Influence of glucosamine on oligochitosan solubility and antibacterial activity

Accepted Manuscript Influence of D-glucosamine on the oligochitosan (short-chain chitosan) solu‐ bility and antibacterial activity Inesa V. Blagodatskikh, Sergey N. Kulikov, Oxana V. Vyshivannaya, Evgeniya A. Bezrodnykh, Igor A. Yamskov, Vladimir E. Tikhonov PII: DOI: Reference:

S0008-6215(13)00323-6 http://dx.doi.org/10.1016/j.carres.2013.08.012 CAR 6551

To appear in:

Carbohydrate Research

Received Date: Revised Date: Accepted Date:

28 May 2013 5 August 2013 14 August 2013

Please cite this article as: Blagodatskikh, I.V., Kulikov, S.N., Vyshivannaya, O.V., Bezrodnykh, E.A., Yamskov, I.A., Tikhonov, V.E., Influence of D-glucosamine on the oligochitosan (short-chain chitosan) solubility and antibacterial activity, Carbohydrate Research (2013), doi: http://dx.doi.org/10.1016/j.carres.2013.08.012

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Influence of D-glucosamine on the oligochitosan (short-chain chitosan) solubility and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

antibacterial activity Inesa V. Blagodatskikh,a Sergey N. Kulikov,b Oxana V. Vyshivannaya,a Evgeniya A. Bezrodnykh,a Igor A. Yamskov,a Vladimir E. Tikhonov*a a

A. N. Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences,

119991, V-334, Vavilov st. 28, Moscow, Russia b

Kazan Scientific Research Institute of Epidemiology and Microbiology, 420015, Bolshaya Krasnaya

st. 67, Kazan, Russia *Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +7-499-1359375; Fax: +7-499-1355085.

Vladimir E. Tikhonov Abstract: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Light scattering studies indicate that oligochitosan (short-chain chitosan) solutions contain aggregates at pH values below the critical pH of phase separation, while at or above this point the gel phase coexists with the aggregate solution. This work demonstrates for the first time that the presence of D-glucosamine in an oligochitosan solution shifts the critical pH to a higher value and improves the oligochitosan antibacterial activity against E. coli, S. aureus and S. epidermis in neutral and slightly alkaline aqueous media. By comparing the results of light scattering studies and antimicrobial assays one can conclude that the antimicrobial activity of oligochitosan is dependent on its unimolecular form, not its supramolecular structures. The widening of the homogeneity region of an oligochitosan solution could lead to promising biomedical applications.

Keywords: Oligochitosan, glucosamine, antibacterial activity, E. coli, S. aureus, S. epidermidis

Vladimir E. Tikhonov 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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1. Introduction The misuse and overuse of antibiotics and the growing number of diseases caused by antibioticresistant invasive bacterial and fungal pathogens have become a global problem.1 An increase in human allergy complaints has also been observed in patients undergoing antibiotic treatment.2 Therefore, new non-toxic biocides with activity against a broad spectrum of the invasive and noninvasive human pathogens are needed to potentially reduce the level at which classic antibiotics are administered. Chemically, chitosan represents a collective name used for a group of polyaminosaccharides derived from chitin, a constituent of the cell walls in most fungi and the shells of insects and crustaceans. Chitosans consisting of glucosamine and N-acetylglucosamine (or glucosamine only) differ in their molecular weights (MW) and degrees of acetyl-groups content (DA). Chitosans can be distinguished by their molecular weight (MW): high molecular weight chitosan (HMW), low molecular weight (LMW) chitosan, and oligochitosan. Although the boundaries between these groups are fluid, the term “oligochitosan” can used for chitosan molecules with fewer than 100 glucosamine units, i.e. MW≈16 kg·mol−1.3 Many reviews of the physicochemical properties, applications and antimicrobial activities of chitosan and oligochitosan against bacteria, fungi and viruses have been published.3-9 As a result of these investigations, chitosan and oligochitosan were shown to be non-toxic, biocompatible and biodegradable.3,10-12 In addition, they provide synergistic/additive effects in combination with antibiotics, indicating that chitosan and oligochitosan can be used to enhance the antimicrobial activity of pharmaceuticals. 13-16 Unfortunately, chitosan applications are restricted in some medical formulations: Chitosan dissolved in an aqueous acidic media is stable below the critical pH of the phase separation point. While chitosan bactericidal activity is high in acidic media, it dramatically decreases as the pH increases above the apparent chitosan conjugate acid dissociation constant (Ka).6,17,18 The neutralization of a chitosan

