Isolation, partial characterization, and biological properties of polysaccharides from crude papain

Two polysaccharides have been isolated from crude papain by precipitation of papain with ammonium sulfate, further precipitation of other proteins with trichloroacetic acid, and chromatography of the supernatant on DEAE-cellulose. The first polysaccharide to be eluted, designated PP-I, contained D-glucuronic acid, D-glucose, D-galactose, L-arabinose, and L-rhamnose, in the approximate molar ratios of 4:1:12:10:4. The other (PP-II), eluted at a higher salt-concentration, contained the same sugars (with about one-third less glucose and more uronic acid) in the approximate molar ratios of 13:1:40:26:12. Reduction of the uronic acid groups of PP-II produced a polysaccharide (PP-II-R) containing the same sugars in the approximate molar ratios of 2:11:37:28:12. Hydrolysis of a mixture of the two polysaccharides yielded an aldobiouronic acid, D-glucosyluronic acid-D-galactose. Neither polysaccharide preparation contained protein. These polysaccharides dramatically affected aggregation and alignment of normal human fibroblasts but had no effect on a mouse embryo fibroblast aneuploid cell-line that does not exhibit contact inhibition of growth or movement. In aggregating cells, these polysaccharides caused the cells to behave as contact-inhibited cells, that is, cell division and nuclear area were decreased.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

羊肚菌多糖提取分离及生物活性研究进展*

羊肚菌(Morchella spp.)是一种珍稀食药用真菌,从羊肚菌中提取的多糖在抗癌、抗氧化、降血糖、降血脂及免疫调节等方面具有良好的生物活性,在食品、药品和保健品开发方面具有广阔的应用前景。羊肚菌多糖的有效提取是对其进行结构解析和生物活性研究的基础,不同的提取方式对羊肚菌多糖的结构和生物活性具有一定影响。羊肚菌多糖的结构特性如分子量、单糖组成、一级结构等,对其生物活性具有很大影响,因此研究羊肚菌多糖的结构对揭示其生物活性及作用机制具有重要意义。针对羊肚菌多糖进行综述,总结羊肚菌多糖提取分离、结构解析及生物活性的研究进展,分析羊肚菌多糖生物活性的作用机制,并对今后研究方向提出展望,以期为羊肚菌多糖的研究与开发提供理论基础。     

Bubble rise velocities and drag coefficients in non-Newtonian polysaccharide solutions.

Microbially produced polysaccharides have properties which are extremely useful in different applications. Polysaccharide producing fermentations start with liquid broths having Newtonian rheology and end as highly viscous non-Newtonian solutions. Since aerobic microorganisms are used to produce these polysaccharides, it is of great importance to know the mass transfer rate of oxygen from a rising air bubble to the liquid phase, where the microorganisms need the oxygen to grow. One of the most important parameters determining the oxygen transfer rate is the terminal rise velocity of air bubble. The dynamics of the rise of air bubbles in the aqueous solutions of different, mostly microbially produced polysaccharides was studied in this work. Solutions with a wide variety of polysaccharide concentrations and rheological properties were studied. The bubble sizes varied between 0.01 mm3 and 10 cm3. The terminal rise velocities as a function of air bubble volume were studied for 21 different polysaccharide solutions with different rheological properties. It was found that the terminal velocities reached a plateau at higher bubble volumes, and the value of the plateau was nearly constant, between 23 and 27 cm/s, for all solutions studied. The data were analyzed to produce the functional relationship between the drag coefficient and Reynolds number (drag curves). It was found out that all the experimental data obtained from 21 polysaccharide solutions (431 experimental points), can be represented by a new single drag curve. At low values of Reynolds numbers, below 1.0, this curve could be described by the modofoed Hadamard-Rybczynski model, while at Re > 60 the drag coefficient was a constant, equal to 0.95. The latter finding is similar to that observed for bubble rise in Newtonian liquids which was explained on the basis of the "solid bubble" approach.     

