Source: UNIV OF MASSACHUSETTS submitted to NRP
ULTRASONIC EXTRACTION AND SONOCHEMICAL MODIFICATION OF CHITIN AND CHITOSAN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0202739
Grant No.
2005-35503-15428
Cumulative Award Amt.
(N/A)
Proposal No.
2004-02310
Multistate No.
(N/A)
Project Start Date
Mar 1, 2005
Project End Date
Feb 28, 2008
Grant Year
2005
Program Code
[71.1]- (N/A)
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
FOOD SCIENCE
Non Technical Summary
Chitin, a carbohydrate from the shells of freshwater and northatlantic shrimp, and its chemical derivative chitosan are of great interest to the food and pharmaceutical industry due to their cholesterol-lowering, antimicrobial, film-forming and metal binding properties. However, extraction of these compounds is expensive, time-consuming and because of the strong acids and alkalines required, not very environmental friendly. Because of this, chitosan is currently very expensive and not readily available to the general consumer. In this research, we will use a novel technology, high-intensity ultrasound to substantially improve the extraction process. We will investigate the potential of ultrasound to greatly reduce the required extraction time, lower the costs of the process and enable the use of less concentrated solvents to make the process more environmental friendly.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5013721202050%
5013722202050%
Goals / Objectives
The long-term goal of this research is to develop the fundamental physicochemical and engineering concepts required to establish high-intensity ultrasonication as a viable stand-alone or add-on unit operation for the food and agricultural industries. Ultimately, the goal is to add ultrasound to the growing list of alternative processing technologies such as application of high-pressure, pulsed-electric fields, irradiation and microwaves. The specific objective of this research project which is the next step towards attaining our long-term goal is to identify the fundamental mechanical and chemical processes that enable ultrasound to aid in the extraction and modification of the functional carbohydrates chitin and chitosan. Chitosan is of great interest to the food and pharmaceutical industry due to its hypocholesterolemic, antimicrobial, film-forming and metal binding properties. Chitosan production is a two step process. In the first step, the raw material, typically crustacean shells, is deproteinized, demineralized and purified to yield chitin. In the second step, chitin is treated with a strong base to remove acetyl side chains to obtain chitosan. Both steps are very time consuming and cost intensive. We suggest that application of high-intensity ultrasound improves the efficiency of the demineralization and deacetylation process of chitin and accelerates the subsequent conversion to chitosan thereby reducing costs and process time requirements. We further propose that prolonged exposure of chitosan to ultrasound may alter its structure (e.g. molecular weight distribution and degree of acetylation) due to initiation of a series of radical-driven chemical reactions. The sonolytic end products may possess novel functional properties not seen in conventionally produced chitosan. Our hypothesis is based on new work conducted in our laboratories that has demonstrated that application of high-intensity ultrasound during the extraction of chitin from crustacean shell fragments improved yield and purity of chitin. In a second study, we found that exposure of acidic chitosan solutions to high-intensity ultrasound reduced its molecular weight, altered the degree of acetylation and yielded additional, not yet identified, sonolytic breakdown products. We recently published similar findings for pectin which suggest that both mechanical and complex chemical processes are responsible for physicochemical changes observed in chitin and chitosan treated with high-intensity ultrasound.
Project Methods
We plan to test our central hypothesis and accomplish the overall research objective of this application by pursuing the following three research objectives. (1) Identify yield, purity and quality of chitin extracted from crustacean shells as a function of key ultrasonic processing parameters. The working hypothesis, based on our preliminary studies, is that the efficiency of the deproteinzation and demineralization process is a function of key factors that govern cavitational events i.e. sonic frequency, sonication time, ultrasonic wave intensity and temperature. (2) Identify the influence of high-intensity ultrasonication on the deacetylation reaction to convert chitin to chitosan and determine the functional properties of ultrasonically deacetylated chitosan. We hypothesize that production of oxygen radicals in the gas phase of the cavitational bubbles and subsequent conversion to hydrogen peroxide in the solvent phase together with the thermal and mechanical effects of high-intensity ultrasound impact the elimination of acetyl side groups in the presence of a strong base. The goal is to develop a reaction kinetics model as a function of ultrasonic processing parameters that can be used to predict degree of acetylation and polymerization of the final product. To achieve this goal we will correlate degree of acetylation and molecular weight distribution of ultrasonically produced chitosan with above mentioned key ultrasonic processing parameters. (3) Identify sonochemical reaction pathways and end products of ultrasonication of acidic chitosan dispersions and determine the functional properties of sonochemically modified chitosan. The goal is to develop a comprehensive understanding of the effects of sonication on chitosan physical, chemical and functional properties.

