Ultrasonication is a highly efficient method to release chitin and chitosan from fungal sources such as mushrooms. Chitin and chitosan must be depolymerized and deacetylated in down-stream processing in order to obtain a high-quality biopolymer. Ultrasonically-assisted depolymerization and deacetylation is a highly efficacious, simple and rapid technique, which results in high-quality chitosans with high molecular weight and superior bioavailability.

Mushroom-Derived Chitin and Chitosan via Ultrasonication

Edible and medicinal mushrooms such as Lentinus edodes (shiitake), Ganoderma lucidum (Lingzhi or reishi), Inonotus obliquus (chaga), Agaricus bisporus (button mushrooms), Hericium erinaceus (lions mane), Cordyceps sinensis (caterpillar fungus), Grifola frondosa (hen-of-the-wood), Trametes versicolor (Coriolus versicolor, Polyporus versicolor, turkeytail) and many other fungus species are widely used as food and for the extraction of bioactive compounds. These mushrooms as well as processing residuals (mushroom waste) can be used to produce chitosan. Ultrasonication not only promotes the release of chitin from the fungal cell wall structure, but also drives the conversion of chitin into valuable chitosan via ultrasonically-assisted depolymerization and deacetylation.

 

Intense ultrasonication using a probe-type ultrasonic system is a technique used to promote the depolymerization and deacetylation of chitin, leading to the formation of chitosan. Chitin is a naturally occurring polysaccharide found in the exoskeletons of crustaceans, insects, and the cell walls of certain fungi. Chitosan is derived from chitin by removing the acetyl groups from the chitin molecule.

Ultrasonic Procedure for Fungal Chitin to Chitosan Conversion

When intense ultrasonication is applied for the production of chitosan from chitin, a chitin suspension is sonicated with high-intensity, low-frequency ultrasound waves, typically in the range of 20 kHz to 30 kHz. The process generates intense acoustic cavitation, which refers to the formation, growth, and collapse of microscopic vacuum bubbles in the liquid. Cavitation generates localized extremly high-shear forces, high temperatures (up to several thousand degrees Celsius) and pressures (up to several hundred atmospheres) in the liquid surrounding the cavitation bubbles. These extreme conditions contribute to the breakdown of the chitin polymer and the subsequent deacetylation.
 

 

Ultrasonic Depolymerization of Chitin

The depolymerization of chitin occurs through the combined effects of mechanical forces, such as microstreaming and liquid jetting, as well as by ultrasonically initiated chemical reactions induced by free radicals and other reactive species formed during cavitation. The high-pressure waves generated during cavitation cause the chitin chains to undergo shear stress, resulting in the scission of the polymer into smaller fragments.

Ultrasonic Deacetylation of Chitin

In addition to depolymerization, intense ultrasonication also promotes the deacetylation of chitin. Deacetylation involves the removal of acetyl groups from the chitin molecule, leading to the formation of chitosan. Intense ultrasonic energy, particularly the high temperatures and pressures generated during cavitation, accelerate the deacetylation reaction. The reactive conditions created by cavitation help break the acetyl linkages in chitin, resulting in the release of acetic acid and the conversion of chitin into chitosan.
Overall, intense ultrasonication enhances both the depolymerization and deacetylation processes by providing the necessary mechanical and chemical energy to break down the chitin polymer and facilitate the conversion to chitosan. This technique offers a rapid and efficient method for the production of chitosan from chitin, with numerous applications in various industries, including pharmaceuticals, agriculture, and biomedical engineering.

Industrial Chitosan Production from Mushroom with Power Ultrasound

Commercial chitin and chitosan production is mainly based on waste of the marine industries (i.e. fishing, shell fish harvesting etc.). Different sources of raw material result in different chitin and chitosan qualities, resulting of production and quality fluctuations due to seasonal fishing variations. Furthermore, chitosan derived from fungal sources offers reportedly superior properties like homogeneous polymer length and greater solubility when compared with chitosan from marine sources. (cf. Ghormade et al., 2017) In order to supply uniform chitosan, the extraction of chitin from fungal species has become a stable alternative production. Chitin and citiosan production from fungi can be easily and reliable achieved using ultrasonic extraction and deacetylation technology. Intense sonication disrupts cell structures to release chitin and promotes mass transfer in aqueous solvents for superior chitin yields and extraction efficiency. Subsequent ultrasonic deacetylation converts the chitin into the valuable chitosan. Both, ultrasonic chitin extraction and deacetylation to chitosan can be linearly scaled to any commercial production level.

