Purification and properties of extracellular chitinases from the parasitic fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (+/-5) for P-1 and 31,134 (+/-6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5-4.0 and 50 degrees C for P-1 and 4.0-4.5 and 40 degrees C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65-74%) = glycol chitin > colloidal chitin = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B > glycol chitin = chitosan 10B > colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48-39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.     

Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0–9.0). The optimum temperature was 60°C and the enzymes were stable up to 50°C. The chitinases exhibited wide substrate specificities for chitin-related compounds.     

Anticoagulant activity of heterochitosans and their oligosaccharide sulfates

Three kinds of partially deacetylated heterochitosans, 90% deacetylated chitosan, 75% deacetylated chitosan and 50% deacetylated chitosan, were prepared from crab chitin by N-deacetylation with 40% sodium hydroxide solution for different durations. Nine kinds of heterochitooligosaccharides (hetero-COSs) with relatively high molecular weights (5,000–10,000 Da; 90-HMWCOSs, 75-HMWCOSs, and 50-HMWCOSs), medium molecular weights (1,000–5,000 Da; 90-MMWCOSs, 75-MMWCOSs, and 50-MMWCOSs), and low molecular weights (below 1,000 Da; 90-LMWCOSs, 75-LMWCOSs, and 50-LMWCOSs) were prepared using an ultrafiltration membrane reactor system, respectively. In addition, their sulfated derivatives were prepared by a method using a trimethylamine-sulfur trioxide, and the anticoagulant properties of the heterochitosans and their COS sulfates with different chain lengths and degrees of deacetylation were investigated. Clotting times in thrombin-time assay were prolonged in the presence of various concentrations of the heterochitosans and their COS sulfates using normal human plasma. The 90% deacetylated chitosan sulfate exhibited the highest anticoagulant activity among all the heterochitosans and their COS sulfates.     

Extraction of chitin from prawn shells and conversion to low molecular mass chitosan

Extraction and depolymerisation of chitin and chitosan from prawn shells was carried out using various chemical procedures. Sodium hydroxide and hydrochloric acid solutions were used for deproteination and demineralisation, respectively, while acetone was used for decolourisation. The amount of chitin and subsequently chitosan obtained was ∼35% and 25% respectively of the dry weight of the shells. The chitin was deacetylated using sodium hydroxide at 100 °C and the influence of the concentration of the reagent and duration of the reaction was investigated. The degree of deacetylation (DD) of the chitosan was evaluated by FTIR and NMR spectroscopy and the molecular mass distribution was determined by Gel Permeation Chromatography. It was found that the final DD was significantly higher using 50% sodium hydroxide solution (73% ± 9%) compared to 25% sodium hydroxide solution (40% ± 5%). It was noted also that the deacetylation reaction was more than 80% completed after 2 h but the chitosan produced had higher molecular mass while chitosan produced after 10 h had lower molecular mass and higher degree of deacetylation. The molecular mass distribution was bimodal for all the samples and consisted of a broad high molecular mass peak (peak 1) and a sharp low molecular mass peak (peak 2). The Mw of peak 1 decreased from ∼1.3 × 10⁶ after 2 h reaction with sodium hydroxide to 3.1 × 10⁵ after 10 h reaction indicating that depolymerisation and deacetylation occurred simultaneously. Peak 2 had a Mw of ∼2.4–9.9 × 10³.     

非晶化甲壳素脱乙酰制备高脱乙酰度壳聚糖

对甲壳素进行超微粉碎处理,通过控制碾磨时间,得到结晶度为80.91%、58.06%、31.94%和8.09%等4种甲壳素细粉,再对其和普通粉碎甲壳素的非均相脱乙酰制备壳聚糖的反应进行对比研究。结果表明,随着甲壳素样品结晶度的降低,在相同的脱乙酰反应条件下制备的壳聚糖的脱乙酰度更高。采用单次碱处理的方式,使用普通粉碎甲壳素得到的壳聚糖脱乙酰度为84%,使用结晶度为31.94%和8.09%的甲壳素细粉,壳聚糖脱乙酰度可达90%以上。动力学研究分析,普通粉碎甲壳素和超微粉碎处理得到的4种甲壳素细粉非均相脱乙酰反应的活化能分别为58.71、47.23、42.30、35.44和31.73 kJ/mol,即反应的活化能随结晶度的降低而降低,表明非晶化处理能增强甲壳素非均相脱乙酰反应活性。     