Progress 03/01/05 to 02/28/08

Outputs
OUTPUTS: - Two Graduate Ph.D. students and one M.S. students graduate from the University of Tennessee and the University of Massachusetts with an Ph.D. degree or M.S. degree, respectively, in Food Science. - In the course of the project, the students were trained in fundamental food science principles as well as specialized analytical techniques that applied to the project, including high-performance liquid chromatography of carbohydrates, Fourier transform infrared spectroscopy, degree of acetylation analysis by direct titration, light scattering and rheology for molecular weight analysis as well as a nonthermal processing of food stuff with high-intensity ultrasound. - The student have since either joined the workforce or are working as Post-Doctoral research workers in other Food Science Labs. The M.S. Student who conducted the initial experiments went on to obtain his Ph.D. degree at the University of Maine after completion of the M.S. degree. - The quality of the research and the students was recognized by peers as two of the students either won or were finalists in poster competitions during presentations at the IFT Annual Meetings - Results of this study were presented at numerous meetings, such as various Annual Meetings of the Institute of Food Technologists (2006, 2007, 2008). Results were published or have been submitted to various peer-reviewed journals including the Journal of Agricultural and Food Chemistry, the Journal of Food Science, the Journal of Food Hydrocolloids and the Journal of Food Chemistry. - Results of the study were also presented to the food industry (namely Primex Inc., the predominant manufacturer of chitosan worldwide) through a series of consulting arrangements. Primex also supported the project by donating raw material such as shrimp waste and purified chitin. - The project lead to a strengthening of international relations with the University of Iceland in Reykjavik, Iceland, where our collaborators were located. Several students from Iceland were visiting the University of Tennessee or the University of Massachusetts as a result of the ongoing collaboration on this project PARTICIPANTS: (1) Jochen Weiss, Associate Professor, Principal Director, University of Massachusetts, Amherst, MA, USA. (2) Svetlana Zivanovic, Associate Professor, Co-Principal Investigator, University of Tennessee, Knoxville, TN, USA. (3) Shari Baxter, M.S. Student, University of Tennessee, Knoxville, TN, USA. (4) Tao Wu, Ph.D. Student, University of Tennessee, Knoxville, TN, USA. (5) Gunnar Kjartansson, M.S. (University of Tennessee) and Ph.D. Student, University of Massachusetts, Amherst, MA, USA. (6) Doug Hayes, Associate Professor, Collaborator, University of Tennessee, Knoxville, TN, USA. (7) Kristberg Kristbergsson, Professor, Collaborator, University of Iceland, Reykjavik, Iceland. (8) United States Freshwater Prawn and Shrimp Growers Association, Non-Profit Organization TARGET AUDIENCES: Target audience for this research includes food process equipment manufacturers and food process engineers (for the development of new high-intensity ultrasound process equipment), chitin and chitosan ingredient producers (for the production of value added chitosan), food chemists (for the development of a new analytical method for the degree of acetylation), the food industry in general, and shrimp or prawn growers in the US or worldwide that may now better utilize and convert their crustacean byproducts. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A new analytical method was developed to accurately determine the degree of acetylation (DA) of chitin (one of the key chemical characteristics that determine the functionality of chitosan) during the entire conversion process of chitin (DA>90) to chitosan (DA<10). High intensity ultrasound applied as a pretreatment at high ultrasonic intensities and at lower temperatures to deacetylate chitin from North Atlantic shrimp (Pandalus Borealis) was found to be an effective mean to aid in the extraction of chitin from crustacean waste by aiding in the removal of proteins and minerals. Pretreatment with high-intensity ultrasound slightly decreased yield but led to a chitin with higher purity and lower crystallinity which could be more rapidly converted to chitosan translating again into time savings for manufacturer and a higher quality chitin. Application of high-intensity ultrasound reduced the time for which the crustacean waste had to be in contact with the solvent thus reducing processing time. Application of high-intensity ultrasound reduces solvent requirements, that is, the conversion can be conducted at lower solvent strength. This in turn minimizes environmental waste issues and contributes to environmental health and safety. Ultrasound was especially useful in the conversion of chitin to chitosan. Here significant time savings could be realized, reducing the required process time from 12-24 hours to a few hours. In this case again, less solvent was required to achieve a full conversion, which lowers the environmental impact of having to discard and dispose the spent or unreacted solvent (concentrated NaOH). Our results have yielded new guidelines for manufacturers: Reaction temperatures need to be carefully controlled when using high-intensity ultrasound as a pretreatment process due to decreases in efficiency and increases in yield losses at higher temperatures. A brief pretreatment with high-intensity ultrasound is better suited to reduce time required for subsequent chitosan conversion. Our results led to new insights into the mechanism of action of high-intensity ultrasound, namely, application of ultrasound promoted the conversion of chitin to chitosan possibly due to better accessibility of solvents to the reactive sites, generation of new surface areas as well as generation of free radicals that contribute to enhanced reactivity of the reagent in radical-mediated reactions. This study thus demonstrates that high intensity ultrasound, a low cost, non-thermal processing technique, can be applied to shorten processing times required to extract chitin from crustaceans as well as yield chitin (and thus subsequently chitosan) of higher purity compared to traditional processing conditions. For the production of chitin and chitosan, ultrasounds thus has the potential to lower production cost, decrease processing time, allow for a better control of the production process and reduce environmental impact of the process waste.

