KTH Royal Institute of Technology
School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH)
Department of Chemistry, Division of Glycoscience
Comparison of Corn and Rye Arabinoxylans for the Production of Bio-based Materials
Chen Chen
Supervisor: Francisco Vilaplana
Co-supervisor: Reskandi C. Rudjito & Secil Yilmaz Turan
TRITA-CBH-GRU-2020:261 10044 Stockholm, Sweden
September 2020
Abstract
Enzymes and subcritical water can be used for the extraction of hemicelluloses from cereal by- products, making the processes eco-friendly. The polysaccharides extracted from cereal by- products can be used as matrices for development of materials for various applications. This includes bio-based materials such as films and hydrogels, which offer alternatives to existing materials produced from petrochemicals. The polymeric structure of cereal hemicelluloses contains functional groups which enable the modification of their structure by cross-linking, resulting in the formation of hydrogels.
This project aims to use subcritical water extraction (SWE) to extract arabinoxylans (AXs) form corn and rye bran meanwhile the enzymatic treatment is done for purifying the samples during both pre- and post-treatment. AXs were further crosslinked by enzyme (laccase) for hydrogel preparation. During the whole project, the characterization included moisture and yield determination, starch and protein content which were tested using a spectrophotometer, monosaccharide content was analyzed by high performance anion exchange chromatography followed by pulsed amperometric detection (HPAEC-PAD) and phenolic acid content was quantified by high performance liquid chromatography (HPLC).
The pretreatment for destarching and SWE process was successful. The result showed that arabinoxylans from corn bran were having higher content of arabino substituents, arabino to xylans ratio and ferulic acid content than rye samples. The enzymatic crosslinking could form strong gels in the condition that the AXs had high ferulic acid content. In terms of forming strong hydrogels or to improving the properties of AXs gel, the pre- and post-treatment should be optimized to increase the purity of the extracted feruloylated AX content.
Keywords:
cereal bran, subcritical water extraction, arabinoxylans, enzymatic crosslinking, hydrogelSammanfattning
Enzymer och subkritiskt vatten kan användas för extraktion av hemicellulosa från spannmålsbiprodukter, vilket gör extraktionen miljövänlig. Polysackariderna extraherade från spannmålsprodukter kan användas som matriser för utveckling av material för diverse applikationer. Detta inkluderar biobaserade material som filmer och hydrogeler, där petrokemikalier kan ersättas som råvara. Den polymera strukturen hos spannmålshemicelluloser innehåller funktionella grupper som möjliggör formation av tvärbindningar vilket resulterar i bildandet av hydrogeler.
Syftet med detta projekt är extraktion av arbinoxylaner (AXs) från majs och råkli genom att använda subkritiskt vatten-extraktion (SWE) där rening under för- och efterbehandling utförs enzymatiskt. AX modifierades därefter enzymatiskt (laccas) med tvärbindningar för hydrogelframställning. Under hela projektet karakteriserades hydrogelen utifrån fuktinnehåll, bestämmelse av utbyte, stärkelse och proteininnehåll som testades med en spektrofotometer, monosackaridhalten analyserades med högpresterande anjonsutbyteskromatografi följt av puls- amperometrisk detektion (HPAEC-PAD), samt kvantifierades fenolsyrahalten med högupplöst vätskekromatografi (HPLC).
Resultatet visade att arabinoxylaner från majskli hade högre innehåll av arabinosubstituenter, där förhållandet mellan arabino och xylans, samt arabino och ferulsyra innehållet var högre än för rågproverna. Den enzymatiska tvärbindningen kunde bilda starka geler i det tillståndet där AX hade en hög ferulsyrahalt. När det gäller att bilda starka hydrogeler eller att förbättra egenskaperna hos AXs-gel, bör för-och efterbehandlingen optimeras för att öka renheten för det extraherade feruloylerade AX-innehållet.
Nyckelord: spannmålskli, extraktion av subkritiskt vatten, arabinoxylaner, enzymatisk tvärbindning, hydrogel
Table of Content
Abstract ... 1
Sammanfattning ... 2
Table of Content ... 1
1. Introduction ... 1
1.1. Biorefinery ... 1
1.2. Polysaccharides ... 1
1.3. Plant cell wall and polysaccharides ... 4
1.3.1. Plant cell wall ... 4
1.3.2. Cell wall polysaccharides ... 5
1.4. Cereal residue and hemicelluloses ... 7
1.4.1. Corn bran, rye bran: Origin, structure and composition ... 7
1.4.2. Preparation methods of bran ... 8
1.4.3. Arabinoxylans from cereal ... 8
1.5. Extraction and characterization of xylans ... 12
1.5.1. Extraction of arabinoxylans ... 12
1.6. Materials produced from arabinoxylans ... 14
1.7. Arabinoxylans hydrogel ... 15
2. Materials and Methods ... 17
2.1. Materials ... 17
2.2. Bioprocess design ... 17
2.2.1. Destarching ... 17
2.2.2. Laboratory scale subcritical water extraction ... 17
2.3. Characterization methods ... 19
2.3.1. Determination of moisture content ... 19
2.3.2. Yield determination ... 19
2.3.3. Starch content ... 19
2.3.4. Polysaccharide hydrolysis and monosaccharide composition ... 20
2.3.5. Quantification of phenolic acids by HPLC ... 21
2.3.6. Determination of protein content ... 21
2.3.7. Molar mass distributions ... 22
2.3.8. Crosslinking of arabinoxylans ... 22
3. Results and Discussion ... 23
3.1. Characterization of initial and pretreated bran ... 23
3.1.1. Moisture content ... 23
3.1.2. Starch content ... 23
3.1.3. Phenolic acid content ... 24
3.1.4. Monosaccharide content of initial bran ... 24
3.1.5. Monosaccharide content of pretreated bran ... 25
3.2. Characterization of the subcritical water extracted fractions ... 26
3.2.1. Extraction yields ... 26
3.2.2. Monosaccharide content of extracts ... 26
3.2.3. Protein content of extracts ... 30
3.2.4. Molar mass distributions ... 31
3.2.5. Phenolic acid content of extracts ... 32
3.3. Characterization of crosslinked AXs ... 33
3.3.1. Enzymatic crosslinking and hydrogel preparation ... 33
3.3.2. Phenolic acid content of crosslinked AXs ... 35
4. Conclusions ... 37
5. Future work ... 38
6. Acknowledgement ... 40
Appendix ... 41 References ... I
1. Introduction
1.1. Biorefinery
As the demand for energy and production continues to increase, the pressure on the supply of traditional non-renewable energy sources based on fossil fuels has also increased. Following this, the development and utilization of new energy has become a hot topic. Among them, the efficient development of material resources is a highly feasible solution. Biorefinery is the sustainable processing of biomass into a variety of marketable bio-based products and bioenergy / biofuels. Utilizing existing biomass resources for food and feed ingredients, pharmaceuticals, chemicals, materials, minerals and short-cycle carbon dioxide [1-3]. Figure 1 gives some examples of bio-based products and energy uses.
Figure 1: energy and value-added chemicals from biomass [1-3].