Concentration of the proteins of skimmed milk by membraneless,isobaric osmosis

The concentration of skimmed milk proteins by polysaccharides such as gum arabic, arabinogalactan and apple pectin with a high content of methoxyl groups was studied. Investigation of the thermodynamic compatibility of skimmed milk proteins with these polysaccharides at different NaCl concentrations and pH has shown that above a certain polysaccharide concentration termed the ‘threshold of complete incompatibility’ the protein is almost completely excluded from the polysaccharide phase. Phase diagrams obtained for the systems: water-skimmed milk proteins-arabinogalactan, water-skimmed milk proteins-gum arabic and water-skimmed milk proteins-pectin, indicate that highly esterified apple pectin is superior to the other polysaccharides for concentrating skimmed milk proteins.The proposed method of concentration which may be called ‘membraneless isobaric osmosis' has a higher productivity and lower energy consumption than other methods of biopolymer concentration.     

Glycerolphosphate-containing cell wall polysaccharides from Streptococcus sanguis

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of l-rhamnose, d-glucose, and N-acetyl d-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is l-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.     

Polysaccharide breakdown by mixed populations of human faecal bacteria

Measurements of polysaccharide-degrading activity in different fractions of human faeces showed that bacterial polysaccharidases and glycosidases were primarily associated with the washed bacterial fractions. Amylase, pectinase and xylanase were the major polysaccharide-hydrolysing enzymes detected, whilst α-L-arabinofuranosidase, β-D-xylosidase, β-D-galactosidase and β-D-glucosidase were the most active glycosidases. Starch and 3 non-starch polysaccharides (NSP; pectin, xylan and arabinogalactan) were fermented by mixed populations of human faecal bacteria in batch culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly degraded substrates and that arabinogalactan and the relatively insoluble polysaccharide xylan were broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the batch culture fermentations showed that the arabinose side chains of pectin, xylan and arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but extracellular polysaccharidase activity increased as the fermentations progressed. Molar ratios of acetate, propionate and butyrate produced in these experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate, propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were 50:22:29, 50:42:8, 82:15:3, and 84:14:2, respectively. The presence of starch did not inhibit the breakdown of arabinogalactan, xylan or pectin by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided with mixed polysaccharide substrates.     

奶油栓孔菌菌丝体多糖的提取工艺优化、结构表征及抗氧化活性

奶油栓孔菌Trametes lactinea是一种生物活性丰富的大型真菌。本研究在单因素试验的基础上,通过响应面法优化其菌丝体多糖的提取工艺,利用DEAE-Cellulose-52阴离子交换柱和Sephadex G-200层析柱对粗多糖进行分离纯化,获得TLMPS-0、TLMPS-1和TLMPS-3均一多糖组分。采用化学组成分析、UV-vis、FTIR、刚果红实验对3种多糖组分进行结构分析,并检测了多糖清除自由基的能力和还原力。结果表明,奶油栓孔菌菌丝体多糖最优提取工艺为：提取温度99℃、料液比1:30 (g/mL)、提取时间5h,提取次数2次。在此工艺条件下,多糖提取率为4.01%。TLMPS-0、TLMPS-1和TLMPS-3的糖醛酸含量分别为12.91%±0.44%、8.24%±0.22%、7.50%±0.66%,硫酸基含量分别为22.24%±1.88%、14.55%±0.56%、18.68%±0.69%,并且证明TLMPS-0是一种α-吡喃型多糖或β-吡喃型多糖,而TLMPS-1是一种β-吡喃型多糖,均不具备三螺旋空间构象,此外,3种多糖组分均具有一定的清除DPPH自由基、ABTS自由基、羟基自由基的能力和铁离子还原能力,其中TLMPS-0抗氧化活性最强。研究结果为奶油栓孔菌多糖的功能研究与挖掘提供了研究基础与理论依据。     