Vladimir E. Tikhonov 4 aqueous solution up to the critical pH causes a loss of most of its positive charges, forming hydrated, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

gel-like particles that precipitate in alkaline media.17, 19-22 The chemical modification of chitosan is an efficient, but tedious approach to improving the water solubility of chitosan derivatives. Chitosan derivatives (like those containing quaternary ammonium or monosaccharide side groups) are more soluble in alkaline aqueous media, but their dramatization significantly alters the nature, physicochemical and biological properties of the chitosan.23-26 Below its critical pH, oligochitosan is more soluble and active than chitosan; however, the aggregative stability of its aqueous solution above that point is insufficient, and an increase in the solution pH over the critical point results in a decrease in the oligochitosan antimicrobial activity. 17,18 Thus, the low solubility of oligochitosan at and above the critical pH represents a major limitation to its use in some medical formulations. This study aimed to evaluate the influence of D-glucosamine on the stability of an oligochitosan solution and its antibacterial activity against E. coli, S. aureus and S. epidermidis over a wide pH range.

2. Materials and methods 2.1. Sample preparation and characterization Two oligochitosan samples were prepared according to the procedure described in the literature. 17 The elemental microanalysis data is as follows: sample 1 (found: % C 36.65, % H 6.15, % N 6.99, % Cl 17.35; calculated: % C 36.79, % H 6.08 , % N 7.08, % Cl 17.42); sample 2 (found: % C 37.04, % H 6.20, % N 7.01, % Cl 16.81; calculated: % C 37.12, % H 6.09 , % N 7.07, % Cl 16.87). The degree of acetylation (DA, mol%) was determined using 1H-NMR.27 The weight-average (Mw) and number-average (Mn) molecular weights were determined via highperformance size-exclusion chromatography (HP-SEC) according to a procedure published in the literature [28]. The apparent conjugated acid dissociation constant (Ka) was determined at 25°C via potentiometric titration in accordance with a method described in the literature.18

Vladimir E. Tikhonov 5 D-(+)-Glucosamine hydrochloride (≥99%), tris(hydroxymethyl)aminomethan (TRIS) and other 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

chemicals were purchased from Sigma-Aldrich. 2.2. Light scattering (LS) studies The static and dynamic light scattering measurements (SLS and DLS) were performed on a PhotoCor Complex spectrometer (PhotoCor Instruments, Russia) equipped with a pseudo cross-correlation system for photon counting with a He–Ne laser as the light source (λ=633 nm, 10 mW) using procedure published previously.28 Aqueous solutions of the oligochitosan hydrochloride (c=1000 μg/ml) were filtered through 0.22-μm PVDF filters (Millipore) prior to measurement and were then titrated with a 3 M TRIS solution filtered through a 0.22-μm PVDF filter using an Anion 4100 (Russia) pH meter. 2.3. Minimal inhibitory concentration (MIC) assay The E. coli ATCC 25922, S. aureus ATCC 35591, and S. epidermidis ATCC 14990 bacterial strains were obtained from the American Type Culture Collection. A stock oligochitosan hydrochloride solution in distilled water was filtered through a 0.22-µm PVDF filter (Millipore) and stored at 4°C until needed. The MICs of the oligochitosan samples were determined by a microdilution assay in accordance with the modified method described in the literature.9 All experiments were carried out in triplicate. The MIC (mean value ±10%) was defined as the lowest concentration required suppressing cell multiplication. 3. Results 3.1. LS studies To evaluate the influence of glucosamine on the aggregation of oligochitosan in an aqueous solution and to determine the critical pH for phase separation, two well-characterized oligochitosan samples (sample 1: Mw =11.50 kg·mol−1 and sample 2: Mw =16.10 kg·mol−1) were prepared and used for the DLS studies. The samples were notable for their low polydispersity indices (sample 1: Mw/Mv=1.44 and sample 2: Mw/Mv=1.61), low DA values (sample 1: 3% and sample 2: 5%), and their equivalent apparent conjugated acid dissociation constants (pKa 6.4). Given that glucosamine has a higher pKa