Publications

  • Kjartansson, G., Wu, T., Zivanovic, S., Weiss, J. 2008. Sonochemically-Assisted Conversion of Chitin to Chitosan, USDA National Research Initiative Principal Investigators Meeting, New Orleans, LA, June 28th.
  • Kjartansson, G., Kristbergsson, K. Zivanovic, S., Weiss, J. 2008. Influence of temperature during deacetylation of chitin to chitosan with high-intensity ultrasound as a pre-treatment, Annual Meeting of the Institute of Food Technologists, New Orleans, LA, June 30th, # 95-18.
  • Kjartansson, G., Kristbergsson, K., Zivanovic, S., Weiss, J. 2008. Influence of high-intensity ultrasound to accelerate the conversion of chitin to chitosan, Annual Meeting of the Institute of Food Technologists, New Orleans, LA, June 30th, # 95-17.
  • Kjartansson, G., Kristbergsson, K., Zivanovic, S., Weiss, J. 2008. FTIR spectroscopy to determine the degree of deacetylation of chitin and chitosan, Annual Meeting of the Institute of Food Technologists, New Orleans, LA, June 30th, # 94-07.
  • Wu, T., Zivanvovic, S., Hayes, D.G., Weiss, J. (2008). Efficient reduction of chitosan molecular weight by high-intensity ultrasound: Underlying mechanism and effect of processing parameters. Journal of Agricultural and Food Chemistry 56(13):5112-5119.
  • Kjartansson, G., Kristbergsson, K., Zivanovic, S., Weiss, J. (2008). On the Use of Infrared Spectroscopy to Determine the Degree of Deacetylation of Chitin and Chitosan. Journal of Agricultural and Food Chemistry, pending.
  • Kjartansson, G., Kristbergsson, K., Zivanovic, S., Weiss, J. (2008). Influence of high-intensity ultrasound as a Pretreatment on degree of deacetylation of chitin and chitosan, Journal of Food Science, pending.
  • Kjartansson, G., Kristbergsson, K., Zivanovic, S., Weiss, J. (2008). Influence of ultrasonic intensity on the conversion of chitin to chitosan. Food Research International, pending.


