Polysaccharides of the fungus Cunninghamella japonica mycelium

Preliminary data on the polysaccharide composition of mycelium of the submerged grown fungus Cunninghamella japonica (synonymous with C. echinulata) were obtained. Mild acid hydrolysis of the mycelium led to formation of glucose, mannose and galactose, whereas acid treatment under drastic conditions afforded glucosamine as the hydrolysis product of chitin and chitosan, the summary content of both glucosaminoglycans being estimated as about 35%. Sequential treatment of the mycelium with hot water, 2% aqueous NaOH and 10% AcOH gave rise to several polysaccharide fractions, which were characterized by their monosaccharide composition. The yield ofchitosan extracted by AcOH was negligible. Additional purification of the fraction obtained by the action of alkali afforded a polysaccharide preparation, which was shown to be a linear (1-->3)-alpha-D-glucopyranan according to the data of chemical methods of structural analysis and NMR spectroscopy. It was concluded that Cunninghamella japonica differs from several other known representatives of Mucorales by the presence of this alpha-D-glucan, as well as by low content of chitosan and polyuronides.     

Polysaccharide composition of mycelium and cell walls of the fungus <Emphasis Type="Italic">Penicillium roqueforti</Emphasis>

Preliminary data on the polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti grown by the method of submerged cultivation have been obtained. Mild acid hydrolysis of both mycelium and cell walls results in formation of glucose, mannose, and galactose, while the treatment with acid under severe conditions results in formation of glucosamine, a product of chitin hydrolysis, the content of which is 19% in the cell walls. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water and 1 M NaOH at room temperature; their monosaccharide composition was characterized. The main fraction extracted by alkali, according to the data of NMR spectroscopy, mass spectrometry, and the chemical methods of structural analysis, is a linear α-D-glucopyranan, where the blocks of (1 → 3)-bound glucose residues are linked by single bonds (1 → 4). Water-soluble polysaccharides contain the linear blocks of (1 → 5)-bound residues of β-galactofuranose, most probably attached to the mannan core. The findings are of interest for chemotaxonomy of Penicillium fungi.     

Polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti

Preliminary data on the polysaccharide composition of mycelium and cell walls of the submergedly grown fungus Penicillium roqueforti were obtained. Mild acid hydrolysis of mycelium and cell walls led to formation of glucose, mannose and galactose, whereas acid treatment under drastic conditions afforded glucosamine as the hydrolysis product of chitin, which content in the cell walls was estimated as 19%. Sequential treatment of the mycelium with hot water and 1 M NaOH at room temperature gave rise to several polysaccharide fractions, which were characterized by their monosaccharide composition. The main fraction obtained by the action of alkali, according to NMR spectroscopy, mass spectrometry and chemical methods of structural analysis data, is a linear alpha-D-glucopyranan containing blocks of (1 --> 3)-linked glucose residues interconnected by (1 --> 4)-linkages. Water-soluble polysaccharides contained linear blocks of (1 --> 5)-linked beta-galactofuranose residues, probably connected with a mannan core. The data obtained may be important for chemotaxonomy of the genus Penicillium.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

Comparative Study of Polysaccharides from Reproductive and Sterile Tissues of Five Brown Seaweeds

Sterile and reproductive tissues of five brown algae (Sargassum pallidum, Silvetia babingtonii, Fucus evanescens, Saccharina japonica, and Alaria ochotensis) from Russian Far East seas were compared for the content and monosaccharide composition of fucoidans as well as the content of laminarans and alginic acids. It was proved that reproduction has an apparent effect on fucoidan content and its monosaccharide composition. Fucoidan content in fertile tissues was shown to be 1.3–1.5 times as high as in sterile ones. Based on the present and previously reported data, we believe that fucoidan accumulation during the development of reproductive structure is a general trend for brown seaweeds. Reproduction also caused changes in the monosaccharide composition. As a result, fertile plants synthesized fucoidan with less heterogeneous monosaccharide composition in comparison with sterile ones. Structural changes of this polysaccharide are species-specific and perhaps depend on the type of the synthesized polysaccharide. The fertile plants of S. babingtonii yielded the highest fucoidan content [25% dry weight (dw)] among the tested species, whereas the maximum alginic acid content was found for the sterile tissues of S. japonica—up to 43% dw. There was no general trend in alginic acid content variation during sporulation. The alginic acid content was much higher in the sterile tissues of S. japonica in comparison with the fertile ones. On the contrary, the fertile tissues of A. ochotensis contained more alginic acid than the sterile ones.     

