Inhibitory Effect Of Chitosan Research Articles

Inhibitory Action of Low-Molecular Chitosan on Growth of Bacteria with Different Tinctorial Properties

Abstract—Inhibitory effect of chitosan (molecular mass 28 kDa, deacetylation 94%) and of its quaternized derivative with 60% substitution on bacteria of various taxonomic groups was investigated. Bacteria differing in the cell wall surface characteristics and affinity to dyes were found to differ in theri sensitivity to chitosan. Correlation dependencies between antibacterial activity of quaternized chitosan and the surface characteristics of bacterial cell walls were revealed.

Addition time plays a major role in the inhibitory effect of chitosan on the production of Pseudomonas aeruginosa virulence factors.

Pseudomonas aeruginosa is a gram-negative bacterium capable of forming persistent biofilms that are extremely difficult to eradicate. The species is most infamously known due to complications in cystic fibrosis patients. The high mortality of cystic fibrosis is caused by P. aeruginosa biofilms occurring in pathologically overly mucous lungs, which are the major cause facilitating the organ failure. Due to Pseudomonas biofilm-associated infections, remarkably high doses of antibiotics must be administered, eventually contributing to the development of antibiotic resistance. Nowadays, multidrug resistant P. aeruginosa is one of the most terrible threats in medicine, and the search for novel antimicrobial drugs is of the utmost importance. We have studied the effect of low molecular weight chitosan (LMWCH) on various stages of P. aeruginosa ATCC 10145 biofilm formation and eradication, as well as on production of othervirulence factors. LMWCH is a well-known naturally occurring agent with a vast antimicrobial spectrum, which has already found application in various fields of medicine and industry. LMWCH at a concentration of 40mg/L was able to completely prevent biofilm formation. At a concentration of 60mg/L, this agent was capable to eradicate already formed biofilm in most studied times of addition (2-12h of cultivation). LMWCH (50mg/L) was also able to suppress pyocyanin production when added 2 and 4h after cultivation. The treatment resulted in reduced formation of cell clusters. LMWCH was proved to be an effective antibiofilm agent worth further clinical research with the potential to become a novel drug for the treatment of P. aeruginosa infections.

Inhibitory Effect of Chitosan and Phosphate Cross-linked Chitosan against Cucumber Mosaic Virus and Pepper Mild Mottle Virus.

Cucumber mosaic virus (CMV) and Pepper mild mottle virus (PMMoV) causes severe economic loss in crop productivity of both agriculture and horticulture crops in Korea. The previous surveys showed that naturally available biopolymer material – chitosan (CS), which is from shrimp cells, reduced CMV accumulation on pepper. To improve the antiviral activity of CS, it was synthesized to form phosphate cross-linked chitosan (PCS) and compared with the original CS. Initially, the activity of CS and PCS (0.01%, 0.05%, and 0.1% concentration) compound against PMMoV infection and replication was tested using a half-leaf assay on Nicotiana glutinosa leaves. The total number of local lesions represented on a leaf of N. glutinosa were counted and analyzed with phosphate buffer treated leaves as a negative control. The leaves treated with a 0.1% concentration of CS or PCS compounds exhibited an inhibition effect by 40–75% compared with the control leaves. The same treatment significantly reduced about 40% CMV accumulation measured by double antibody sandwich enzyme-linked immunosorbent assay and increased the relative expression levels of the NPR1, PR-1, cysteine protease inhibitor gene, LOX, PAL, SRC2, CRF3 and ERF4 genes analyzed by quantitative reverse transcriptase-polymerase chain reaction, in chili pepper plants.