Effect of chitin and chitosan on gelling properties of surimi from barred garfish (Hemiramphus far)

The addition of chitin/chitosan significantly increased the breaking force and deformation of gels prepared from barred garfish surimi (P < 0.05). Addition of 7B chitosan with 65.6% degree of deacetylation (% DD) at the level of 15 mg g⁻¹ resulted in the maximum increases in both breaking force and deformation of suwari and kamaboko gels compared to the control and gels containing chitin or chitosan with other % DD (P < 0.05). A chitosan concentration of 10 mg g⁻¹ was found to render the highest breaking force of kamaboko gel compared to other concentrations tested (P < 0.05). Kamaboko gel containing chitosan had an increased breaking force as the calcium chloride concentration increased (P < 0.05), indicating the role of endogenous transglutaminase in cross‐linking of protein–protein and protein–chitosan conjugates. Therefore the incorporation of chitosan and calcium chloride greatly improved the gelling properties of surimi from barred garfish without changes in colour. © 2000 Society of Chemical Industry     

Chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables

Chitosan has recently gained more interest due to its applications in food and pharmaceutics. Among others, the antimicrobial activity of chitosan has been pointed out as one of its most interesting properties of chitosan.The aim of this study was threefold: (1) the quantification of the antimicrobial effect of chitosan with a deacetylation degree of 94% and a molecular weight of 43 kDa on different psychrotrophic spoilage organisms and food pathogens. (2) The determination of the influence of different food components (starch, whey protein, NaCl and oil) on the antimicrobial effect of chitosan and (3) the investigation of the effects of chitosan coatings on controlling decay of minimally processed fruits and vegetables (strawberry and lettuce). For the first aim several bacteria and yeast were exposed to chitosan concentrations varying from 40 to 750 mg/l. Generally, Gram-negative bacteria seemed to be very sensitive for the applied chitosan (MIC0.006% (w/v)) while the sensitivity of Gram-positive bacteria was highly variable and that of yeast was intermediary (0.01% (w/v)). To achieve the second aim, the media, with one of these components added, were inoculated with Candida lambica (±2 log cfu/ml) and were incubated at 7°C until the yeast reached the stationary phase. Starch, whey proteins and NaCl had a negative effect on the antimicrobial activity. Oil conversely had no influence. For the third aim, the chitosan coating was formed by dipping the products in a chitosan–lactic acid/Na-lactate solution from which the pH was adjusted to the pH of the products. These products were equilibrium modified atmosphere (EMA)-packaged, stored at 7°C and during storage sensorially and microbiologically evaluated. A chitosan coating on strawberries was applicable while on mixed lettuce the chitosan coating was not applicable due to the development of a bitter taste. The microbiological load on the chitosan-dipped samples was lower for both products. The antimicrobial effect of chitosan on lettuce disappeared after 4 days of storage, while it maintained on the strawberries during 12 days.     

Characterisation of 2-Cys peroxiredoxin isozyme (Prx1) from Taiwanofungus camphorata (Niu-chang-chih): Expression and enzyme properties

Peroxiredoxins (Prxs) are a family of antioxidant peroxidases. The functions of Prxs comprises of cell protection against oxidative stress and regulation of cell proliferation. A putative 2-Cys Prx isozyme (Prx1) cDNA was cloned from Taiwanofungus camphorata (commonly known as Niu-chang-chih in Taiwan). The deduced amino acid sequence is conserved amongst the reported Prxs. A 3-D homology structure was created for this Prx1. To characterise the T. camphorata Prx1, the coding region was subcloned into a pAVD10 and transformed into Escherichia coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni²⁺-nitrilotriacetic acid sepharose. The purified enzyme showed two forms using a 15% SDS–PAGE. The enzyme retained 60% activity at 60 °C for 2.5 min. The enzyme was stable under a broad pH range from 5 to 11. The enzyme showed 57% activity after 40 min of incubation at 37 °C with trypsin. The ability of the enzyme to protect intact supercoiled plasmid DNA from ^·OH induced nicking was demonstrated.     

