1. Introduction
Chitin is the second most abundant polysaccharide in nature after cellulose. Chitin is found mainly in the exoskeleton of crustaceans and insects, in addition to bacteria, fungi, and mushrooms [
1]. The partial deacetylation of chitin promotes the attainment of chitosan, and the difference between them is in the acetyl group. Chitin contains mainly units of N-acetyl-
d-glucosamine (GlcNAc), while chitosan consists mainly of
d-glucosamine (GlcN). The units that form both chitin and chitosan are linked by β (1 → 4) glycosidic bonds,
Figure 1. In this context, it is understood that the greater the number of deacetylated units (GlcN) in chitosan, the greater the degree of deacetylation [
1,
2].
Chitosan has received attention as a functional biopolymer due to its cationic nature, biocompatibility, biodegradability, non-toxicity, and adsorption properties [
2]. Its main characteristics are the molecular weight (Mw) and the degree of acetylation (DA) or degree of deacetylation (DDA), which correspond to the molar fractions of GlcNAc and GlcN. Most commercial chitosans have molecular weights ranging from 50–2000 kDa, with an average DDA of 50–100% (commonly 80–90%) [
3]. Based on molecular weight, chitosan can be grouped into low molecular weight (<100 kDa), medium molecular weight (100–1000 kDa), and high molecular weight (>1000 kDa) [
4].
The high molecular weight and the high degree of polymerization (DP) of chitosan result in low solubility at neutral pH. The high viscosity in solution is the main limitation in the food, cosmetics, agriculture, and health industry [
5]. Therefore, to obtain chitosan with a more uniform molecular size and easy solubility, it is necessary to convert chitosan into oligomers. Chitosan with DP <20 and a molecular weight less than 3.9 kDa is called chitosan oligomers, chitooligomers, or chitooligosaccharides (COS) [
3].
Chitooligosaccharides are the products of the hydrolysis of chitosan, and because they are soluble in water, they have several applications, such as antimicrobial, antioxidant, anti-tumor, and agricultural purposes [
6,
7,
8,
9,
10,
11,
12].
Oligomers obtained from the hydrolysis of chitosan can be classified into homo-chitooligosaccharides, which are formed exclusively by GlcN or GlcNAc units, and also hetero-chitooligosaccharides. These latter are formed by units of GlcN and GlcNAc with varying degrees of deacetylation. Additionally, they can have different degrees of polymerization (number of monomer units within an oligomer) [
13]. Regarding solubility, hetero-chitooligomers with DP <10 are considered soluble in water. However, water solubility with DP greater than 10 depends on the degree of deacetylation and the pH solution [
14].
The way to obtain chitooligosaccharides can be by acid hydrolysis [
4,
15], oxidative [
16,
17], microwaves [
18,
19], gamma radiation [
11,
20], and enzymatic methods [
21,
22]. These hydrolysis methods provide chitosan with different molecular weights and degrees of deacetylation, which influences its composition, yield, and functionality. These methods have advantages and limitations, described in
Figure 2.
Among hydrolysis techniques, acid hydrolysis has emerged as a convenient method for depolymerizing polysaccharides. However, acid hydrolysis generally requires severe treatment, as the rigid crystalline region in the chitosan granules inhibits acid penetration. For this reason, a high concentration of acid, ranging from 5 to 12 M, is used in most studies [
9,
23,
24,
25]. However, excessive acid loading can cause glucosamine degradation, which significantly reduces yield and generates major waste deposition problems [
15,
26]. In recent studies, a smaller amount of acid only made it possible to obtain medium molecular weight chitosan in a short hydrolysis time [
4]. In high time, low molecular weight chitosan and chitooligosaccharides were obtained. However, yield decreased due to soluble products such as glucosamine monomers and dimers [
15,
27]. Therefore, current studies seek to develop the technique using different types and concentrations of acids associated with ionic liquids, induced electric fields, or other hydrolysis methods.
Oxidative hydrolysis, using hydrogen peroxide, is considered an easy and non-dangerous method. Because it is a slow method, most studies use this type of hydrolysis associated with other techniques [
17,
20]. In addition, to reduce the production cost, process variables are also studied [
12,
16].
The hydrolysis of chitosan by radiation has gained considerable attention because it is a relatively simple process, does not need to use a chemical reagent, is carried out at room temperature, and can be applied on a large scale [
8,
28]. This type of hydrolysis can have a high production cost if high doses of radiation are used. In this case, studies seek to use radiation associated with other reagents, especially hydrogen peroxide and acetic acid, to increase the efficiency of the process [
11,
29].
