High-throughput methods to assess lipophilic and hydrophilic antioxidant capacity of food extracts in vitro

Assays comprising three probes for different mechanisms of antioxidant activity in food products have been modified to allow better comparison of the contributions of the different mechanisms to antioxidant capacity (AOC). Incorporation of a common format for oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), and iron(II) chelating activity (ICA) assays using 96-well microplates provides a comprehensive and high-throughput assessment of the antioxidant capacity of food extracts. The methods have been optimized for aqueous extracts and validated in terms of limit of quantification (LoQ), linearity, and precision (repeatability and intermediate reproducibility). In addition, FRAP and ORAC assays have been validated to assess AOC for lipophilic extracts. The relative standard deviation of repeatability of the methods ranges from 1.2 to 6.9%, which is generally considered to be acceptable for analytical measurement of AOC by in vitro methods. Radical scavenging capacity, reducing capacity, and iron chelating properties of olive mill wastewaters (OMWW), oregano, and parsley were assessed using the validated methods. OMWW showed the highest radical scavenging and reducing capacities, determined by ORAC and FRAP assays, respectively, followed by oregano and parsley. The ability to chelate Fe (2+) was, in decreasing order of activity ( p > 0.05) parsley congruent with oregano > OMWW. Total phenol content, determined by the Folin-Ciocalteu method, correlated to the radical scavenging and reducing capacities of the samples but not to their chelating properties. Results showed that the optimized high-throughput methods provided a comprehensive and precise determination of the AOC of lipophilic and hydrophilic food extracts in vitro.     

Effects of epilactose on calcium absorption and serum lipid metabolism in rats

Epilactose (4-O-beta-galactopyranosyl-D-mannnose) is a rare disaccharide in cow milk that can be synthesized from lactose by the cellobiose 2-epimerase of Ruminococcus albus. In this study, we examined the biological activities of epilactose using male Wistar-ST rats. The apparent rates of calcium and magnesium absorption of rats fed epilactose and fructooligosaccharide diets were greater than those fed control and lactose diets, accompanied by greater weight gain of the cecal wall and higher levels of short-chain fatty acids and other organic acids. Epilactose also increased the calcium absorption in everted small intestinal sacs. In addition, the levels of plasma total cholesterol and nonhigh-density lipoprotein cholesterol were lower in epilactose-fed rats. These results indicate that epilactose promotes calcium absorption in the small intestine and possibly lowers the risk of arteriosclerosis. Cecal microbes may efficiently utilize epilactose and contribute to these biological activities.     

Isoflavone-free soy protein prepared by column chromatography reduces plasma cholesterol in rats

To know whether isoflavones are responsible for the hypocholesterolemic effect of soy protein, the effect on plasma cholesterol of isoflavone-free soy protein prepared by column chromatography was examined in rats. Five-week-old male Sprague-Dawley rats were fed cholesterol-enriched AIN-93G diets containing either 20% casein (CAS), 20% soy protein isolate (SPI), 20% isoflavone-free SPI (IF-SPI), 19.7% IF-SPI + 0.3% isoflavone-rich fraction (isoflavone concentrate, IC), or 20% CAS + 0.3% IC for 2 weeks. Plasma total cholesterol concentrations of rats fed SPI and IF-SPI were comparable and were significantly lower than that of rats fed CAS. The addition of IC to the CAS and IF-SPI did not influence plasma cholesterol level. Fecal steroid excretion of the three SPI groups was higher than that of the two CAS groups, whereas the addition of IC showed no effect. Thus, a significant fraction of the cholesterol-lowering effect of SPI in rats can be attributed to the protein content, but the isoflavones and other minor constituents may also play a role.     