1.2. Polysaccharides
Polysaccharides play important roles in all living organisms. The most well-known examples for the functions of polysaccharides are energy storage and formation of biological structures.
As for the structure, polysaccharides are formed of multiple monosaccharide molecules which undergo dehydration polymerization and are then connected by glycosidic bonds. Depending on the types, they can be linear or branched long chains. A polysaccharide chain may be built up by different monomers, and the glycosidic linkages between the different sugar units can be different, which lead to different anomeric configurations [4]. Moreover, non-carbohydrate groups may also appear in polysaccharides which provides a wealth of biological functions and structural properties, e.g. ferulic acid groups can form cross-linkage in hemicellulose.
The sources of polysaccharides are wide and rich. They can be divided into three types by their functions: energy storage polysaccharides, structural polysaccharides and mucosubstances.
Starch and glycogen are the two main storage polysaccharides. Starch functions as the energy storage polysaccharide in plants, which consists of two macromolecular populations: amylose and amylopectin, Figure 2 shows the structures of them. Both of them are formed by the condensation of α-D-glucose (pyranose forms). Amylose is in the form of a continuous linear but helically arranged chain, α-D-glucose are linked together by 1-4 α-linkages; Amylopectin has much more branched chain and, mostly, 1-4 α-linkages connect the majority units in the linear part while 1-6 α-linkages (also include a few 1-3 α-linkages) initiate the branching.
Glycogen is a polysaccharide that functions as the energy reserve in animals, bacteria and fungi.
It has a branched structure made of glucose that are linked together by 1-4 α-linkages and 1-6 α-linkages, which is quite similar to amylopectin. The differences are that glycogen has more and longer branches than those of amylopectin [5].
a) b)
Figure 2: Structures of a) amylose and b) amylopectin.
The second type, structural polysaccharides, includes chitin, cellulose, hemicellulose and pectin;
the structures of them are in Figure 3. In chitin, the basic unit is a nitrogen containing glucose (known as N-acetyl glucosamine) and these monomers are connected together by 1-4 β- linkages. Cellulose and hemicellulose are the most important and major components of cell wall. Differently, cellulose is a fibrous homopolysaccharide with linear glucose (1-4 β-linkages) chains; hemicellulose has shorter but more complicated structures instead. Different monomers can form hemicelluloses. which can be in the form of beta-glucans, xyloglucans, xylans, mannans, galactans and glucomannans. Xylans, very common in nearly all kinds of plants, which includes several different types, such as glucuronoxylan, arabinoxylan, and glucuronoarabinoxylan; the types depend on the sources. Mannans, another common hemicellulose could be found both in soft wood and hard wood which often occur with other monosaccharides; purer mannans (> 95%) could be found from some seeds [6]. Galactans, normally with the arabinose as the common side group, could be easily found in larch wood. In this project, arabinoxylans is the focus hemicellulose.
O OH
O HO
OH O
O OH
HO OH
O
O OH
HO OH
O O
OH
HO
OH O
O OH
O HO
OH
O O
OH
HO OH
O
O O
HO OH
O O
OH
HO OH
O O
OH
HO OH O
a) b)
c)
Figure 3: Structures of a) chitin, b) cellulose and c) pectin.
Mucosubstances are slimy substances or mucilages which are heterogeneous branched polysaccharides. They are quite common in plant cells and can also be found in animal body.
They possess acidic or aminated polysaccharides which are formed from galactose, mannose, sugar derivatives and uronic acids [7]. In cell walls, they bind proteins and connective tissue;
on the other hand, mucopolysaccharides can hold water in the interstitial spaces water, which keeps moisture to protect cells. In animal body, mucopolysaccharides can work as the bio lubricant and increase the elasticity in cartilage.
Polysaccharides can originate from a great number of species, and the structures between different polysaccharides are quite different, so the physical and chemical properties of these polysaccharides vary. Most of these structures have common ground: 1. water-soluble or hydratable; and 2. Forming intermolecular and intramolecular associations. However, influenced by intermolecular interaction, such as hydrogen bond, these properties are not universal. The unbranched, lightly branched and unsubstituted homopolymer will form hydrogen bonds between different molecules, which leads to insolubility at normal temperature, e.g. cellulose, mannan, and crystalline amylose [4]. Some structures are slightly branched or substituted, which can easily form crosslinking network and bind the structure tightly, for example, xylans or arabinoxylans. In order to increase the water solubility of such insoluble polysaccharides, a small number of substituents, such as phenolic acid group can achieve this purpose. Most polysaccharides, like hemicellulose and pectin, can form gel by forming hydrogen bond, ionic interactions, ionotropic gels, by hydrophobic interactions or need certain substituted groups, such as ferulic acid. The formation of cross linkage can improve the mechanical properties of polysaccharide-based materials and these cross-linked complexes can be used in various applications such as drug delivery and water treatment.
O
O O
NH
OH O OH
NH HO
HO
n
CH3 O
CH3 O
O
O O
OH
OH O OH
OH HO
HO
n
O CO2CH3 O
HO
OH O
O CO2CH3 HO
OH O
1.3. Plant cell wall and polysaccharides
1.3.1. Plant cell wall
The plant cell wall is involved in maintaining a certain shape of the cell, enhancing the mechanical strength of the cell, and also relating to the physiological activity of the cell. There are different layers in the plant cell wall; from outer layer to inner layer, these are: middle lamella, primary cell wall and secondary cell wall (Figure 4).
Figure 4: the model of cell wall structure [8]
The composition of the cell wall varies from species to species and also depends on the cell type and the stage of the cell development. For terrestrial plants, the main composition of their cell wall are polysaccharides in the form of cellulose, hemicellulose, pectin, as well as lignin and a few kinds of proteins. Though the content of various components varies depending on their location and functions of the cell wall, the majority is always the cellulose. Hemicellulose content is the second most abundant, including several types. Cellulose forms microfibrils and these microfibrils will be tightly “packed” with the hemicelluloses that are also known as cross- linking glycans. Lignin can form a complex network in the cell wall and tightly connect xylem vessels and wood tissues together Uzman [8], which increases the mechanical strength of the cell wall and makes the cell wall rigid. In the middle lamella, where it is rich in lignin, adjacent plant tissues / cells can be linked and fixed together. Pectin in the primary cell walls has the function to form the matrix to embed the cellulose network. Hemicellulose and pectin work as the structural materials and have the function of increasing the interface between cellulose and lignin, maintaining the ordered spacing of cellulose and regulating the wall porosity and strength.
1.3.2. Cell wall polysaccharides
For polysaccharides in the cell wall, cellulose, hemicellulose and pectic substances should be concerned about. In each family of these polysaccharides, there are various members which can contain features in common. Figure 5 lists the common monosaccharides which could found in cell wall polysaccharides.
Figure 5: Some common monomers found in plant cell wall.
