Chitosan Hydrolysate Research Articles

Effects of chitosan hydrolysate on control of postharvest infection caused by Botrytis cinerea and physiological responses of wounded tomato fruit

Chitosan is considered an eco-friendly plant protection agent. Chitosan hydrolysate (CH) with an average molecular weight (MW) of the main fraction of 135 × 103 Da, with a deacetylation degree (DD) of 93 % is an unfractionated product of nitric acid hydrolysis of high molecular weight chitosan. The effect of the CH on gray mold caused by Botrytis cinerea in tomato fruit stored at 25 °C was investigated. Chitosan provided effective control of B. cinerea on tomato fruit. The CH treatment of wounded tomato fruit had a protective effect, reducing the percentage of infected fruit on the 7 d after treatment to 50 %, compared with untreated wounded fruit (69 %) and water-treated fruit (88 %). The protective effect of the CH in tomato fruit may be due to a direct fungitoxic action against the pathogen. It was found that the CH induced an immune response in tomato fruit via the accumulation of phenolic compounds. The level of phenolic compounds in the CH group was higher than in the untreated wound group from day 1 to 5, with a maximum reached on day 3 (113 mg-eq GA kg-1). No significant differences were found in the activities of peroxidase, phenylalanine ammonia lyase, polyphenol oxidase, β-1,3-glucanase and chitinase. Thus, the CH affects the processes occurring during fruit damage and contributes to the preservation of the consumer qualities of fruit.

Phenylalanine Ammonia-Lyase-Mediated Differential Response of Tomato (Solanum lycopersicum L.) Cultivars with Different Stress Tolerance to Treatment with Low-Molecular-Weight Chitosan

The latest research has shown that chitosan acts as a growth stimulator and elicitor in plants, including resistance to biotic and abiotic factors. However, increasing concentrations could possibly make chitosan a source of stress for plants. In this study, we investigated the effect of low-molecular-weight chitosan hydrolysate on the root development of tomato (Solanum lycoperscum) cultivars Red Cherry, Lel, and Tytan. The growth rate change, together with total phenolic content, phenylalanine ammonia-lyase (PAL) activity, and gene expression, were studied in relation to tomatoes. High concentrations of chitosan negatively affect the growth of tomato seedlings and contribute to changes in the tropism of the seedling roots. After the addition of chitosan hydrolysate, the PAL activity and the total phenolic content decreased 24 h later. PAL is a key enzyme in the biosynthesis of many plant stress factors. An analysis of the tomato PAL gene family was carried out. The SlPAL gene expression in the seedlings of cv. Cherry increased 1.5 times after 48 h, while in cv. Lel, the expression stably decreased in the presence of chitosan. The obtained results are supposed to aid our understanding of the mechanisms underlying the effects of chitosan on plant development and further its successful application in agriculture as well as in research on plant stress.

A synthetic Tn-BSA conjugate vaccine bearing chitotriose as built-in adjuvant

Chitotriose (CTS), the hydrolysate of chitosan, is readily soluble in water because of the shorter chain lengths of the oligomers and the free amino groups in the d-glucosamine units. In the current study, we report the synthesis of novel conjugate vaccine Tn-BSA-CTS with chitotriose as built-in adjuvant, along with an evaluation of the effect of adjuvant chitotriose (CTS). Immunological evaluations of the resultant conjugate vaccine revealed that Tn-BSA-CTS could provoke the highest titers of IgG antibodies (102,400). The Tn-BSA-CTS conjugate remarkably enhanced both humoral and cellular immunity. The obtained results demonstrate the potential of CTS as a novel vaccine adjuvant in the development of antitumor vaccine and the covalent linkage of tumor vaccine to CTS might be available strategy to increase the efficacy against cancer.

Protective Properties of Copper-Loaded Chitosan Nanoparticles against Soybean Pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens.

