**4. Chitooligosaccharides**

Smart polymers have been studied to develop GOS production processes. Poly-N-isopropyl acrylamide is a thermo-responsive poly-N-isopropyl acrylamide (PNIPAAm), which presents good solubility in water and distinct phase transition at its lower critical solution temperature (LCST). It is applied in different areas, such as medicine, biotechnology, and engineering [115,116]. Based on these advantages, Palai et al (2014) [117] developed a useful bioconjugate between PNIPAAm and β-galactosidase. The constructed PNIPAAm-β-galactosidase (PNbG) can be used in catalysis and, after that; it can be easily separated from the solution by heating at a temperature above its LCST. Further on, Palai et al (2015) [118] continued the GOS production research using this bioconjugate. A maximum GOS yield of 35 % was obtained at pH 6 and 40°C. An increase in GOS yield was observed when the temperature was risen from 30 to 40°C. At 45°C or above, after prolonged time, enzyme deactivation occurred. Moreover, bioconjugates could be reutilized at least ten times; and the separation was done by simple

The use of resting or living cells for GOS production appears to be interesting due to its low cost when compared to the use of purified enzyme. Despite the complexity of biocatalysis processes involving whole cells, glucose and galactose can be consumed by them. The consumption of the monosaccharides is interesting because their presence in foods is unde‐ sirable, since they do not exhibit prebiotic effect, increase caloric value of food, and can inhibit

Nevertheless, the use of whole cells can be exploited in order to selectively improve GOS production [120]. Beta-galactosidase form *Aspergillus oryzae* was used to produce GOS from lactose, followed by fermentation with *Kluyveromyces marxianus* cells, that consumed mono and disaccharides. GOS with 95% purity containing mostly tri- and tetrasaccharides were obtained [120]. Association of β-galactosidase and cells can be applied to develop GOS enriched food products. During yogurt manufacturing, GOS was produced by addition of a commercial β-galactosidase, since starter and probiotic culture were not able to provide it. Thus, this yogurt with low lactose content can be useful for lactose intolerant people. Moreover, GOS was stable during storage, probably because it was not metabolized by microbial culture

Products containing GOS were launched for the first time in Japan in the 1980s. Due to their various and important health benefits, applications of GOS gradually increased worldwide. These oligosaccharides can be found in diverse products such as yogurt, bakery products, beverages, snack bars among others [122]. GOS are able to stimulate the growth of bifidobac‐ teria and lactobacilli in the lumen despite other members of the microbiota that were consid‐ ered potentially harmful. These oligosaccharides can prevent bacterial adherence due to their properties of mimicking host cell receptors in which bacterial adhesion occurs [123]. GOS can hinder the development of colon cancer, effect which can be attributed to their capacity of delaying fermentation processes, and reducing the activity of genotoxic bacterial enzymes associated with this disease [124]. Mineral absorption can be stimulated by GOS administra‐ tion, and their effect on calcium absorption was verified. GOS can be used to alleviate constipation, which is relatively common in elderly people and pregnant women. It occurs due to increased bacterial growth and fecal weight; besides this, short fatty acids stimulate

decantation after addition of 0.05 M NaCl and heating at 40°C.

the activity of certain β-galactosidases [119].

42 Food Production and Industry

and enzyme was inactivated by yogurt pH [121].

In the last years, studies of production and application of chitooligosaccharides (COS) have increased due to their biodegradability, biorenewability, biocompatibility, physiological inertness and hydrophilicity, properties that serve as a basis for the use of COS as functional food or to preserve food from degradation.

Chitin is one of the most abundant natural compounds on earth and its production is mainly based on the extraction from marine species (shrimps, crabs, lobsters, krills, etc.) [126]. Chitin is a copolymer of N-acetyl-D-glucosamine and D-glucosamine units linked by β-(1,4) glyco‐ sidic bonds, where N-acetyl-D-glucosamine units are predominant in the polymeric chain as shown in Figure 6A [127]. Chitin obtained from natural sources has a complex composition, containing several minerals, proteins, lipids, pigments and other compounds. Chitosan, an important derivative from chitin, is the deacetylated form of chitin, where N-acetyl groups are removed by chemical methods (Figure 6B).