Vladimir E. Tikhonov 6 (7.75) 29 than the oligochitosans, one would expect to observe a shift in the phase separation point of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

oligochitosan in solutions of higher pH in the presence of glucosamine because the primarily linkages that stabilise the inter-chain contacts in the oligochitosan aggregates at acidic pH (and in the gel phase at neutral or slightly alkaline pH) are thought to be hydrogen bonds. Thus, the aggregation and phase separation effects that occur in an oligochitosan solution (c =1000 μg/ml; 0.005 M glucosamine units) were investigated both with and without 1 M glucosamine hydrochloride at various pH values. The oligochitosan/glucosamine mixture (a solution volume ratio of 1:1) was filtered through 0.22-μm PVDF membranes and titrated with an equally filtered 3 M TRIS solution until a gel-like precipitate appeared. The solution pH was constantly monitored. Measurements were taken at various pH values, and the intensity of the scattered light was measured. In the presence of glucosamine, a remarkable shift was noted in the phase separation point up to pH 7.20 (Fig. 1a) in the solution containing the oligochitosan with a M w of 11.50 kg·mol−1, while this shift was observed at pH 6.80 in the solution containing the oligochitosan with a Mw of 16.10 kg·mol−1 (Fig. 1b). The drastic increase in the LS intensity at a slightly alkaline pH was due to the appearance of microparticles with Rh≈1–2 μm (DLS data not shown). Only traces of the gel-like precipitate were found after one day of storage at pH 7.25 and pH 6.85, whereas at pH values below 7.25 and 6.80, neither precipitation nor an increase in the LS intensity were observed in the solutions. These results indicated that the pH range of oligochitosan solubility could be widened to include slightly basic media by the addition of D-glucosamine to an oligochitosan solution. However, unlike glucosamine, the addition of other kosmotropic and chaotropic agents (1 M glucose, 1 M sorbitol, 1 M N,N,Ntrimethylglycine, 1 M urea, 1 M N-acetylglucosamine, and 3 M TRIS) had no effect on the critical pH values. 3.2. Antimicrobial activity of oligochitosan The susceptibility of the gram-negative E. coli and gram-positive S. aureus and S. epidermidis bacterial cells toward these two oligochitosan samples with closely related Mw values but different aggregative properties was tested in vitro. The MIC values of the samples were determined at various solution pH values in the absence and presence of glucosamine (Fig. 2, 3). The preliminary

Vladimir E. Tikhonov 7 experiments indicated that glucosamine had no bacteriostatic activity against these bacterial cells 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(data not shown). The as-obtained results demonstrated that the presence of glucosamine could affect the bacteriostatic activities of the oligochitosan samples at and above the critical pH for phase separation, and the increase in activity was more notable at slightly alkaline pH values (pH 7.00–7.75). In contrast, no differences in the MIC values were noted at pH 6.50–6.75 for the E. coli or S. aureus or for the S. epidermidis at pH 6.50–7.25. In addition, a difference was noted between the oligochitosan samples: the bacteriostatic activity of the oligochitosan with a Mw of 11.50 kg/mol against the E. coli and S. aureus decreased more slowly with increasing pH in both the absence and presence of glucosamine (Fig. 2a, b/Fig. 3a, b, respectively) than the oligochitosan sample with a M w of 16.10 kg·mol−1. This difference was more pronounced at pH≥7 and could have been caused by the differences in the MW values of the samples or their phase separation pH, which increased with increasing MW.24 However, the dependence of the oligochitosan MIC values on solution pH was more notable for the E. coli and S. aureus than for the S. epidermidis due to the higher susceptibility of the S. epidermidis cells (Figs. 2c and 3c). Because the oligochitosan concentrations required to suppress the growth of the S. epidermidis cells were significantly lower, glucosamine’s effect on the oligochitosan bacteriostatic activity toward the S. epidermidis cells was not noted at pH 6.50–7.25 due to the reduced tendency of the oligochitosans to aggregate with the reduction in their concentrations [30].