1.3.2.1. Cellulose
As mentioned before, cellulose is the major component of the plant cell walls, its amount is considered the highest of organic compounds on Earth. The structure of cellulose is ß 1-4 D
glucan as shown in the Figure 6. Cellulose has a linear structure, and in most cases, the degree of polymerization (DP) can reach around 10000. Hydrogen bond can be easily formed both intermolecularly and intramolecularly. Between several layers of cellulose, Van der Waals interactions can also be found. With the long linear structure and interaction, crystal structures will be easily created [4].
O
OH OH
OH OH
Xylose
O
OH OH OH CH2OH
Arabinose
O
OH OH
OH OH
Fucose 5-Carbon Sugers
6-Carbon Sugers
O OH
OH
CH2OH OH
OH
O CH2OH
OH
O CH2OH
OH
O
OH OH
OH HO OH
OH
OH OH
OH CH3
OH
Glucose Mannose Galactose Rhamnose
O COOH
OH
O COOH
OH
O
OH OH OH
OH OH
Glucuronic acid Galacturonic acid 4-O-Methyl Glucurnic acid OH
OH OH
COOH
OCH3 OH
Figure 6: Inter- and intro-hydrogen bonds between cellulose chains 1.3.2.2. Hemicellulose
There are several types of hemicelluloses that can be found. Hemicelluloses are short (DP is around 500-3000) and branched chains with several types of monomers, such as: 5-carbon sugars (xylose, arabinose, fucose), 6-carbon sugars (glucose, mannose, galactose, rhamnose) and some acidic groups (glucuronic acid, galacturonic acid). With the branched structures and side groups , hemicellulose can easily form crosslinking networks, which contributes to form a matrix for cellulose microfibrils to be embedded in. Scheller and Ulvskov [9] group suggested that hemicellulose should be described as the family of polysaccharides in which their backbones have the β (1-4) linkages with an equatorial configuration [9]. As for the branches to form, there are several sites, such as O-2 linkage, O-3 linkage and C5 and C6 of the monomer units. The substitutions of hemicelluloses, such as ferulic acid group, can cross-link hemicelluloses to each other. Hemicellulose has several biological functions: tethering glycans (binding to cellulose microfibrils), structural polysaccharides, source of signal molecules, and seed storage carbohydrates. Also, due wo the nutritional, health, and economic importance of hemicelluloses, separation and modification of hemicellulose can be an expansive subject.
1.3.2.3. Pectic substances
Pectic substances are rich in the primary cell walls and intercellular regions of higher plants.
The main structure of pectic substances is a linear chain of D-galacturonic acid. The differences between pectic species is the varying proportions of the acid groups which are present as methyl
O
O
O
O O
O
O
O
O
HO OH
OH
HO OH
HO
OH OH
HO HO HO
OH
O
O
O
O O
O
O
O
O
HO OH
OH
HO OH
HO
OH OH
HO HO HO
OH
esters [10].
1.4. Cereal residue and hemicelluloses
1.4.1. Corn bran, rye bran: Origin, structure and composition
Cereals are the main ingredient of our diet. It is a natural source of dietary fiber. They contain both insoluble and soluble fibers, and their proportion will depend on the grain, variety, place of origin and processing methods. By far the most concerned are wheat, barley, rye, corn and oats. Among these, bran contains a lot of dietary fiber, but it is not well used. “Cereal bran” is relatively vague and refers to various milling products [11]; generally, bran is the hard-outer layers and cell walls from endosperm of cereal grain, normally produced from the by-product of crops such as wheat, barley, rye and corn. Bran is mainly composed of the outer pericarp layers which are rich in both cellulose and lignin, aleurone / sub-aleurone layers which are rich in hemicellulose [11, 12].
Traditional usage of bran is for animal feed. It is known that bran is rich in dietary fiber, starch, protein and essential fatty acids; its potential application makes it a strong research and development value. On the other hand, ferulic acid (FA) is a phenolic acid, which plays an antioxidative role in inhibiting lipid and protein oxidation [12]; making cereal bran a potential source for use in both food industry and health care, cereal bran which is ferulic acid-rich has a greater potential for development.
Figure 7: the structure of corn model [13]
Corn bran (shown in Figure 7) is the fiber residue that is produced from the corn kernel by milling, both dry milling and wet milling. Compared to other cereal brans, corn bran has the highest concentrations of dietary fibers and phenolics [14]. Most insoluble dietary fibers from corn bran include 20 to 28% cellulose, around 70% hemicelluloses and 1% lignin [12].
Compared to other cereals, corn arabinoxylans have higher ferulic acid, di-ferulic acid, tri- ferulic acid [15] and degree of substitution. The percentage of unsubstituted, mono-substituted, and di-substituted xylose in corn arabinoxylans may be 24 %, 52 % and 24 % of the total substitution groups, respectively [16].
Rye bran is similarly produced as corn bran, but not as easy to separate the aleurone and pericarp from the endosperm. Dietary fiber concentration in rye is around 20.4% to 25.2%, and in bran this concentration is higher than in flour. Earlier studies found that the rye bran from Nordic countries contain dietary fibers from 41 to 48%, while starch content is from 13 to 28%; AX can account for half of the total concentration of dietary fibers in rye bran, protein content is around 14 to 18% and the rest is a mixture of cellulose, lignin, β-glucan and fructan [11, 17, 18].
1.4.2. Preparation methods of bran
For cereal products preparation, milling or grinding is a main step. Milling procedures can be divided into wet or dry milling.
As a part of a high fiber content, bran not only has food value, but can also be used as a raw material for extracting hemicellulose. Separating the bran from the starchy endosperm is usually accomplished by grinding. As a rough separation method, dry milling cannot obtain high-purity bran, and usually a small amount of germ cannot be separated, so it is often used in feed preparation [19]. The corn bran and rye bran used in this project was prepared by dry milling.
Another milling method is wet milling. Comparing with dry milling, cereal should be macerated and during this step, the endosperm is much easier to separate from the bran. That is because the maceration step changes the physical and chemical properties of the basic components such as starch, protein and cell wall materials [20]. Wet milling can usually obtain a product with more complete separation, and the product has a smaller size and a larger surface area. Normally corn and wheat would be wet milled during production.
1.4.3. Arabinoxylans from cereal
Most hemicelluloses from bran are complex heteroxylan. For the heteroxylans found in bran and other types of cereal, most of them have the D-xylopyranosyl backbone and L- arabinofuranosyl side groups [21]. The basic backbone units of xylans are linked by β-1,4 bond, and several types of monosaccharide units, such as arabinose, mannose, xylose, and galactose.
form branches. Besides, some structures function as crosslinkers, such as ferulic acid groups.
The degree of crosslinking is related to the concentration of ferulic acid in the heteroxylan.
