhan
Dilipkumar Pal
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur,
Chhattisgarh, India
Kunal Pal
Ashok R. Patel
Guangdong Technion Israel Institute of Technology, Shantou, China
Rehan Ali Pradhan
Canada
Sushant Prajapati
Dilshad Qureshi
K.J. Rao
Department of Biotechnology, Veltech University, Chennai, Tamil Nadu, India
Sirsendu S. Ray
Sai Preetham Reddy Peddireddy
xxvi Contributors
Fiona Concy Rodrigues

Sai S. Sagiri
Department of Chemistry and Biochemistry, The City College of New York, New York, NY, United
States
Lanny Sapei
Department of Chemical Engineering, University of Surabaya, Raya Kalirungkut, Surabaya, East
Java, Indonesia
Nandini Sarkar
Department of Biotechnology and Medical Engineering, National Institute of Technology
Rourkela, Rourkela, Odisha, India
Preetam Sarkar
Angana Sarkar
Alok Saxena
Amity Institute of Food Technology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India
Iis Septiana
Pyeongchang-gun, Gangwon-do, Republic of Korea
Kumakshi Sharma
Health, Safety and Environment Branch, National Research Council Canada, Edmonton, AB,
Canada
Loveleen Sharma
Amity Institute of Food Technology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India
Abhinay Kumar Singh
Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
Vinay K. Singh
Research and Development, Intas Pharmaceuticals Limited, Ahmedabad-Gujarat, India
Agustin Wulan Suci Dharmayanti
University of Jember, Jember, East Java, Indonesia
Yumewo Suzuki
DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan
Irshaan Syed
Contributors xxvii
Akira Tabuchi
Goutam Thakur
Aman Ullah
Canada
Rituja Upadhyay
School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China;
School of Food Science, University of Idaho, Moscow, ID, United States
Madan L. Verma
Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC, Australia
Lei Wang
Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE,
United States
Varsha Wankhade
Department of Zoology, Savitribai Phule Pune University, Pune, Maharashtra, India
Hiroyuki Yamada
Kazuhiko Yamatoya
Yue Zhang
College of Food & Biology Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang,
China; Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln,
NE, United States
Muhammad Zubair
Canada
CHAPTER
Introduction of biopolymers: food

and biomedical applications
1
Dilshad Qureshi1, Suraj Kumar Nayak1, Arfat Anis2, Sirsendu S. Ray1, Doman Kim3,
Thi Thanh Hanh Nguyen3, Kunal Pal1
1
Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India;
2
Department of Chemical Engineering, King Saud University, Riyadh, Saudi Arabia; 3Department of International
Agricultural Technology & Institute of Green BioScience and Technology, Seoul National University, Pyeongchang,
Gwangwon-do, Republic of Korea
1. Introduction
Polymers are macromolecules which usually have monomeric units (same or different) that combine in
different ways (Mills and White, 2012). The polymers may be either of synthetic or natural origin (Deb
et al., 2019). Nowadays, humankind has become more dependent on the use of synthetic polymers
(Buggy, 2016). Unfortunately, the use of these synthetic polymers is associated with a large number of
environmental and health issues (Kaushik et al., 2016). This can explain the search of natural polymers
by the researchers, which can efficiently be used to replace the synthetic polymers for different ap-
plications. The natural origin polymers are also regarded as “biopolymers” (Numata and Kaplan,
2011). Hence, biopolymers can be described as naturally occurring macromolecules which are usually
produced by living systems including plants, animals, and microorganisms (Yadav et al., 2015). In
recent years, there is a growing tendency to use more of natural polymers for developing various food
and biomedical products (Babu et al., 2013; Davidenko et al., 2014; Verbeek, 2012). It is noteworthy to
mention over here that the mankind has been using biopolymers not only for food and biomedical
applications, where it has found numerous applications, but also in textile, cosmetics, pharmaceuticals,
and paper industries (Anwunobi and Emeje, 2011).
As previously mentioned biopolymers contain repeating units of monomers and are usually derived
from plants, animals, and microorganisms. In general, the repeating units of a biopolymer may either
be sugars, amino acids, or fermentative products like aliphatic polyesters (Prameela et al., 2018).
These biopolymers may have different functional groups like hydroxyl, amino, amide, carboxyl,
phosphate, phenolic, etc. (Li et al., 2012), which impart to their different biological activities (Kim,
2016). The biopolymers are usually broadly classified into three groups, namely, polysaccharides,
proteins, and polynucleotides (Mohan et al., 2016). Polysaccharides are generally made up of sugar
moieties which are covalently bonded with each other via glycosidic linkages (Garcı́a, 2018). Removal
of one water molecule occurs with the formation of each glycosidic bond. On the basis of charge,
polysaccharides can be neutral (dextran, pullulan), polycationic (chitosan), and polyanionic (alginate)
Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00001-1

Copyright © 2020 Elsevier Inc. All rights reserved.
1
2 Chapter 1 Introduction of biopolymers
(Harding et al., 2015). Polysaccharides are homogenous (containing one type of monomers such as
glycogen) or heterogeneous (containing different sugar units, e.g., xanthan gum, gellan gum)
(Johnson-Green, 2002). On the other hand, proteins may be defined as the polymers that consist of
amino acid moieties as the monomeric unit. These amino acids are joined together by amide linkages
resulting in the formation of three-dimensional (3D) structure (Wool and Sun, 2011). This polyamide
chain is the basic level in the hierarchy of the protein structure. Various molecular interactions such as
hydrogen bonding, salt and disulfide bridges, and hydrophobic and hydrophilic interactions are
responsible for the folding of the chain into secondary structures (e.g., a-helix, b-pleated sheets). The
structure is further packed closely through the aforementioned interactions into tertiary structures.
Interactions among different protein subunits result in the formation of a quaternary structure (Pollock,
2007). Interestingly, the other class of biopolymers, i.e., polynucleotides (such as DNA, RNA),
comprise of as many as 13 or more nucleotide monomers (Davidenko et al., 2014). These two het-
eropolymeric molecules exhibit significance in the living nature. They have a distinct biological
function regarding the storage, replication, and discerning of the genetic information. The backbone
framework of the two nucleic acids comprises of a phosphate group, sugar moiety, and four nitrog-
enous bases with few differences (Frank-Kamenetskii, 2005).
Due to the availability of the biopolymers with different chemistries, it is possible to formulate
various food and biomedical products that have varied structural and physicochemical properties. This
is possible because of the presence of different functional groups on the polymeric chains of the
biopolymer (SenGupta, 2007). These functional groups help the biopolymers to interact with
the different components which are present in the products (Maleki, 2008; Shishir et al., 2018). Despite
the aforementioned advantages, the biopolymeric materials have been reported to have inadequate
mechanical properties, which make them unsuitable for use in designing specific products (Vieira
et al., 2011). To circumvent this problem, many authors have proposed the use of cross-linkers, which
are multifunctional chemical agents, and have the ability to form covalent bonds with the polymer
chains (Reddy et al., 2015). Further, the employed cross-linking technique may also result in the
formation of products with varied properties as the chemical reaction may take place in different ways
when environmental conditions of the cross-linking reaction are changed (Akakuru and Isiuku, 2017;
Thakur et al., 2017).
Considering the above discussion in mind, in this chapter, we will be discussing about the various
cross-linking strategies for the biopolymers, the properties of some of the commonly used poly-
saccharide and protein-based biopolymers, and finally their food and biomedical applications.
2. Cross-linking methods employed to design biopolymer-based