Research Results for Ultrasonic Chitin and Chitosan Deacetylation

Zhu et al. (2018) conclude in their study that ultrasonic deacetylation has proven to be a crucial breakthrough, converting β-chitin into chitosan with 83–94% deacetylation at reduced reaction temperatures. The picture left shows a SEM image of ultrasonically deacetylated chitosan (90 W, 15 min, 20 w/v% NaOH, 1:15 (g: mL) (picture and study: © Zhu et al., 2018)
In their protocol, NaOH solution (20 w/v %) was prepared by dissolving NaOH flakes in DI water. The alkali solution was then added to GLSP sediment (0.5 g) at a solid-liquid ratio of 1:20 (g: mL) into a centrifuge tube. Chitosan was added to NaCl (40 mL, 0.2 M) and acetic acid (0.1 M) at a 1:1 solution volume ratio. The suspension was then subjected to ultrasound at a mild temperature of 25°C for 60 min using a probe-type ultrasonicator (250W, 20kHz). (cf Zhu et al., 2018)
 
Pandit et al. (2021) found that the rate of degradation for chitosan solutions is seldom affected by the concentrations of acid utilized to solubilize the polymer and largely depends upon the temperature, intensity of ultrasound waves, and ionic strength of the media used to dissolve the polymer. (cf. Pandit et al., 2021)
 
In another study, Zhu et al. (2019) used Ganoderma lucidum spore powders as fungal raw material and investigated ultrasonically‐assisted deacetylation and the effects of processing parameters such as sonication time, solid‐to‐liquid ratio, NaOH concentration, and irradiation power on the degree of deacetylation (DD) of chitosan. The highest DD value was obtained at the following ultrasonic parameters: 20 min sonication at 80W, 10% (g:ml) NaOH, 1:25 (g:ml). The surface morphology, chemical groups, thermal stability, and crystallinity of the ultrasonically obtained chitosan were examined using the SEM, FTIR, TG, and XRD. The research team reports a significant enhancement of the degree of deacetylation (DD), dynamic viscosity ([η]) and molecular weight (Mv¯) of the ultrasonically produced chitosan. The results underlined the ultrasonic deacetylation technique of fungi a highly potent production method for chitosan, which is suitable for biomedical applications. (cf. Zhu et al., 2019)

Superior Chitosan Quality with Ultrasonic Depolymerization and Deacetylation

Ultrasonically-driven processes of chitin / chitosan extraction and depolymerization are precisely controllable and ultrasonic process parameters can be adjusted to the raw materials and the targeted end product quality (e.g., molecular weight, degree of deacetylation). This allows to adapt the ultrasound process to external factors and to set optimum parameters for superior outcome and efficiency.
Ultrasonically deacetylated chitosan shows excellent bioavailability and biocompatibility. When ultrasonically prepared chitosan biopolymers are compared to thermally derived chitosan regarding biomedical properties, the ultrasonically produced chitosan exhibits significantly improved fibroblast (L929 cell) viability and enhanced antibacterial activity for both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).
(cf. Zhu et al., 2018)
 

High-Performance Ultrasonic Equipment for Chitin and Chitosan Processing

The fragmentation of chitin and the decetylation of chitin to chitosan requires powerful and reliable ultrasonic equipment that can deliver high amplitudes, offers precise controllability over the process parameters and can be operated 24/7 under heavy load and in demanding environments. Hielscher Ultrasonics product range fulfils these requirements reliably. Besides outstanding ultrasound performance, Hielscher ultrasonicators boast high energy efficiencies, which is a significant economical advantage – especially when employed on commercial large-scale production.
Hielscher ultrasonicators are high-performance systems that can be equipped with accessories such as sonotrodes, boosters, reactors or flow cells in order to match your process needs in an optimal manner.
 With digital color display, the option to preset sonication runs, automatic data recording on an integrated SD card, remote browser control and many more features, Hielscher ultrasonicators ensure highest process control and user-friendliness. Paired with robustness and heavy load-bearing capacity, Hielscher ultrasonic systems are your reliable work horse in production. 
Chitin fragmentation and deacetylation requires powerful ultrasound to obtain the targeted conversion and a final chitosan product of high-quality. Especially for the fragmentation of the chitin flakes and the depolymerization / deacetylation steps, high amplitudes and elevated pressures are crucial. Hielscher Ultrasonics industrial ultrasonic processors easily deliver very high amplitudes. Amplitudes of up to 200µm can be continuously run in 24/7 operation. For even higher amplitudes, customized ultrasonic sonotrodes are available. The power capacity of Hielscher ultrasonic systems allow for efficient and fast depolymerization and deacetylation in a safe and user-friendly process.
 