Arabinoxylans (AXs) are one of the most common hemicelluloses in cereals. The AXs are found in many different natural sources such as wheat, rye, corn, oats, millet. Depending on which source the AXs originates from, the branches on the structure differ [11]. The molecular weight (Mw) of AXs can vary depending on the source of the polysaccharide and the extraction method used (time, pH and temperature). The average Mw of AX is estimated to be 10 to 10,000 kDa [22]. Also, the functionality and quantity of AXs can vary across species but the composition percentage is predominantly constant amongst most plants. However, in the case of wheat and corn bran, different arabinose contents have been observed. The arabinoxylan structure generally consists of highly disperse chains of a β-(1-4) glycosidically linked d- xylopyranosyl (Xylp) units that are often mono-/di-/unsubstituted with Araf residues at the oxygen bound to the second and or third carbon of the Xylp residue. The substitutions indicate the presence of four major structural units, which are monosubstituted Xylp at O-3 position, monosubstituted Xylp at O-2 position, disubstituted Xylp at O-2,3 position, and unsubstituted Xylp (Figure 8). The concentration and distributions of specific repeating units can vary depending on the plant species observed. However, according to previous studies, there can be a specific region distribution in certain species where regions are composed of highly substituted AX units followed by mono- or unsubstituted regions, thus creating a modified
“block” distribution of units as seen in wheat endosperm[23].
a) b)
c)
d) O
HO
OH OH O
O O OH
O
O
HO
OH OH O
O O O
HO
Araf Xylp
O
HO
OH OH O
O O O
O
Araf Xylp
O
HO
OH
Araf OH
O O OH
O Xylp
HO
e)
Figure 8: Structural elements present in arabinoxylans: (a) monosubstituted Xylp at O-3 position, (b) monosubstituted Xylp at O-2 position, (c) disubstituted Xylp at O-2,3 position, (d) unsubstituted Xylp, (e) the general structure of an arabinoxylan molecule.
In this project, the AXs would be extracted from corn and rye bran. Comparing with rye bran, corn bran arabinoxylans are heavily branched; almost 80% of the xylopyranosyl units of the backbone are substituted by side chains [21]. The majority of the Araf substitutions are small, usually containing only one linked unit. However, some of them are considerably larger. For example, esterified residues of ferulic acid can be attached to the Araf residue which may result in the formation of cross-links in the AXs structure. The structure of an AX molecule is illustrated in Figure 8 (e). Arabinoxylans from corn sources often have the other substitutes, including Gal, GlcA. OMeGlcA, which results in them being called glucuronoarabinoxylan (GAX) [24]. The AXs and GAX structure from rye and corn, respectively, are shown in Figure 9.
a) AXs structure from rye bran
b) GAX structure from corn bran
Figure 9: The structure of (a) arabinoxylan (AX) and (b) glucuronoarabinoxylan (GAX).
Based on analyses of the composition of different species, different moieties can be observed as well. As a matter of fact, corn bran arabinose residues can carry several types of six- membered sugars such as galactopyranose and α-d-glucuronic acid [23]. The residues can confer specific traits that translate into important structural seed characteristics. The higher quantity of feruloylated AXs that covalently binds to lignin and other cell wall material causing greater rigidity in the resulting structure and leading to the structural integrity of the protective coat of the endosperm. Such a process is particularly observed in wheat species, where there is a high moiety concentration in the pericarp layer as opposed to the aleurone layer, which contains minor feruloylated AX content [14]. In the case of wheat bran, there is a high ratio of arabinose/xylose (Ara/Xyl) observed in the outer pericarp, which indicates high substitution of Xylp and possibly greater concentration of FA moieties, whereas, in corn bran, the ratio between the monosaccharide Ara and the Xyl residues was found to be A/X: 0.5 [25]. However, given recent studies, the mean content of esterified FA is much higher in maize compared to other species, such as wheat [26].
1.5. Extraction and characterization of xylans
1.5.1. Extraction of arabinoxylans
The cell walls of cereal are mainly composed of three main components and these are cellulose, hemicellulose, and lignin. The concentrations of these components differ both from different kinds of cereals and in the different parts and layers. For example, grain contains several components: fiber, starch, fat, protein and pectin. The individual chemical composition of the grain varies with the type of grain. For biobased materials, extraction and purification of the initial materials are very important steps. In this experiment, AXs would be used as a cross- linking substrate to prepare hydrogel and purity of the AXs is necessary. There are several ways to extract and separate components effectively from initial materials, and these would be discussed in more detail in the following paragraphs.
1.5.1.1. Subcritical water extraction, SWE
Subcritical water is water that remains at liquid state when heated to a temperature between 373 K and the critical point of 474 K, and pressured above atmospheric pressure [27, 28]. At these conditions, several water properties change, such as the viscosity, density, and surface tension. These parameters change due to the fact that high pressure and high temperature decrease the degree of hydrogen bonding, which influences the viscosity of the subcritical water.
In addition, the relative permittivity of subcritical water has been reduced a lot; at this time, the polarity of subcritical water is reduced. According to the principle of similar compatibility, above the critical temperature, this condition becomes conducive to the dissolution of organic matter.
When conducting subcritical water extraction (SWE), it is important to consider the type of
components present in the material that is to be extracted. Both water-soluble and water- insoluble components could be separated by SWE. In this study, the aim is to extract hemicelluloses, which is a relatively water-soluble component; by using SWE, these AXs should be easily extracted in laboratory conditions. Due to the presence of other water-soluble components present in corn fibers and rye bran, such as starch, a pretreatment of the sample may be necessary to improve extraction efficiency. This can, for example, be done with an enzymatic treatment. Overall, SWE process is considered as an environmentally friendly and green option to extraction due to the solvents that are otherwise generally involved in traditional extractions. The subcritical water can replace otherwise potentially harmful organic solvents and other than being safer and less costly, it ultimately allows for a process that is more affine to green chemistry [28].
SWE has been successfully used for the extraction of xylose hemicellulose from oil palm fronds. The study analyzed the effect of different reaction times (5-20 minutes), pressures (3.45- 5.52 MPa) and temperatures (443-473 K). The most successful reaction conditions which gave the highest yield were obtained at 10 minutes, 4.14 MPa and 463 K [29].
1.5.1.2. Ultrasound-enhanced subcritical water extraction (USWE)
In a study about SWE enhanced by ultrasound the extraction of polysaccharides from the plant Lycium barbarum was performed. In the experiment, four different independent parameters were examined which were the time, temperature of the extraction, the ratio of liquid to solid, and the electric power of the ultrasound. The highest yield was obtained with the reaction conditions of 373 K, 53 min extraction, liquid to solid ratio of 26 cm3/g and the power of 160 W. The study stipulated USWE to be of great value due to its high yield, easy pretreatment, and reduced heating requirements compared to other extraction methods and SWE [30].
1.5.1.3. Alkaline extraction
Alkaline extraction of hemicellulose can be performed in multiple ways [31]. An example of such method is the direct extraction by sodium hydroxide. The treatment of the biomass with sodium hydroxide makes it possible to extract the hemicellulose of interest. During the extraction, a reducing agent such as sodium borohydride (NaBH4) is added to the solution to prevent peeling reactions from occurring. Thereby the degradation of the polymeric hemicellulose structure is prevented. Through centrifugation of the solution, the insoluble residues are isolated from the liquid. Further treatment of the supernatant liquid leads to precipitation of hemicellulose which by freeze-drying can be fully extracted and measured.