Otherwise, microwave hydrolysis is considered less efficient when compared to other high-energy radiation, such as ultraviolet and gamma. However, due to the lower environmental impact, microwave hydrolysis presents a viable alternative to obtain polysaccharides with molecular weights of interest [
30]. In addition, due to the cost of the process, it is necessary to use less power and less hydrolysis time. For this, studies have associated this method with the use of hydrochloric acid, acetic acid, or ionic liquid [
17,
18,
19]. However, it is necessary to be careful with the addition of these solvents in high concentrations, as they can generate residues and, consequently, a more significant environmental impact.
Enzymatic hydrolysis has received much attention because of its lower environmental risk. However, despite the enzymatic process taking place under mild conditions, the hydrolysis rate is slow in viscous solutions. Thus, a low substrate concentration (1% m/v) is used in most studies. The low substrate concentration causes an increase in the solution’s volume and the need for more enzymes. Additionally, specific enzymes have high prices and limited availability. Alternatively, low-cost enzymes from the hydrolases group can degrade chitosan [
31,
32,
33].
Enzymatic hydrolysis is the only method that has association restrictions with other types of hydrolysis due to the sensitivity of the enzymes related to pH, high temperature, and radiation. Thus, most studies seek to optimize process variables (enzyme/substrate ratio, temperature, pH, and hydrolysis time) as an alternative to reduce costs [
6,
22,
31,
34]. Despite the disadvantages, enzymatic hydrolysis still seems to be the best alternative due to its selectivity. This selectivity favors the obtaining of chitosan chains with a specific average molecular weight [
33].
Global interest in chitosan hydrolysis products has been steadily increasing. Based on the references cited in this review, the diversity of countries in which this theme has been highlighted was verified (
Appendix A).
This review will present the main hydrolysis techniques of chitosan and analyze the main factors that influence the obtaining and characteristics of low molecular weight chitosan and chitooligosaccharides effectively at a lower cost.
5. Oxidative Hydrolysis with Hydrogen Peroxide
Hydrogen peroxide (H
2O
2) is used for the hydrolysis of polysaccharides because it is easy to handle, readily available, and environmentally friendly [
16]. This method is based on the formation of reactive hydroxyl radicals by dissociating hydrogen peroxide. Hydroxyl radicals, which are robust oxidizing species, can attack β-
d- glycosidic bonds (1 → 4), resulting in the hydrolysis of chitosan [
48].
The depolymerization of chitosan by hydrogen peroxide provides a breakdown of the 1,4-β-
d-glucoside bonds of the polysaccharide chain, leading to a decrease in molecular weight [
49]. Chito-oligosaccharides obtained by oxidative hydrolysis are mainly composed of monosaccharides to pentasaccharides. The most significant amount found is disaccharides and trisaccharides, with or without acetylation [
17]. Tian et al. [
49] present the hydrolysis mechanism described below:
The hydroperoxide anion is very unstable and is easily decomposed into a highly reactive hydroxyl radical (HO·).
Eventually, the hydroxyl radical (OH·) attacks the glycosidic bond of chitosan to produce the chitosan oligomer according to the reactions below:
During hydrolysis, R-NH2 reacts preferentially with H+ to produce R-NH3+, which causes a decrease in H+ and an increase in pH. Additionally, HOO− is rapidly decomposed into HO ·, which means that H2O2 is continuously decomposed. These radicals undergo other reactions quickly to form low molecular weight water-soluble oxidation products.
This type of hydrolysis is mainly influenced by the concentration of hydrogen peroxide, time, and temperature of hydrolysis,
Table 4.
5.1. Concentration of Hydrogen Peroxide
The concentration of hydrogen peroxide is directly proportional to the rate of hydrolysis. However, its high concentration can influence the elimination of hydroxyl radicals and reduce the reaction efficiency. In this sense, the concentration of 2% H
2O
2 is ideal for chitosan hydrolysis [
48].
In addition to the hydrolysis rate, the yield is also maximum (62%) when the H
2O
2 concentration reaches 2%. From this concentration, the yield decreases to 58% and 55% at concentrations of 2.5% and 3%, respectively. This decrease can be attributed to the oligosaccharides production with a shallow degree of polymerization that makes ethanol precipitation difficult [
12].