The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

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Synergistic Chitin Treatment Improved by Ultrasonication

In order to overcome the drawbacks (i.e., low efficiency, high energy cost, long processing time, toxic solvents) of traditional chemical and enzymatic chitin deacetlytion, high-intensity ultrasound has been integrated into chitin and chitosan processing. High intensity sonication and the resulting effects of acoustic cavitation lead to a rapid scission of polymer chains and reduce the polydispersity, thereby promoting the synthesis of chitosan. Furthermore, ultrasonic shear forces intensify mass transfer in the solution so that chemical, hydrolytic, or enzymatic reaction are enhanced. Ultrasonic chitin treatment can be combined with already existing chitin processing techniques such as chemical methods, hydrolysis or enzymatic procedures.

Ultrasonically-Assisted Chemical Deacetylation and Depolymerization

Since chitin is a non-reactive and insoluble biopolymer, it must undergo the process steps of demineralization, deproteinization and depolymerization / deacetylation in order to obtain soluble and bioacessible chitosan. These process steps involve treatments with strong acids such as HCl and strong bases such as NaOH and KOH. As these conventional process steps are inefficient, slow, and demand high energies, process intensification by sonication improves chitosan production significantly. The application of power-ultrasound increases chitosan yields and quality, reduces the process from days to a few hours, allows for milder solvents, and makes the whole process more energy-efficient.

Ultrasonically Improved Deproteinization of Chitin

Vallejo-Dominguez et al. (2021) found in their investigation of chitin deproteinization that the “application of ultrasound for the production of biopolymers reduced the protein content as well as the particle size of chitin. Chitosan of high deacetylation degree and medium molecular weight was produced through ultrasound assistance.”

Ultrasonic Hydrolysis for Chitin Depolymerization

For chemical hydrolysis, either acids or alkalis are used to deacetylate chitin, however alkali deacetylation (e.g., sodium hydroxide NaOH) is more widely used. Acid hydrolysis is an alternativ method to the traditional chemical deacetylation, where organic acid solutions are used to depolymerize chitin and chitosan. The method of acid hydrolysis is mostly used when the molecular weight of chitin and chitosan must be homogeneous. This conventional hydrolysis process is known as slow and energy- and cost-intensive. The requirement of strong acids, high temperatures and pressures are factors which turn the hydrolytic chitosan process into a very expensive and time-consuming procedure. The acids used require downstream processes such as neutralization and desalting.
With the integration of high-power ultrasound into the hydrolysis process, the temperature and pressure requirements for the hydrolytic cleavage of chitin and chitosan can be significantly lowered. Furthermore, sonication allows for lower acid concentrations or the use of milder acids. This makes the process more sustainable, efficient, cost-effective and environmental-friendlier.

Ultrasonically-Assisted Chemical Deacetylation

Chemical disintegration and deacteylation of chitin and chitosan is mainly achieved out by treating chitin or chitosan with mineral acids (e.g., hydrochloric acid HCl), sodium nitrite (NaNO₂), or hydrogen peroxide (H₂O₂). Ultrasound improves the deacetylation rate thereby shortening the reaction time required to obtain the targeted degree of deacetylation. This means sonication reduces the required processing time of 12-24 hours to a few hours. Furthermore, sonication allows for significantly lower chemical concentrations, for example 40% (w/w) sodium hydroxide using sonication whilst 65% (w/w) are required without the use of ultrasound.

Ultrasonic-Enzymatic Deacetylation

Whilst enzymatic deacetylation is a mild, environmentally-benign processing form, its efficiency and costs are uneconomic. Due to complex, labour-intense and expensive downstream isolation and purification of enzymes from the end product, enzymatic chitin deacetylation is not implemented in commercial production, but only used in scientific research lab.
Ultrasonic pre-treatment before enzymatic deacetlytation fragments chitin molecules thereby enlarging the surface area and making more surface available for the enzymes. High-performance sonication helps to improve enzymatic deacetylation and makes the process more economic.