Another hemicellulose alkaline extraction method is used for is direct alkaline hydrogen peroxide extraction, which is slightly different but still follows the same principles. The advantage of this method is that the resulting product will be very pure, but some drawbacks to this method appear. Alkaline extraction is more aggressive than water-based extractions and alkaline solution releases hemicellulose by swelling cellulose, breaking the hydrogen bond between cellulose and hemicellulose and breaking the chemical bond between hemicellulose and lignin, such as ester bonds and ether bonds; so the degradation of the hemicellulose will
occur [31] and that it often will cause loss of the ferulic acid thereby a reduction of the antioxidant activity of the hemicellulose.
1.5.1.4. Enzymatic extraction
Another method to extract hemicellulose is by enzymatic extraction. This can for example be done by endoxylanases, but other enzymes can also be used depending on where the specific cleaving of moieties is desired. The negative aspect of this method is that it results in a lower yield and molecular weight than what is obtained through other chemical extraction methods.
Nonetheless, it has also benefited such as being more environmentally friendly and may be used for applications in the food industry [32].
1.6. Materials produced from arabinoxylans
Studies on the use of AXs hydrogels are progressively emerging. AXs are natural, non-toxic, highly biocompatible and biodegradable. It can thus be used in medical applications for instance as a matrix for drug delivery. AXs are also environmentally friendly and there is a large quantity of AXs that are not fully used. These include by-products from the production of commercial cereals. The availability and eco-friendly properties of AXs allow for its development into new uses of AXs.
The AXs-based materials are neutral in flavor. They are also ionic stable and thermostable and are not sensitive to pH, and temperature. These properties give AXs-based materials plenty of marketing interest. As for films, arabinoxylans have been used to produce these products because of their ability to form cohesive matrixes, and their neutral taste and odor. The mechanical properties barrier properties of arabinoxylan films are of the same order of magnitude as other types, like methylcellulose films, methylcellulose films, or starch films.
AXs gels have a great number of applications on medical uses. They not only have the similar mechanical properties as other types of hydrogels, but also other functional properties, such as antioxidant and anticancer agents [33]. Additionally, simple and fast preparation method, and high-water absorption performance are the other benefits. Feruloylated arabinoxylans are easily modified with other functional groups, and that is the reason why researches would like use AXs for medical uses.
However, at present, the extraction and purification of AXs is relatively expensive, compared to cellulose. Due to the fact that most bran is not fully utilized as a product, the extraction cost is often too high, product requirements and functional restrictions also constrain the industrial production of AXs. Other materials with similar functions, such as cellulose, are still more competitive in the market. Many of the special functions for hemicellulose development and use are still in the laboratory stage.
1.7. Arabinoxylans hydrogel
Arabinoxylans can form covalent hydrogels. FA on the AXs chains could covalently bond with FAs to form dimers or trimers during crosslinking process. Previous research has shown that not only FA content but also the molecular weight of AXs and concentration of AXs used during hydrogel formation would have great influence on gel properties and gel forming ability [34].
The most common way for forming AXs hydrogels can be both chemical treatment and enzymatic treatment. Ferric chloride, ammonium persulphate can be used for chemical crosslinking treatment; as for enzymatic crosslinking, commeon enzymatic oxidizing agents used include.laccase / O2, peroxidase / H2O2, and oxidants linoleic acid / lipoxygenase. In this project, enzymatic crosslinking with laccase would be used for preparing the hydrogel. Figure 10 shows the reaction between FA on AXs during the crosslinking process with laccase. During crosslinking, there are 5 kinds of FA dimers structures that could be formed: 8-5', 8-O-4', 5-5', 8-5', and 8-8' di-FA. Previous research showed that 8-5' and 8-O-4' di-FA are the most abundant [35]. As mentioned before, AXs are rich in FA; so that the hydrogel prepared by AXs could also have the interesting properties: antioxidant and prebiotic properties.
Figure 10: Schematic representation of FA reaction for crosslinking with laccase and O2 O
OH OMe
O
O
OMe C
O
O
OMe
CH O
O
OMe O2
Laccase
Ferulic acid
Phenoxy radicals
O
MeO
HC
O CH
O O
MeO
CH
O O
MeO
O
MeO
HC
O di-FA
2. Materials and Methods
2.1. Materials
Corn bran and rye bran were used as arabinoxylans (AXs) sources in this project. The preparation and extraction methods are described in this section.
The corn bran was purchased from a farm and prepared by dry milling from a family farm in Mongolia, China; and rye bran was provided by Lantmännen (Stockholm, Sweden). Due to the larger particle size of the corn bran, additional milling was done by a blender to improve the efficiency of subsequent destarching and extraction.
2.2. Bioprocess design
2.2.1. Destarching
The destarching of corn and rye bran, which was the pretreatment, was performed enzymatically using α-amylase (Type IV−B from porcine pancreas). In order to maintain the high efficiency of the enzyme, the reaction was carried out at 37 ˚C in sodium phosphate buffer (PBS) buffer (pH 7.0).
Pre-weighed bran samples were suspended in a flask in 50 mM PBS buffer at a 1:10 (w/v) solid to liquid ratio. Then the samples were incubated at 90 ˚C for 8 minutes to gelatinize the starch.
After cooling the samples to room temperature, the gelatinized samples were destarched with α-amylase (16 U/g initial bran) by incubating at 37˚C for approximately 5 h under constant stirring. Then a second batch of α-amylase (16 U/g initial bran) was added and incubated under the same condition overnight for another 17 h. The pretreated samples were centrifuged (8000 rpm, 30 min, 4˚C) 3 times and washed in between with water twice and with absolute ethanol (95%) once. Finally, the pretreated brans were dried in the oven at 60˚C overnight [36].
2.2.2. Laboratory scale subcritical water extraction
Subcritical water extraction (SWE) was done to extract arabinoxylans from the pretreated brans using an accelerated solvent extractor Dionex ASE 350 (Thermo Fisher Scientific Inc., USA).
The extraction model is shown in Figure 11.
Figure 11: SWE process design
The pretreated corn and rye brans were weighed to 6.5 g and 3.75 g, respectively, and placed into extraction cells. Inside the extraction cell, the samples were sandwiched between ASE Extraction Cellulose Filters (Thermo Fisher Scientific Inc., USA) to prevent incompatible particles from entering the pipeline and the extraction liquid. With the Dionex ASE Prep DE Diatomaceous Earth (Thermo Fisher Scientific Inc., U.S.A.) as the inert material, which was only used during rye bran extraction, the bran was added into the extraction cell. Based on the different sources and production methods of the two types of bran, their particles were different sizes, resulting in different degrees of looseness during the extraction process. The total amount of 6.5g of pretreated corn bran or rye bran (including inner materials) was added in each extraction cell [36]. The SWE was performed using pressure mode with PBS buffer (100mM, pH 7.0) at 160˚C for sequential times of 5, 15, 30, 60 min. The solid to liquid ratio used for corn bran and rye bran was 1:8 (w/v) and 1: 9.5 (w/v), respectively. In order to obtain purer arabinoxylans, the 5-min extracts were removed and the 15-, 30- and 60-min extracts were pulled together and used for further analyses. The extracts contained a high amount of glucans despite the pretreating. The removal of these glucans was achieved by an additional enzymatic treatment using a combination of 16 U/g α-amylase (Type IV−B from porcine pancreas) and 5 µL glucosidase (Celluclast) at 37˚C for 4 hours under constant stirring. After that, the extracted AXs were frozen in the fridge to inhibit the enzyme activity until the ethanol precipitation step.