polymeric architectures
Biopolymers are being investigated thoroughly to design matrices for food and biomedical applica-
tions. Some of the common applications include controlled release of bioactive molecules (e.g., fla-
vors, proteins, probiotics, vitamins, drugs, mammalian cells including stem cells etc.), scaffold
fabrication for regenerative medicine, gels for food, pharmaceuticals, and biomedical applications
(Shit and Shah, 2014; Udenni Gunathilake et al., 2017). In many of the aforesaid applications,
designed polymeric architectures are expected to undergo degradation under certain conditions (Jain
et al., 2017). It is important that the products generated from the degradation of these polymeric
2. Cross-linking methods employed to design biopolymer-based polymeric 3
architectures
architectures are harmless to the human body (Ulery et al., 2011). Such a requirement is necessary for
ensuring good biocompatibility of the polymeric architectures. In this section, we will discuss about
the different physical and chemical cross-linking strategies that are used for designing polymeric
architectures for different applications. It is important to note that the chemical cross-linking strategies
help in designing polymeric architectures with very good mechanical stability (Ozbolat, 2016). Un-
fortunately, the cross-linking agents (the chemical compounds used for cross-linking) have mostly
been reported to be toxic (Ozbolat, 2016) and can also react with the bioactive agents which are loaded
in the polymeric architectures, thereby rendering them inactive. Such disadvantage can be easily
avoided if the physical cross-linking strategies are applied (Puoci, 2014). The common cross-linking
strategies employed have been discussed in this section.
2.1 Physically cross-linked gels

Recent years has seen an increased interest in employing physical cross-linking strategies to design
polymeric architectures (de Oliveira et al., 2019; Ebara et al., 2014; Montaser et al., 2019). This is
mainly because the physical cross-linking strategies help in eliminating the use of chemical cross-
linking agents. In this section, various physical cross-linking strategies for biopolymers will be
discussed.
2.1.1 Cross-linking by ionic interactions

Alginate is a commonly used biopolymer that can be easily cross-linked by ionic interactions. The
biopolymer consists of b-D-mannuronic acid and a-L-guluronic acid residues. These acidic residues
can ionically interact with multivalent cations like calcium (Ca2þ) ions. Guluronate acid residues play
a prominent role in the cross-linking of alginate by interacting with Ca2þ ions and forming a char-
acteristic “egg-box” structure (Bruchet and Melman, 2015) (Fig. 1.1).
In many cases, alginate beads are being prepared by adding aqueous solutions of sodium alginate
(SA) or potassium alginate into an aqueous solution of Ca2þ ions prepared using calcium chloride salt.
The cross-linking of the alginate polymer occurs very fast resulting in the formation of globular gels.
Unfortunately, this method results in the formation of gel beads with varying cross-linking density and
a polymer concentration gradient, thereby resulting in the formation of gel beads with different
physical stabilities (Kuo and Ma, 2001) (Fig. 1.2).
In an alternative method, calcium carbonate (CaCO3) powder (having very low solubility in water)
is homogeneously dispersed in the alginate solution. The gelation of the alginate phase is induced by
releasing Ca2þ with the addition of acids (e.g., acetic acid, Glucono-a-lactone) (Connon and Hamley,
2014; Thakur et al., 2017). Since the CaCO3 is homogeneously dispersed within the alginate solution,
the cross-linking of the alginate matrices occurs in a near homogeneous manner (Rehm, 2009). Hence,
the gels produced by this method are usually uniform gels. The use of calcium sulfate (CaSO4) has
been explored for the cross-linking of the alginate polymer by many authors. Unfortunately, it is very
difficult to control the gelation kinetics of alginate phase when CaSO4 is used as the ionically cross-
linking material. In the year 2001, Kuo and Ma reported that the mixtures of CaCO3 and D-glucono-d-
lactone, and CaCO3, D-glucono-d-lactone, and CaSO4 were able to reduce the rate of gelation
significantly which allowed them to prepare alginate matrices having structurally uniform properties
(Kuo and Ma, 2001) (Fig. 1.3).
FIGURE 1.1
Structural framework of the alginate backbone.
Alginate has been recognized as an important ingredient in food industries as a thickening agent,
gelling agent, film forming, stabilizing, and emulsifying agent (Qin et al., 2018). Along with this,
alginate has wide applications in pharmaceutics for controlled drug delivery, and in biomedical sci-
ences in cell culture, tissue regeneration, and wound healing (Lee and Mooney, 2012).
architectures
FIGURE 1.2
Schematic diagram of the external gelation of the alginate solution using calcium chloride salt solution.
In a similar manner, polycationic polymers like chitosan can be ionically cross-linked using pol-
yanions (Wu et al., 2014). One such example is chitosan. It consists of b-(1 / 4)-linked glucosamine
units. The solutions of hydrated glycerol phosphate disodium salt and sodium triphosphate are two of
the commonly used physical cross-linkers of the chitosan (Patil, 2008; Saharan and Pal, 2016). Further,
it is important to note that when the solution of polymeric anions (e.g., alginate) and polymeric cations
(e.g., chitosan) are mixed together, the oppositely charged ionic groups present in the polymers
FIGURE 1.3
Structural diagram of the internal gelation of the alginate solution using calcium carbonate and glucono-d-
lactone.
ionically interact with each other to form a gelled network (Kulig et al., 2016). Unfortunately, these
gels usually form structurally inhomogeneous matrices (Kuo and Ma, 2008). Due to this reason,
matrices formed as per this methodology is not commonly used. Preparation of matrices using this
methodology is carried out very carefully. This polymer-polymer complexation process has mainly
been restricted to the coating of one of the polymer with the other. As for example, Gandomi et al.
(2016) coated porous alginate matrices with a chitosan layer, which resulted in the formation of a
semipermeable membrane across the alginate matrix (Gandomi et al., 2016). It is surprising to note
that many neutral biopolymers (which do not have any ionic groups) can also result in the formation of
gelled structures in the presence of ions. One such example is dextran biopolymer. This polymer can
form gelled structure through crystallization by forming hydrogen bonds or in the presence of po-
tassium (Kþ) ions. Although being neutral, the glucose residues in the polymer chains of the dextran
molecules form a cage-like structure. The Kþ ions are suitably placed within the cage. This phe-
nomenon results in the formation of a complex cross-linked structure (Mishra, 2017).
Though ionic cross-linking allows us to avoid the use of generally toxic chemical cross-linkers
(Grumezescu, 2018b), these structures are usually unstable in nature in aqueous/physiological envi-
ronment (Kuo and Ma, 2008). They get destabilized and undergo structural breakdown when placed in
the aforesaid conditions (Grumezescu, 2018a). This phenomenon appears to be useful in some food
applications but in many biomedical applications such phenomenon is undesirable. Hence, for
biomedical applications usually a second-stage cross-linking is carried out after the initial ionic cross-
linking process (Peppas, 2010; Thakur and Thakur, 2015).
2.1.2 Self-assembly of hydrophobized polysaccharides