Progress 03/01/06 to 03/01/07

Outputs
Deacetylation of chitin to chitosan and influence of high intensity ultrasound as a pretreatment step was investigated. For deacetylation the most commonly used method is with aqueous alkali solvents, were the most frequent alkali is NaOH. Typical deacetylation processes involve, use of 40 to 50% (w/w) NaOH solutions at 100 to 115C for 0.5 to 6 hrs. The extent of deacetylation is governed by the alkali concentration, the temperature, time of reaction and particle size and density. Deacetylation in excess of 80-85% is rarely achieved with a one single process e.g. soaking the chitinious material in NaOH for a period of time. To ensure DDA>85% , it is necessary to filter wash and resuspend in fresh NaOH. Commercial produced chitin flakes (2.0 g) were soaked in NaOH solutions (1:20, flakes:solvent) with concentrations from 40, 45 and 50% (w/w) over a period of 0-300 minutes for deacetylation at 95C. Samples were withdrawn at 20 minute intervals for analysis. Samples were washed, frozen and freeze dried and grounded. To determine the effect of high intensity ultrasound both short and long term, chitin flakes (2.0g) were soaked in 120mL, 0.25M NaOH and sonicated at 90C/65Wcm-2 for 2-14 minutes for short term treatments and 30-60 minutes for long-term treatments. Sonicated flakes were washed and freeze-dried. Flakes were then resuspended in 45% and 50% of NaOH (95C) for 0-300 minutes for deacetylation. Samples were withdrawn at 30 minute intervals for analysis. Yield was determined gravimetrically. Degree of deactylation (DDA) of washed, lyophilized, and ground samples was determined by direct titration and FTIR. For 50% NaOH chitosan yield decreased from 96.2% to 93.4% for 2-minutes sonicated samples and from 67.2% to 59.4% for 60-minutes sonicated samples after pretreatment in 0.25M NaOH for 240 minutes possibly due to production of water-soluble fragments. DDA analysis of samples after sonication and dispersion in NaOH showed that the deacetylation proceeded more rapidly compared to conventional deacetylation. For example, DDA of untreated samples increased from 17.5% to 65.3% and 80.1% after suspension in 50% NaOH for 180 and 210 minutes while DDA of 2-minutes sonicated samples increased to 72.1% and 82.3% and 60 minutes samples to 86.4% and 87.1%. Conversion of untreated samples with 45% NaOH was significantly less efficient (slower deacetylation), e.g. samples were only deacetylated to 65.22% after 240 min with conventional method. For short term sonication the DDA of 2 minutes sonicated samples increased 67.3% and 84.4% for 240 and 280 minutes and for long term, 60 minutes samples to 87.1% and 88.6%. Application of high intensity ultrasound improved access of the reagent to the crystalline regions of the chitinous matter and increased the swelling of the material. High intensity ultrasound may also have disrupted the complex formed between chitin and NaOH and thereby reverting remaining N-acetylated units back to the more reactive form.