The extracts were precipitated in 95% ethanol (1:4 w/v) at 4˚C overnight until the polysaccharides were fully precipitated. 0.05% (w/v) sodium acetate was added to improve the precipitation of corn glucuronoarabinoxylans (GAX), as the polysaccharide from corn bran had more uronic sugars and acetyl groups. The precipitate was then centrifuged (20 minutes, 2300 rpm, 3 times) with washing using 75% ethanol in between. The precipitate was dried in an oven at 60˚C until all of the ethanol had been evaporated. Then the extracts were resuspended in water and mixed well and freeze-dried for 72 hours prior to analysis.
2.3. Characterization methods
2.3.1. Determination of moisture content
The moisture content of the initial brans and pretreated brans were determined gravimetrically.
The moisture content value was calculated by the difference between the original weight and dry weight (weight after freeze-drying for 2 ~3 days), according to equation 1:
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (%) = !!"#$#%&' # !(")
!!"#$#%&' × 100% Equation 1
2.3.2. Yield determination
The yields of arabinoxylans extracts were determined gravimetrically and calculated based on the dry weight according to the following Equation 2. The total AXs yield comparing with the initial bran was calculated by Equation 3.
𝑌𝑖𝑒𝑙𝑑$!% = !&*+," ,.+"&/+#!%
!0,*!", ,.+"&/+#!% × 100% Equation 2
𝑌𝑖𝑒𝑙𝑑&'&() = !(,1+&"/2,( *#0,"
!#%#+#&' 0"&% × 𝑌𝑖𝑒𝑙𝑑$!% × 100% Equation 2
2.3.3. Starch content
The starch content of the initial and pretreated (destarched) bran, and extracts were determined using the Total Starch Assat Kit (Megazyme, Wicklow, Ireland). Due to the large size of the initial corn bran, it was re-milled using a blender to decrease the size of the bran, therefore making the starch molecules more accessible for the enzymes. For the starch content analysis, 1 mg of samples was wetted with 20 µL of aqueous ethanol (80% v/v) in Eppendorf tubes. After adding 300 µL of thermostable α-amylase, which was diluted 1:30 by sodium acetate buffer (100 mM, pH 5), the Eppendorf tubes were incubated in a boiling water bath at 100°C for total 12 min with vortexing at every 4 min. 10 µL of the amyloglucosidase (AMG, 330U) was then added onto the samples and the samples were incubated at 50 ˚ C for 30 min with stirring. The reaction followed the total starch analysis procedure from Megazyme (AOAC method 996.11).
𝑠𝑡𝑎𝑟𝑐ℎ + 𝐻*𝑂&+,-.'/&(0), 1#(.2)(/,, 45#655˚8
;⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯= 𝑚𝑎𝑙𝑡𝑜𝑑𝑒𝑥𝑡𝑟𝑖𝑛𝑠
𝑚𝑎𝑙𝑡𝑜𝑑𝑒𝑥𝑡𝑟𝑖𝑛𝑠9:;, <5˚8
;⎯⎯⎯⎯⎯⎯= 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
The volume of the samples was adjusted to 1 mL using 670 µL distilled water. The supernatant obtained after centrifugation (1500 rpm, 20 min) was used for subsequent starch analysis. The preparation of the samples is described in detail in the appendix. The samples were analyzed using a spectrophotometer at a wavelength of 595 nm and quantified using a calibration curve each time.
2.3.4. Polysaccharide hydrolysis and monosaccharide composition
The samples after each treatment (initial bran, pretreated brans and extract AXs) were hydrolyzed by sulfuric acid method or TFA method. The monosaccharide compositions of the certain samples were determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), respectively.
2.3.4.1. Sulfuric acid hydrolysis
Approximately 1 mg of initial and pretreated bran samples were weighed in glass tubes and 125 µL of 72% sulfuric acid was added. The samples were incubated at room temperature for 3 h and stirred frequently to enable the complete soaking of samples in sulfuric acid. 1375 µL of water was then added to each tube followed by further hydrolysis at 100 ˚C for 3 h in the thermo-block. When the hydrolysis was finished the samples were cooled down to room temperature and then diluted 1:10 with water. The samples were filtered through 0.2 µm Chromacol 17-SF-02(N) filters into the HPAEC-PAD vials followed by analysis [37].
2.3.4.2. TFA hydrolysis
Approximately 1 mg of extracts were weighed in glass tubes and hydrolyzed by 1 mL of 2 M trifluoroacetic acid (TFA) at 120 ˚C for 3 h in the thermo-block. When the hydrolysis was finished, 100 µL of the hydrolyzed samples was transferred into new glass tubes and dried under compressed air overnight. The hydrolysates were then resuspended in 1000 µL of ultra- pure water and filtered through 0.2 µm Chromacol 17-SF-02(N) filters into the HPAEC-PAD vials followed by analysis[38].
2.3.4.3. HPAEC-PAD analysis for monosaccharides content
The prepared hydrolysates were injected into an HPAEC-PAD system (ICS3000, Dionex, Sunnyvale, CA) using a CarboPac PA1 column maintained at 30°C with a flow rate of 1 mL min−1. The standard preparation was detailed in the appendix. The detection and quantification of the monosaccharides was performed according to McKee et al. [39], where the gradient was employed using 4 different solvents (Solvent A: Milli-Q water; solvent B: 300 mM sodium hydroxide; solvent C: 200 mM sodium hydroxide + 170 mM sodium acetate; solvent D: 1 M
sodium acetate). Two different analysis methods were used for neutral monosaccharides and uronic acids. For the analysis of neutral monosaccharides, the equilibration step for the column was (i) inject 60 % (v/v) solvent B and 40 % (v/v) solvent C for 7 min; (ii) ramp the column to 100 % (v/v) solvent A for 1 min; (iii) inject 100 % (v/v) solvent A for 6 min; and (iv) elution over 20 min with 100% (v/v) solvent A with a post-column addition [39]. For the analysis of uronic acids, the equilibration step for the column was 10 % (v/v) solvent B for 5 min prior to injection and then elution over 30 min with 10 % (v/v) solvent B and 40 % (v/v) solvent D [39].
2.3.5. Quantification of phenolic acids by HPLC
The phenolic acid content of the samples was determined by high performance liquid chromatography (HPLC). The method reference for the retention time of each phenolic acid peaks are around:17 min for caffeic acid peak, 24 min for p-coumaric acid peak, 25 min for ferulic acid peak, 39 min for 5-5' dimer ferulic acid (5-5' AcFE) peak and 40 min for cinnamic acid peak. Only the cinnamic acid was measured at the 270 nm wavelength, while others were measured at 325 nm wavelength [40].