Gelation in water-based systems using amphiphilic molecules has been widely studied. In many of
these systems, the amphiphilic molecules undergo self-assembly to form a network-like structure,
which, in turn, induces the gelation of the aqueous phase (Malo de Molina and Gradzielski, 2017).
Many authors have explored this technique for designing polysaccharide-based physical hydrogels
(Fig. 1.4). Hydrophobic modifications of the polysaccharide chains can help in achieving this. The
FIGURE 1.4
Schematic diagram of the cross-linking of hydrophobized polysaccharides through self-assembly.
architectures
hydrophobic modification renders the polysaccharide as amphiphiles. In other words, the biopolymers
become polymer amphiphiles (Alhaique et al., 2015). The self-assembly or the aggregation of these
polymer amphiphiles to form gelled structures has been greatly explored for pharmaceutical and
biotechnological applications (Hassani et al., 2012; Thomas et al., 2018). Some of the common
polysaccharides which have been successfully modified for designing hydrogels include carboxy-
methylcellulose (CMC), hyaluronic acid, guar gum, chitosan (Camponeschi et al., 2015), starch,
alginate, agarose (Ahmed, 2015), dextran, pullulan, and carboxymethyl curdlan (Maitra and Shukla,
2014). The hydrophobization of the polysaccharides are usually achieved by preparing palmitoyl,
cholesterol, and polyester side-chain-substituted derivatives of the previously mentioned poly-
saccharides (Liu et al., 2016). Modification of the chitosan using polyacrylic acid and poly-N-
isopropylacrylamide polymers has also been proposed for designing pH- and temperature-sensitive
hydrogels, respectively (Kim et al., 2000; Yazdani-Pedram et al., 2000). Further, the modification
of carboxymethyl dextran with poly-N-isopropylacrylamide to design thermosensitive hydrogels has
also been proposed (Huh et al., 2000).
2.1.3 Cross-linking by the crystallization of the polysaccharides

The crystallization of the polysaccharides, which results in the formation of a network structure, is
prepared by freeze-thaw technique. The hydrogels prepared by this method are also reported as
“physical hydrogels.” In these gels, a specific type of gelation is induced by cryogenic treatment of the
polysaccharide solution. It is reported that during the freezing and subsequent storage of the poly-
saccharide, solution in the frozen state results in the formation of side-by-side associations of the
polysaccharide chains during the crystallization (freezing) of the solvent (in this case water) (Fig. 1.5).
When this frozen structure is thawed the polysaccharide network which has been formed during the
crystallization step maintains its structural integrity (Zhang et al., 2013). This methodology has been
used to design hydrogels using various polysaccharide including maltodextrins, locus bean gum,
starch, agarose, hyaluronan, and xanthan (Bhatia, 2016).
FIGURE 1.5
Schematic diagram of the cross-linking of polysaccharides using freeze-thaw method.
2.1.4 Cross-linking by induction of hydrogen bonds among the polysaccharide

molecules
Hydrogen bonding among the polysaccharide molecules is one of the major mechanisms that is
employed for the gelation of the aqueous phase. The gelation of the agarose solution in water is one
such example. Agarose remains insoluble in cold water. The agarose gels are formed by dissolving the
agarose powder in near boiling water, which is subsequently cooled down to room temperature to
induce gelation (Zucca et al., 2016). The gelation is induced by the formation of inter- and intra-
molecular hydrogen bonds, which are responsible for maintaining the 3D structure of the agarose gels
(Tako and Nakamura, 1988) (Fig. 1.6). This structure can be destabilized by disrupting the hydrogen
bonds by supplying external thermal energy. This results in the conversion of the gelled structure into a
liquid state (Bansal and Boccaccini, 2012). Another example of cross-linking by induction of
hydrogen bonds is the gelation of the chitosan molecules in a pH-dependent fashion. Chenite et al.
(2000) reported the formation of such hydrogels by adding glycerol phosphate salts to aqueous so-
lutions of chitosan (Chenite et al., 2000). The gels were produced by heating the solution to body
temperature. They reported that the thermal reversibility of gelation is dependent on the pH of the
chitosan and glycerol phosphate solutions. If the final mixture was at a pH value in the range of
6.9e7.2, then the gels formed were partially thermoreversible. On the contrary, completely ther-
moreversible hydrogels could be produced when the pH values were in the range of 6.5 and 6.9. The
complete thermoreversibility of the gels whose solution was having pH in the range of 6.5 and 6.9 was
attributed to the inhibition of the hydrogen bond formation with the consequent higher electronic
repulsion within the polymeric chains of the chitosan molecules (Chenite et al., 2000). Hydrogen
bonding also plays an important role in the preparation of the protein-based gels including gelatin gels
(Lean, 2006). It is worthy to note that in the gels prepared by hydrogen bonding, hydrophobic in-
teractions also play an important role to a certain extent, and vice versa (Chang et al., 2018b; Karino
et al., 2002).
FIGURE 1.6
Schematic diagram of hydrogen bondingeinduced cross-linking of polysaccharide chains.
architectures
2.1.5 Cross-linking using protein molecules

The interactions between the specific peptide domains have been reported to synthesize gels. In such a
method, two peptide domains that show positive interactions are incorporated separately into nonin-
teracting polymeric chains. When these two protein-engineered polymeric solutions are mixed
together, the positive interactions between the peptide domains incorporated within the polymer chains
serve as the cross-linking points. This results in the formation of a 3D network structure with a
capability to immobilize water molecules (Fig. 1.7). The main advantage in this type of hydrogels is
that there is no need to alter the pH, temperature, and ionic strength of the polymeric solution during
the formation of the hydrogels. This is beneficial when various living biological entities, e.g., microbial
and mammalian cells, and biologically active agents are incorporated within the hydrogel matrix (Foo
et al., 2009).
2.2 Chemical cross-linking

Physical cross-linking methods provide a good opportunity to improve the properties of the bio-
polymeric structures. Unfortunately, in many cases where an alteration in the environmental conditions
(e.g., pH, temperature, and ionic strength) occurs during the application stage, the polymeric archi-
tectures may lose their structural integrity (Saini, 2017). Though in many applications, this property
may be useful, but in many applications this property is undesirable. Hence, it has been found that even
FIGURE 1.7
Schematic diagram of the cross-linking of polymers using proteins as cross-linking agents.
after carrying out the physical cross-linking, the polymeric architectures are further being chemically
cross-linked so as to consolidate the structural properties of the physically cross-linked polymeric
architectures (Parhi, 2017). Also, many biopolymers/biopolymer mixtures cannot be physically cross-
linked. In such cases, chemical cross-linking remains the only available strategy for improving the
structural properties of the biopolymeric architectures. The chemical cross-linkers are basically
multifunctional chemically reactive molecules which can form covalent bonds with the functional
groups present in the biopolymers (Shen et al., 2016). In this way, chemical cross-linkers form
interconnecting bridges among the biopolymeric molecules. During the process of chemical cross-
linking, there is an increase in the molecular weight with the corresponding increase in the me-
chanical and the structural ability of the polymeric architectures (Maitra and Shukla, 2014).
Though the chemical cross-linkers can provide the aforesaid advantages, they are not devoid of the
disadvantages. The primary concern with the use of cross-linkers is the toxicity due to the presence of
unreactive monomers within the polymeric architectures (Patel and Mequanint, 2011). Hence, proper
care should be taken to eliminate the unreactive monomers after the cross-linking process is over.
Further, the sites for the degradation of the polymeric structures can be significantly altered due to the
chemical reaction occurring during the cross-linking process. This may significantly reduce the
biodegradability of the biopolymeric architectures (Azeredo and Waldron, 2016). Also, there is a
decrease in the number of functional groups within the biopolymeric architectures. This may alter the
environment-sensitive properties of some of the biopolymers, which, in turn, will compromise the
environment-sensitive properties of the final product (He et al., 2012).
The researchers have explored various chemical cross-linkers and cross-linking strategies. Some of
the common cross-linkers used for the cross-linking of biopolymers include glutaraldehyde, poly-
carboxylic acids like citric acid and maleic acid, EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide/N-hydroxysuccinimide), epichlorohydrin, STMP (trisodium metaphosphite),
polysaccharide dialdehydes like dextran dialdehyde and alginate dialdehyde, genipin, proanthocya-
nidin (PA), and glyoxal. In this section, we will be discussing in brief the cross-linking mechanism of
the aforesaid cross-linking agents.
2.2.1 Glutaraldehyde
Glutaraldehyde is a dialdehyde and is very commonly used for its ability to react with both protein and
polysaccharides. It can stabilize protein and polysaccharide-based biopolymeric architectures with a
very high efficiency. This cross-linking agent is a nonspecific cross-linker and the cross-linking can
happen both between inter- and intramolecular functional groups. The aldehydic group present in the
glutaraldehyde molecules has the capability to react with the free amino groups present in the poly-
peptide chain (Fan et al., 2018). Further, glutaraldehyde can also react with the hydroxyl groups which
are present in both proteins and polysaccharide molecules (W. Wang et al., 2016). The mechanism of
the chemical reaction has been provided in Fig. 1.8. It is noteworthy to mention that all the
glutaraldehyde-based cross-linking occurs at low pH. Hence, the addition of acids in the reaction
mixture is an essential step in glutaraldehyde-based cross-linking.
Apart from mixing the glutaraldehyde reagent in the solution form to the polymeric solutions to
induce cross-linking, many authors have reported vapor-phase cross-linking using glutaraldehyde
(Destaye et al., 2013; Lu et al., 2015). In this process, glutaraldehyde solutions are made in different
concentrations. The gels/matrices that need to be cross-linked are placed at the top of the container,
which contains glutaraldehyde solutions of definite concentration using suitable attachments.
architectures
FIGURE 1.8
Schematic representation for the cross-linking of the polymer using glutaraldehyde.
Thereafter, the container is completely sealed. This arrangement is incubated at a particular temper-
ature for a definite time period. During the incubation process, the glutaraldehyde vapors are released
from the solution, which then interacts with the samples to be cross-linked. It has been found that an
alteration in the concentration of the glutaraldehyde solutions results in the differential cross-linking
density (Lu et al., 2015).
2.2.2 Polycarboxylic acids