Soybeans are a valuable food product, containing 40% protein and a large percentage of unsaturated fatty acids ranging from 17 to 23%. Pseudomonas savastanoi pv. glycinea (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are harmful bacterial pathogens of soybean. The bacterial resistance of soybean pathogens to existing pesticides and environmental concerns requires new approaches to control bacterial diseases. Chitosan is a biodegradable, biocompatible and low-toxicity biopolymer with antimicrobial activity that is promising for use in agriculture. In this work, a chitosan hydrolysate and its nanoparticles with copper were obtained and characterized. The antimicrobial activity of the samples against Psg and Cff was studied using the agar diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The samples of chitosan and copper-loaded chitosan nanoparticles (Cu2+ChiNPs) significantly inhibited bacterial growth and were not phytotoxic at the concentrations of the MIC and MBC values. The protective properties of chitosan hydrolysate and copper-loaded chitosan nanoparticles against soybean bacterial diseases were tested on plants in an artificial infection. It was demonstrated that the Cu2+ChiNPs were the most effective against Psg and Cff. Treatment of pre-infected leaves and seeds demonstrated that the biological efficiencies of (Cu2+ChiNPs) were 71% and 51% for Psg and Cff, respectively. Copper-loaded chitosan nanoparticles are promising as an alternative treatment for bacterial blight and bacterial tan spot and wilt in soybean.

Effect of Seed Priming with Chitosan Hydrolysate on Lettuce (Lactuca sativa) Growth Parameters.

Seed priming increases germination, yield, and resistance to abiotic factors and phytopathogens. Chitosan is considered an ecofriendly growth stimulant and crop protection agent. Chitosan hydrolysate (CH) is an unfractionated product of hydrolysis of high-molecular-weight crab shell chitosan with a molecular weight of 1040 kDa and a degree of deacetylation of 85% with nitric acid. The average molecular weight of the main fraction in CH was 39 kDa. Lettuce seeds were soaked in 0.01-1 mg/mL CH for 6 h before sowing. The effects of CH on seed germination, plant morphology, and biochemical indicators at different growth stages were evaluated. Under the 0.1 mg/mL CH treatment, earlier seed germination was detected compared to the control. Increased root branching was observed, along with 100% and 67% increases in fresh weight (FW) at the 24th and 38th days after sowing (DAS), respectively. An increase in the shoot FW was found in CH-treated plants (33% and 4% at the 24th and 38th DAS, respectively). Significant increases in chlorophyll and carotenoid content compared to the control were observed at the 10th DAS. There were no significant differences in the activity of phenylalanine ammonia-lyase, polyphenol oxidase, β-1,3-glucanase, and chitinase at the 24th and 38th DAS. Seed priming with CH could increase the yield and uniformity of plants within the group. This effect is important for commercial vegetable production.

Oligochitosan recovered from shrimp shells through subcritical water hydrolysis: Molecular size reduction and biological activities

Oligochitosan (OC) from shrimp shells derived chitosan was prepared using subcritical water hydrolysis (SWH) and their physicochemical and bio-functional properties were observed in this study. Reaction time of SWH was varied from 15 min to 60 min and the temperature from 140 °C to 170 °C. Gel permeation chromatography (GPC) confirmed that the molecular weight of the chitosan hydrolysate (CH) at 170 °C/15 min was obtained at 3.06 kDa. Fourier transform infrared and X-ray diffraction spectroscopy confirmed the conversion of OC from CH. Bio-functionality of CH was tested on the basis of antioxidant, antimicrobial, and anticancer activities. The ability of OC to act against the pancreatic cancer cell line Panc 1 was analyzed by MTT assay and Syto 9/PI fluorescent techniques. The MTT assay indicated that the maximum cytotoxicity was revealed by OC (170 °C/15 min) at 500 µg/mL. This research revealed that SWH can be an efficient method for the production of OC.