10 mg of dry samples were weighed into 1.5 mL dark Eppendorf tubes and mixed with 500 µL of 2 M NaOH for saponification. The samples were flushed with a gentle stream of N2 and the saponification was performed at 30 ˚C in the dark under stirring overnight. Samples after saponification were acidified using 12 M HCl (37%) to pH 2 which were measured by the paper pH indicator. The samples were then extracted by partitioning in ethyl acetate to a total volume of 3000 µL in the dark Eppendorf tubes. After drying the pulled samples under a gentle stream of N2, the samples were resuspended in 1000 µL of 2% acetic acid-MeOH (1:1, v/v) mixture which would give a 10 mg/mL sample concentration. The prepared samples were then diluted 10 times using the 2% acetic acid - MeOH mixture and then transferred into the glass HPLC vials. The phenolic acid standards included caffeic acid, p-coumaric acid, ferulic acid, t- cinnamic acid, and 5-5' AcFE which were dissolved in methanol. The steps are shown into detail in the appendix.
2.3.6. Determination of protein content
The soluble protein content of the extracts was determined by the dye-binding Bradford protein assay [41]. By binding with Coomassie dye under acidic condition, the protein molecules will change color from brown to blue. The absorbance at 595 nm was measured for the samples which should be tested after 2 min and before 1 hour. With the calibration curve which should be tested together with the samples by spectrophotometer in 595 nm wavelength, the protein content could be calculated. The calibration curve is shown below in Figure 12.
Figure 12: Calibration curve for BSA standard.
2.3.7. Molar mass distributions
The molar mass distributions of the extracts were analyzed using size exclusion chromatography (SECcurity 1260, Polymer Standard Services, Mainz, Germany) coupled to a refractive index detector (SECcurity 1260, Polymer Standard Services, Mainz, Germany) according to Ruthes et al. [27] . SEC analysis was performed at 60°C with a flow rate of 0.5 mL/min using standard calibration by pullulan. The samples were prepared at a concentration of 3 mg/mL in DMSO containing 0.5% lithium bromide.
2.3.8. Crosslinking of arabinoxylans
The enzymatic crosslinking of the extracted arabinoxylans was performed using laccase from Trametes versicolor. Two different methods were used. For method 1: AX samples were weighed to 100 mg and mixed with 5 mL water followed by the addition of 2 mL laccase solution (1 mg/mL) in a glass bottle. The mixture was incubated at 37˚C for 24 h. These samples were named as neutral gels. For the acidic gels, the laccase crosslinked AXs were freeze dried and then resuspended in HCl solution at pH 2.0 at a concentration of 5% (w/v). For method 2, two different arabinoxylans extracted by SWE from another source of corn bran was used as controls, i.e. one high feruloylated AX (HFA-corn bran AX) and low feruloylated (LFA-corn bran AX) and crosslinked as described above; by directly mixing the AXs (50 mg) and 400 or 800 µL of laccase solution (1mg/mL), the gel formation step was done in the incubator at 37˚C for 24 h. The crosslinking was carried out in duplicate and the gelation process was evaluated visually.
y = 1.049x + 0.0402 R² = 0.9959
0 0.2 0.4 0.6 0.8 1 1.2
0 0.2 0.4 0.6 0.8 1
Abs 595
BSA (mg/mL)
BSA Std Curve
3. Results and Discussion
3.1. Characterization of initial and pretreated bran
The main contents of the initial and pretreated bran are shown in Table 1:
Table 1. Chemical composition of the initial and pretreated bran from corn and rye.
Initial corn bran
(In-C)
Initial rye bran
(In-R)
Pretreated corn bran
(De-C)
Pretreated rye bran
(De-R)
Moisture [%] 11.01 10.23 2.95 2.03
Starch content [%] 23.35 17.72 2.24 2.07
Total carbohydrate [µg/mg DW]
707.11 647.63 860.03 882.40
Ara [% of total carbohydrate] 9.50 8.45 11.43 11.98
Xyl [% of total carbohydrate] 29.24 31.09 47.00 55.49
A/X ratio 0.32 0.27 0.24 0.22
Glc [% of total carbohydrate] 54.34 52.93 29.23 22.69
Uronics* [% of total carbohydrate]
2.93 4.48 7.80 6.58
Other monosaccharides* [%
of total carbohydrate]
0.41 3.05 0.33 3.26
Phenolic acid content (µg/mg DW)
28.42 3.57 32.92 6.62
* Add here what are the composition of the uronics included in the table
3.1.1. Moisture content
The moisture content of the initial bran and pretreated bran from both corn and rye were calculated after freeze-drying. Before pretreatment, the moisture content of initial corn bran was 11.01% and rye bran was 10.23%; after pretreatment, the moisture content of the pretreated corn bran was 2.95% and 2.03% for rye bran, as shown in Table 1.
The initial bran had a higher moisture than the pretreated bran, because the pretreated bran was dried in the oven at 60 ˚C after the pretreating step.
3.1.2. Starch content
Starch is one of the major components in bran, which will affect purity of extracts in the
following process. Therefore, before analyzing the total D-glucose content, the starch in the bran should be estimated. D-glucose content was measured to make sure that most of the starch was removed by the pretreating step. In order to reduce the impact of starch on the extraction step, the starch content was aimed to be less than 2% after pretreating [36]. Table 1 shows the starch content of the initial and pretreated bran, which were analyzed using the total starch assay kit (see section 3.2).
From the calculation result, the pretreating step was successful as it removed most of the starch and decreased the final starch content to 2% in the pretreated brans. As for the initial bran, corn bran was prepared from the dry milling production, whereby germ and other components form the corn were likely mixed together with the bran sample. This led to a lower purity of the corn bran, which could explain its higher starch content as compared to the initial rye bran. Due to the pieces of the initial corn bran being quite large, even after the bran was re-milled using a blender, the reagents and enzymes were more difficult to react with the inner structure, especially the structure of bran which was closely packed and different molecular chains including starch were entangled with each other. From this perspective, the starch content of initial corn bran could be higher than the calculated result.
3.1.3. Phenolic acid content
Before subcritical water extraction of the samples, the initial bran and pretreated bran had a relatively high content of phenolic acids. Among all phenolic acids (Table 1), the content of ferulic acid was the highest in the initial and pretreated brans. The data indicated that the content of phenolic acids in the corn bran was much higher than that of rye bran. For phenolic acid content of initial bran, corn sample was 28.42 µg/mg and rye sample was 3.57 µg/mg. After the pretreatment, the content of the total phenolic acids rose by comparing with that of the initial brans, increasing to 32.92 µg/mg for pretreated corn bran and 6.62 µg/mg for pretreated rye bran. The hemicellulose from corn bran is rich in phenolic acids, especially ferulic acid [42].
The test results obtained were consistent with previous studies [42, 43].
3.1.4. Monosaccharide content of initial bran
The monosaccharide composition of both the initial corn and rye bran are reported in Table 1.