Comparative study of the chemical composition of ethanol extracts from brown algae and their effects on seedling growth and productivity of soya <Emphasis Type="Italic">Glycine max</Emphasis> (L.) MERR

A comparative analysis of the chemical composition of brown algae extracts has been performed. It was found that L. cichorioides is characterized by a high content of mannitol, glutaminic acid, and iodine. In the extracts obtained from C. costata, high levels of mineral substances and free amino acids were revealed. Extracts obtained from F. evanescens were rich with proteins and phenol compounds. Extracts obtained from L. japonica were characterized by uniquely high iodine and free amino acid contents, among which glutaminic acid prevailed. All studied extracts were shown to stimulate soy seedling growth and increase its productivity. The strongest stimulatory effect on seedling growth was from the extract obtained from L. japonica in a concentration of 100 μg/ml, whereby the growth of root and stem was increased by 20 and 26%, respectively, compared to the control values. Treatment of seeds with the extract of L. japonica in a concentration of 2 mg/ml led to an increase in productivity by 15.6%. Brown algae extracts can be recommended for agriculture to stimulate growth and prepare seeds for sowing.     

An improved method of protein isolation and proteome analysis with <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales) incubated under different pH conditions

The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.     

Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.     

Optimization of extraction process by response surface methodology and preliminary structural analysis of polysaccharides from defatted peanut (Arachis hypogaea) cakes

In this work, response surface methodology was used to determine optimum conditions for extraction of polysaccharides from defatted peanut cake. A central composite design including independent variables, such as extraction temperature (x₁), extraction time (x₂), and ethanol concentration (x₃) was used. Selected response which evaluates the extraction process was polysaccharide yield, and the second-order model obtained for polysaccharide yield revealed coefficient of determination of 97.81%. The independent variable with the largest effect on response was ethanol concentration (x₃). The optimum extraction conditions were found to be extraction temperature 48.7 °C, extraction time 1.52 h, and ethanol concentration of 61.9% (v/v), respectively. Under these conditions, the extraction efficiency of polysaccharide can increase to 25.89%. The results of structural analysis showed that the main composition of defatted peanut cake polysaccharide was α-galactose.     

The concentration of polysaccharides and proteins in EPS of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and <Emphasis Type="Italic">Aureobasidum pullulans</Emphasis> as revealed by <Superscript>13</Superscript>C CPMAS NMR spectroscopy

Extracellular polymeric substances were extracted from the bacterial strain Pseudomonas putida and the fungal species Aureobasidium pullulans using three different methods (formaldehyde–NaOH, ethylenediaminetetraacetic acid (EDTA) and cation-exchange-resin). The composition of the extracellular polymeric substances (EPS) was analysed by biochemical and high-resolution solid state ¹³C nuclear magnetic resonance (NMR) spectroscopic methods. The EPS yield was strongly dependent on the extraction method, with the formaldehyde–NaOH method showing the best extraction efficiency. The NMR method revealed that when using the EDTA extraction method, about 40% of the EDTA accumulated in the EPS and that was responsible for the apparent high extraction yields. EPS protein content determined by the NMR method was up to 30% higher than the protein content determined using the biochemical (Lowry) method for P. putida and for A. pullulans. The average protein carbon content determined by the NMR method was approximately 70% of the total carbon content. NMR results could be supported by elemental analysis, which showed a high nitrogen content (~10%) in the EPS. The carbohydrate carbon content detected with both methods in the cell aggregates and the EPS was approximately 20% in each. In this study, quantitative ¹³C cross-polarisation magic angle spinning NMR spectroscopy was conducted on unlabeled cell strains, and EPS and could be used to quantify protein and carbohydrate of different samples.     