Polycarboxylic acids like citric acid, maleic acid, succinic acid, and tricarballylic acid, which have
more than three carboxylic groups, have been used to cross-link cellulose and starch molecules. These
polycarboxylic acids are reported to react with the hydroxyl groups present in the cellulose and the
starch molecules. During the cross-linking process, an intermediate polycarboxylic acid anhydride is
reported to be formed (Fahmy and Fouda, 2008). A plausible mechanism for the reaction process using
polycarboxylic acids has been provided in Fig. 1.9.
2.2.3 EDC coupling

EDC, chemically known as carbodiimide, has been used to couple carboxyl groups to primary amines.
The coupling reaction results in the formation of an intermediate compound, which is known as amine-
reactive O-acylisourea. This intermediate product is stabilized using NHS or sulfo-NHS to form an
amine-reactive NHS or sulfo-NHS ester. Additionally, the O-acylisourea intermediate may also
chemically react with another amine group which is present in a separate biopolymeric chain. This
leads to the cross-linking of biopolymers having carboxyl and amine groups. It has been reported that
the removal of the excess EDC may be done easily using dialysis and gel filtration techniques. This is
possible due to the water-soluble nature of EDC (Conde et al., 2014). The probable mechanism of
EDC/NHS cross-linking mechanism has been provided in Fig. 1.10.
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Samuel Chase,
William Paca,
Maryland.
Thomas Stone,
Charles Carroll, of Carrollton.
George Wythe,
Richard Henry Lee,
Thomas Jefferson,
Virginia. Benjamin Harrison,
Thomas Nelson, jr.,
Francis Lightfoot Lee,
Carter Braxton.
William Hooper,
North Carolina. Joseph Hewes,
John Penn.
Edward Rutledge,
Thomas Heyward, jr.,
South Carolina.
Thomas Lynch, jr.,
Arthur Middleton.
Button Gwinnett,
Georgia. Lyman Hall,
George Walton.
Resolved, That copies of the Declaration be sent to the several
assemblies, conventions, and committees or councils of safety, and to
the several commanding officers of the Continental Troops: That it
be PROCLAIMED in each of the United States, and at the Head of the
Army.—[Jour. Cong., vol. 1, p. 396.]
Articles of Confederation.
Done at Philadelphia on the 9th day of July, 1778.

[While the Declaration of Independence was under consideration
in the Continental Congress, and before it was finally agreed upon,
measures were taken for the establishment of a constitutional form
of government; and on the 11th of June, 1776, it was “Resolved, That
a committee be appointed to prepare and digest the form of a
confederation to be entered into between these Colonies;” which
committee was appointed the next day, June 12, and consisted of a
member from each Colony, namely: Mr. Bartlett. Mr. S. Adams, Mr.
Hopkins, Mr. Sherman, Mr. R. R. Livingston, Mr. Dickinson, Mr.
McKean, Mr. Stone, Mr. Nelson, Mr. Hewes, Mr. E. Rutledge, and
Mr. Gwinnett. On the 12th of July, 1776, the committee reported a
draught of the Articles of Confederation, which was printed for the
use of the members under the strictest injunctions of secrecy.
This report underwent a thorough discussion in Congress, from
time to time, until the 15th of November, 1777; on which day,
“Articles of Confederation and Perpetual Union” were finally agreed
to in form, and they were directed to be proposed to the Legislatures
of all the United States, and if approved by them, they were advised
to authorize their delegates to ratify the same in the Congress of the
United States; and in that event they were to become conclusive. On
the 17th of November, 1777, the Congress agreed upon the form of a
circular letter to accompany the Articles of Confederation, which
concluded with a recommendation to each of the several Legislatures
“to invest its delegates with competent powers, ultimately, and in the
name and behalf of the State, to subscribe articles of confederation
and perpetual union of the United States, and to attend Congress for
that purpose on or before the 10th day of March next.” This letter
was signed by the President of Congress and sent, with a copy of the
articles, to each State Legislature.
On the 26th of June, 1778, Congress agreed upon the form of a
ratification of the Articles of Confederation, and directed a copy of
the articles and the ratification to be engrossed on parchment;
which, on the 9th of July, 1778, having been examined and the
blanks filled, was signed by the delegates of New Hampshire,
Massachusetts Bay, Rhode Island and Providence Plantations,
Connecticut, New York, Pennsylvania, Virginia, and South Carolina.
Congress then directed that a circular letter be addressed to the
States whose delegates were not present, or being present, conceived
they were not authorized to sign the ratification, informing them how
many and what States had ratified the Articles of Confederation, and
desiring them, with all convenient dispatch, to authorize their
delegates to ratify the same. Of these States, North Carolina ratified
on the 21st and Georgia on the 24th of July, 1778; New Jersey on the
26th of November following; Delaware on the 5th of May, 1779;
Maryland on the 1st of March, 1781; and on the 2d of March, 1781,
Congress assembled under the new form of government.]
ARTICLES OF CONFEDERATION.
To all to whom these presents shall come,

