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Microbial Exo-Polysaccharides for Biomedical Applications
Abstract
The productions and applications of various microbial exopolysaccharides have been under intensive researches over the past few decades. Some of these exopolysaccharides are commercial available and some are currently under intensive development; they include ionic heteropolysaccharide and neutral homopolysaccharide. These extracellular polymers constitute a structurally diverse class of biological macromolecules with a wide range of physiochemical properties which are the basis for the different applications in the broad fields of pharmacy and medicine. They have found applications in such diverse biomedical fields as ophthalmology, orthopedic surgery, tissue engineering, implantation of medical devices and artificial organs, prostheses, dentistry, bone repair and drug delivery.
... Moreover, they are highly susceptible to natural biodegradation and are less harmful than synthetic polymers. Their applications are diverse, ranging from the laboratory through clinical to tableting; they have found applications in such diverse bio-medical fields as ophthalmology, orthopedic surgery, tissue engineering, implantation of medical devices and artificial organs, prostheses, dentistry, bone repair and many other medical fields [5]. In addition, they have therapeutic and pharmaceutical uses in that they enable the controlled, slow-release of drugs into the body. ...
... In addition, they have therapeutic and pharmaceutical uses in that they enable the controlled, slow-release of drugs into the body. They also make possible targeting of drugs into sites of inflammation or tumors for disease treatment, and they can be used for skin rejuvenation and wound healing [5,32]. Several of these microbial polysaccharides are commercial industrial products, whereas others are in various stages of developments. ...
This review provides a comprehensive summary of the most recent developments of various aspects (i.e., production, purification, structure, and bioactivity) of the exopolysaccharides (EPSs) from Paenibacillus spp. For the production, in particular, squid pen waste was first utilized successfully to produce a high yield of inexpensive EPSs from Paenibacillus sp. TKU023 and P. macerans TKU029. In addition, this technology for EPS production is prevailing because it is more environmentally friendly. The Paenibacillus spp. EPSs reported from various references constitute a structurally diverse class of biological macromolecules with different applications in the broad fields of pharmacy, cosmetics and bioremediation. The EPS produced by P. macerans TKU029 can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics. However, the relationships between the structures and activities of these EPSs in many studies are not well established. The contents and data in this review will serve as useful references for further investigation, production, structure and application of Paenibacillus spp. EPSs in various fields.
... Polysaccharides cause a multicomponent defensive response that alters resistivity levels (Moscovici 2015), and these natural polysaccharides are less hazardous than synthetic polymers because they are biodegradable. In current years, considerable development has been made in looking for microbial exopolysaccharides (EPSs) for their novelty and high functional properties (Shih 2010). Bacterial exopolysaccharides (EPS) are water-soluble polymeric sugars exported from the bacteria and linked to the external surface or adhered to the outer cell membrane. ...
Microorganisms produce a diverse group of biomolecules having amphipathic nature (amphiphiles). Microbial amphiphiles, including amyloids, bio-surfactants, and other exo-polymeric substances, play a crucial role in various biological processes and have gained significant attention recently. Although diverse in biochemical composition, these amphiphiles have been reported for common microbial traits like biofilm formation and pathogenicity due to their ability to act as surface active agents with active interfacial properties essential for microbes to grow in various niches. This enables microbes to reduce surface tension, emulsification, dispersion, and attachment at the interface. In this report, the ecological importance and biotechnological usage of important amphiphiles have been discussed. The low molecular weight amphiphiles like biosurfactants, siderophores, and peptides showing helical and antimicrobial activities have been extensively reported for their ability to work as quorum-sensing mediators. While high molecular weight amphiphiles make up amyloid fibers, exopolysaccharides, liposomes, or magnetosomes have been shown to have a significant influence in deciding microbial physiology and survival. In this report, we have discussed the functional similarities and biochemical variations of several amphipathic biomolecules produced by microbes, and the present report shows these amphiphiles showing polyphyletic and ecophysiological groups of microorganisms and hence can `be replaced in biotechnological applications depending on the compatibility of the processes.