蛹皮壳聚糖的制备及在毛织物艾蒿染色中的应用

以柞蚕蛹皮为原料制取壳聚糖,并用该壳聚糖预处理羊毛织物进行染色,探讨壳聚糖预处理对羊毛织物染色性能的影响。柞蚕蛹皮采用稀盐酸、稀氢氧化钠进行脱钙、脱脂肪制备甲壳素,采用浓氢氧化钠溶液脱乙酰基,双氧水脱色工艺,制备脱乙酰度90%的乳白色壳聚糖产品,并用正交试验法优化脱乙酰基工艺。结果表明,甲壳素脱乙酰基的最佳工艺为:氢氧化钠质量分数50%,处理温度100℃,时间9 h,甲壳素与碱液质量比1∶20;经壳聚糖预处理后,羊毛织物艾蒿染色的染色深度明显提高,皂洗和摩擦色牢度基本不变。     

A novel thermostable chitinase (PJC) from pomegranate (Punica granatum) juice

A novel chitinase was isolated and purified to its homogeneity from pomegranate juice by a combination of ammonium sulphate precipitation and ion-exchange chromatography. The pomegranate juice chitinase (PJC) was purified to specific activity of 14.5 U/mg and a recovery of 34%. The monomeric protein migrated on SDS–PAGE at 29 kDa. The enzyme was found to be glycosylated (7.2%). It exhibited optimal activity at pH 4.5 and 70 °C. The enzyme was stable in the pH range 3.0–9.0 and up to 65 °C. The internal peptide sequence results suggest that the purified PJC shared high homology with class III chitinases of other known plant chitinases. The purified enzyme could hydrolyse colloidal chitin to its oligomers. It did not exhibit any antifungal activity.     

Effect of lactic acid on Listeria monocytogenes and Edwardsiella tarda attached to catfish skin

This study evaluated the use of lactic acid to decontaminate Listeria monocytogenes andEdwardsiella tarda attached to catfish skin with or without mucus. At the highest inoculum levels (10⁴–10⁵cfu skin⁻¹), lactic acid (0·5–2·0%) exposure for 10 min reduced counts of L. monocytogenes firmly attached to catfish skin by 0·9–>1·9 log₁₀cfu skin⁻¹and cells loosely attached by 2·7–>3·7 logs. Counts of E. tarda firmly attached to catfish skin were reduced by 0·9–>3·0 logs and cells loosely attached by 1·5–>3·5 logs. Overall bacterial numbers of lactic acid-treated cells that were firmly attached to skin with mucus were higher than on skin without mucus. Firmly attached L. monocytogenes was more resistant to lactic acid than was firmly attached E. tarda. Catfish skin mucus decreased the antimicrobial effect of lactic acid against attached L. monocytogenes and E. tarda.     

超声波预处理醇碱体系进行甲壳素脱乙酰工艺的研究

以自溶法脱蛋白、EDTA·Na2螯合脱钙制备的甲壳素为原料,采用超声波预处理醇碱体系进行甲壳素脱乙酰工艺的研究.以脱乙酰度(D.D.)为考察指标,依据单因素实验结果,采用L9(34)正交试验考察乙醇浓度(A)、氢氧化钠质量浓度(B)、反应时间(C)及超声波处理时间(D)对脱乙酰度(D.D.)的影响.实验结果表明脱乙酰工...     

Synthesis and partial characterization of octenylsuccinic starch from Phaseolus lunatus

Phaseolus lunatus starch was modified by esterification with octenyl succinic anhydride (OSA) and reaction effect evaluated in terms of chemical composition, gelatinization, pasting and emulsification properties. Succinylation was done using a 2³ factorial design with four replicates of the central treatment. Evaluated factors and levels were OSA concentration (1% and 3%), pH (7 and 9) and reaction time (30 and 60 min). Succinyl group percentage was the response variable. The optimum treatment was a reaction with 3% OSA at pH 7 for 30 min, which produced 0.5083% succinyl groups and 0.0083° of substitution. No significant changes were observed in proximate composition between the native and derivative starches. Apparent amylose level decreased notably from 32.4% to 23.6% due to OSA inclusion. Succinylation decreased starch gelatinization temperature (75.3–64.6 °C), decreased enthalpy (10.7–9.7 J/g), increased viscosity (700–1000 BU), increased emulsifying capacity (0.47–0.53 ml oil/ml sample), and made emulsions more stable over time. Starch modification did not, however, improve stability in heating–cooling processes.     