We, the undersigned, delegates of the States affixed to our names,
send greeting:
Whereas the delegates of the United States of America in Congress
assembled did, on the fifteenth day of November, in the year of our
Lord one thousand seven hundred and seventy-seven, and in the
second year of the independence of America, agree to certain Articles
of Confederation and Perpetual Union between the States of New
Hampshire, Massachusetts Bay, Rhode Island and Providence
Plantations, Connecticut, New York, New Jersey, Pennsylvania,
Delaware, Maryland, Virginia, North Carolina, South Carolina, and
Georgia, in the words following, viz:
Articles of Confederation and Perpetual Union between the States
of New Hampshire, Massachusetts Bay, Rhode Island and
Providence Plantations, Connecticut, New York, New Jersey,
Pennsylvania, Delaware, Maryland, Virginia, North Carolina,
South Carolina, and Georgia.
Article I. The style of this Confederacy shall be, “The United
States of America.”
Article II. Each State retains its sovereignty, freedom, and
independence, and every power, jurisdiction, and right, which is not
by this confederation expressly delegated to the United States in
Congress assembled.
Article III. The said States hereby severally enter into a firm
league of friendship with each other for their common defense, the
security of their liberties, and their mutual and general welfare;
binding themselves to assist each other against all force offered to, or
attacks made upon them, or any of them, on account of religion,
sovereignty, trade, or any other pretense whatever.
Article IV. The better to secure and perpetuate mutual friendship
and intercourse among the people of the different States in this
Union, the free inhabitants of each of these States, paupers,
vagabonds, and fugitives from justice excepted, shall be entitled to all
privileges and immunities of free citizens in the several States; and
the people of each State shall have free ingress and regress to and
from any other State, and shall enjoy therein all the privileges of
trade and commerce, subject to the same duties, impositions, and
restrictions, as the inhabitants thereof respectively: Provided, That
such restrictions, shall not extend so far as to prevent the removal of
property imported into any State to any other State, of which the
owner is an inhabitant: Provided, also, That no imposition, duties, or
restriction shall be laid by any State on the property of the United
States or either of them.
If any person guilty of or charged with treason, felony, or other
high misdemeanor, in any State, shall flee from justice, and be found
in any of the United States, he shall, upon demand of the governor or
executive power of the State from which he fled, be delivered up, and
removed to the State having jurisdiction of his offense.
Full faith and credit shall be given in each of these States to the
records, acts, and judicial proceedings of the courts and magistrates
of every other State.
Article V. For the more convenient management of the general
interests of the United States, delegates shall be annually appointed
in such manner as the Legislature of each State shall direct, to meet
in Congress on the first Monday in November, in every year, with a
power reserved to each State to recall its delegates or any of them, at
any time within the year, and to send others in their stead for the
remainder of the year.
No State shall be represented in Congress by less than two nor by
more than seven members; and no person shall be capable of being a
delegate for more than three years in any term of six years; nor shall
any person, being a delegate, be capable of holding any office under
the United States, for which he, or another for his benefit, receives
any salary, fees or emolument of any kind.
Each State shall maintain its own delegates in a meeting of the
States, and while they act as members of the committee of these
States.
In determining questions in the United States in Congress
assembled, each State shall have one vote.
Freedom of speech and debate in Congress shall not be impeached
or questioned in any court or place out of Congress; and the
members of Congress shall be protected in their persons from arrests
and imprisonments during the time of their going to and from, and
attendance on, Congress, except for treason, felony, or breach of the
peace.
Article VI. No State, without the consent of the United States in
Congress assembled, shall send any embassy to, or receive any
embassy from, or enter into any conference, agreement, alliance, or
treaty with any King, prince, or state; nor shall any person holding
any office of profit or trust under the United States, or any of them,
accept of any present, emolument, office or title of any kind whatever
from any King, prince, or foreign state; nor shall the United States in
Congress assembled, or any of them, grant any title of nobility.
No two or more States shall enter into any treaty, confederation, or
alliance whatever between them without the consent of the United
States in Congress assembled, specifying accurately the purposes for
which the same is to be entered into, and how long it shall continue.
No State shall lay any imposts or duties, which may interfere with
any stipulations in treaties entered into by the United States in
Congress assembled with any King, prince, or state, in pursuance of
any treaties already proposed by Congress to the Courts of France
and Spain.
No vessels of war shall be kept up in time of peace by any State,
except such number only as shall be deemed necessary by the United
States in Congress assembled, for the defense of such State, or its
trade; nor shall any body of forces be kept up by any State in time of
peace, except such number only, as, in the judgment of the United
States, in Congress assembled, shall be deemed requisite to garrison
the forts necessary for the defense of such State; but every State shall
always keep up a well regulated and disciplined militia, sufficiently
armed and accoutered, and shall provide and constantly have ready
for use, in public stores, a due number of field-pieces and tents, and
a proper quantity of arms, ammunition, and camp equipage.
No State shall engage in any war without the consent of the United
States in Congress assembled, unless such State be actually invaded
by enemies, or shall have received certain advice of a resolution
being formed by some nation of Indians to invade such State, and the
danger is so imminent as not to admit of a delay till the United States
in Congress assembled can be consulted; nor shall any State grant
commissions to any ships or vessels of war, nor letters of marque or
reprisal, except it be after a declaration of war by the United States in
Congress assembled; and then only against the kingdom or state, and
the subjects thereof, against which war has been so declared, and
under such regulations as shall be established by the United States in
Congress assembled, unless such State be infested by pirates, in
which case vessels of war may be fitted out for that occasion, and
kept so long as the danger shall continue, or until the United States
in Congress assembled shall determine otherwise.
Article VII. When land forces are raised by any State for the
common defense, all officers of, or under the rank of colonel, shall be
appointed by the Legislature of each State respectively by whom such
forces shall be raised, or in such manner as such State shall direct;
and all vacancies shall be filled up by the State which first made the
appointment.
Article VIII. All charges of war, and all other expenses that shall
be incurred for the common defense or general welfare and allowed
by the United States in Congress assembled, shall be defrayed out of
a common treasury, which shall be supplied by the several States, in
proportion to the value of all land within each State, granted to, or
surveyed for, any person, as such land and the buildings and
improvements thereon shall be estimated, according to such mode as
the United States in Congress assembled shall, from time to time,
direct and appoint.
The taxes for paying that proportion shall be laid and levied by the
authority and direction of the Legislatures of the several States,
within the time agreed upon by the United States in Congress
assembled.
Article IX. The United States in Congress assembled shall have
the sole and exclusive right and power of determining on peace and
war, except in the cases mentioned in the sixth article; of sending
and receiving embassadors; entering into treaties and alliances:
Provided, That no treaty of commerce shall be made whereby the
legislative power of the respective States shall be restrained from
imposing such imposts and duties on foreigners as their own people
are subjected to, or from prohibiting the exportation or importation
of any species of goods or commodities whatsoever; of establishing
rules for deciding, in all cases, what captures on land or water shall
be legal, and in what manner prizes taken by land or naval forces in
the service of the United States, shall be divided or appropriated; of
granting letters of marque and reprisal in times of peace; appointing
courts for the trial of piracies and felonies committed on the high
seas, and establishing courts for receiving and determining finally,
appeals in all cases of captures: Provided, That no member of
Congress shall be appointed a judge of any of the said courts.
The United States in Congress assembled shall also be the last
resort on appeal in all disputes and differences now subsisting, or
that hereafter may arise between two or more States concerning
boundary, jurisdiction, or any other cause whatever; which authority
shall always be exercised in the manner following: Whenever the
legislative or executive authority or lawful agent of any State in
controversy with another, shall present a petition to Congress,
stating the matter in question, and praying for a hearing, notice
thereof shall be given by order of Congress to the legislative or
executive authority of the other State in controversy, and a day
assigned for the appearance of the parties by their lawful agents, who
shall then be directed to appoint, by joint consent, commissioners or
judges to constitute a court for hearing and determining the matter
in question; but if they cannot agree, Congress shall name three
persons out of each of the United States, and from the list of such
persons each party shall alternately strike out one, the petitioners
beginning, until the number shall be reduced to thirteen; and from
that number not less than seven nor more than nine names, as
Congress shall direct, shall, in the presence of Congress, be drawn
out by lot; and the persons whose names shall be so drawn, or any
five of them, shall be commissioners or judges, to hear and finally
determine the controversy, so always as a major part of the judges
who shall hear the cause, shall agree in the determination; and if
either party shall neglect to attend at the day appointed, without
showing reasons which Congress shall judge sufficient, or, being
present, shall refuse to strike, the Congress shall proceed to