Isolation and chemical characterization of algal polysaccharides from the green seaweed Ulva clathrata (Roth) C. Agardh

In order to obtain information on the content and composition of the water-soluble polysaccharides from Ulva clathrata, an extraction at 60°C, in different media, was performed: water, EDTA and HCl (F-I), each followed by a sequential extraction in NaOH 0.1 M (F-II). The extracts were recovered and analyzed for total carbohydrates, proteins, rhamnose, uronic acids and sulfate content. Differences were obtained in the yield and composition in both fractions of the different media (F-I and F-II). Higher yields resulted in the first fraction on all media. HCl extraction was the best in both fractions (14.83 ± 1.5% and 5.96 ± 1.1%, F-I and F-II, respectively). In all cases, F-I was more sulfated ranging from 27.87% to 35.8% and higher in rhamnose content, whereas F-II had higher protein and slightly higher uronic acid content. FTIR spectra showed that soluble polysaccharides from the green seaweed U. clathrata are sulfated polysaccharides, similar to ulvan obtained from other Ulva species and confirmed by the ¹ H-NMR spectrum, where the characteristic signal for the deoxy sugar (rhamnose) is present.     

Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Study of antioxidant activities of sulfated polysaccharides from Laminaria japonica

The composition, molecular weight and in vitro antioxidant activity of various sulfated polysaccharides obtained by anion exchange chromatography, acid hydrolysis and radical process degradation of the crude sulfated polysaccharide extracted from Laminaria japonica were compared. The low sulfated F-A2, with a peak-molecular weight (Mp) of 5–15 kDa, 14.5% sulfated ester and 21.8% glucuronic acid, exhibited a very strong antioxidant activity on superoxide and hydroxyl radicals, with activity even higher than that of large molecular weight fractions F-A and F-B. However, highly sulfated fractions with a peak-molecular weight below 15 kDa had much lower antioxidant activities than other fractions. These results indicated that the sulfate group of the low molecular weight fractions represents a physical block for the reaction with oxygen radicals. The chemical properties and antioxidant activities of sulfated polysaccharide fractions obtained by radical process degradation of crude sulfated polysaccharide were quite different from those obtained by acid hydrolysates. By radical process degradation, the high molecular weight was decreased to give LM2 (Mp 8 kDa) and LM1 (Mp 1.5 kDa), with a yield of 40% and 15%, respectively. LM2 was enriched with fucose and sulfated ester, while containing low amounts of glucuronic acid. The antioxidant activity showed that LM2 was unable to scavenge either superoxide or hydroxyl radical, which suggested that radical process degradation targeted mainly ascopyllan-like species rich in glucuronic acid, while the fraction rich in sulfated l-fucose remained unchanged. However, LM1 with Mp 1.5 kDa still retained apparent scavenging ability for superoxide radical, although it contained no glucuronic acid and certain amounts of galactose and mannose as main neutral sugars. These result suggest that the antioxidant activity of sulfated polysaccharides is apparently related not only to molecular weight and sulfated ester content, as previously determined, but also to glucuronic acid and fucose content.     