A considerable amount of residues from processing of fish and crustaceans, rich in chitin and chitosan, are considered hazardous wastes and at the same time have high potential commer‐ cial value as raw material [128]. It is possible to obtain chitooligosaccharides from those residues, after prior demineralization and deproteinization by acid and alkali treatments [129].

Chitooligosaccharides are produced by chemical methods or by enzymatic methods from chitosan, produced by alkaline N-deacetylation. At industrial scale, the chemical route is used to produce chitooligosaccharides; however, this methodology presents several disadvantages such as high cost, low yield due to indiscriminate breaks of the polymer chain, production of toxic compounds due to modification on the chitin structure, as well as, corrosion and environmental hazards [130].

The enzymatic process is an attractive solution to overcome the above-mentioned disadvan‐ tages, due to their specific action on the substrate, despite the economic costs. Enzymatic hydrolysis of chitin or chitosan involves several enzymes: chitinase, chitosanase, lysozyme and cellulase [131]

According to Mourya *et al.* (2014) [132], various specific enzymes as chitosanases, chitinases and other nonspecific enzymes can hydrolyze chitin and chitosan. Action of chitinases and chitosanases are related to the degree of acetylation of the biopolymers. A novel flow chart for COS production from chitin employing chitinases and chitosanases has been reported (Figure 7) [130].

Chitnases are chitinolytic enzymes hydrolyzing the glycoside bonds between the sugars, which have the unique capacity to hydrolyze the GlcNAc-GlcNAc (2-acetamido-2-deoxy-β-Dglucose) links. Pre-treatment with acid solution is necessary to break down the crystalline structure of chitin and increase the availability of substrate to the action of enzymes. Chitosa‐ nases are enzymes that hydrolyze chitosan, classified according to the substrate specificity towards chitosan, which act specifically on the deacetylated (D–D) bonds [133].

In recent years, many scientific papers reported the application of chitinolytic enzymes, from different microorganisms, for the hydrolysis of chitin and chitosan. Enzymes for hydrolysis can be free or immobilized in non-toxic and inert supports.

Fernandes de Assis *et al*. 2010 [134] reported that COS yields of 54% were obtained after 10 minutes of hydrolysis reaction. Initial concentration of chitosan was 1% and the final oligomers concentration was 5.43 mg/mL. Production yields decreased when hydrolysis reaction time exceeded 10 minutes.

Gao *et al.* 2012 [135] determined that the optimal enzyme/chitosan ratio was 7.3 U/mg chitosan at 55°C to produce COS from chitosan employing chitinases from *Bacillus cereus*, achieving a hydrolysis yield of 76%. The yields of COS (GlcN)2, (GlcN)3 and (GlcN)4 were 13.2; 32.6 and 30.2%, respectively.

Ming *et al*. 2006 [136] producing chitooligosaccharides, reported pH range 4.5-6 as the optimal for chitinase activity, reaching 20 g/L of chitooligossacharides from an initial concentration of 50g/L of chitosan, which means a system with 40% of yield in the conversion of chitosan into chitooligosaccharides. Also, employing free and immobilized chitosanase from *Bacillus*

A considerable amount of residues from processing of fish and crustaceans, rich in chitin and chitosan, are considered hazardous wastes and at the same time have high potential commer‐ cial value as raw material [128]. It is possible to obtain chitooligosaccharides from those residues, after prior demineralization and deproteinization by acid and alkali treatments [129]. Chitooligosaccharides are produced by chemical methods or by enzymatic methods from chitosan, produced by alkaline N-deacetylation. At industrial scale, the chemical route is used to produce chitooligosaccharides; however, this methodology presents several disadvantages such as high cost, low yield due to indiscriminate breaks of the polymer chain, production of toxic compounds due to modification on the chitin structure, as well as, corrosion and

The enzymatic process is an attractive solution to overcome the above-mentioned disadvan‐ tages, due to their specific action on the substrate, despite the economic costs. Enzymatic hydrolysis of chitin or chitosan involves several enzymes: chitinase, chitosanase, lysozyme