Impacts
Results of this part of the study have shown dramatic reductions in required process time and solvent concentrations of chitin from shrimp and prawn shells to the highly valuable, functional chitosan. Ultrasound pretreatments even if brief (less than 10 minutes) can lead to a doubling of the degree of deacetylation. Costs associated with purchase of ultrasonic processing equipment can be expected to be quickly offset by reductions in purchasing costs of solvents. In addition, less solvents will reduced costs associated with disposal of wasted reagents. In conclusion, ultrasonic assisted deacetylation will have substantial economic and environmental benefits over the classical process. Results of this project thus contribute to maintaining and sustaining the US agricultural sector both on the producers side (shrimp and prawn farmers) and the processing side (production of chitin and chitosan - value added).

Publications

  • Kjartansson, G., S. Zivanovic, K. Kristbergsson and J. Weiss (2006). "Sonication Assisted Extraction of Chitin from North Atlantic Shrimps (Pandalus Borealis)." Journal of Agricultural and Food Chemistry in print.
  • Kjartansson, G. T., K. Kristbergsson, S. Zivanovic and J. Weiss (2006). Deacetylation of crustacean chitin with high intensity ultrasound. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
  • Kjartansson, G. T., K. Kristbergsson, S. Zivanovic and J. Weiss (2006). Determination, characterization and comparison of chemical and morphological changes in ultrasonically extracted chitin from the shells of fresh water prawns (M. rosenbergii) and North Atlantic shrimp (P. borealis). IUFOST Iceland - Natural Science Symposium 2006, Reykjavik, Iceland.
  • Weiss, J. (2006). Physicochemical Effect of High-Intensity Ultrasonication on Food proteins and Carbohydrates. 231st American Chemical Society National Meeting, Alanta, GA.


Progress 03/01/05 to 02/28/06

Outputs
The effect of sonication during extraction of chitin on yield, purity, and crystallinity of chitin fresh water prawn (FWP) shells (Macrobrachium rosenbergii) and NorthAtlantic shrimp shells was investigated. Yield of chitin from FWP decreased from 8.28 % to 5.02 %, which was attributed to increased concentrations of depolymerized materials in the wash water. Removal of minerals was not affected by sonication. Application of ultrasound enhanced removal of proteins for from 44.01 % to 12.55 %, 10.59 % and 7.45 % after 0, 1 and 4 hrs of sonication treatments. Glucosamine content slightly decreased with sonication. The crystallinity index of chitin decreased from 91.6% to 83.7% to 80.6 % after 1h and 4h of sonication, yielding chitosans with crystallinity indices of 86.4 %, 83.7 % and 73.1 % after deacetylation, respectively. FTIR scans indicated that degree of acetylation of chitins was unaffected by sonication but that degree of acetylation of chitosans produced from sonicated chitin decreased from 70.0 % to 68.7 % and 61.4 %. Yield of chitin from NAS decreased from 16.5 % to 11.4 %, which was attributed to increased concentrations of depolymerized materials in the wash water. Removal of minerals was not affected by sonication. Application of ultrasound enhanced removal of proteins from 39.8 % to 10.6 %, 8.3 % and 7.3 % after 0, 1 and 4 hrs of sonication treatments. Sonication had no effect on glucosamine content. The crystallinity index of chitin decreased from 87.6% to 79.1% and 78.5 % after 1 and 4 hrs of sonication, yielding chitosans with crystallinity indices of 76.7%, 79.5% and 74.8% after deacetylation, respectively. FTIR scans indicated that degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from Fresh Water Prawns indicated that more impurities were left in the NAS-chitin. Finally, studies in progress on deacetylation of chitin to chitosan indicated a much more pronounced effect of sonication, that is a pretreatment of 30 minutes in 0.25N NaOH resulted in a deacetylation of chitin by 40% and accelerated subsequent deacetylation in 12N NaOH to 80% by four times.

Impacts
Results of this study show that use of high-intensity ultrasonication can enhance removal of proteins required for extraction of chitin. Ultrasound should not be used during the demineralization step because of extensive depolymerization. The use of high-intensity ultrasound during deacetylation is most promising as a pretreatment and can lead to substantial reductions in processing time and savings in solvent requirements.

Publications

  • No publications reported this period