Application of enzymatic hydrolyzate of chitosan as a plant growth promoter

The natural polymer chitosan is widely used in various fields of agriculture, industry and medicine. The properties of a polymer largely depend on its molecular weight and supramolecular structure. In particular, to enhance the beneficial properties of chitosan, its low-molecular derivatives are obtained. The presented work shows the possibility of using enzymatic chitosan hydrolysates as a biostimulator of plants. It has been established that pretreatment of pea seeds (Pisum sativum) variety “Zhegalova 112” with solutions of chitosan hydrolyzate in all studied concentrations increases the seeds and soil germination. In the course of the work, the highest efficiency of chitosan hydrolysate was confirmed when growing pea seeds in the soil, namely higher soil germination, significantly faster growth of green biomass of plants, acceleration of leaf formation. A pronounced effect of chitosan hydrolysates on plants under stress conditions was discovered. So, cultivation of pretreated with chitosan hydrolysate cucumber seeds (Cucumis sativus) in an unsuitable period for growth promotes their germination. At the same time, seed germination is completely absent in the control group.

Cytotoxicity on Cancer Cells according to Average Molecular Weight of Chitosan Hydrolysates

To confirm the molecular characteristics of chitooligosaccharides (COS) derived from the hydrolysis of high molecular weight chitosan (HMWC), measuring average molecular weight (MW) using gel permeation chromatography (GPC) and their cytotoxicity was confirmed by treatment with several cancer cells. To evaluate the cytotoxicity of Chi-S according to different MW on various cancer cells, two types of chitooligosaccharide (COS) named Chi-S and COS-S were subjected in a concentration-dependent manner. It was divided into chitinous active substance (Chi-S) containing N-acetylglucosamine and chitosan oligosaccharide substance (COS-S), respectively. The average MW of Chi-S obtained by enzymatic treatment of HMWC for 24 h was confirmed to be about 2.7 kDa. Chi-S showed no cytotoxicity against all treated cancer cells. However, rather than showing cytotoxicity in immune macrophages RAW264.7, significant cell proliferation was observed. On the other hand, COS-S (average MW of approximately 7.3-kDa) obtained by enzymatic treatment of HMWC for 12 h showed significant cytotoxicity against all cancer cells including immune macrophages RAW264.7 at a high concentration range of 100 mg/mL or more. Furthermore, no significant changes were observed in breast cancer cells and normal cells treated with the control group. Therefore, based on the results, we estimate that Chi-S may be involved in indirect anticancer activity by selective or adaptive immunological activity according to the molecular characteristics. Our data will be a good guide for subsequent studies that can focus on the particular activity of molecules according to the difference between chitosan MW and the deacetylation degree (DD) of HMWC.

Matrix Sensitivity and Interpretation for Precise Molecular Weight Analysis of Chitosan Hydrolysates

The biological activity of chitosan differs depending on the molecular weight and degree of deacetylation, so more precise molecular weight determination has become more important. It is difficult to determine the absolute molecular weight size due to the lack of precision, whereas the approximate molecular size is determined using HPLC. Therefore, the hydrolysates using acid or enzyme were subjected to precise molecular weight analysis by using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF). For analysis, two types of matrices were used in this study. The results showed slight differences in sensitivity but were confirmed to be similar to each other. In addition, it was confirmed that any matrix used in this study did not significantly affect the results. In conclusion, the results of this study will provide very important scientific clues in the investigation of the biological activities of chitosan such as antibacterial, anticancer, immune, and antioxidant activity.

꾸지뽕나무 열매 추출물의 Chlorogenic Acid, Rutin 함량 분석 및 생리활성

This study was carried out to very precisely understand the effect of solvent selection on the molecular properties in the investigation of the biological activity according to the molecular size of chitosan. Chitosan hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) were prepared in enzymatic hydrolysis under solvent selective conditions. Chitosan hydrolysates were characterized by HPLC and Matrix-Assisted Laser Desorption/ Ionization-Time of Flight (MALDI-TOF) mass spectrometry analyses, respectively. HPLC analysis did not show a significant difference in the size of hydrolysates according to the type of chitosan. On the other hand, it was confirmed that each molecular size was significantly different from the results presented by HPLC with MALDI-TOF mass spectrometry analysis. In addition, the effect on the antibacterial activity of the hydrolysates of chitosan, designated to “active molecular chitosan (AMC)”, by the solvent selected for the enzymatic reaction also showed a significant difference. Therefore, it is intended to suggest that the selection of solvents for the enzymatic reaction for chitosan hydrolysis and their antibacterial activity is very important.