The concentration of reducing sugars is also maximum (14.8%) when using 2% H
2O
2. However, from the concentration of 2.5% and 3%, it was observed that the amount of reducing sugars reduced to 14% and 12%. This occurred due to the aldehyde oxidation when there is an excess of H
2O
2 [
16].
5.2. Association with Other Types of Hydrolysis
The formation of radical groups is practically inefficient when hydrogen peroxide is used alone. For this reason, to improve the efficiency of hydrolysis, several studies associate hydrogen peroxide with other patterns of degradation. Among them are some works previously discussed using gamma radiation [
7,
8,
20,
40,
42] and microwaves [
17,
45].
In addition to these methods, chitosan is also effectively hydrolyzed by hydrogen peroxide under ultraviolet irradiation. In this case, when chitosan is hydrolyzed only in the presence of hydrogen peroxide, viscosity decreases by 20% and 63% at 30 and 180 min, respectively. However, when hydrogen peroxide is combined with ultraviolet irradiation, viscosity decreases by 84% and 92% for the same analyzed times [
48].
5.3. Association with Other Reagents
Another way to increase the efficiency of the process is to hydrolyze chitosan using hydrogen peroxide under the catalysis of phosphotungstic acid. Phosphotungstic acid is a heteropolytic acid that presents simple preparation and high reactivity and is non-corrosive, in addition to having acid resistance and relatively high thermal stability [
16,
50]. The rate of hydrolysis of chitosan at 70 °C for 30 min without the catalyst was only 43%. This indicates that degradation is inefficient when H
2O
2 is used alone. However, when 0.1% phosphotungstic acid was used, the rate was 99.32% [
50].
The concentration of phosphotungstic acid influences the hydrolysis product. There is a sharp increase in the concentration of reducing sugars with a higher concentration of phosphotungstic acid from 0.04% to 0.1%, without an additional increase from 0.1% [
16].
When associated with acetic acid, its molar ratios influence the recovery of chitooligosaccharides. Therefore, the higher the concentration of hydrogen peroxide about acetic acid, the higher the yield of chitooligosaccharides (dimers–decamers), with a yield of 36%, 22%, 18%, and 14%, for molar ratios of 5.7, 2.8, 1.9, and 1.7 respectively, in addition to obtaining chitosan with lower molecular weight, 6.61, 7.7, 9.29, and 9.03 kDa, respectively [
46].
5.4. Effect of Time
As with other types of hydrolysis, time will also influence when using hydrogen peroxide. In chitosan hydrolyzed by H
2O
2 in HCl solution (0.9%) for 0.5–8 h, there was a reduction in molecular weight with the increase in hydrolysis time. A rapid decrease from 480 to 50 kDa in 0.5 h has been shown, with a 95% yield [
51]. In the study using hydrogen peroxide and acetic acid, the yields of chitooligosaccharides increased considerably with increasing hydrolysis time, reaching an optimal yield of 62.42% in 4 h [
12].
The reaction rate is higher when H
2O
2 hydrolyzes the chitosan under the catalytic action of phosphotungstic acid with an ideal time of 30 min of hydrolysis. In these conditions, a higher concentration of reducing sugars was obtained in 30 min and no further increase after 40 min [
16]. Additionally, there is a reduction in the molecular weight of chitosan from 700 to 4.7 and 4.3 kDa in 30 and 120 min, respectively. This shows that after 30 min, under the catalysis of phosphotungstic acid (0.1%), there will be no significant reduction in molecular weight [
50].
5.5. Effect of Temperature
Temperature is another variable that influences the reaction, since the temperature is directly proportional to the hydrolysis rate. This behavior was observed in 498 kDa chitosan, hydrolyzed in H
2O
2 solution (0.3%) for 2 h at 30, 50, and 90 °C. The obtainment of medium and low molecular weight chitosan of 200, 25 Da, and 5 kDa, respectively, was observed [
51].
Additionally, the yield of chitosan after H
2O
2 hydrolysis is proportional to the increase in temperature, with a maximum concentration of reducing sugars at 65 °C. However, when the reaction temperature exceeds 65 °C, browning occurs in the reaction mixture and a decrease in the concentration of reducing sugars due to oxidation of the aldehyde. Therefore, the most suitable maximum reaction temperature is 60 °C [
12,
16,
49].