Synergistic Inhibitory Effect of Chitosan and Amphotericin B on Planktonic and Biofilm Populations of C. albicans, C. parapsilosis and C. krusei

Synergistic Inhibitory Effect of Chitosan and Amphotericin B on Planktonic and Biofilm Populations of C. albicans, C. parapsilosis and C. krusei

Hydrocolloid effects on Nε-carboxymethyllysine and acrylamide of deep-fried fish nuggets

The safety of breaded and deep-fried foods has received more attention recently. The purpose of this study was to investigate the effects of hydrocolloids on the contents of Nε-carboxymethyllysine (CML) and acrylamide (AA) in breaded fish nuggets, and the relationship between the AA, CML and physicochemical properties. The CML and AA were found mainly in the fish crust. For the interior of fish nuggets, CML content was far lower than the crust and no AA was detected. Chitosan (CH) and gum Arabic (GA) could significantly reduce the CML content of fish crust by ~40–50%. However, the use of xanthan gum (XG) significantly increased the CML content of fish crust (p < 0.05). All hydrocolloids had an inhibitory effect on acrylamide (p < 0.05), of which 0.5% GA or CH had the greatest effects. The correlation analysis showed that the inhibitory effect of CH and GA on CML might be attributed to their higher moisture and lower oil contents. The inhibitory effect of all hydrocolloids on AA might be attributed to the decreased extent of Maillard reactions. The addition of 0.5 and 1% CH and GA significantly reduced both the AA and CML content of the fish crust (p < 0.05). This provided a basis for improving the nutritional quality and reducing the safety concerns when consuming breaded and deep-fried foods.

Study on the Effect of Prawn (Machrobrachuim rosenbergii) Chitosan Coating on Peeled Shallot (Allium ascalonicum)

Shallot (Allium ascalonicum) is the most important commercial vegetable crop grown in India. Shallot peeling is tough and consumes time. The availability of peeled shallots in markets will reduce the time consumed on peeling shallots for cooking. But peeled shallot does not have longer shelf life. Edible coating for peeled shallots can reduce spoilage and can inhibit microbial growth too. The Chitosan coating of shallots was based on the dipping method of coating fruits and vegetables. The study on coating chitosan in shallots and different parameters with varying composition were analyzed. The physio-chemical parameters like protein content, carbohydrate content, calcium content and total phenols content and weight loss shows that there is a significant difference with coated and uncoated Shallot stored at 4C for a period of 15 days. The microbial studies and physical characteristic studies show that number of microbes and decay is significantly less for coated onion samples compared with uncoated samples respectively. The inhibitory effect of chitosan produced from prawn shell against fungi was studied by adding 0.2% oregano oil, 0.4% of Tween 80 (polysorbate 80) and 0.7% of Glycerol. The Zone of inhibition was obtained has which were found to be 3.1cm, 1.5cm and 2.9cm for Oregano (Or), Thyme (Th) and Mint (M) leaves. Addition of 0.1% Tween 80 to 1.5 % chitosan in acetic acid solution improved the effectiveness of coating on the Shallot skin. The above study revealed that Chitosan coatings were in effect on successful quality improvement and extending shelf life of Shallot by delayed ripening, lowered respiration rate, regulated gas exchange, decrease in transpiration rate, retaining fruit firmness, reduction in weight loss etc.

Inhibition of melanin synthesis and melanosome transfer by chitosan biomaterials.

Decreasing skin pigmentation is desirable for various medical or cosmetic conditions. Although numerous pharmaceutical agents are currently available, their depigmentation effects are still not satisfactory. In this study, we investigated the effects of chitosan, a natural marine product, on melanin synthesis and melanosome transfer. Treating B16F10 melanoma cells caused the inhibitory effect of chitosan on melanogenesis to be more prominent under α-melanocyte-stimulating hormone (α-MSH) stimulation. Chitosan samples of different molecular weights inhibited melanogenesis to a comparable extent, whereas increasing the deacetylation of chitosan enhanced its depigmentation effects. Chitosan was found to effectively reduce basal or α-MSH-stimulated melanogenesis by suppressing the expression of melanogenic-related proteins (microphthalmia transcription factor, tyrosinase, and tyrosinase-related protein-1 and protein-2) as well as inhibiting tyrosinase activity. Moreover, the inhibitory effect of chitosan on melanogenesis in human melanocytes was confirmed. A transwell coculture system using permeable inserts was designed to allow the contact of human melanocytes and human HaCaT keratinocytes through the tiny holes on the membrane. When chitosan was added to this melanocyte-keratinocyte coculture system, we observed decreased melanosome release from melanocytes. Reduced melanosome uptake by keratinocytes was also observed, and was probably mediated by inhibiting protease-activated receptor 2 expression. Many skin-whitening agents can modulate the process of melanogenesis, but few have been shown to inhibit the melanosome transfer and uptake process. We demonstrated that chitosan exhibits a robust effect on depigmentation by inhibiting melanogenesis as well as melanosome transfer and uptake. Therefore, chitosan represents a potential therapeutic agent for hyperpigmentation disorders.