不同供氯水平对烤烟生长发育和养分吸收的影响

采用田间小区试验研究了不同供氯水平对烤烟生长发育和养分吸收的影响.结果表明,施氯能促进烤烟生长发育,尤其当供氯水平为90.0 kg·hm~(-2)时,烤烟株高明显优于对照和其余处理.供氯水平在45.0～90.0 kg·hm~(-2)的范围内,烟叶对氮的吸收量明显高于其他处理,且在供氯水平为90.0 kg·hm~(-2)时达到峰值.施氯促进烤烟对钾的吸收,但烟叶中K_2O/Cl随供氯水平增加而降低.氯与磷之间存在着拮抗关系.在22.5～67.5 kg·hm~(-2)施氯范围内,烟叶含钙量随着氯水平增加而增加.在22.5～45.0 kg·hm~(-2)施氯范围内,施氯促进烟叶对镁的吸收.烟叶各叶位对氯的吸收量则表现出随供氯水平增加而增加的趋势,且各叶位氯吸收量表现为下部叶>中部叶>上部叶.供氯水平在22.5 kg·hm~(-2)时,上中下部烟叶的氯含量均在最佳范围(0.3%～0.8%)内.     

Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.     

Use of fluorogenic probes to differentiate between hydrophilic and lipophilic antioxidant activity in a fish cell line

In finfish aquaculture, dietary antioxidants have been shown to improve indicators of general fish health and to inhibit the oxidative deterioration of polyunsaturated fatty acids. To facilitate the characterization of novel antioxidants or antioxidant mixtures, we developed assays for antioxidant activity in a fish cell line. We used 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) to determine the protective effects of a panel of representative antioxidant compounds against the formation of reactive oxygen species (ROS) under conditions that promote oxidative stress, whereas protective effects against lipid peroxidation were measured using the thiobarbituric acid reactive substances (TBARS) assay and a novel implementation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C(11)-BODIPY(581/591)). We found that the highly hydrophilic antioxidant, sodium ascorbate, inhibited H(2)DCFDA oxidation but had no effect on lipid peroxidation, whereas the highly hydrophobic antioxidant, α-tocopherol, potently inhibited lipid peroxidation but did not prevent H(2)DCFDA oxidation. The data suggest that a single assay is not sufficient for estimating antioxidant activity in cultured fish cells.     

不同人工施氮量对荆条氮素利用的影响

为了解植物在不同氮生境下的响应,以太行山丘陵区荆条群落为对象,采用人工施氮的方法,研究不同施氮水平下荆条氮素利用特征。结果表明:1)人工施氮后,成熟绿叶和枯叶中氮质量分数随施氮量的增加而增加,并在施氮量为32 g/m2时达到最大值,施氮对成熟绿叶磷质量分数也有明显的影响,当施氮量为8 g/m2时,叶中磷质量分数最高,约为对照的2.29倍,施氮量与枯叶中磷质量分数无显著相关关系;2)施氮能显著降低荆条的氮素回收率,随施氮量增加,氮素回收率整体上表现出负相关,平均降幅约为18.57%,施氮量在4～8 g/m2之间时,氮素回收率无显著差异;3)随施氮量提高,土壤氮和磷的质量分数呈现升高的趋势,土壤磷质量分数在施氮量为4 g/m2时达到最大值,之后随施氮量的增加而下降,土壤有机质和土壤含水量与氮素回收率呈负相关关系。     

Synthesis of lipophilic clovamide derivatives and their antioxidative potential against lipid peroxidation

Some N-(hydroxycinnamoyl)-L-tyrosine and L-DOPA alkyl esters were synthesized and evaluated as a variation of the clovamide (N-caffeoyl-L-3,4-dihydroxyphenylalanine) structure, a known antioxidant found in red clover. The amides were prepared in good yields starting from methyl and dodecylesters of L-tyrosine and L-DOPA by reacting with the N-hydroxysuccinimidyl esters of ferulic, sinapic, and acetyl-protected caffeic acid, respectively. In the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and superoxide radical quencher assays they showed radical scavenging activity equal to or higher than those of the standard antioxidants ascorbic acid and tocopherol. The antioxidative potentials of the clovamide derivatives against bulk lipid oxidation, as determined by the accelerated autoxidation of oils, were equal to or higher than those of the standard antioxidants; some of the compounds were able to protect an emulsion of linoleic acid/beta-carotene against oxidation. N-Caffeoyl L-tyrosine methyl ester and the N-cinnamoyl L-DOPA alkyl esters especially were potent antioxidants in bulk lipids and moderate protectants in emulsions.     