nominate three persons out of each State, and the Secretary of
Congress shall strike in behalf of such party absent or refusing; and
the judgment and sentence of the court to be appointed in the
manner before prescribed shall be final and conclusive; and if any of
the parties shall refuse to submit to the authority of such court or to
appear or defend their claim or cause, the court shall, nevertheless,
proceed to pronounce sentence or judgment, which shall, in like
manner, be final and decisive; the judgment or sentence, and other
proceedings, being in either case transmitted to Congress, and
lodged among the acts of Congress for the security of the parties
concerned: Provided, That every commissioner, before he sits in
judgment, shall take an oath, to be administered by one of the judges
of the supreme or superior court of the State, where the cause shall
be tried, “well and truly to hear and determine the matter in
question, according to the best of his judgment without favor,
affection, or hope of reward:” Provided, also, That no State shall be
deprived of territory for the benefit of the United States.
All controversies concerning the private right of soil claimed under
different grants of two or more States, whose jurisdictions, as they
may respect such lands, and the States which passed such grants, are
adjusted, the said grants or either of them being at the same time
claimed to have originated antecedent to such settlement of
jurisdiction, shall, on the petition of either party to the Congress of
the United States, be finally determined, as near as may be, in the
same manner as is before prescribed for deciding disputes respecting
territorial jurisdiction between different States.
The United States in Congress assembled shall also have the sole
and exclusive right and power of regulating the alloy and value of
coin struck by their own authority, or by that of the respective States;
fixing the standard of weights and measures throughout the United
States; regulating the trade and managing all affairs with the
Indians, not members of any of the States: Provided, That the
legislative right of any State within its own limits, be not infringed or
violated; establishing and regulating post-offices from one State to
another, throughout all the United States, and exacting such postage
on the papers passing through the same, as may be requisite to
defray the expenses of the said office; appointing all officers of the
land forces in the service of the United States, excepting regimental
officers; appointing all the officers of the naval forces, and
commissioning all officers whatever in the service of the United
States; making rules for the government and regulation of the said
land and naval forces, and directing their operations.
The United States in Congress assembled shall have authority to
appoint a committee to sit in the recess of Congress, to be
denominated “a Committee of the States,” and to consist of one
delegate from each State, and to appoint such other committees and
civil officers as may be necessary for managing the general affairs of
the United States, under their direction; to appoint one of their
number to preside; provided that no person be allowed to serve in
the office of president more than one year in any term of three years;
to ascertain the necessary sums of money to be raised for the service
of the United States, and to appropriate and apply the same for
defraying the public expenses; to borrow money or emit bills on the
credit of the United States, transmitting every half-year to the
respective States, an account of the sums of money so borrowed or
emitted; to build and equip a navy; to agree upon the number of land
forces, and to make requisitions from each State for its quota, in
proportion to the number of white inhabitants in such State, which
requisitions shall be binding; and thereupon the Legislature of each
State shall appoint the regimental officers, raise the men, and clothe,
arm, and equip them in a soldier-like manner, at the expense of the
United States; and the officers and men so clothed, armed, and
equipped, shall march to the place appointed, and within the time
agreed on by the United States in Congress assembled; but if the
United States in Congress assembled shall, on consideration of
circumstances, judge proper that any State should not raise men, or
should raise a smaller number than its quota, and that any other
State should raise a greater number of men than the quota thereof,
such extra number shall be raised, officered, clothed, armed, and
equipped in the same manner as the quota of each State, unless the
Legislature of such State shall judge that such extra number cannot
be safely spared out of the same; in which case they shall raise,
officer, clothe, arm, and equip as many of such extra number as they
judge can be safely spared. And the officers and men so clothed,
armed, and equipped shall march to the place appointed, and within
the time agreed on by the United States in Congress assembled.
The United States in Congress assembled shall never engage in a
war, nor grant letters of marque and reprisal in time of peace, nor
enter into any treaties or alliances, nor coin money, nor regulate the
value thereof, nor ascertain the sums and expenses necessary for the
defense and welfare of the United States or any of them, nor emit
bills, nor borrow money on the credit of the United States, nor
appropriate money, nor agree upon the number of vessels of war to
be built or purchased, or the number of land or sea forces to be
raised, nor appoint a commander-in-chief of the Army or Navy,
unless nine States assent to the same; nor shall a question on any
other point, except for adjourning from day to day, be determined,
unless by the votes of a majority of the United States in Congress
assembled.
The Congress of the United States shall have power to adjourn to
any time within the year, and to any place within the United States,
so that no period of adjournment be for a longer duration than the
space of six months; and shall publish the journal of their
proceedings monthly, except such parts thereof relating to treaties,
alliances, or military operations, as in their judgment require
secrecy; and the yeas and nays of the delegates of each State on any
question, shall be entered on the journal, when it is desired by any
delegate; and the delegates of a State, or any of them, at his or their
request, shall be furnished with a transcript of the said journal,
except such parts as are above excepted, to lay before the Legislature
of the several States.
Article X. The committee of the States, or any nine of them, shall
be authorized to execute, in the recess of Congress, such of the
powers of Congress as the United States in Congress assembled, by
the consent of nine States, shall, from time to time, think expedient
to vest them with: Provided, That no power be delegated to the said
committee, for the exercise of which, by the Articles of
Confederation, the voice of nine States in the Congress of the United
States assembled is requisite.
Article XI. Canada, acceding to this confederation, and joining in
the measures of the United States, shall be admitted into, and
entitled to, all the advantages of this Union; but no other colony shall
be admitted into the same, unless such admission be agreed to by
nine States.
Article XII. All bills of credit emitted, moneys borrowed, and
debts contracted, by or under the authority of Congress, before the
assembling of the United States, in pursuance of the present
confederation, shall be deemed and considered as a charge against
the United States, for payment and satisfaction whereof the said
United States and the public faith are hereby solemnly pledged.
Article XIII. Every State shall abide by the determinations of the
United States in Congress assembled, on all questions which by this
confederation are submitted to them. And the articles of this
confederation shall be inviolably observed by every State, and the
union shall be perpetual; nor shall any alteration at any time
hereafter be made in any of them, unless such alteration be agreed to
in a Congress of the United States, and be afterwards confirmed by
the Legislatures of every State.
And whereas it has pleased the Great Governor of the world to
incline the hearts of the Legislatures we respectively represent in
Congress, to approve of, and to authorize us to ratify the said articles
of confederation and perpetual union: Know ye, That we, the
undersigned delegates, by virtue of the power and authority to us
given for that purpose, do, by these presents, in the name and in
behalf of our respective constituents, fully and entirely ratify and
confirm each and every of the said Articles of Confederation and
Perpetual Union, and all and singular the matters and things therein
contained. And we do further solemnly plight and engage the faith of
our respective constituents, that they shall abide by the
determinations of the United States, in Congress assembled, on all
questions which, by the said confederation, are submitted to them;
and that the articles thereof shall be inviolably observed by the States
we respectively represent; and that the union shall be perpetual.
In witness whereof we have hereunto set our hands, in Congress.
Done at Philadelphia, in the State of Pennsylvania, the ninth day
of July, in the year of our Lord one thousand seven hundred and
seventy-eight, and in the third year of the Independence of
America.
On the part and behalf of the State of New Hampshire.—Josiah
Bartlett, John Wentworth, jr., August 8, 1778.
On the part and behalf of the State of Massachusetts Bay.—John
Hancock, Samuel Adams, Elbridge Gerry, Francis Dana, James
Lovell, Samuel Holten.
On the part and in behalf of the State of Rhode Island and
Providence Plantations.—William Ellery, Henry Marchant, John
Collins.
On the part and behalf of the State of Connecticut.—Roger
Sherman, Samuel Huntington, Oliver Wolcott, Titus Hosmer,
Andrew Adams.
On the part and behalf of the State of New York.—Jas. Duane, Fra.
Lewis, Wm. Duer, Gouv. Morris.
On the part and in behalf of the State of New Jersey.—Jno.
Witherspoon, Nath. Scudder, Nov. 26, 1778.
On the part and behalf of the State of Pennsylvania.—Robt.
Morris, Daniel Roberdeau, Jona. Bayard Smith, William Clingan,
Joseph Reed, July 22d, 1778.
On the part and behalf of the State of Delaware.—Thos. McKean,
Feb. 13, 1779, John Dickinson, May 5, 1779, Nicholas Van Dyke.
On the part and behalf of the State of Maryland.—John Hanson,
March 1, 1781, Daniel Carroll, March 1, 1781.
On the part and behalf of the State of Virginia.—Richard Henry
Lee, John Banister, Thomas Adams, Jno. Harvie, Francis Lightfoot
Lee.
On the part and behalf of the State of North Carolina.—John
Penn, July 21, 1778, Corns. Harnett, Jno. Williams.
On the part and behalf of the State of South Carolina.—Henry
Laurens, William Henry Drayton, Jno. Mathews, Richard Hutson,
Thomas Heyward, Jr.
On the part and behalf of the State of Georgia.—Jno. Walton, July
24, 1778, Edw. Telfair, Edw. Langworthy.
Ordinance of 1787.
An Ordinance for the Government of the Territory of the United