According to Mourya *et al.* (2014) [132], various specific enzymes as chitosanases, chitinases and other nonspecific enzymes can hydrolyze chitin and chitosan. Action of chitinases and chitosanases are related to the degree of acetylation of the biopolymers. A novel flow chart for COS production from chitin employing chitinases and chitosanases has been reported (Figure

Chitnases are chitinolytic enzymes hydrolyzing the glycoside bonds between the sugars, which have the unique capacity to hydrolyze the GlcNAc-GlcNAc (2-acetamido-2-deoxy-β-Dglucose) links. Pre-treatment with acid solution is necessary to break down the crystalline structure of chitin and increase the availability of substrate to the action of enzymes. Chitosa‐ nases are enzymes that hydrolyze chitosan, classified according to the substrate specificity

In recent years, many scientific papers reported the application of chitinolytic enzymes, from different microorganisms, for the hydrolysis of chitin and chitosan. Enzymes for hydrolysis

Fernandes de Assis *et al*. 2010 [134] reported that COS yields of 54% were obtained after 10 minutes of hydrolysis reaction. Initial concentration of chitosan was 1% and the final oligomers concentration was 5.43 mg/mL. Production yields decreased when hydrolysis reaction time

Gao *et al.* 2012 [135] determined that the optimal enzyme/chitosan ratio was 7.3 U/mg chitosan at 55°C to produce COS from chitosan employing chitinases from *Bacillus cereus*, achieving a hydrolysis yield of 76%. The yields of COS (GlcN)2, (GlcN)3 and (GlcN)4 were 13.2; 32.6 and

Ming *et al*. 2006 [136] producing chitooligosaccharides, reported pH range 4.5-6 as the optimal for chitinase activity, reaching 20 g/L of chitooligossacharides from an initial concentration of 50g/L of chitosan, which means a system with 40% of yield in the conversion of chitosan into chitooligosaccharides. Also, employing free and immobilized chitosanase from *Bacillus*

towards chitosan, which act specifically on the deacetylated (D–D) bonds [133].

can be free or immobilized in non-toxic and inert supports.

environmental hazards [130].

and cellulase [131]

44 Food Production and Industry

exceeded 10 minutes.

30.2%, respectively.

7) [130].

**Figure 7.** Global flow chart for production of COS by enzymatic hydrolysis of chitin and chitosan (adapted from Jung and Park 2014 [130]).

*pumilus*, Kuriowa *et al*. 2009 [137], produced chitooligosaccharides in batch and continuous systems. In a system with free enzyme at batch conditions a concentration of 2.8 g/L was achieved, from an initial concentration of 5g/L after 40 minutes of treatment. Another system used was a membrane reactor with cutoff 2000Da. Enzyme concentration of 940 U/L, 40 minutes of residence time and 35°C were reported as the optimal conditions to attain 2.6 g/L of chitooligosaccharides. The membrane bioreactor with the free enzyme was able to maintain a constant rate of chitooligosaccharides production for 96 hours, after that time concentration decreased due to inactivation of enzymes. In order to extend the period of operation, the use of immobilized enzymes was evaluated in the membrane bioreactor. The maximum total concentration of chitooligosaccharides was 2.3 g/L with 620 U/L of immobilized chitinase during 1 month, however it is important to point out that yield was 46%, lower when compared to free enzyme tests. *figure\_7*

COS can be applied as food preservatives due to their antimicrobial activity and as functional food, mainly in prebiotics and to help the absorption of important minerals, as calcium. Antimicrobial activity of COS depends on the degree of polymerization (DP) and the degree of deacetylation (DD) as summarized in Table 1.

Inhibitory effects of COS were tested on both Gram (-) and Gram (+) bacteria, including *Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium, Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus brevis* and *Lactobacillus bulgaricus* [138]. Solutions containing 1% (w/v) COS with different molecular weights inhibited bacterial growth by 1-5 log cycles. For Gram (-) bacteria the antimicrobial activity was inversely proportional to the molecular size of oligomers, which means higher antibacterial activity was found with lower molecular weight of oligomers (1 kDa). This phenomenon was not observed for Gram (+) bacteria.