The Effect of Chitosan Hydrolysate on Solanum Lycopersicum Plant Growth

Chitosan is a well-known subjectof researchbecause of its beneficial properties, including its antibacterial and fungicidal activity, as well as its effect on plant physiology. One of the maindifficulties in agricultural chitosan implementation is the poor solubility of high molecular chitosan in water. Reducing themolecular weight of chitosan by acid hydrolysis increases its solubility. This research studied the effect of chitosan hydrolysate on the germination and development ofSolanum lycopersicum L. plants. Theeffects of chitosan hydrolysate on seed germination, shoot development in the first week of development and in the first month of development were evaluated. According to the results, high concentrations of chitosan hydrolysate completely inhibited seed germination. However, short-term treatment by high concentrations of chitosan hydrolysate stimulatedthe development of seedlings, leading to substantially longerroots. Regular root application of high concentrations of chitosan hydrolysate inhibited plant development.
 Keywords: chitosan hydrolysate, chitosan, plant growth regulators, plant germination

THE EFFECT OF CHITOSAN HYDROLYSATE ON THE FUNGI ALTERNARIA SOLANI, FUSARIUM SOLANI AND RHIZOCTONIA SOLANI IN VITRO

Chitosan hydrolysate was obtained using nitric acid; the prevailing fraction had a molecular weight of 30 kDa and a deacetylation degree of 95%. The effect of chitosan hydrolysate when added to potato dextrose agar (PDA) in different concentrations (0.5, 1, 1.5, 2, 4, 6 and 8 mg/mL) was studied on the growth of the fungi Alternaria solani Sorauer, Fusarium solani (Mart.) Sacc. and Rhizoctonia solani J.G. Kühn. A. solani was the most sensitive to the addition of chitosan hydrolysate to PDA in radial growth experiments. On days 3 and 7 of incubation, the antifungal activity of the phytopathogen growth was 69%-92% and 69%-88%, respectively, in the concentration range of 0.5-2 mg/ml.

Biocidal and resistance-inducing effects of chitosan on phytopathogens

In connection with the of the law adoption on organic products in the Russian Federation, as well as the appearance of fungicide-resistant forms of phytopathogens, the search for new preparations of biological origin for plant protection from disease remains relevant. Chitosan is a promising substance obtained from shells of crustaceans, zygomycete fungi: on the one hand, it prevents the pathogen's penetration into the plant inducing a horizontal resistance, and on the other, it has a bactericidal effect against some bacteria and fungi. This article shows the biocidal effect of chitosan hydrolysate with a molecular weight of the main fraction of 33.7 kDa in relation to 5 phytopathogenic bacteria. The minimum bactericidal concentration ranged from 0.25 to 0.5%. It was noted that the substance acts more actively against Gram-negative bacteria than in relation to Gram-positive. Biological substance efficacy on artificial infectious background of cucumber downy mildew during preventive treatment amounted to the average of 55.4% compared to control with no treatment.

Comparison of determination of sugar-PMP derivatives by two different stationary phases and two HPLC detectors: C18 vs. amide columns and DAD vs. ELSD

Reducing sugars, especially aldoses with their ring structures, have strong reducibility to react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives detected by ultraviolet (UV) detector at 248 nm. In this study, several carbohydrates and some of their alcohols, including 1). seven main monosaccharides, 2). three common disaccharides, 3). two oligosaccharides, and 4). some sweeteners like sugar alcohols were investigated for their reactivities with PMP. Analytes were separated chromatographically by two common high performance liquid chromatography (HPLC) columns and subsequently detected by two tandem detectors. Results pointed out that C18 column had a stronger capacity to separate the reducing sugar-PMP derivatives rather than the sugar themselves. While amide column could only effectively separate the original sugars. These phenomena demonstrated that polarity of reducing sugars decreased after their PMP derivatization. Moreover, both diode array detector (DAD) and evaporative light scattering detector (ELSD) were able to detect the reducing sugar-PMP derivatives, including the PMP-derivative of the oligosaccharide hydrolysate of chitosan, though the DAD exhibits a higher sensitivity than ELSD. In conclusion, carbohydrates themselves are more likely to be efficiently separated by an amide column and detected by ELSD, while DAD combined with a C18 column has more power to determine carbohydrates derivatives.