Effect of dietary lipid-lowering drugs upon plasma lipids and egg yolk cholesterol levels of laying hens.

To evaluate the effect of lipid-lowering agents upon egg quality, reproductive performance, plasma lipids, and egg yolk cholesterol levels, 30-week-old Shaver laying hens were fed a basal diet (commercial ration) supplemented with 0.1% probucol (PROB), 0.025% gemfibrozil (GEMF), or lovastatin at 0.0005% (LOV1), 0.001% (LOV2), or 0.0015% (LOV3) for a 12-week experimental period. It was observed that the supplementation of the drugs did not impair albumen and shell quality. Hen performance was not adversely affected. The depression in triglyceride concentrations approached statistical significance only in LOV2 (38.5%), and total cholesterol was significantly depressed in LOV2 (36.0%), LOV3 (36.8%), PROB (29.6%), and GEMF (30.4%) treatments. Egg cholesterol content, expressed per gram of yolk, was significant lowered in LOV1 (7.5%) and LOV3 (12. 7%).     

Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol

Hydroxytyrosol, a naturally occurred o-phenolic compound exhibiting antioxidant properties, was synthesized by a three-step high-yielding procedure from natural and low-cost compounds such as tyrosol or homovanillyl alcohol. First, the efficient chemoselective protection of the alcoholic group of these compounds was performed by using dimethyl carbonate (DMC) as reagent/solvent; second, the oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and in situ reduction with sodium dithionite (Na2S2O4) allowed the preparation of carboxymethylated hydroxytyrosol; finally, by a mild hydrolytic step, hydroxytyrosol was obtained in high yield and purity, as confirmed by NMR spectra and HPLC profile. By using a similar methodology, lipophilic hydroxytyrosol derivatives, utilized as additives in pharmaceutical, food, and cosmetic preparations, were prepared. In fact, at first the chemoselective protection of the alcoholic group of tyrosol and homovanillyl alcohol was performed by using acyl chlorides without any catalyst to obtain the corresponding lipophilic derivatives, and then these compounds were converted in good yield and high purity into the hydroxytyrosol derivatives by oxidative/reductive pathway with IBX or DMP and Na2S2O4.     

Accelerators increase permeability of cuticles for the lipophilic solutes metribuzin and iprovalicarb but not for hydrophilic methyl glucose

Effects of diethylsuberate (DESU), tributyl phosphate (TBP), and monodisperse ethoxylated alcohols (EAs) on rate constants of penetration (k) of model solutes across astomatous cuticular membranes isolated from Madagascar ivy (Stephanotis floribunda) and pear (Pyrus communis) leaves were studied. Model solutes (selected on the basis of their octanol/water partition coefficients, K(ow)) were iprovalicarb (log K(ow) = 3.18), metribuzin (log K(ow) = 1.60), and methyl glucose (MG) (log K(ow) = -3.0). K(ow) varied by more than 6 orders of magnitude. Accelerators had wax/water partition coefficients (log K(ww)) ranging from 1.75 (DESU) to 4.32 (C(12)E(2)), and their equilibrium concentrations in Stephanotis wax varied from 0 to about 160 g kg(-)(1). Accelerators increase solute mobility in cuticles by increasing fluidity of cutin and waxes. This effect was quantified by plotting log k versus the accelerator concentration in wax. With the lipophilic solutes metribuzin and iprovalicarb, these plots were linear. Slopes of these plots characterize the intrinsic activities of the accelerators, and they decreased in the order DESU (0.029) > TBP (0.015) > EAs (0.01). Using these intrinsic activities, the effects of accelerators on rate constants of penetration can be calculated for any accelerator concentration in wax. For instance, at 50 g kg(-)(1), rate constants for lipophilic solutes increased by factors of 28 (DESU), 5.6 (TBP), and 3.2 (C(12)E(n)()), respectively. Permeability of cuticles for the hydrophilic MG was not increased by DESU, TBP, C(12)E(2), and C(12)E(4), while C(12)E(6) and C(12)E(8) increased it. Small hydrophilic solutes such as MG can access aqueous pores in cuticles, and this pathway is not affected by changes in fluidity of amorphous waxes. After rate constants of penetration of ionic CaCl(2) were compared with those for nonionic MG, it was concluded that 60% of the MG diffused across aqueous pores, while 40% used an alternative pathway. Because the solubility of MG in wax is extremely low, it is unlikely that MG diffused along the lipophilic pathway used by metribuzin and iprovalicarb. This agrees with the observation that DESU and TBP had no effect on rate constants for MG. An alternative pathway of unknown properties is suggested. It is speculated that C(12)E(6) and C(12)E(8) sorbed in cuticles might have generated a polar pathway for MG.     