4. Discussion Chitosan consists of glucosamine and N-acetylglucosamine units, and its chemical structure includes both hydrophilic and hydrophobic sites. The intra- and interchain interactions lead to an aggregation of chitosan macromolecules in chitosan solutions. This aggregation has been confirmed experimentally by many authors who demonstrated the aggregation of chitosan in solutions below its pK a. Hydrophobic interactions and hydrogen bonding are commonly accepted as the primary mechanism of interchain aggregation.20-22,31,32 An attempt to disrupt the chitosan aggregates by adding urea as a conventional chaotropic agent indicated that even 7 M urea was unable to completely prevent the aggregation. This result confirmed that hydrogen bonding and/or hydrophobic interactions were

Vladimir E. Tikhonov 8 responsible for this phenomenon.32 The incomplete breakage of the hydrogen bonds by the urea was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

in agreement with the results reported with other polysaccharides.33,34 Oligochitosan is known to have several advantages over HMW and LMW chitosans in biomedical and industrial applications. In particular, due to its lower viscosity and higher compatibility with many additives, the application of oligochitosan has no undesirable impact on the physicochemical properties of consumer products. Additionally, oligochitosan has some advantages over HMW and LMW chitosans in its biological activity and biomedical applications.3,17,35-37 However, most structural studies of chitosan solutions have been performed for HMW and LMW chitosans only, while the associative behavior of oligochitosan has thus far received little attention.17,28 The LS and microscopy studies of dilute acidic oligochitosan solutions have demonstrate the presence of aggregates with wide structural diversity. In addition, oligochitosan (short-chain chitosan) with a Mw below 12 kg·mol−1 was shown to form aggregates of constant size in aqueous acidic media. This behavior, unlike those of the HMW and LMW chitosan solutions, allowed the oligochitosans to be selected in a series according to aggregate size and mass increase with increasing Mw above 12 kg·mol−1.28 Given the data obtained for this series of oligochitosans with low DA values, one could hypothesize that hydrogen bonds formed the primary linkages stabilizing the inter-chain contacts in the oligochitosan aggregates at acidic pH (and in the gel phase at neutral or slightly alkaline pH). Therefore, partial substitution of such bonds between the oligochitosan subunits with those between the subunits and glucosamine might diminish the intra- and interchain bridging, the aggregation of the oligochitosan macromolecules in aqueous media and the formation of a gel phase at the critical pH. The data obtained in this work are in agreement with this supposition. The LS studies and antibacterial tests indicated that the addition of glucosamine to an oligochitosan solution promoted the oligochitosan solubility at pH values above its critical point. The interaction between the oligochitosan macromolecules and glucosamine is believed to aid in maintaining a virtual charge through a specific complexation via hydrogen bonding with glucosamine, which has a higher pK a than oligochitosan, to improve solubility. In addition, glucosamine increased the oligochitosan bacteriostatic activity toward the tested cells in neutral and slightly alkaline aqueous media. The as-obtained results indicated that

Vladimir E. Tikhonov 9 the antimicrobial activity of the oligochitosan is connected with its unimolecular form, not its 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

supramolecular structures. 5. Conclusions The LS studies indicate that the addition of glucosamine to an oligochitosan solution can shift its critical pH for phase separation to a higher value. The antibacterial assay demonstrates that the glucosamine does not alter the oligochitosan bacteriostatic activity in acidic media in which oligochitosan is highly soluble, but increases its activity in neutral and slightly alkaline media. These effects are more pronounced for oligochitosans with MW above 12 kg·mol−1. The demonstrated effect of the glucosamine on the phase separation point is consistent with the hypothesis regarding the considerable impact of interchain hydrogen bonding on both the aggregation and phase separation phenomena. Given that oligochitosan can be broken down and digested in the human body by specific (lysozyme, chitinase, and chitotriosidase) and unspecific (lipase and α-amylase) human enzymes that exist in various human body fluids and tissues and that oligochitosan can be converted to lower chitooligosaccharides, glucosamine and N-acetyl glucosamine,