Effect of chitosan oligosaccharide-conjugated selenium on improving immune function and blocking gastric cancer growth

Selenium (Se) is a potential chemopreventive or chemotherapeutic agent against malignant tumor. Selenium-oligosaccharides are important selenium source of dietary supplementation. Due to the insufficient natural production, it is therefore urgent to develop selenium-oligosaccharides by artificial synthesis. Chitosan, the N-deacetylated derivative of chitin, has been applied widely in biomedical field, owing to its nontoxicity, hydrophilicity, biocompatibility, and biodegradation. While chitosan is water insoluble at neutral pH, limiting its application in physiological conditions. Chitosan oligosaccharide (COS), the hydrolysate of chitosan, is readily soluble in water because of the shorter chain lengths of the oligomers and the free amino groups in the D-glucosamine units. This study was aimed at preparing COS-conjugated selenium (COS–Se) and examining the toxicity and ability on improving immune function and blocking gastric cancer growth. Our results demonstrated that COS–Se displayed directly co-mitogenic and mitogenic actions on mouse splenocytes proliferation in vitro. Besides, COS–Se treatment could effectively elevate phagocytosis and increase the secretion of anti-inflammatory cytokine in mouse peritoneal macrophages. Further in vivo experiments showed that COS–Se exhibited immuno-enhancing effects through promoting the phagocytic index, spleen index and thymus index with no obvious toxicity to Kunming mice. Moreover, COS–Se inhibited proliferation and metastasis of human gastric cancer cells, with non-toxic effects on the normal fibroblast cells in vitro. COS–Se supplementation could significantly repress the growth of gastric adenocarcinoma through reducing levels of CD34, vascular endothelial growth factor and matrix metalloproteinase-9 of nude mice. In conclusion, COS–Se was non-toxic and showed great potential as a functional food ingredient in cancer prevention.

Synergistic Antimicrobial Activity of Chitosan Hydrolysates derived fromthe Hydrolysis of High Molecular Weight Chitosan using Immobilized Enzyme

The features of antibacterial activity of chitosan with different molecular weight and with antibiotics are discussed in this study. Depolymerization of high molecular weight chitosan(HMWC) mediated by enzymatic effects was observed in the concentration of enzyme immobilized onto a matrix and time dependent manner. Antibacterial activity of chitosan hydrolysates CTSN-P8 and CTSN-B3 in the presence or absence of antibiotics was investigated. Synergistic antibacterial activity of CTSN-P8 with Kanamycin against Staphylococcus aureus and E. coli was observed based on the dependence of molecular weight of chitosan. On the other hand, significant antifungal activity of CTSN-B3 was observed in morphological changes of Penicillium italicum. Our data demonstrate the critical importance of microbial species and the size of chitosan that can provide some potent biologically activity without significant depressive activity of antibiotics.

A new oligosaccharide-filgrastim conjugate prepared by enzymatic method: Preparation and physicochemical characterization

Modification of recombinant proteins using conjugation techniques is a useful and easy tool to improve the stability and pharmacokinetic properties of pharmaceutical protein products. In this study, we prepared a new conjugate by using natural oligopolysaccharides via an enzymatic method. Microbial transglutaminase was used for the enzymatic conjugation of filgrastim with hydrolysates of chitosan, which is a natural polymer in the polysaccharide structure. Conjugate formation and physicochemical properties of the modified filgrastim were demonstrated analytically. The results indicated that the conjugates protected the active substance filgrastim, both at 5 °C ± 3 °C and 25 °C ± 2 °C for four months. The in vitro bioactivity of the conjugate solution on NFS-60 cells was compared with that of the commercial reference filgrastim solution, and the relative potency was observed to be higher than that of the reference.