Effects of estrogen and estrus cycle on pharmacokinetics, absorption, and disposition of genistein in female sprague-dawley rats

Genistein is an active soy isoflavone with anticancer activities, but it is unknown why it has a higher oral bioavailability in female than in male rats. Our study determined the effects of estrus cycle on genistein's oral bioavailability. Female rats with various levels of estrogen were orally administered with genistein or used in a four-site rat intestinal perfusion experiment. Rats in "proestrus" group (with elevated estrogen) had significantly reduced (57% decrease, p < 0.05) oral bioavailability of total genistein (aglycone + conjugates) than those in "metoestrus" group (with basal level of estrogen). Female ovariectomized rats, due to lack of estrogen, showed oral bioavailability of total genistein similar to the "metoestrus" group but higher (155% increase, p < 0.05) than the "proestrus" group. On the basis of intestinal perfusion studies, the increased bioavailability was partially attributed to the higher (>100% increase, p < 0.05) hepatic disposition via glucuronidation and possibly more efficient enterohepatic recycling of genistein in the "metoestrus" group. Furthermore, chronic exogenous supplementation of estradiol in ovariectomized rats significantly reduced (77%, p < 0.05) the oral bioavailability of total genistein, mostly via increased sulfation (>10-fold) in liver, to a level comparable to those in the "proestrus" group. In conclusion, the oral bioavailability of total genistein was inversely proportional to elevated estrogen levels in female rats, which is partially mediated through the regulation of hepatic enzymes responsible disposition of genistein.     

Effects of water-soluble hemicellulose from soybean hull on serum antibody levels and activation of macrophages in rats

Effects of soybean hull water-soluble hemicellulose (WSHC) on serum immunoglobulin (Ig) concentration and production of NO and IL-1beta from peritoneal macrophages were examined and compared with those of Agaricus blazei in the rat system. WSHC consisted of arabinose, galactose, xylose, glucose, and rhamnose, and the molecular weight was approximately 500000. Rats were ip administrated each sample at a dose of 0.67, 13.4, or 26.9 mg/kg/day for 14 days. The administration of WSHC resulted in significantly higher productions of IgM (p < 0.01 on day 6, p < 0.05 on day 14) and IgG (p < 0.05 on day 6) than those in other groups. When peritoneal macrophages were stimulated with various concentrations of sample (0.67, 13.4, or 26.9 mg/mL), WSHC significantly increased both NO and IL-1beta productions only at the concentration of 13.4 (mg/mL) compared with those of a saline group. These findings demonstrate that WSHC enhances humoral immunity and activation of macrophages, thereby leading to the augmentation of immune responses in rats.     

Effects of wheat antioxidants on oxygen diffusion-concentration products in liposomes and mRNA levels of HMG-CoA reductase and cholesterol 7alpha-hydroxylase in primary rat hepatocytes

Three wheat antioxidant fractions were investigated for their potential effects on oxygen diffusion-concentration products in liposomes prepared with egg yolk phosphatidycholine (yolk PC) and rat liver PC (liver PC), using the electron spin resonance (ESR) oximetry method with 2,2'-azobis(2-aminopropane) dihydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) as radical generators. Both water-soluble wheat antioxidant (WWA) and the second lipophilic antioxidant (LWA2) fractions were able to inhibit oxygen diffusion-concentration product induced by either AAPH or AMVN. The first lipophilic wheat antioxidant (LWA1) fraction only showed antioxidant activity in yolk PC liposomes with AAPH as the radical initiator but had pro-oxidant activity under other testing conditions. Both liposome composition and radical initiator altered the antioxidative properties of WWA, LWA1, and LWA2. WWA also showed the strongest DPPH(*) scavenging capacity on a per grain weight basis. HPLC analysis showed that WWA had a much higher level of total phenolic acids, which may partially explain their antioxidant properties. In addition, wheat antioxidants significantly down-regulated the mRNA of HMG-CoA reductase, the key enzyme for cholesterol biosynthesis, and up-regulated the mRNA of cholesterol 7alpha-hydroxylase (CYP7A1), the key enzyme for cholesterol metabolism, in primary rat hepatocytes. These data indicated the potential of wheat antioxidants in reducing the risk of atherosclerosis through multimechanisms.     

土壤肥力和施氮量对小麦根系氮同化及子粒蛋白质含量的影响

采用田间土柱栽培的方法,研究了不同土壤肥力条件下施氮量对小麦根系氮素吸收、同化及子粒产量和蛋白质含量的影响。结果表明,小麦浅层根系衰老开始的时间比深层根系早,深层根系对小麦生育后期氮素吸收具有重要的作用。增施氮肥或土壤肥力高有利于小麦根系的生长发育,提高了根系活力和氮素同化关键酶的活性,延缓了根系的衰老,提高了小麦子粒产量和蛋白质含量。     

Changes in hydrophilic and lipophilic antioxidant activity in relation to their phenolic composition during the chamber drying of red grapes at a controlled temperature

The purpose of this work was to study the variation of phenol compounds, as measured by HPLC, during the chamber drying under controlled temperature conditions of red grapes of the Merlot and Tempranillo varieties in relation to antioxidant activity. Both lipophilic and hydrophilic antioxidant activities in these grapes increased during the drying process; the former was measured via proton transfer in the coupled oxidation reaction between linoleic acid and β-carotene, and the second via electron transfer in the DPPH assay. The hydrophilic component was invariably greater in Tempranillo grapes, and so was the lipophilic component in Merlot grapes. Only the increase in hydrophilic antioxidant activity obtained a significant correlation with the phenolic compounds during the drying process. However, based on the phenolic fraction analysis, this result was primarily due to phenolic polymers and, to a lesser extent, also to phenolic acids, flavans, and some flavonols and anthocyans.     

高基施比例条件下施氮量对覆膜种植烤烟氮素利用的影响

2009年采用大田小区试验,在广东南雄烟区探讨了高基肥比例条件下(无机肥料N基追比为7:3)施N量对覆膜种植烤烟N素利用的影响。试验结果表明:①饼肥和猪粪(N=64.50 kg/hm2,其中饼肥N 42.00%,不施用无机肥料N)有较强的持续供N能力,在烤烟移栽后30~75天使烟株根际土壤NO3--N浓度仍能维持在550.00 mg/kg以上;②在基施饼肥和猪粪(N=64.50 kg/hm2)的基础上,增施48~168 kg/hm2的无机N肥可显著提高烤烟移栽后75天前烟株根际土壤NO3--N含量,但在移栽75天后烟株根际土壤NO3--N积累效应不显著;③试验无机肥料供N范围内(48~168 kg/hm2),施N量对烤烟产量、产值、等级结构和内在化学品质无显著影响,但48 kg/hm2的施N量处理其N肥利用率和农学利用率极显著高于108kg/hm2和168 kg/hm2处理;④在广东南雄烟区地膜覆盖栽培条件下,基施64.50 kg/hm2有机N、配施48 kg/hm2无机N(基追比为7:3)可有效满足烤烟N素营养需求,提高N肥利用率和烟农的经济效应。     

外源一氧化氮对镉胁迫下长春花质膜过氧化、ATPase及矿质营养吸收的影响

重金属污染已成为全球范围的主要问题之一,其中土壤镉（Cd）污染已成为当今社会普遍关注的问题。镉是植物生长发育的非必需元素,极小浓度即可产生较大危害。一氧化氮（NO）是一种氧化还原信号分子和活性氮（RNS）, 参与植物对重金属镉胁迫的应答。长春花（Catharanthus roseus）是我国广泛栽培的兼具园林绿化和抗癌药源等有重要价值的多年生草本花卉植物。为了解镉胁迫下外源NO 对园林地被植物生理响应的调控机制,采用盆栽试验研究了外源NO（硝普钠SNP）对镉胁迫下长春花幼苗生长、 活性氧代谢、 质膜ATPase酶和5'-核苷酸酶活性以及矿质营养元素吸收的影响。结果表明, 25 mg/kg 镉胁迫严重抑制长春花幼苗的生长,显著增加地上部和根系镉的富集量,抑制对大量元素和微量元素的吸收。施加0.45、 0.90、 1.80 mg/kg 的SNP显著降低镉从根系向地上部的转运,缓解因镉胁迫对钾（K）、 钙（Ca）、 镁（Mg） 和 铁（Fe）、 铜（Cu）、 锌（Zn） 吸收产生的抑制效应,降低镉胁迫的毒害作用,促进植物生长。镉胁迫下,丙二醛（MDA）含量和活性氧（O2和H2O2）水平显著升高。施加低浓度 SNP 能够显著缓解细胞质膜过氧化,降低硫代巴比妥酸反应产物（TBARS）堆积,且对抗氧化酶和ATPase酶具有相同作用。添加0.45、 0.90、 1.80 mg/kg 的SNP 可提高镉胁迫下长春花地上部和根系的抗氧化酶［过氧化氢酶（CAT）、 超氧化物歧化酶（SOD）、 过氧化物酶（POD）］活性与抗氧化物（还原型谷胱甘肽GSH）含量,诱导质膜H+-ATPase、 Ca2+-ATPase和 5-AMPase 活性提升到正常水平（对照CK）。添加1.80 mg/kg 的SNP对镉毒害的缓解作用最有效,而添加3.60、 7.20 mg/kg 的SNP的处理则无明显效果。     

Effects of hydrophilic plasticizers on mechanical, thermal, and surface properties of chitosan films

Chitosan films were plasticized with four hydrophilic compounds, namely, glycerol (GLY), ethylene glycol (EG), poly(ethylene glycol) (PEG), and propylene glycol (PG). Our objective was to investigate the effect of plasticizers on mechanical and surface properties of chitosan films. The stability of plasticized films was observed by storage for 3 and 20 weeks in an environmental chamber at 50 +/- 5% RH and 23 +/- 2 degrees C. Plasticization improves the chitosan ductility, and typical stress-strain curves of plasticized films have the features of ductile materials, except the film made with 5% PG that exhibits as a brittle polymer and shows an antiplasticization effect. In most cases, the elongation of plasticized films decreases with the storage time, which might be due to the recrystallization of chitosan and the loss of moisture and plasticizer from the film matrix. Although at the beginning the mechanical properties of films made with PG, at high plasticizer concentration, are comparable to those of films made with EG, GLY, and PEG, their stability is poor and they tend to become brittle materials. The surface properties, analyzed by contact angle measurement, reveal that plasticization increases film hydrophilicity. It is found that GLY and PEG are more suitable as chitosan plasticizers than EG and PG by taking into account their plasticization efficiency and storage stability. Furthermore, a plasticizer concentration of 20% (w/w) with GLY or PEG seemingly is sufficient to obtain flexible chitosan film with a good stability for 5 months of storage.