Effect of chitosan coating on the shelf life of ready-to-eat bovine meatballs and the control of Listeria monocytogenes growth on their surface during refrigeration storage

Edible chitosan coating on the surface of ready-to-eat (RTE) bovine meatballs was evaluated for its effect on their shelf life and the control of Listeria monocytogenes at 5 °C. L. monocytogenes was inoculated onto the surface of RTE bovine meatballs with and without edible chitosan coating. The samples were stored at 5 °C. Total aerobic viable count (TVC) and the bacterial counts of L. monocytogenes, lactic acid bacteria and Enterobacteriaceae were determined on days 0,1,7,14,21 and 28. The sensory characteristics were also evaluated at the same time spots by semi trained panelists. The results of the microbiological analysis depicted that the use of edible chitosan membranes reduced all of the microbial populations that were enumerated, and retarded their growth leading to the conclusion that they can prolong the shelf life of these products by 14 days. Moreover, the population of the inoculated L. monocytogenes was about 2 log CFU/g lower in the meatballs coated with chitosan, indicating an inhibitory effect of chitosan in the growth of L. monocytogenes. The sensory analysis showed that the samples coated with chitosan were satisfactorily accepted by the panelists even at day 28, in contrast to the samples without chitosan (control samples) which were unacceptable at day 14. These results indicate that edible chitosan coatings represent a potential agent in controlling L. monocytogenes on the surface of RTE meatballs as well as other RTE meat products, prolonging their shelf life without affecting their sensory characteristics.

Effect of chitosan coating in the shelf life and control of Listeria monocytogenes growth on the surface of ready-to-eat bovine meatballs during chilled storage

Edible chitosan coating on the surface of ready-to-eat (RTE) bovine meatballs was evaluated for its effect in the shelf life of them and the control of Listeria monocytogenes at 5 °C. L. monocytogenes was inoculated onto the surface of RTE bovine meatballs with and without edible chitosan coating. The samples were stored at 5 °C. Total aerobic viable count (TVC) and the bacterial counts of L. monocytogenes , lactic acid bacteria and Enterobacteriaceae were determined on days 0,1,7,14,21 and 28. The sensory characteristics were also evaluated at the same time spots by semi trained panelists. The analysis depicted that the use of edible chitosan membranes reduced all of the microbial populations that were enumerated, and retard their growth leading to the conclusion that they can prolong the span life of these products by 14 days. Moreover, the population of the inoculated L. monocytogenes was about 2 log CFU/g lower in the meatballs coated with chitosan, indicating an inhibitory effect of chitosan in the growth of L. monocytogenes . The sensory analysis showed that the coated with chitosan samples were satisfactorily accepted by the panelists even at day 28, in contrast to the samples without chitosan (control samples) which were unacceptable at day 14. These results indicate that edible chitosan coatings represent a potential agent in controlling L. monocytogenes on the surface of RTE meatballs as well as other RTE meat products, prolonging their shelf life without affecting their sensory characteristics.