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO₂. The sensitivity to 20% CO₂ was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO₂ was combined with 2% O₂ and in 3 weeks when combined with “0”% O₂ (the remaining atmosphere was made up from N₂). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m²/24 h and the atmospheres were 2% O₂/20% CO₂ and “0”% O₂/20% CO₂. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h and “0”% O₂/20 % CO₂) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m²/24 h and 2% O₂ resulted in cereulide concentrations of 0.816 – 1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h, “0”% O₂/20 % CO₂) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production.Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.     

Inactivation of Escherichia coli O157:H7 on surface-uninjured and -injured green pepper (Capsicum annuum L.) by chlorine dioxide gas as demonstrated by confocal laser scanning microscopy

Cells of Escherichia coli O157:H7 on uninjured and injured surfaces of green pepper were inactivated by 0·15–1·2 mg l⁻¹ClO₂gas treatments. A membrane-surface-plating method was used for resuscitation and enumeration of E. coli O157:H7 treated with ClO₂. The location and viability ofE. coli O157:H7 on uninjured and injured green pepper surfaces after ClO₂gas treatments were visualized using confocal laser scanning microscopy (CLSM). Live and dead cells of E. coli O157:H7 on pepper surfaces were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively. A 7·27 log reduction of E. coli O157:H7 on uninjured green pepper surfaces was obtained with a 0·60 mg l⁻¹ClO₂gas treatment for 30 min at 20°C under 90–95% relative humidity. For injured surfaces, a 6·45 log reduction was achieved with a 1·2 mg l⁻¹ClO₂gas treatment. Each ClO₂gas treatment (0·15–1·2 mg l⁻¹ClO₂) for inoculated bacteria on uninjured surfaces showed significantly more reductions (1·23–4·24 log) than for those on injured surfaces (P<0·05). The microphotographs of CLSM showed that bacteria preferentially attached to injured surfaces and those bacteria could be protected from bacterial reduction by the injuries. This study indicates that ClO₂gas treatment can be a potential effective method of pathogen reduction for fresh fruits and vegetables.     

球磨、超声和盐酸处理对几丁质的微观结构和酶促脱乙酰效率的影响

几丁质是地球上第二丰富的天然多糖,可经过脱乙酰生成壳聚糖。但是,其结晶度高、溶解性差,使其酶促脱乙酰效率很低。为研究不同处理对几丁质的微观结构和酶促脱乙酰效率的影响,本实验分别对几丁质进行了球磨、超声和盐酸改性处理。首先确定球磨和超声改性的最佳条件;然后使用傅里叶变换红外光谱、元素分析、X射线衍射、热重-差示扫描量热法和扫描电子显微镜对改性几丁质的微观结构进行表征;最后测定红球菌11-3的几丁质脱乙酰酶对改性几丁质的脱乙酰效率。结果表明,球磨和超声改性的最佳条件分别为1 800 r/min、45 min和400 W、45 min。3种几丁质改性方法中,球磨几丁质改性效果最好,与原几丁质相比,黏均分子质量降低了88.14%;几丁质分子间氢键网络被破坏,部分糖苷键断裂;脱乙酰度有所增加;结晶度由96.30%降低至73.04%;热稳定性被破坏;结构呈现堆叠现象,变得疏松多孔。此外,几丁质脱乙酰酶对球磨几丁质的脱乙酰效率更高,乙酸产量较原几丁质提高了2.40倍。综上,球磨处理可以更有效地改变天然几丁质的理化性质和微观结构,从而提高几丁质的酶促脱乙酰效率。     