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the discovered effect may be

promising for biomedical applications of oligochitosan. 6. References 1. Davies, J.; Davies, D. Microbiol. Mol. Biol. Rev. 2010, 74, 417–433. 2. Rouveix, B. Int. J. Antimicrob. Agents 2003, 21, 215-221. 3. Aam, B.B.; Heggset, E.B.; Norberg, A.L.; Sørlie, M.; Vårum, K.M.; Eijsink, V.G.H. Mar. Drugs 2010, 8, 1482-1517. 4. Rinaudo, M. Prog. Polym. Sci. 2006, 31, 603–632. 5. Kumar, M.N.V. React. Func. Polym. 2000, 46, 1-27. 6. Kong, M.; Chen, X.G.; Xing, K.; Park, H.J. Int. J. Food Microbiol. 2010, 144, 51-63. 7. Vinsova, J.; Vavrikova, E. Curr. Pharm. Design 2008, 14, 1311-1326. 8. Bernkop-Schnürch, A.; Dünnhaupt, S. Eur. J. Pharm. Biopharm. 2012, 81, 463-469. 9. Raafat, D.; Bargen, K.; Haas, A.; Sahl, H.G. Appl. Environ. Microbiol. 2008, 74, 3764–3773. 10. Baldrick, P. Regul. Toxicol. Pharmacol. 2010, 56, 290–299.

Vladimir E. Tikhonov 11. Kean, T.; Thanou, M. Adv. Drug Deliv. Rev. 2010, 62, 3–11. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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12. Qin, C.; Gao, J.; Wang, L.; Zeng, L.; Liu, Y. Food Chem. Toxicol. 2006, 44, 855–861. 13. Tin, S.; Sakharkar, K.R.; Lim, C.S.; Sakharkar, M.K. Int. J. Biol. Sci. 2009, 5, 153-160. 14. Tin, S.; Lim, C.S.; Sakharkar, M.K.; Sakharkar, K.R. Lett. Drug Des. Discov. 2010, 7, 31-35. 15. Ballal, N.V.; Kundabala, M.; Bhat, K.S.; Acharya, S.; Ballal, M.; Kumar, R.; Prakash, P.Y. Aust. Endod. J. 2009, 35, 29–33. 16. Şenel, S. J. Drug Deliv. Sci. Technol. 2010, 20, 23-32. 17. Kulikov, S.; Tikhonov, V.; Blagodatskikh, I.; Bezrodnykh, E.; Lopatin, S.; Khairullin, R.; Philippova, Y.; Abramchuk, S. Carbohydrate Polymers 2012, 87, 545-550. 18. Qin, C.; Li, H.; Xiao, Q.; Liu, Y.; Zhu, J.; Du, Y. Carbohydrate Polymers 2006, 63, 367-374. 19. Buhler, E.; Rinaudo, M. Macromolecules 2000, 33, 2098 – 2106. 20. Sorlier, P.; Rochas, C.; Morfin, I.; Viton, C.; Domard, A. Biomacromolecules 2003, 4, 1034– 1040. 21. Popa-Nita, S.; Alcouffe, P.; Rochas, C.; David, L.; Domard, A. Biomacromolecules 2010, 11, 6–12. 22. Korchagina, E.V., Philippova, O.E. Biomacromolecules 2010, 11, 3457–3466. 23. Alves, N.M.; Mano, J.F. Int. J. Biol. Macromol. 2008, 43, 401-414. 24. Dash, M.; Chiellini, F.; Ottenbrite, R.M.; Chiellini, E. Prog. Polym. Sci. 2011, 36, 981-1014. 25. Stepnova, E.A.; Tikhonov, V.E.; Babak, V.G.; Babushkina, T.A.; Lopatin, S.A.; Yamskov, I.A. Eur. Polym. J. 2007, 43, 2414-2424. 26. Chung, Y.C.; Kuo, C.L.; Chen, C.C. Bioresour. Technol. 2005, 96, 1473-1482. 27. Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87-94. 28. Blagodatskikh, I.V.; Bezrodnykh, E.A.; Abramchuk, S.S.; Muranov, A.V.; Sinitsyna, O.V.; Khokhlov, A.R.; Tikhonov, V.E. J. Polym. Res. 2013, 20, DOI: 10.1007/s10965-013-0073-0. 29. Zimmerman Jr., H.K. J. Phys. Chem. 1958, 62, 963-965. 30. Schatz, C.; Pichot, C.; Delair, T.; Viton, C.; Domard, A. Langmuir 2003, 19, 9896-9903. 31. Buhler, E.; Rinado, M. Macromolecules 2000, 33, 2098-2106.