Chitosan disrupts biofilm formation and promotes biofilm eradication in Staphylococcus species isolated from bovine mastitis

Staphylococci are the main pathogens associated with hard-to-control intramammary infections in dairy cattle, and bacterial biofilms are suspected to be responsible for the antimicrobial resistance and persistence of this disease. Biofilms have the ability to resist to higher levels of antibiotics and reduce their efficacy. It is thus necessary to develop strategies targeted to bacterial biofilm infections. Chitosan is a polysaccharide with a proven broad spectrum of antimicrobial activity against fungi and bacteria. The aim of this study was assess the effect of low molecular weight (LMW) chitosan against biofilm hyperproducer Staphylococcus spp. (S. aureus and S. xylosus) strains usually involved in chronic bovine mastitis, and to test their efficacy in biofilm formation and eradication. The results obtained showed that LMW chitosan is able to inhibit S. aureus and S. xylosus planktonic growth in a dose-dependent manner and reduce bacterial viability. LMW chitosan inhibits biofilm formation, reduces biofilm viability and disrupts established biofilm. These results indicate the inhibitory effects of chitosan on biofilm formation, and these effects are observed at lower concentrations for S. xylosus. Our studies show the potential of this biopolymer to be used as an effective antibiofilm agent able to act upon staphylococcal infections.

Effect of chitosan, and bacteriocin – Producing Carnobacterium maltaromaticum on survival of Escherichia coli and Salmonella Typhimurium on beef

The aim of this study was to investigate the synergistic effect of chitosan and bacteriocins against Escherichia coli and Salmonella in media and in lean beef. The inhibitory effects of chitosan and bacteriocins against E. coli AW1.7 and S. enterica Typhimurium in media were determined by a critical dilution assay. The efficacy a bacteriocin-producing strain of Carnobacterium maltaromaticum and high molecular weight chitosan (HMWC) in inactivation of E. coli AW1.7 and S. Typhimurium was evaluated on beef. Current interventions applied in the beef industry, steaming coupled with lactic acid, were used as reference. HMWC demonstrated higher antibacterial activity than water soluble chitosan (WSC) or chitosan oligosaccharides (COS) in media, and the addition of partially purified bacteriocins from C. maltaromaticum UAL307 increased the activity of the chitosan in vitro. The hurdle combinations associated with HMWC inactivated E. coli AW1.7 and S. enterica Typhimurium more effectively on lean beef when compared to steam or steam coupled with lactic acid. When used on beef, addition of bacteriocins and chitosan did not increase the antibacterial efficacy. Cell counts of S. enterica were further reduced during storage in presence of C. maltaromaticum and chitosan; however, this decrease was not dependent on bacteriocin production. In conclusion, addition of chitosan alone or in combination with C. maltaromaticum UAL 307 as protective culture significantly reduces cell counts of E. coli and Salmonella on beef. Results will be useful to improve pathogen intervention treatments in beef processing.

Inhibitory effects of chitosan on Cronobacter malonaticus cells and biofilm formation

Cronobacter malonaticus is one of foodborne pathogens associated with life-threating infections in newborns and adults. Controlling C. malonaticus in food samples is important to achieve microbial safety. In this study, inhibitory effects of chitosan with low molecular weight (LMW) and high molecular weight (HMW) on growth of C. malonaticus cells and biofilm formation were determined. Results indicated that minimum inhibitory concentration (MICs) of 0.165% (w/v) and 0.225% (w/v) for LMW and HMW were observed respectively. Compared to control group, both LMW and HMW with 1/4 MIC and 1/2 MIC could significantly inhibit growth of C. malonaticus cells, and caused the release of intracellular nuclear acid of C. malonaticus cells. Furthermore, the cell shrinkage, injury of cell membrane, the disruption of cell integrity, and deformation of cells were also found by scanning electron microscope (SEM) and transmission electron microscope (TEM) after treatment of 1/2 MIC of LMW and HMW chitosan. Finally, we demonstrated that chitosan with LMW and HMW significantly inhibited the biofilm formation in C. malonaticus compared to control groups. The findings provide valuable information for application of chitosan to control C. malonaticus in food samples.