The proposed mechanism of antibacterial activity for COS with DP>12 was cellular lysis [139]. This would be due to the cationic charges of COS that could link to the negative charges present in the cell walls, leading to the formation of large bacterial clusters, which might block the nutrition transport across the bacterial cell and result in death of the bacteria. Highly deace‐ tylated COS were shown to be more effective at inhibiting the growth of *Staphylococcus aureus*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Streptococcus fecalis* and *Samonella typhimurium* than COS with low degree of deacetylation [140].

It has been suggested that COS are able to pass through the bacterial cell wall and be incor‐ porated in the cytoplasm of Gram (+) bacteria [141]. Those low molecular weight compounds can have importance in gene expression related to regulation of stress, autolysis and energy metabolism.

Chitooligosaccharides with DP 4 were demonstrated to have higher antimicrobial effect on four bacteria species (*Escherichia coli*, *Staphylococcus aureus*, *Streptococcus lactis*, *Bacillus subtilis*) and six fungi (*Saccharomyces cerevisiae*, *Rhodotorula bacarum*, *Mucor circinelloides*, *Rhizopus apiculatus*, *Penicillium charlesii*, *Aspergillus niger*) [142]. At the same time, degrees of deacetyla‐ tion over 90% were shown to be more efficient in the inhibition of microbial growth. In addition, chitooligosaccharides with low molecular weight were able to cross the cell wall and interact with DNA in the cytoplasm suppressing the growth of microorganisms. Highly deacetylated COS have many free amines, which can bond to negatively charged residues at the cell wall, leading to formation of aggregates of microorganisms. Those aggregates precip‐ itate, resulting in death of the microorganism.

Chitooligosaccharides can be employed as preservatives due to their antioxidative properties. Antioxidant activity of chitooligosaccharides depends on their degree of deacetylation and molecular weights [143]. It was shown that 90% deacetylated medium molecular weight COS have the highest free radical scavenging activity for DPPH, hydroxyl, superoxide and carbon centered radicals [144]. Antioxidant properties are closely related to the amino and hydroxyl groups, which can react with unstable free radicals to form stable macromolecule radicals [145,146].

According to Halden *et al*. 2013 [147] COS could be applied as feed additives or hypocholes‐ terolemic agents. Based on their study, hypercolesterol concentration in blood is directly related to the generation of reactive oxygen species. Thus, chitooligosaccharides can be used to scavenge the free radicals on the body, triggering the enhanced synthesis of catalase and superoxide dismutase and decreasing lipid peroxidation.

COS were conjugated with phenolic acid (PAC-COS) to improve the antioxidant properties of the oligosaccharides in the presence of reactive oxygen species (2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH) and nitric oxide (NO)) [148]. The increase on the antioxidant activity is associated to the structure of phenolic acids and the substitutions on the aromatic ring of the side chain.

Chitooligosacharides can be considered as prebiotics because they are non-digestible food ingredients with beneficial effects on probiotic bacteria (*Lactobacillus* and *Bifidobacterium*) present in the gastrointestinal tract [5]. In fact, prebiotic activities of COS preparations (0.1 to 0.5%) with varying degree of polymerization (2 to 8) were reported [149]. Assays were conducted with three strains of probiotic bacteria, *Bifidobacterium bifidum KCTC 3440*, *Bifido‐ bacterium infantis KCTC 3249* and *Lactobacillus casei KCTC 3109*.

However, an opposite effect was shown on the population of *Lactobacillus* and *Bifidobacteri‐ um* when chitooligosaccharides were tested as prebiotic agents in healthy rats [150]. Chitooli‐ gosaccharides have been demonstrated to have a weaker prebiotic effect over *Lactobacillus* and *Bifidobacterium* when compared with other oligosaccharides as fructooligosaccharides, mannanoligosaccharides; and galactooligosaccharides [151].

Chitooligosaccharides from marine species, mainly shrimps and crabs, are produced and commercialized by several companies (Table 2), such as:

