Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Antibacterial activity and action mechanism of a novel chitosan oligosaccharide derivative against dominant spoilage bacteria isolated from shrimp Penaeus vannamei

With the aim of exploring the potential application of a novel chitosan oligosaccharide derivative (COS-All-Tio) in shrimp preservation, six dominant spoilage bacteria in the spoiled shrimp (Penaeus vannamei) were isolated and identified as Shewanella putrefaciens (RMS1), S. putrefaciens (S2), Pseudomonas weihenstephanensis (P1), P. gessardii (P2), Aeromonas bestiarum (A1) and Aeromonas molluscorum (A2). The antibacterial effect of COS-All-Tio against the six bacterial isolates were studied. Bacterial inhibition zone determination, and minimum inhibitory concentration and minimum bactericidal concentration assays indicated that the antibacterial activity of COS-All-Tio was greatly improved when compared to that of chitosan oligosaccharide (COS). The antibacterial mechanism investigation against S. putrefaciens (RMS1) revealed that COS-All-Tio could inhibit bacterial growth by influencing of membrane integrity. Such disturbance of membrane structure resulted in the leakage of intracellular substance of the bacteria. A strong synergistic antibacterial effect against S. putrefaciens (RMS1) was observed when COS-All-Tio was used in combination with food preservatives (e.g. ε-polylysine hydrochloride). Therefore, COS-All-Tio might have potential in shrimp preservation.     

Preparation, characterization, and antibacterial activity of oleic acid-grafted chitosan oligosaccharide nanoparticles

An oleic acid-grafted chitosan oligosaccharide (CSO-OA) with different degrees of amino substitution (DSs) was synthesized by the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. Fourier transform infrared spectroscopy (FT-IR) suggested the formation of an amide linkage between amino groups of chitosan oligosaccharide and carboxyl groups of oleic acid. The critical aggregation concentrations (CACs) of CSO-OA with 6%, 11%, and 21% DSs were 0.056, 0.042, and 0.028 mg·mL⁻¹, respectively. Nanoparticles prepared with the sonication method were characterized by means of transmission electron microscopy (TEM) and Zetasizer, and the antibacterial activity against Escherichia coli and Staphylococcus aureus was investigated. The results showed that the CSO-OA nanoparticles were in the range of 60–200 nm with satisfactory structural integrity. The particle size slightly decreased with the increase of DS of CSO-OA. The antibacterial trial showed that the nanoparticles had good antibacterial activity against E. coli and S. aureus.     

Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group

A novel fiber-reactive chitosan derivative was synthesized in two steps from a chitosan of low molecular weight and low degree of acetylation. First, a water-soluble chitosan derivative, N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC), was prepared by introducing quaternary ammonium salt groups on the amino groups of chitosan. This derivative was further modified by introducing functional (acrylamidomethyl) groups, which can form covalent bonds with cellulose under alkaline conditions, on the primary alcohol groups (C-6) of the chitosan backbone. The fiber-reactive chitosan derivative, O-acrylamidomethyl-HTCC (NMA-HTCC), showed complete bacterial reduction within 20 min at the concentration of 10ppm, when contacted with Staphylococcus aureus and Escherichia coli (1.5-2.5 x 10(5) colony forming units per milliliter [CFU/mL]).     

Hydroxyl radicals scavenging activity of N-substituted chitosan and quaternized chitosan

N-substituted chitosan and quaternized chitosan were synthesized and their antioxidant activity against hydroxyl radicals was assessed, respectively. Compared with the antioxidant activity of chitosan, the results indicated that the two kinds of chitosan derivatives had different scavenging ability on hydroxyl radicals, which should be related to the form of amido in the two kinds of chitosan derivatives.     

Preparation and antimicrobial activity of hydroxypropyl chitosan

Water-soluble hydroxypropyl chitosan (HPCS) derivatives with different degrees of substitution (DS) and weight-average molecular weight (Mw) were synthesized from chitosan and propylene epoxide under basic conditions. Their structure was characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis, which showed that both the OH groups at C-6 and C-3 and the NH2 group of chitosan were alkylated. The DS value of HPCS ranged from 1.5 to 3.1 and the Mw was between 2.1x10(4) and 9.2x10(4). In vitro antimicrobial activities of the HPCS derivatives were evaluated by the Kirby-Bauer disc diffusion method and the macrotube dilution broth method. The HPCS derivatives exhibited no inhibitory effect on two bacterial strains (Escherichia coli and Staphylococcus aureus); however, some inhibitory effect was found against four of the six pathogenic fruit fungi investigated. Some derivatives (HPCS1, HPCS2, HPCS3, HPCS3-1, and HPCS4) were effective against C. diplodiella and F. oxysporum. HPCS3-1 is the most effective one with MIC values of 5.0, 0.31, 0.31, and 0.16mg/mL against A. mali, C. diplodiella, F. oxysporum, and P. piricola, respectively. Antifungal effects were also observed for HPCS2 and HPCS3-1 against A. mali, as well as HPCS3 and HPCS3-1 against P. piricola. The results suggest that relatively lower DS and higher Mw value enhances the antifungal activity of HPCS derivatives.     

鲤鱼鱼精蛋白的提取及抑菌活性研究

以鲤鱼的成熟精巢为原料,经0.15 mol/L NaCl溶液浸提,硫酸解离,并分别经乙醇、丙酮分离提取鱼精蛋白。测定了鱼精蛋白对细菌和真菌的最低抑制浓度以及在不同pH条件下的抑菌特性,试验结果表明鱼精蛋白具有较好的热稳定性,与EDTA复合使用可以增强抑菌效果,在酱油中可以代替苯甲酸的防腐作用。     

壳寡糖对大肠杆菌抑菌活性研究

分析壳寡糖对大肠杆菌抑菌效果的影响因素.采用摇瓶法和ELISA板法对不同浓度的壳寡糖进行抑菌试验;比较不同pH、不同脱乙酰度的壳寡糖对大肠杆菌抑菌效果的差异;比较不同聚合度的单一聚合度壳寡糖抑菌效果的差异.壳寡糖浓度大于5 mg/mL时抑菌效果与同浓度苯甲酸钠相近;pH为4时,0.156 mg/mL的壳寡糖溶液抑菌活性即能超过90%;pH为7时,5 mg/mL的壳寡糖才能达到90%抑菌活性.脱乙酰度为95%时,5 mg/mL的壳寡糖溶液抑菌活性能超过97%;脱乙酰度为45%时,40 mg/mL的壳寡糖溶液抑菌活性仅有56%;聚合度大于4的单一聚合度壳寡糖40 mg/mL时抑菌活性能达到99%.结果表明:提高壳寡糖溶液浓度、降低pH、提高脱乙酰度,能提高壳寡糖的抑菌活性,单一聚合度壳寡糖聚合度越高,对大肠杆菌的抑制作用越强.此外,采用ELISA板的方法进行实验,即节省试药又方便快捷.     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts

Aims: The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. Methods and Results: A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4‐β‐glucosidase activity, but a strain dependent β‐glucanase activity was observed. Some Brettanomyces species did show 1,4‐β‐glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Conclusions: Pronounced exo‐β‐glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. Significance and Impact of the Study: The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo‐β‐glucanase activity. Higher activities can be found in Brettanomyces species with β‐glucosidase activity.     

Antimicrobial properties of essential oil of Satureja montana L. on pathogenic and spoilage yeasts

The essential oil of Satureja montana L. had a broad-spectrum of antimicrobial activity against 46 species of yeasts. This high and diffused activity could be used to control potential pathogenic and spoilage yeasts. The assay of MIC toward some pathogenic and spoilage yeasts showed a range values from 0.10 to 0.25 l ml^–1. The MIC and growth rate reduction assay were effective tests for quantitative evaluation of antimicrobial activity.     

Film-forming process and biocide assessment of high-molecular-weight chitosan as determined by combined ATR-FTIR spectroscopy and antimicrobial assays

This pioneering study reported about the film-forming properties of high-molecular-weight chitosan as followed in situ by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and has implications in fields such as biomedical, pharmaceutical, packaging, and coating applications. From the results, it was observed that immediately after dissolution in an acetic acid aqueous solution and subsequent casting over the ATR crystal, the formed carboxylate antimicrobial (-NH3+ -OOCH) species are not stable in the film formulation and become reduced over time; further assays confirmed previous research, which suggested that the presence and stability of these groups is strongly dependent, among other factors, on storage conditions. As-received chitosan and chitosan neutralized in NaOH films did not exhibit biocide performance towards Staphylococcus aureus. The antimicrobial tests were also found to strongly relate the presence of a sufficient quantity of these carboxylate groups to the chitosan activity as a biocide agent. Moreover, a novel methodology based on the use of a normalized infrared band centered at 1405 cm(-1) is proposed which can be correlated with the antimicrobial character of the biopolymer.     

Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity

Currently, novel antibiotics are urgently required to combat the emergence of drug‐resistant bacteria. Antimicrobial peptides with membrane‐lytic mechanism of action have attracted considerable interest. Anoplin, a natural α‐helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin‐4 composed of d ‐amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin‐4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin‐4 treatment relative to anoplin. In conclusion, anoplin‐4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chitosan potentiates the antimicrobial action of sodium benzoate on spoilage yeasts

AIMS: The objective of this study was to determine whether low concentrations of chitosan and benzoate in combination could be used to enhance the antimicrobial action of either compound alone against three spoilage yeasts in saline solutions. METHODS AND RESULTS: Saccharomyces exiguus, Saccharomycodes ludwigii and Torulaspora delbrueckii were suspended in 0.05 and 0.005% chitosan glutamate and 0.025% sodium benzoate, alone or in combination, in 0.9% saline solutions at pH 6.2 and 4.5. Survivor curves were constructed from viable counts determined periodically for up to 120 min. Chitosan at 0.005% almost doubled the extent of death caused by 0.025% benzoate alone, from about 1-2 log to about 2-4 log cfu ml(-1), depending on pH and target organism. CONCLUSIONS: Chitosan (0.005%) and 0.025% sodium benzoate acted synergistically against spoilage yeasts in saline solutions. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the natural compound chitosan may be useful as an adjunct in the potentiation of the biocidal efficacy of antimicrobial compounds such as benzoates.     

Selective toxicity of wyerone and other phytoalexins to gram-positive bacteria

The antibacterial activities of isoflavonoid (kievitone and phaseollin), flavonoid (hydroxyflavans), furanoacetylenic (wyerone), and sesquiterpenoid (capsidiol and rishitin), phytoalexins against eight Gram-negative and six Gram-positive bacteria were examined using the paper-disc antibiotic assay method. With the exception of capsidiol, which was inactive at the highest concentration tested (200,μg/disc) all of the phytoalexins were selectively toxic towards Gram-positive species. Wyerone and kievitone were generally more toxic than other phytoalexins; rishitin was the least active inhibitor.     

Antifungal properties of Schiff bases of chitosan, N-substituted chitosan and quaternized chitosan

Schiff bases of chitosan, N-substituted chitosan, and quaternized chitosan were synthesized and their antifungal properties were analyzed against Botrytis cinerea Pers. (B. cinerea pers.) and Colletotrichum lagenarium (Pass) Ell.et halst (C. lagenarium (Pass) Ell.et halst) based on the method of D. Jasso de Rodríguez and co-workers. The results showed that quaternized chitosan had better inhibitory properties than chitosan, Schiff bases of chitosan, and N-substituted chitosan.     

The influence of molecular weight of quaternized chitosan on antifungal activity

Quaternized chitosan derivatives with different molecular weights were synthesized in the laboratory. Subsequent experiments were conducted to test their antifungal activities against Botrytis cinerea Pers. (B. cinerea pers.) and Colletotrichum lagenarium (Pass) Ell.et halst (C. lagenarium (Pass) Ell.et halst). Our results indicate that quaternized chitosan derivatives have stronger antifungal activities than chitosan. Furthermore, quaternized chitosan derivatives with high molecular weight are shown to have even stronger antifungal activities than those with low molecular weight.     

Influences of disulfide connectivity on structure and antimicrobial activity of tachyplesin I

Tachyplesin I is a potent antimicrobial peptide with broad spectrum of antimicrobial activity. It has 2 disulfide bonds and can form 3 disulfide bond isomers. In this study, the structure and antimicrobial activity of 3 tachyplesin I isomers (tachyplesin I, 3C12C, 3C7C) were investigated using molecular dynamic simulations, circular dichroism structural study, as well as antimicrobial activity and hemolysis assay. Our results suggest that in comparison to the native peptide, the 2 isomers (3C12C, 3C7C) have substantial structural and activity variations. The native peptide is in the ribbon conformation, while 3C12C and 3C7C possess remarkably different secondary structures, which are referred as “globular” and “beads” isomers, respectively. The substantially decreased hemolysis effects for these 2 isomers is accompanied by significantly decreased anti‐gram‐positive bacterial activity.