Chitin and Chitosan Production from Mushrooms

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

Chitin and Chitosan from Mushrooms

Edible and medicinal mushrooms such as Lentinus edodes (shiitake), Ganoderma lucidum (Lingzhi or reishi), Inonotus obliquus (chaga), Agaricus bisporus (button mushrooms), Hericium erinaceus (lion’s 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 chition into valuable chitosan via ultrasonic depolymerization.

Ultrasonic deacetylation of chitin to chitosan

Deacetylation of chitin to chitosan is promoted by sonication

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Ultrasonic extractor UIP4000hdT for extraction en deacetylation of chitin from mushrooms

Ultrasonication is used to extract chitin from mushrooms. Furthermore, ultrasound promotes the deacetylation of chitin in order to obtain chitosan.

Ultrasonication is a fast and mild extraction method to produce high-quality mushroom extract. In the video, a UP400St is used for the extraction of polysaccharides from edible mushrooms.

Cold Mushroom Extraction using UP400St with 22mm probe

Chitin, which is an N-acetylglucosamine polymer (poly-(β-(1–4)-N-acetyl-D-glucosamine), is a naturally occurring polysaccharide widely found in the exoskeleton of invertebrates such as crustacean and insects, the inner skeleton of squid and cuttlefish as well as the cell walls of fungi. Embedded into the structure of mushroom cell walls, chitin is responsible for the shape and rigidity of the fungal cell wall. For many applications, chitin is converted to its deacetylated derivative, known as chitosan via a depolymerization process.
Chitosan is the most common and most valuable derivative of chitin. It is is a high molecular weight polysaccharide linked by b-1,4 glycoside, composed from N-acetyl-glucosamine and glucosamine.
Chitosan can be derived through chemical or enzymatic N-deacetylation. In the chemically driven deacetylation process, the acetyl group (R-NHCOCH3) is cleaved off by strong alkali at high temperatures. Alternatively, chitosan can be synthesized via enzymatic deacetylation. However, on industrial production scale chemical deacetylation is the preferred technique, since enzymatic deacetylation is significantly less efficient due to the high cost of the deacetylase enzymes and the low chitosan yields obtained. Ultrasonication is used to intensify the chemical degradation of the (1→4)-/β-linkage (depolymerization) and effect the deacetylation of chitin to obtain high-quality chitosan. When sonication is applied as pre-treatment for the enzymatic deacetylation, chitosan yield and quality is improved, too.

Industrial Chitosan Production from Mushroom with 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.

Ultrasonic extraction and deacetylation  of fungal chitin give high-quality chitosan.

Sonication intensifies the production of fungal chitosan and makes the production more efficient and economical.
(picture and study: © Zhu et al., 2019)

Ultrasonic chitin extraction from mushrooms with the UP400ST probe-type ultrasonicator (400W, 24kHz)

Ultrasonicator UP400St for mushroom extraction: Sonication gives high yields of bioactive compounds such as the polysaccharides chitin and chitosan

Highly Efficient Chitosan Synthesis via Sonication

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.

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.

Research Results for Ultrasonic Chitin and Chitosan Deacetylation

Sonochemically deacetylated chitin results in high-quality chitosan.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)

Chitins and chitosans from mushroom can be efficiently extracted using probe-type ultrasonication.

SEM images of chitins and chitosans from two mushroom species: a) Chitin from L. vellereus; b) Chitin from P. ribis; c) Chitosan from L.vellereus; d) chitosan from P. ribis.
picture and study: © Erdoğan et al., 2017

Industrial ultrasonic tank reactor with high-performance ultrasonic probe for chitin deacetylation

Ultrasonic reactor with 2000W ultrasound probe (sonotrode) for chitin extraction from mushrooms and subsequent depolymerization / deacetylation

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Superior Chitosan Quality with Ultrasonic 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)

How Does Ultrasonic Extraction and Deacetylation of Chitin Work?

When power ultrasound waves are couples into a liquid or slurry (e.g., a suspension consisting of chitin in a solvent), the ultrasonic waves travel through the liquid causing alternating high-pressure / low-pressure cycles. During low-pressure cycles, minute vacuum bubbles (so-called cavitation bubbles) are created, which grow over several pressure cycles. At a certain size, when the bubbles cannot absorb more energy, they implode violently during a high-pressure cycle. The bubble implosion is characterised by intense cavitational (or sonomechanical) forces. These sonomechanical conditions occur locally in the cavitational hot-spot and are characterized by very high temperatures and pressures of up to 4000K and 1000atm, respectively; as well as corresponding high temperature and pressure differentials. Furtehrmore, micro-turbulences and liquid streams with velocities of up to 100m/s are generated. Ultrasonic extraction of chitin and chitosan from fungi and crustaceans as well as chitin depolymerization and deacetylation are mainly caused by sonomechanical effects: the agitation and turbulences disrupt cells and promote mass transfer and can also cut polymer chains in combination with acidic or alkaline solvents.
Working principle of chitin extraction via ultrasonication: Ultrasonic extraction efficiently breaks the cell structure of mushrooms and releases the intracellular compounds from the cell wall and cell interior (i.e., polysaccharides such as chitin and chitosan and other bioactive phytochemicals) into the solvent. Ultrasonic extraction is based on the working principle of acoustic cavitation. The effects of ultrasonic / acoustic cavitation are high-shear forces, turbulences and intense pressure differentials. These sonomechanical forces break cellular structures such as the chitinous mushroom cell walls, promote mass transfer between fungus biomaterial and solvent and result in very high extract yields within a rapid process. Additionally, sonication promotes the sterilization of extracts by killing bacteria and microbes. Microbial inactivation by sonication is a result of the destructive cavitational forces to the cell membrane, the production of free radicals, and localized heating.
Working principle of depolymerization and deacetylation via ultrasonication: The polymer chains are caught in the shear field around a bubble and the chain segments of the polymer coil near a collapsing cavity will move at a higher velocity than those further away. Stresses are then produced on the polymer chain due to the relative motion of the polymer segments and solvents and these are sufficient to cause cleavage. The process is thus similar to other shearing effects in polymer solutions ~2° and gives very similar results. (cf. Price et al., 1994)

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High-Performance Ultrasonic Equipment for Fungal Chitin and Chitosan Processing

Ultrasonic deacetylation of chition to chitosan

Scanning electron microscopy (SEM) images in a magnification of 100× of a) gladius, b) ultrasound-treated gladius, c) β-chitin, d) ultrasound-treated β-chitin, and e) chitosan (source: Preto et al. 2017)

4kW ultrasonicator for industrial chitin / chitosan processing from crustacean and fungiThe 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, highest process control and user-friendliness are ensured. 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 deacetylation in a safe and user-friendly process.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Batch Volume Flow Rate Recommended Devices
1 to 500mL 10 to 200mL/min UP100H
10 to 2000mL 20 to 400mL/min UP200Ht, UP400St
0.1 to 20L 0.2 to 4L/min UIP2000hdT
10 to 100L 2 to 10L/min UIP4000hdT
n.a. 10 to 100L/min UIP16000
n.a. larger cluster of UIP16000

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