States Northwest of the Ohio River. [In Congress, July 13, 1787.]
Be it ordained by the United States in Congress assembled, That
the said Territory, for the purposes of temporary government, be one
district; subject, however to be divided into two districts, as future
circumstances may, in the opinion of Congress, make it expedient.
Be it ordained by the authority aforesaid, That the estates both of
resident and non-resident proprietors in the said Territory, dying
intestate, shall descend to and be distributed among their children,
and the descendants of a deceased child, in equal parts; the
descendants of a deceased child or grandchild to take the share of
their deceased parent in equal parts among them; and where there
shall be no children or descendants; then in equal parts to the next of
kin, in equal degree; and among collaterals, the children of a
deceased brother or sister of the intestate shall have, in equal parts
among them, their deceased parents’ share; and there shall, in no
case, be a distinction between kindred of the whole and half blood;
saving in all cases to the widow of the intestate, her third part of the
real estate for life, and one-third part of the personal estate; and this
law relative to descents and dower shall remain in full force until
altered by the Legislature of the district. And until the governor and
judges shall adopt laws as hereinafter mentioned, estates in the said
Territory may be devised or bequeathed by wills in writing, signed
and sealed by him or her, in whom the estate may be, (being of full
age,) and attested by three witnesses; and real estates may be
conveyed by lease and release, or bargain and sale, signed, sealed,
and delivered by the person, being of full age, in whom the estate
may be and attested by two witnesses, provided such wills be duly
proved, and such conveyances be acknowledged, or the execution
thereof duly proved and be recorded within one year, after proper
magistrates, courts, and registers shall be appointed for that
purpose; and personal property may be transferred by delivery,
saving, however, to the French and Canadian inhabitants, and other
settlers of the Kaskaskies, Saint Vincent’s, and the neighboring
villages, who have heretofore professed themselves citizens of
Virginia, their laws and customs now in force among them, relative
to the descent and conveyance of property.
Be it ordained by the authority aforesaid, That there shall be
appointed, from time to time, by Congress, a governor, whose
commission shall continue in force for the term of three years, unless
sooner revoked by Congress; he shall reside in the district, and have
a freehold estate therein, in one thousand acres of land, while in the
exercise of his office.
There shall be appointed, from time to time, by Congress, a
secretary, whose commission shall continue in force for four years,
unless sooner revoked; he shall reside in the district, and have a
freehold estate therein, in five hundred acres of land, while in the
exercise of his office; it shall be his duty to keep and preserve the acts
and laws passed by the Legislature, and the public records of the
district, and the proceedings of the governor in his executive
department; and transmit authentic copies of such acts and
proceedings every six months to the secretary of Congress. There
shall also be appointed a court, to consist of three judges, any two of
whom to form a court, who shall have a common law jurisdiction,
and reside in the district, and have each therein a freehold estate, in
five hundred acres of land, while in the exercise of their offices, and
their commissions shall continue in force during good behavior.
The governor and judges, or a majority of them, shall adopt and
publish in the district such laws of the original States, criminal and
civil, as may be necessary, and best suited to the circumstances of the
district, and report them to Congress, from time to time, which laws
shall be in force in the district until the organization of the general
assembly therein, unless disapproved of by Congress; but afterwards
the legislature shall have authority to alter them as they shall think
fit.
The governor for the time being shall be commander-in-chief of
the militia; appoint and commission all officers in the same below
the rank of general officers. All general officers shall be appointed
and commissioned by Congress.
Previous to the organization of the General Assembly, the governor
shall appoint such magistrates and other civil officers in each county
or township as he shall find necessary for the preservation of the
peace and good order in the same. After the General Assembly shall
be organized, the powers and duties of magistrates and other civil
officers shall be regulated and defined by the said assembly; but all
magistrates and other civil officers, not herein otherwise directed,
shall, during the continuance of this temporary government, be
appointed by the governor.
For the prevention of crimes and injuries, the laws to be adopted
or made shall have force in all parts of the district, and for the
execution of process, criminal and civil, the governor shall make
proper divisions thereof; and he shall proceed from time to time, as
circumstances may require, to lay out the parts of the district in
which the Indian titles shall have been extinguished, into counties
and townships, subject, however, to such alterations as may
thereafter be made by the Legislature.
So soon as there shall be five thousand free male inhabitants of full
age in the district, upon giving proof thereof to the governor, they
shall receive authority, with time and place, to elect representatives
from their counties or townships, to represent them in the General
Assembly; Provided, That for every five hundred free male
inhabitants, there shall be one representative; and so on,
progressively, with the number of free male inhabitants, shall the
right of representation increase, until the number of representatives
shall amount to twenty-five; after which the number and proportion
of representatives shall be regulated by the Legislature: Provided,
That no Person be eligible or qualified to act as a representative
unless he shall have been a citizen of one of the United States three
years, and be a resident in the district, or unless he shall have resided
in the district three years; and in either case, shall likewise hold in
his own right, in fee simple, two hundred acres of land within the
same: Provided, also, That a freehold in fifty acres of land in the
district, having been a citizen of one of the States, and being resident
in the district, or the like free hold and two years’ residence in the
district, shall be necessary to qualify a man as an elector of a
representative.
The representatives thus elected shall serve for the term of two
years; and in case of the death of a representative, or removal from
office, the governor shall issue a writ to the county or township for
which he was a member to elect another in his stead, to serve for the
residue of the term.
The General Assembly, or Legislature, shall consist of the
governor, legislative council, and a house of representatives. The
legislative council shall consist of five members, to continue in office
five years, unless sooner removed by Congress, any three of whom to
be a quorum; and the members of the council shall be nominated
and appointed in the following manner, to wit: As soon as
representatives shall be elected, the governor shall appoint a time
and place for them to meet together, and when met, they shall
nominate ten persons, residents in the district, and each possessed of
a freehold in five hundred acres of land, and return their names to
Congress; five of whom Congress shall appoint and commission to
serve as aforesaid; and whenever a vacancy shall happen in the
council, by death or removal from office, the house of representatives
shall nominate two persons, qualified as aforesaid, for each vacancy,
and return their names to Congress; one of whom Congress shall
appoint and commission for the residue of the term. And every five
years, four months at least before the expiration of the time of
service of the members of the council, the said house shall nominate
ten persons, qualified as aforesaid, and return their names to
Congress; five of whom Congress shall appoint and commission to
serve as members of the council five years, unless sooner removed.
And the governor, legislative council, and house of representatives,
shall have authority to make laws in all cases for the good
government of the district, not repugnant to the principles and
articles in this ordinance established and declared, and all bills