Vladimir E. Tikhonov 11 32. Philippova, O.V.; Korchagina, E.V.; Volkov, E.V.; Smirnov, V.A.; Khokhlov, A.R.; Rinado, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

M. Carbohydrate Polymrs 2012, 87, 687-694. 33. Erlander, S.R.; Purvinas, R.M.; Griffin, H.L. Cereal Chem. 1968, 45,140-153. 34. Erlander, R.S., Tobin, R.J. J. Macromol. Sci. 1968, A2, 1521-1542. 35. Tikhonov, V.; Stepnova, E.; Lopatin, S.; Varlamov, V.; Il’ina, A.; Yamskov, I. The unusual bell-like dependence of the activity of chitosan against Penicillium vermoesenii on chitosan molecular weight. In Chitosan: Manufacture, Properties, and Usage. Biotechnology in Agriculture, Industry and Medicine. Davis, S.P., Ed.; Nova Science Publishers: NY, USA, 2011, chapter 17, pp.315-326. 36. Muzzarelli, R.A.A. Enhanced biochemical efficacy of oligomeric and partially depolymerized chitosans. In: Focus on Chitosan Research, Ferguson, A.N., O’Niell A.G., Eds.; Nova Science Publishers: NY, USA, 2011, pp. 115-140. 37. Chae, S.Y.; Jang, M.K.; Hah, J.W. J. Control Release 2005, 102, 383-394. 38. Nordtveit, R.J.; Vårum, K.M.; Smidsord, O. Carbohydr. Res. 1994, 23, 253-260. 39. van Eijk, M.; van Roomen, C.P.A.; Renkema, G.H.; Bussink, A.P.; Andrews, L.; Blommaart, E.F.C.; Sugar, A.; Verhoeven, A.J.; Boot, R.G.; Aerts, J.M.F.C. Int. Immunol. 2005, 17, 15051512. 40. Lee, K.Y.; Ha, W.S.; Park, W.H. Biomaterials 1995, 16, 1211-1216. 41. Boot, R.G.; Blommaart, E.F.C.; Swart, E.; Ghauharali-van der Vlugt, K.; Bijl, N.; Moe, C.; Place, A.; Aerts, J.M.F.C. J. Biol. Chem. 2001, 9, 6770–6778. 42. Muzzarelli, R.A.A.;Terbojevich, M.; Cosani, A. Unspecific activities of lipase and amylase on chitosans. In: Chitin Enzymology, Muzzarelli, R.A.A., Ed.; Atec Edizioni, Ancona, Italy, 1996, Vol.2, pp.69-82.

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Captures to Figures: Figure 1. Variations of the LS intensity (scattering angle 90°) of oligochitosan Mw 11.50 kg·mol−1(a) and oligochitosan 16.10 kg·mol−1(b) solutions in the absence (1) and presence (2) of glucosamine as the functions of pH. Symbol sizes along both axes are within the measurement accuracies. Figure 2. MIC (mean value ±10%) of oligochitosan (Mw 11.50 kDa) sample against E. coli (a), S. aureus (b) and S. epidermidis (c) in the absence (white columns) and presence (black columns) of glucosamine.

Figure 3. MIC (mean value ±10%) of oligochitosan (Mw 16.50 kDa) sample against E. coli (a), S. aureus (b) and S. epidermidis (c) in the absence (white columns) and presence (black columns) of glucosamine

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Highlights 1. Oligochitosan solution contains aggregates below the critical pH value of phase separation; 2. The addition of glucosamine to the solution shift the critical point to a higher value; 3. Glucosamine improves the oligochitosan antibacterial activity at pH above the critical point.

*Graphical Abstract (for review)