The purpose of the research was to explore different ways through which an existing chitin digestion protocol could be improved Chitin is a naturally abundant mucopolysaccharide commonly found in crustacean shells, insect shells, and fungal cell walls. Chitin is known to be highly insoluble in both water and organic solvents which presents a challenge both in measuring small masses of chitin and in digestion procedures.

1. A Systematic Study of Chitin
Digestion Protocols
Arjuna Karikaran, John Tang, Parastoo Azadi
Complex Carbohydrate Research Center (CCRC), UGA, Athens, GA
Abstract
• The purpose of the research was to explore different ways through which
an existing chitin digestion protocol could be improved
• Chitin is a naturally abundant mucopolysaccharide commonly found in
crustacean shells, insect shells, and fungal cell walls. Chitin is known to be
highly insoluble in both water and organic solvents which presents a
challenge both in measuring small masses of chitin and in digestion
procedures.
• Some examples of attempted procedural modifications include using
trimethylsilyl (TMS) groups instead of O-acetyl groups and to use a less
harsh acid to prevent degradation of the standard.
• GC analysis was used to determine the efficiency of various digestion
methods of chitin. Through analysis of the produced spectrographs.
• Appropriate quantification proved to be very difficult and improving the
methods by which percent digestion was measured ended up being the
main focus of the work conducted so far.
Methods: Original Protocol
1. 300- 400 µg of sample was weighed and placed in a glass tube
2. The chitinase solution was prepared. 5 µg of chitinase per sample were
added. Samples would then be placed at 37 ° C overnight
3. Samples were then frozen in the -10 ° C freezer and lyophilized
4. 400 µL of 4M HCL was added to hydrolyze samples
5. 200 µL of MeOH, 100 µL of pyridine and 100 µL of acetic anhydride was
added in this order.
6. The samples were vortexed and sat for 30 min, then dried down.
7. 200µl of TMS was added and the samples were placed at 80 °C for 30 min
8. The samples were dried down. The samples were redissolved in 500 µL of
hexane, vortexed and centrifuged.
9. The supernatant from vortexing was collected and dried down to ~100µL of
hexane. The remaining solution was transferred to a GC vial for GC analysis.
10. Following extraction with hexane, GC/MS analysis of TMS methyl
glycosides was performed on an Agilent 7890A GC interfaced to a 5975C
MSD, using a Supelco Equity-1 fused silica capillary column (30 m x 0.25
mm ID).
Results: Digestion Rates
Fig. 3 Early experiments with varying
sample weights to determine best weight.
Some other parameters were varied such
as type of sample and volume of chitinase.
References
Chen, X. (2015). Human Milk Oligosaccharides (HMOS). Advances in Carbohydrate Chemistry and Biochemistry, 72:113-190. doi:10.1016/bs.accb.2015.08.002
Mcguire, M. K., et al (2017). What’s normal? Oligosaccharide concentrations and profiles in milk produced by healthy women vary geographically. The American Journal of Clinical Nutrition 105(5):1086-1100. doi:10.3945/ajcn.116.139980
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Conclusions and future work
Background: Structural Changes
Methods: GC Analysis Figures
• While the original goal of increasing the efficiency of the chitinase
procedure in order was not explicitly achieved, many insights into the
procedure were gained.
• Better standard procedure were created to prevent degradation of
standards in acid steps
• Conclusions based upon statistically significant results are unable to be
drawn from these experiments as the digestion protocol took a weeks time
• Future work hopes to expand upon the trends seen in these experiments to
produce results that can be statistically proven.
• Different solvents will be used in the future to try and alleviate the issues
with sample weighing from the early steps of the experiment
• Specific directions for future work include doing the same steps in larger
quantities with different solvents to provide a larger volume of data
Results: Fungal Wall Chromatogram; Sugars Labeled
Fig. 6 Chromatogram produced from FW1 trial with sugars labeled. Fungal wall
samples could not be directly compared to chitin. Unlike other samples fungal wall
contains other sugars. The rate of digestion from the analysis of chitin to GlcNAc is
divided by the digestion rate in fungal wall to provide a normalized rate of digestion
Fig. 1 An example of the GC analysis software.
The GC machine used was the Agilent Technologies 5975C inert MSD,
7890A GC system, and 7693 autosampler.
Fig. 2 Sequence list in GC software.
The injection volume was 1 µL and the method
was “AMINOZHIRUI” a sequence developed by the Azadi Lab
Date Name
Sample weight
(µg) Step altered
Percent
Digestion
16-Sep Exp1 210 Std 31.7
16-Sep exp2 240
40 chitinase vs 20
chitinase 89.07
16-Sep exp3 270 Chitosan 23.64
23-Sep exp4 190 40 chitinase 53.4
23-Sep exp5 220 80 Chitinase 61.7
23-Sep exp6 220 20 Chitinase 28.4
30-Sep exp7 210 Fungal wall 14.3 (32.66)
30-Sep exp8 170 Chitin stds 43.78
7-Oct exp9 240 Chitin 56.77
7-Oct exp10 210 Chitobiose 65.63
14-Oct exp11 100
.1mg chitobiose
(dif stds) Over 100
14-Oct exp12 150
.15mg Chitobiose
(dif stds) Over 100
21-Oct exp13 150 Chitobiose Over 100
21-Oct exp14 310 FW Over 100
Date Name
Sample weight
(µg) Step altered Percent Digestion
4-Nov Chitin 1 150 Over 100
4-Nov Chitin 2 100 Over 100
4-Nov Fw1 150 Over 100
4-Nov FW2 100 Over 100
11-Nov Crab1 150 44.08
11-Nov Crab2 100 34.49
11-Nov
Shrimp
1 150 46.13
11-Nov
Shrimp
2 100 39.43
18-Nov FW1 300 0.02(.18)
18-Nov FW2 300 0.03
18-Nov Chitn 1 100 11.1
18-Nov Chitin 2 100 3.2
Fig. 4 Later experiments with consistent
sample weights. Experiments that have a
digestion over 100 revealed issues with the
standards that likely contributed to higher
digestions in earlier experiments
Fig. 5 Examples of chromatograms
produced by Enhanced Data
Analysis, the program used to
determine percent digestion after GC
analysis. Peak area was determined
via manual integration and
compared to the inositol levels in
standards to produce percent
digestions.
Fig. 7 Structure of Chitin and Chitosan.
Chitosan is a partially deacetylated form of
Chitin. Chitosan samples produced similar
digestion rates to the chitin samples run
with them.
Fig. 8 Structure of N-
Acetylglucosamine
(GlcNAc). The final
monosaccharide product of
the experiment
• Chitin’s polysaccharide chain is composed of 2-acetamido-2-deoxy-β-D-
glucose, each unit is linked via a β-1,4 link. The additional amine group
allows for better hydrogen bonding, this contributes to Chitins strength
• These linkages are hydrolyzed to produce GlcNac
• The dimer of a β-1,4 linked glucosamine units is known as Chitobiose was
also tested in these experiments. Only one linkage needs to be broken it
is a more sensitive indicator of successful changes to procedure.
Discussion
• Many steps of the procedure were changed yielding slightly better results,
for example the changes to the acid hydrolysis step and the chitinase ratio
• When Digestion levels are listed as over 100 It means that The GlcNAc
standard peak was smaller than the sample GlcNAc as this is impossible it
led to an examination of the procedures for the standards