having passed by a majority in the house, and by a majority in the
council, shall be referred to the governor for his assent; but no bill or
legislative act whatever shall be of any force without his assent. The
governor shall have power to convene, prorogue, and dissolve the
General Assembly when in his opinion it shall be expedient.
The governor, judges, legislative council, secretary, and such other
officers as Congress shall appoint in the district, shall take an oath or
affirmation of fidelity and of office, the governor before the President
of Congress, and all other officers before the governor. As soon as a
Legislature shall be formed in the district, the council and house
assemble, in one room, shall have authority, by joint ballot, to elect a
delegate to Congress, who shall have a seat in Congress, with a right
of debating, but not of voting during this temporary government.
And for extending the fundamental principles of civil and religious
liberty, which form the basis whereon these republics, their laws and
constitutions, are erected; to fix and establish those principles as the
basis of all laws, constitutions, and governments, which forever
hereafter shall be formed in the said Territory; to provide, also, for
the establishment of States, and permanent government therein, and
for their admission to a share in the Federal councils on an equal
footing with the original States, at as early periods as may be
consistent with the general interest:
It is hereby ordained and declared, by the authority aforesaid,
That the following articles shall be considered as articles of compact,
between the original States and the people and States in the said
Territory, and forever remain unalterable, unless by common
consent, to wit:
Article 1. No person, demeaning himself in a peaceable and
orderly manner, shall ever be molested on account of his mode of
worship or religious sentiments, in the said Territory.
Art. 2. The inhabitants of the said Territory shall always be
entitled to the benefits of the writ of habeas corpus, and of the trial
by jury; of a proportionate representation of the people in the
Legislature, and of judicial proceedings according to the course of
the common law. All persons shall be bailable, unless for capital
offenses, where the proof shall be evident or the presumption great.
All fines shall be moderate; and no cruel or unusual punishments
shall be inflicted. No man shall be deprived of his liberty or property
but by the judgment of his peers, or the law of the land; and should
the public exigencies make it necessary for the common preservation
to take any person’s property, or to demand his particular services,
full compensation shall be made for the same. And, in the just
preservation of rights and property, it is understood and declared
that no law ought ever to be made, or have force in the said Territory,
that shall, in any manner whatever, interfere with, or affect, private
contracts or engagements, bona fide and without fraud, previously
formed.
Art. 3. Religion, morality, and knowledge, being necessary to good
government and the happiness of mankind, schools and the means of
education shall forever be encouraged. The utmost good faith shall
always be observed toward the Indians; their lands and property
shall never be taken from them without their consent; and in their
property, rights, and liberty they shall never be invaded or disturbed,
unless in just and unlawful wars authorized by Congress; but laws
founded in justice and humanity shall, from time to time, be made
for preventing wrongs being done to them, and for preserving peace
and friendship with them.
Art. 4. The said Territory, and the States which may be formed
therein, shall ever remain a part of this confederacy of the United
States of America, subject to the Articles of Confederation, and to
such alterations therein as shall be constitutionally made; and to all
the acts and ordinances of the United States in Congress assembled,
conformable thereto. The inhabitants and settlers in the Territory
shall be subject to pay a part of the Federal debts, contracted or to be
contracted, and a proportional part of the expenses of Government,
to be apportioned on them by Congress, according to the same
common rule and measure by which apportionments thereof shall be
made on the other States; and the taxes for paying their proportion
shall be laid and levied by the authority and direction of Legislatures
of the district or districts, or new States, as in the original States,
within the time agreed upon by the United States in Congress
assembled. The Legislatures of those districts, or new States shall
never interfere with the primary disposal of the soil by the United
States in Congress assembled, nor with any regulations Congress
may find necessary for securing the title in such soil to the bona fide
purchasers. No tax shall be imposed on lands the property of the
United States; and in no case shall non-resident proprietors be taxed
higher than residents. The navigable waters leading into the
Mississippi and St. Lawrence, and the carrying places between the
same, shall be common highways, and forever free, as well to the
inhabitants of the said Territory as to the citizens of the United
States, and those of any other States that may be admitted into the
confederacy, without any tax, impost, or duty therefor.
Art. 5. There shall be formed in the said Territory not less than
three, nor more than five States; and the boundaries of the States, as
soon as Virginia shall alter her act of cession, and consent to the
same, shall become fixed and established as follows, to wit: The
western State in the said territory shall be bounded by the
Mississippi, the Ohio, and Wabash Rivers; a direct line drawn from
the Wabash and Post Vincents, due north, to the territorial line
between the United States and Canada; and by the said territorial
line to the Lake of the Woods and Mississippi. The middle States
shall be bounded by the said direct line, the Wabash, from Post
Vincents to the Ohio, by the Ohio, by a direct line drawn due north
from the mouth of the Great Miami to the said territorial line, and by
the said territorial line. The eastern State shall be bounded by the
last mentioned direct line, the Ohio, Pennsylvania, and the said
territorial line: Provided, however, And it is further understood and
declared that the boundaries of these three States shall be subject so
far to be altered, that, if Congress shall hereafter find it expedient,
they shall have authority to form one or two States in that part of the
said Territory which lies north of an east and west line drawn
through the southerly bend or extreme of Lake Michigan. And
whenever any of the said States shall have sixty thousand free
inhabitants therein, such State shall be admitted, by its delegates,
into the Congress of the United States, on an equal footing with the
original States in all respects whatever; and shall be at liberty to form
a permanent constitution and State government: Provided, The
constitution and government so to be formed shall be republican,
and in conformity to the principles contained in these articles; and,
so far as it can be consistent with the general interest of the
confederacy, such admission shall be allowed at an earlier period,
and when there may be a less number of free inhabitants in the State
than sixty thousand.
Art. 6. There shall be neither slavery nor involuntary servitude in
the said Territory, otherwise than in the punishment of crimes,
whereof the party shall have been duly convicted: Provided always,
That any person escaping into the same, from whom labor or service
is lawfully claimed in any one of the original States, such fugitive

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Biopolymer-based Formulations:

Biomedical and Food Applications 1st

For information on all Elsevier publications visit our website at

Publisher: Matthew Deans

Fiona Concy Rodrigues

Introduction of biopolymers: food

Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00001-1

2. Cross-linking methods employed to design biopolymer-based

2.1 Physically cross-linked gels

2.1.1 Cross-linking by ionic interactions

2.1.2 Self-assembly of hydrophobized polysaccharides

2.1.3 Cross-linking by the crystallization of the polysaccharides

2.1.4 Cross-linking by induction of hydrogen bonds among the polysaccharide

2.1.5 Cross-linking using protein molecules

2.2 Chemical cross-linking

2.2.2 Polycarboxylic acids

2.2.3 EDC coupling

Done at Philadelphia on the 9th day of July, 1778.

To all to whom these presents shall come,

An Ordinance for the Government of the Territory of the United

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