甲壳素传统法与微波法制备壳聚糖的比较

针对甲壳素脱乙酰制备壳聚糖采用的两种方法——微波法与传统法,从反应时间、壳聚糖的得率和品质(脱乙酰度和粘均分子量)等方面进行了比较分析。结果表明,甲壳素传统法一次脱乙酰反应很难制备脱乙酰度大于78%高粘均分子量的壳聚糖;与传统方法相比,甲壳素微波法脱乙酰制备壳聚糖不仅大大减少了反应时间,同时还能避免高温长时间处理导致壳聚糖产品的分子量和粘度下降,从而提高壳聚糖的质量指标。     

Response surface methodology for optimization of extraction yield, viscosity, hue and emulsion stability of mucilage extracted from Lepidium perfoliatum seeds

Response surface methodology was used to determine the optimum processing conditions that give maximum extraction yield, viscosity, hue and emulsion stability, as well as, minimum protein content for the gum extracted from Lepidium perfoliatum seed. Temperature (45–75 °C), processing time (1.5–3.5h), pH (5–8) and water to seed ratio (30:1–60:1) were the factors investigated. Experiments were designed according to Central Composite Rotatable Design with these four factors, including central and axial points. For each response, a second-order polynomial model was developed using multiple linear regression analysis. Applying desirability function method, optimum operating conditions were found to be extraction temperature of 48.1 °C, pH of 8, water to seed ratio of 30:1 and process time of 1.5 h. At this optimum point, extraction yield, viscosity, protein content, hue and emulsion stability were found to be 17.36%, 463.07 mPa s, 2.84%, 60.47 and 88.96 %, respectively.     

Heat-stable proteases from psychrotrophic pseudomonads: secondary structure and heat stability

Circular dichroism (CD) spectral analysis of a purified protease of Pseudomonas fluorescens T16 was carried out at different temperatures to determine the relationship between its secondary structure and its heat-stability. The protease protein was found to be 33% β-structure and 67% random coil. The protein lacked α-helical structure. Unfolding of the enzyme molecule was increased from 25°C, with maximum unfolding at 45°C. The enzyme exhibited maximum inactivation (low temperature inactivation [LTI] phenomenon) at temperatures between 45 and 55°C. At higher temperatures (60–95°C) the protease appears to undergo an additional conformational change to a more ordered stable structure. Metal ions such as Ca²⁺ appeared to be involved in structural stabilization and are required for protection against heat inactivation. The comparisons of amino acid composition, sequence homology, hydrophobicity index and polarities of heat-stable proteases were made to predict their heat-stabilities.     

Inactivation of Listeria monocytogenes during drying and storage of peach slices treated with acidic or sodium metabisulfite solutions

This study evaluated whether treating inoculated peach slices with metabisulfite or acidic solutions enhanced inactivation of Listeria monocytogenes during dehydration and storage. Inoculated (five strain mixture of L. monocytogenes, 7.9 log cfu/g) peach slices were treated, dried for 6 h at 60°C and stored aerobically at 25°C for 14 d. Predrying treatments of inoculated peach slices included: (1) no treatment (control); or 10 min immersion in: (2) sterile water, (3) 4.18% sodium metabisulfite, (4) 3.40% ascorbic acid, or (5) 0.21% citric acid solutions. Samples were plated on tryptic soy agar with 0.1% pyruvate (TSAP) and PALCAM agar for enumeration of surviving bacteria. Immersion in sterile water reduced bacterial populations on peach slices by 0.7 log cfu/g (TSAP and PALCAM). Immersion in the sodium metabisulfite solution reduced populations by 1.5–2.0 log cfu/g, while acidic pretreatments reduced populations by 0.5–0.8 log cfu/g. After 6 h of dehydration, populations on control or water immersed slices were reduced by 3.2–3.4 log cfu/g, whereas populations on slices treated with sodium metabisulfite or acidic solutions were reduced by 4.3–5.1 log cfu/g (TSAP) and 5.3–6.2 log cfu/g (PALCAM), respectively. Bacteria were detectable by direct plating at 14 d of storage, except on acid treated slices. Immersion in acidic or metabisulfite solutions, before dehydration, should enhance inactivation of L. monocytogenes contamination on peach slices during dehydration and storage.