The corn and rye bran were selected for comparison because their composition and structure of hemicellulose/arabinoxylans are different. The total carbohydrate content of the initial corn and rye bran was 707.11 µg/mg and 647.63 µg/mg (on dry weight basis), respectively, which was within the acceptable range [44, 45]. Impurities that may affect the proportion of the carbohydrates are fat and protein, especially for the corn bran that was produced by dry milling.
A large content of germ could be unremoved as a result of dry milling, causing a high amount of fat in the corn bran, which was likely present in our corn bran [14]. This resulted in a slightly low total carbohydrate content. The glucose content of both corn and rye bran were relatively high, with 54.34% and 52.93%, respectively. This high proportion of glucose was attributed to
the high content of starch (α-glucan), β-glucan and cellulose in the cell walls. The analysis of the glucose content can be used not only as an evaluation of the pretreating process, but also for the purity of samples. Furthermore, the GAX content of the initial corn bran was 44.95%
and the AX content of the initial rye bran was 39.54%. This principally showed that the content of hemicelluloses (arabinoxylan) in corn bran was higher than that of rye bran. To compare the difference of the corn bran and rye bran, the monomer of Gal, GlcA and 4OMeGlcA of corn bran were higher than the rye bran. The reason causes the higher content of these monosaccharides is, as mentioned earlier, the different AXs structures from corn and rye where the side groups of GAX gives a reason of rich in Gal, GlcA and 4OMeGlcA. Another parameter that needed attention was the A/X ratio. The A/X ratio of corn bran was higher (0.32) than that of rye bran (0.27), as corn bran AX contains higher number of branching points as in agreement with previous studies [44, 45]. Corn bran AX has a more branched structure composed of mainly arabinose, but also galactose, glucuronic acid and 4-O-methyl glucuronic acid. As mentioned earlier, the hemicellulose in corn bran is mainly GAX, and as for rye bran, it is AX.
Although in GAX these branch structures are mostly monomer substitutions, and some oligomer substitutions could be found in GAX structure, the content of arabinose and A/X ratio are normally higher than that from rye bran AX.
The monosaccharide composition was tested using the HPAEC-PAD. For monosaccharide analysis, both acidic hydrolysis and enzymatic hydrolysis can be used. In this project, acidic hydrolysis was chosen. There are usually three ways of acidic hydrolysis: Sulphuric hydrolysis, methanolysis and TFA hydrolysis. In terms of hydrolytic strength, sulphuric hydrolysis is the most aggressive method, which is mostly used for hydrolyzing cellulose structure with large amount of crystal structure and hydrogen bonds. The other two kinds of acidic hydrolysis methods, namely TFA hydrolysis and methanolysis, are mostly used for hydrolyzing hemicellulose structures. Among the three commonly used methods, only sulphuric hydrolysis can hydrolyze cellulose completely but, at the same time, it will be too strong and can excessively hydrolyze monosaccharides, which is not suitable for the hydrolysis of hemicelluloses. As for the TFA hydrolysis, it could cause the incomplete cleavage of some uronic acids however, the total time for the hydrolysis period is shorter. Compared with the extracts, the initial and pretreated brans had a more complex macrostructure, in which different components (such as cellulose, hemicellulose and pectin) interacted with each other and the cellulose accounts for a large proportion followed by hemicelluloses. Therefore, for the initial and pretreated brans, sulphuric hydrolysis was used before cellulose determination and for the SWE extracted fractions, TFA hydrolysis method was well-suited and chosen for hydrolyzing the samples.
3.1.5. Monosaccharide content of pretreated bran
The total carbohydrate content and monosaccharide composition of the pretreated corn and rye bran is shown in Table 1. The proportion of glucose content in the pretreated bran decreased greatly, to around 29.23% for pretreated the corn bran and to 22.69% for the pretreated rye bran.
Most of the remaining glucose came from the insoluble cellulose bran, while a small amount
of starch and mixed-linkage β-glucan remained from the incomplete removal. For the total dry weight of carbohydrates, both the pretreated corn and rye bran were in agreement with previous studies [36], and enriched to compared to the initial brans 860.03 µg/mg for destarched corn bran (De-C) and 882.40 µg/mg for destarched rye bran (De-R)) . This result was due to the removal of starch and protein during pretreating, the washing and centrifugation steps after the pretreating process. However, with the increasing amounts of arabinose and xylose in the pretreated brans, the A/X ratio decreased to 0.27 for the pretreated corn bran and to 0.22 for the pretreated rye bran. It could be a speculated that different populations of AX oligomers could be remained after the destarching process resulting in a decrease in the measured A/X ratio.
3.2. Characterization of the subcritical water extracted fractions
3.2.1. Extraction yields
The yields of the extracted AXs from both corn and rye bran using SWE were calculated by equation 2. As for the yield of SWE process (comparing with pretreated bran and extracts), for the corn and rye extracts were 21.33% and 23.91%, respectively. Comparing with initial bran and extracts, the total yield of AXs for corn was 11.61%, and for rye sample was 11.53%.
3.2.2. Monosaccharide content of extracts
The analysis result of monosaccharide content of the extracts tested by HPAEC-PAD are shown in Table 2 and Table 3, and Figure 13-16 compared the monosaccharide content of the initial bran (In-), pretreated (destarched) bran (De-) and extracts (Ex-).
Table 2: Monosaccharide composition of the corn extracts (Ex-C) from HPAEC-PAD analysis
Extracts (Ex-C)
Total carbohydrate [µg/mg DW] 316.7
GAX * [% of total carbohydrate] 82.74
Ara [% of total carbohydrate] 28.13
Xyl [% of total carbohydrate] 44.63
A/X ratio 0.63
Glc [% of total carbohydrate] 9.52
Uronics **[% of total carbohydrate] 9.35
Other monosaccharides* [% of total carbohydrate]
0.04
* GAX is the summary of Ara, Xyl, Gal, GlcA and OMeGlcA, specifically used for corn samples; Uronics is composed of 4OmeGlcA, GalA and GlcA.
Figure 13: Monosaccharide content of In-C, De-C & and Ex-C
Figure 14: GAX & GalA of In-C, De-C & Ex-C
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
In-C De-C Ex-C
µg/mg
Fucose Arabinose Rhamnose Galactose Glucose
Xylose Mannose 4OMeGlcA GalA GlcA
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00
In-C De-C Ex-C
GAX GalA
T
able 3: Monosaccharide composition of the rye bran extracts (Ex-R) from HPAEC-PAD analysisExtracts (Ex-R)
Total carbohydrate [µg/mg DW] 734.2
AX [% of total carbohydrate] 67.06
Ara [% of total carbohydrate] 20.07
Xyl [% of total carbohydrate] 46.99
A/X ratio 0.43
Glc [% of total carbohydrate] 28.81
Uronics* [% of total carbohydrate] 1.19
Other monosaccharides* [% of total carbohydrate]
2.94
*Uronics is composed of 4OmeGlcA, GalA and GlcA.
Figure 15: Monosaccharide content of In-R, De-R & and Ex-R
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
In-R De-R Ex-R
µg/mg
Fucose Arabinose Rhamnose Galactose Glucose
Xylose Mannose 4OMeGlcA GalA GlcA