Pressurized water extraction of β‐glucan enriched fractions with bile acids‐binding capacities obtained from edible mushrooms

A pressurized water extraction (PWE) method was developed in order to extract β‐glucans with bile acids‐binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly β‐glucans (including chitooligosaccharides deriving from chitin hydrolysis), α‐glucans, and other PSCs (hetero‐/proteo‐glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids‐binding capacities of some extracts were similar to a β‐glucan enriched fraction obtained from cereals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:391–400, 2014     

Improvement in tannase recovery using enzymatic disruption of mycelium in combination with reverse micellar enzyme extraction

Summary A high activity tannase (tannin acyl hydrolase EC 3.1.1.20) is synthetized in high yield by Aspergillus niger LCF 8. At the production optimum, the tannase is strongly bound to the mycelium and detachment of the enzyme by classical physical and chemical means, largely failed. Enzymatic hydrolysis of cell walls using a chitinase from Streptomyces griseus followed by reverse micellar tannase extraction resulted in a recovery of 43% active enzyme, i.e. an improvement in yield of 2.5 from a previous process. Best conditions were enzymatic hydrolysis of mycelium with chitinase at pH 6.0 and 25°C for 2.5 h followed by tannase extraction at pH 7.5 with isooctane containing 80 mM cetyl trimethyl ammonium bromide and stripping at pH 4.0 in the presence of 0.35 M NaCl.     

A Suspension Culture Method for the Rapid Mass Culture of Cistella japonica Mycelium

Different methods were investigated for the rapid mass culture of Cistella japonica by using water extracts of some nutritional sources. In an agar culture test, there was little difference in mycelial growth in water extracts of wheat bran, rice bran and potato. In suspension culture with wheat bran extract, which is easily and cheaply available, the mycelium of C. japonica increased seven times more than that in agar culture after a month's incubation. C. japonica from suspension culture was pathogenic to Chamaecyparis obtusa. These results suggest that suspension culture in water extract of wheat bran can be adopted for the rapid mass culturing of C. japonica for use in inoculation tests.     

Seasonal variation of agar from <Emphasis Type="Italic">Gracilaria vermiculophylla,</Emphasis> effect of alkali treatment time,and stability of its Colagar

Gracilaria vermiculophylla, from Baja California Sur, Mexico, was studied in order to determine the seasonal variation of yield and quality of native and alkaline agar during 2007–2008. The highest alkaline agar yield was obtained in summer (17%) and the highest gel strength in spring (1,132 g cm⁻²). The highest melting temperature was 98°C (winter). The highest gelling temperature was 68°C (summer). The values obtained are within the range of the most important Gracilaria species harvested worldwide. During the agar extraction step, the best results were obtained after 30 min of alkali treatment with sodium hydroxide (7%), after which the quality decreased significantly. We produced Colagar from G. vermiculophylla which consists of the seaweeds treated with sodium hydroxide and dried. The yield and quality of the agar obtained from the Colagar shows stability in both yield and quality during 1 year of storage, suggesting that alkali treatment is a good method of avoiding agar hydrolysis during storage.     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Structural investigation of a polysaccharide released by the cyanobacterium <Emphasis Type="Italic">Nostoc insulare</Emphasis>

The structural investigation of an extracellular polysaccharide released during photoautotrophic growth by the cyanobacterium Nostoc insulare is reported. After 60 days of cultivation, an average yield of purified, desalted, and freeze-dried released polysaccharide (RPS) of 0.9 g L⁻¹ medium was obtained. The apparent hydrodynamic volume, determined for RPS, was 1.1 × 10⁶ Da, and the average molecular weight was 2.8 × 10⁶ Da. No sulfate and only traces of pyruvate and acetate groups were detectable. A protein content of only 0.7% indicates a high degree of purity of RPS. The following constituent uronic acids and sugars were identified: glucuronic acid (GlcA), glucose (Glc), arabinose (Ara), and for the first time, cyanobacterial RPSs 3-O-methyl-arabinose (3-O-Methyl-Ara). Adapted from linkage analyses of untreated RPS and of RPS treated by means of reduction of uronic acids, mild acid hydrolysis with oxalic acid, or lithium degradation, respectively, the following partial structure of RPS is proposed, which possesses an arborisation built by 1,3,4-Glcp and a side chain built by 3-O-Methyl-Araf: →1)-Glcp-(3→1)-Glcp-[(3→1)-3-O-Methyl-Araf](4→1)-GlcAp-(4→).