Inhibitory effect of chitosan on growth of the fungal phytopathogen, Sclerotinia sclerotiorum, and sclerotinia rot of carrot

The antifungal activity of chitosan on a common fungal phytopathogen, Sclerotinia sclerotiorum, and the control effect on sclerotinia rot of carrot were investigated. Mycelial growth and fungal biomass were strongly inhibited by chitosan. Using propidium iodide stain combined with fluorescent microscopy, the plasma membrane of chitosan-treated S. sclerotiorum mycelia was observed to be markedly damaged. Concomitantly, protein leakage and lipid peroxidation was also found to be significantly higher in chitosan-treated mycelia compared to the control. Chitosan provided an effective control of sclerotinia rot of carrot, with induction of activity of defense-related enzymes including polyphenoloxidase and peroxidase. These data suggest that the effects of chitosan on sclerotinia rot of carrot may be associated with the direct damage to the plasma membrane and lipid peroxidation of S. sclerotiorum, and the elicitation of defense response in carrot.

Postharvest efficacy of resistance inducers for the control of green mold on important Sicilian citrus varieties

The disease reducing activity of acibenzolar-S-methyl (ASM), chitosan and β-aminobutyric acid (BABA) against postharvest green mold (GM) of citrus fruit, Penicillium digitatum, was investigated by tests performed in in vitro and in vivo conditions. The inhibition of the mycelial growth of P. digitatum by ASM, chitosan and BABA at different concentrations was evaluated on potato dextrose agar and on orange peel extract agar. The pathogen was totally inhibited (100% growth inhibition) by chitosan at concentrations ≥ 0.1% and by BABA at 1000 mmol and only poorly inhibited by ASM at 0.5% (47% growth inhibition). Inhibitory effects of chitosan and BABA were confirmed by P. digitatum conidial germination tests. The performances of these chemical compounds were evaluated in vivo on artificially inoculated orange, lemon and grapefruit cultivars. On the whole, chitosan significantly reduced GM decay on ‘Valencia’ and ‘Washington Navel’ orange, ‘Femminello’ lemon and grapefruit cv. Marsh Seedless at the concentration of 0.5% (6–20% disease incidence), and BABA provided the best results on ‘Tarocco Scire’and ‘Valencia’ orange and grapefruit cv. Marsh Seedless at the concentration of 1000 mmol (12–16% disease incidence). ASM did not show any efficacy. This study demonstrated that the effects of chitosan and BABA on GM in citrus fruits may be associated with their direct fungitoxic properties against the pathogen. Treatments with chitosan and BABA could be recommended for inclusion in postharvest decay management programs for citrus packinghouses and their use may be an effective method to improve the integrated pest management strategy.

Inhibitory effects of chitosan in combination with antibiotics on Listeria monocytogenes biofilm

Microorganisms on living or inert surfaces usually form biofilms which makes microbes highly resistant to antibiotics and immune clearance. Herein we investigated the synergistic effect of several antibiotics with chitosan on eradication biofilms built by Listeria monocytogenes, a causative organism of the serious foodborne illness in a wide variety of mammalian species including human and food animals. Our data demonstrated that chitosan combined with the aminoglycoside antibiotic such as amikacin, but not clindamycin, vancomycin and erythromycin was more effective in inhibition or disruption of L. monocytogenes biofilms than the antibiotic alone did. To elicit an optimal anti-biofilm and bactericidal efficiency, chitosan with specialized molecular size (∼13 kDa) and high N-deacetylation degree (∼88%) was required in this combination. Further evidence showed that this strategy was effective in removing biofilms built by multiple L. monocytogenes strains and other Listeria species. Thus, our data highlighted that chitosan/amikacin combination might be useful for the treatment of Listeria biofilms and help prevent the spread of antibiotic resistance through improving antibiotic effectiveness.

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(®), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.

Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement

Chitosan associated to various scaffolds has been shown to promote growth and mineral rich matrix deposition by osteoblasts in vitro, whereas its influence on osteoclast differentiation, which plays also a central role in bone remodeling, has never been described. The purpose of this study was to investigate the differentiation and activity of human preosteoclastic cells on calcium phosphate cement containing 2% chitosan (Cementek®/chitosan) compared to the Cementek® alone. Human primary osteoclast precursors were cultured directly on both biomaterials in the presence of rhM-CSF and rhRANK-L for 7 days. Using LIVE/DEAD fluorescent assay, tartrate-resistant acid phosphatase staining, scanning electron microscopy and quantitative RT-PCR, we demonstrated that incorporation of chitosan to Cementek® does not affect the proliferation and adhesion of preosteoclasts but inhibits the formation of TRACP positive cells and prevents the osteoclastic resorption of the composite biomaterial compared to Cementek® alone. This inhibitory effect of chitosan on osteoclast resorption activity should have important implications on bone formation and bone remodeling after in vivo implantation. Indeed, based on the positive results obtained in vivo by several investigators, one can suggest that this property of chitosan can be beneficial for bone regeneration.

Inhibitory Effect of Gamma-Irradiated Chitosan on the Growth of Denitrifiers

In order to find an environmentally benign substitute to hazardous inhibitory agents, the inhibitory effect of γ-irradiated chitosans against a mixed culture of denitrifying bacteria was experimentally evaluated. Unlike other studies using pure aerobic cultures, the observed effect was not a complete inhibition but a transient inhibition reflected by prolonged lag phases and reduced growth rates. Raw chitosan under acid conditions (pH 6.3) exerted the strongest inhibition followed by the 100 kGy and 500 kGy irradiated chitosans, respectively. Therefore, because the molecular weight of chitosan decreases with the degree of γ-irradiation, the inhibitory properties of chitosan due to its high molecular weight were more relevant than the inhibitory properties gained due to the modification of the surface charge and/or chemical structure by γ-irradiation. High dosage of γ-irradiated appeared to increase the growth of mixed denitrifying bacteria in acid pH media. However, in neutral pH media, high dosage of γ-irradiation appeared to enhance the inhibitory effect of chitosan.

Inhibitory effects of chitosan on superoxide anion radicals and lipid free radical

With the electron spin resonance (ESR) technique, the inhibitory effects of chitosan on superoxide anion radicals and linoleic acid lipid radicals were found. The inhibitory ratio E for these two kinds of radicals is in proportion to the concentration of chitosan. It was also observed that E for linoleic acid lipid radicals increased with the increase of the degree of deacetylation and decreased with the increase of the molecular weight of chitosan.

Antifungal Activity of Chitosan and Its Preservative Effect on Low-Sugar Candied Kumquat

The inhibitory effect of chitosan, a deacetylated form of chitin, on the growth of Aspergillus niger and the aflatoxin production of Aspergillus parasiticus was evaluated. The inhibitory effect of chitosan against A. niger was increased as the chitosan concentration was increased from 0.1 to 5.0 mg/ml (pH 5.4). At concentrations of 4.0 or 5.0 mg/ml, chitosan was less effective than potassium sorbate in inhibiting the growth of A. niger. The greatest inhibitory effect of chitosan against A. parasiticus was found at 3.0–5.0 mg/ml. In addition, chitosan could completely prevent aflatoxin production by A. parasiticus at the concentration of 4.0–5.0 mg/ml. Chitosan (2.0 and 5.0 mg/ml) induced considerable leakage of UV-absorbing and proteinaceous material of A. niger at pH 4.8. Using the response surface methodology, a second order polynomial model was derived and used to predict the number of days to obtain visible mold growth under various combinations of chitosan concentrations and °Brix in candied kumquat. The results showed that there was no significant difference in shelf-life extension of candied kumquat at chitosan concentration of 3.5–6.5 mg/ml. However, °Brix had a significant effect on shelf life. Candied kumquat with 6.0 mg/ml chitosan concentration and 61.9° Brix had a predicted mold-free shelf life of 65.3 d.