Anti-Oxidant Activity and Dust-Proof Effect of Chitosan with Different Molecular Weights.

High molecular weight chitosan (HMWC) was degraded to prepare chitosan with different molecular weight based on the fenton reaction, which can produce aggressive OH-radicals produced from hydrogen peroxide in the presence of catalytic metal ions. The relative molecular weight, anti-oxidant activity, and fine dust removal effect of chitosan hydrolysates were elucidated to define their molecular weight and their potent biological activity. Our results demonstrate that chitosan hydrolysates derived from the hydrolysis of HMWC may possess significant free-radical scavenging activity as good anti-oxidants against the radical scavenging activity of DPPH and ABTS, respectively. Furthermore, chitosan hydrolysates can effectively eliminate fine dust, which may contain some particulate matter (PM) and unknown species of microorganisms from the air, suggesting that our data provide important information for producing air filters, dust-proof masks and skin cleaner for the purpose of human healthcare and well-being.

Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, the incidence of cardiotoxicity compromises its therapeutic index. Oxidative stress and apoptosis are believed to be involved in DOX-induced cardiotoxicity. Chitosan oligosaccharides (COS), the enzymatic hydrolysates of chitosan, have been reported to possess diverse biological activities including antioxidant and anti-apoptotic properties. The objective of the present study was to investigate the potential role of COS against DOX-induced cardiotoxicity, and the effects of COS on apoptosis and oxidative stress in rats and H9C2 cells. Furthermore, we also shed light on the involved pathways during the whole process. For this purpose, first, we demonstrated that COS exhibited a significant protective effect on cardiac tissue by not only inducing a decrease in body and heart growth but also ameliorated oxidative damage and ECG alterations in DOX-treated rats. Second, we found that COS reversed the decrease of cell viability induced by DOX, reduced the intracellular reactive oxygen species (ROS), increased the mitochondrial membrane potential (MMP) and Bcl-2/Bax ratio. COS treatment also results in reduced caspase-3 and caspase-9 expressions, and an increase in the phosphorylation of MAPKs (mitogen-activated protein kinases) in DOX-exposed H9C2 cells. Additionally, cellular homeostasis was re-established via stabilization of MAPK mediated nuclear factor erythroid 2-related factor 2/antioxidant-response element (Nrf2/ARE) signaling and transcription of downstream cytoprotective genes. In summary, these findings suggest that COS could be a potential candidate for the prevention and treatment of DOX-induced cardiotoxicity.

Oral administration with chitosan hydrolytic products modulates mitogen-induced and antigen-specific immune responses in BALB/c mice.

The aim of this study was to investigate whether oral administration in BALB/c mice with chitosan hydrolytic products including chitosan hydrolysate, LMWC and a chitooligosaccharide mixture (oligomixture), modulates mitogen-induced and antigen-specific immune responses. A water-soluble chitosan hydrolysate was obtained via cellulase degradation of chitosan, and a LMWC and the oligomixture were separated from this hydrolysate. In non-immunized mice, both the chitosan hydrolysate and oligomixture significantly increased the phagocytic activity of peritoneal macrophages. Three chitosan hydrolytic products significantly increased the mitogen-induced proliferation of splenocytes and Peyer's patch (PP) lymphocytes. LMWC and oligomixture up-regulated IFN-γ secretion, and induced predominantly Th1 cytokine secretion in splenocytes. In antigen-specific immunity, similar effects of the chitosan hydrolytic products were observed on augmenting ovalbumin (OVA)-, as well as mitogen-, induced proliferation of splenocytes harvested from OVA-immunized mice. Interestingly, oligomixture was the most potent chitosan hydrolytic product to elicit OVA-specific IgM, IgG, and IgA production, while LMWC was the most potent one to elevate splenic IFN-γ production and IFN-γ/IL-4 (Th1/Th2) ratio. These results provide the distinct immunomodulatory properties of chitosan hydrolytic products in response to mitogens and specific antigen, paving the way for further development and application of dietary chitosan hydrolytic products against immune disorders and infection.