Preparation of Acetylated Grapefruit Peel Polysaccharide

Grapefruit peel polysaccharide has antioxidant, antitumor, hypoglycemic and other biological activities, and chemical modification can further improve the properties of the polysaccharide. Acetylation modification of polysaccharides has the advantages of simple operation, low cost and little pollution, and is widely used at present. Different degrees of acetylation modification have different effects on the properties of polysaccharides, so it is necessary to optimize the preparation technology of acetylated grapefruit peel polysaccharides. In this article, acetylated grapefruit peel polysaccharide was prepared by acetic anhydride method. With the degree of acetyl substitution as the evaluation index, combined with the analysis of sugar content and protein content in the polysaccharide before and after modification, the effects of three feeding ratios of 1:0.6, 1 : 1.2 and 1 : 1.8 (polysaccharide: acetic anhydride, mass/volume) on acetylation modification were explored through single factor experiments. The results showed that the optimum ratio of material to liquid for acetylation modification of grapefruit peel polysaccharide was 1:0.6. Under these conditions, the degree of substitution of acetylated grapefruit peel polysaccharide was 0.323, the sugar content was 59.50 % and the protein content was 1.038 %. The results provide some reference for the study of acetylated grapefruit peel polysaccharide.     

Pullulan from peat hydrolyzate fermentation kinetics

The Luedeking-Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, alpha, and beta varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant-feed fed-batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed-batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed-batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.     

Immunology of bacterial polysaccharide antigens

Carbohydrates in the form of capsular polysaccharides and/or lipopolysaccharides are the major components on the surface of bacteria. These molecules are important virulence factors in many bacteria isolated from infected persons. Immunity against these components confers protection against the disease. However, developing vaccines based on polysaccharides is difficult and several problems have to be solved. First of all, most of the bacterial polysaccharides are T-lymphocyte independent antigens. Anti-polysaccharide immune response is characterised by lack of T-lymphocyte memory, isotype restriction and delayed ontogeny. Children below 2 years of age and elderly respond poorly to polysaccharide antigens. Secondly, the wide structural heterogeneity among the polysaccharides within and between species is also a problem. Thirdly, some bacterial polysaccharides are poor immunogens in humans due to their structural similarities with glycolipids and glycoproteins present in man. The T-lymphocyte independent nature of a polysaccharide may be overcome by conjugating the native or depolymerised polysaccharide to a protein carrier. Such neoglycoconjugates have been proven to be efficient in inducing T-lymphocyte dependent immunity and to protect both infants as well as elderly from disease. Another approach to circumvent the T-lymphocyte independent property of polysaccharides is to select peptides mimicking the immunodominant structures. Several examples of such peptides have been described.     

皱皮木瓜多糖的分离、纯化及抗氧化活性研究

为了研究木瓜多糖的提取、分离、纯化与抗氧化活性,采用水提醇沉法提取皱皮木瓜中的多糖,得多糖Ⅰ;利用Sevag法除去多糖中的蛋白质后得多糖Ⅱ;以30%H_2O_2脱除色素后再次醇沉得到精制多糖Ⅲ;透析除去小分子后利用AB-8大孔树脂进行分离以水、30%、50%、70%和95%乙醇洗脱,其中水洗脱部分多糖为Ⅳ。用苯酚-硫酸法测定多糖含量。多糖Ⅰ得率为9.83%,多糖含量(纯度,下同)为64.45%;脱蛋白后多糖Ⅱ中多糖含量为78.23%;经脱色后多糖Ⅲ含量达88.39%;大孔树脂水洗脱部分多糖Ⅳ含量为89.74%。以DPPH(2,2-二苯基-1-苦肼基)清除率和Fe~(3+)还原力方法测定木瓜多糖的抗氧化活性,木瓜多糖均体现出一定的抗氧化作用,呈浓度依赖性增强,其中多糖Ⅰ、Ⅱ表现出更好的作用。     

Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides

Abstract The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-β-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolli (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.     

Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity

Elaboration of a capsule composed of one of a range of acidic polysaccharides is a common feature of many bacteria, particularly those capable of causing serious infections in humans. Biochemical and genetical analyses of capsule biogenesis in Escherichia coli are beginning to reveal new aspects of polysaccharide biosynthesis. Genes have been identified which are thought to encode products responsible for the translocation of these high molecular-weight polysaccharides across the cytoplasmic and outer membranes, and the organization of exported polysaccharide into a capsule. Their further analysis should provide new insights into membrane biology, particularly since the genes in question are absent from the often used laboratory strains of E. coli. Genetic analysis of capsule diversity is beginning to suggest possible mechanisms for the generation of the structural diversity of polysaccharides.     

Physical characteristics and antioxidant effect of polysaccharides extracted by boiling water and enzymolysis from Grifola frondosa

Grifola frondosa has been widely consumed in China and other Asian countries. Recent studies on G. frondosa have focused on the activities of polysaccharides extracted by water, and the activities of polysaccharides extracted by enzymolysis have not been studied. In this work, the relationship between the physical properties and antioxidant activity of polysaccharides extracted from G. frondosa by boiling water and enzymolysis was studied. Five polysaccharide extracts from the fruit body of G. frondosa were prepared by different extracting methods including boiling water, single enzyme enzymolysis with three different single enzymes (cellulose, pectinase, and pancreatin), and combined enzyme enzymolysis (cellulose:pectinase:pancreatin; 2:2:1). Characteristics such as the viscosity, Mw, polysaccharide content, protein content, infrared spectra, and antioxidant activities of the extracts were evaluated. The highest antioxidant activity was exhibited by the extracts prepared by combined enzyme extraction. The correlation analysis between antioxidant activity and polysaccharide content, protein content, Mw or viscosity indicated that the Mw had a more important role in antioxidant activity. Overall, the results indicate that the combined enzyme polysaccharide extracts can be developed as a new potential natural antioxidant.     

Molecular origin of two polysaccharides of Campylobacter jejuni 81116

The nature of the polysaccharide molecules of the human enteric pathogen Campylobacter jejuni has been the subject of debate. Previously, C. jejuni 81116 was shown to contain two different polysaccharides, one acidic (polysaccharide A) and the other neutral (polysaccharide B), occurring in a 3 : 1 ratio, respectively. The aim of this study was to determine the molecular origin of these polysaccharides. Using a combination of centrifugation, gel permeation chromatography, chemical assays, and (1)H-NMR analysis, polysaccharide B was shown to be derived from lipopolysaccharide and polysaccharide A from capsular polysaccharide. Thus, C. jejuni 81116 produces both lipopolysaccharide-like molecules and capsular polysaccharide.     

食药用真菌多糖及复合多糖生物活性研究

食药用真菌多糖有多种生物学功能,在抗肿瘤、免疫调节作用、抗衰老、降血脂等方面发挥着重要的生物活性.对正常细胞无毒副作用是食药用真菌多糖的突出优点.合适剂量食药用真菌多糖配伍使用时,各多糖间药理作用呈现协同性,可以提高在抗肿瘤、免疫调节等方面的药效.     

Polysaccharide lyases: recent developments as biotechnological tools

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.     

Structure and molecular weight of Asian lacquer polysaccharides

Structural analysis of Asian lacquer polysaccharides in Vietnam, Myanmar, Cambodia, Taiwan, and Japan was carried out by a combination of chemical and physical methods, and then their structures were compared with that of a Chinese lacquer polysaccharide reported previously. It was found that the structure of polysaccharides in China and Japan, Taiwan and Vietnam, Myanmar and Cambodia, was similar to each other. The polysaccharides in Myanmar and Cambodia had larger amounts of -arabinose and -rhamnose than those in other Asian lacquer polysaccharides. In addition, the degradation process of lacquer polysaccharide was revealed for the first time by the time-course of GPC measurements of polysaccharide in Aizu, Japan. The results suggest that the molecular weight of polysaccharide in lacquer tree had around with narrow molecular weight distribution and then decreased gradually into two molecular weight fractions of and 23×10³ in the proportion of 25 and 75 mol% isolated after 3 weeks.