... In medicine, levan has shown promise as a plasma substitute, drug activity prolongator, and antihyperlipidemic agent [19]. Although a number of microorganisms are capable of producing levan, including Bacillus polymyxa [20], Acetobacter xylinum [21], Lactobacillus sanfranciscensis [22], Leuconostoc mesenteroides [23], Microbacterium laevaniformans [24], Zymomonas mobilis [25], B. subtilis [26], B. amyloliquefaciens [27], and Pseudomonas syringae [28]; relatively few have been demonstrated to produce levan suitable for food use or in yields of necessary to support commercial production. These restrictions have limited the application of levan. ...
Background:
We have previously isolated Bacillus subtilis HMNig-2 and MENO2 strains, from honey and the honeybee gut microbiome respectively, and demonstrated these strains to produce levansucrase with potential probiotics characteristics. Here we report their complete genome sequence and comparative analysis with other and other B. subtilis strains.
Results:
The complete genome sequences of Bacillus subtilis HMNig-2 and MENO2 were de novo assembled from MiSeq paired-end sequence reads and annotated using the RAST tool. Whole-genome alignments were performed to identify functional differences associated with their potential use as probiotics.
Conclusions:
The comparative analysis and the availability of the genome sequence of these two strains will provide comprehensive analysis about the diversity of these valuable Bacillus strains and the possible impact of the environment on bacterial evolution.
Significance and impact of study:
We introduce complete genome sequence of two new B. subtilis strains HMNig-2 and MENO2 with probiotic potential and as cell factories for the production of levan and other valuable components for pharmaceutical and industrial applications.
... Natural polysaccharides can be derived from renewable biomass like algae or plants, or from the fermentation of bacterial or fungal cultures which are harvested as microbial polysaccharides (Moscovici, 2015). Compared to algal or plant sources, microbial sources are increasingly favoured for their high yielding commercial production procedures (Shih, 2010). ...
Hydrogels as artificial biomaterial scaffolds offer a much favoured 3D microenvironment for tissue engineering and regenerative medicine (TERM). Towards biomimicry of the native ECM, polysaccharides from Nature have been proposed as ideal surrogates given their biocompatibility. In particular, derivatives from microbial sources have emerged as economical and sustainable biomaterials due to their fast and high yielding production procedures. Despite these merits, microbial polysaccharides do not interact biologically with human tissues, a critical limitation hampering their translation into paradigmatic scaffolds for in vitro 3D cell culture. To overcome this, chemical and biological functionalization of polysaccharide scaffolds have been explored extensively. This review outlines the most recent strategies in the preparation of biofunctionalized gellan gum, xanthan gum and dextran hydrogels fabricated exclusively via material blending. Using inorganic or organic materials, we discuss the impact of these approaches on cell adhesion, proliferation and viability of anchorage-dependent cells for various TERM applications.’
... In recent years, considerable progress has been achieved in the search for microbial exopolysaccharides (EPSs) that possess unique and highly functional properties (Shih, 2010). Compared with synthetic polymers, natural polysaccharides are eco-friendly, because they are biocompatible and highly susceptible to biodegradation. ...
Paenibacillus polymyxa 92, isolated from wheat roots, produced large amounts (38.4 g L-1) of exopolysaccharide (EPS) in a liquid nutrient medium containing 10 % (w/v) sucrose. The EPS was precipitated from the culture broth with cold acetone and was purified by gel filtration and anion-exchange chromatography. The molecular mass of the EPS was 2.29-1.10 × 105 Da. Diffuse reflectance infrared Fourier transform and nuclear magnetic resonance spectra showed that the EPS was a linear β-(2→6)-linked fructan (levan). Aqueous EPS solutions showed pseudoplastic behavior when shear stress was applied at different temperatures. By using the Ostwald-de Waele model, the rheological characteristics of the EPS solution were ascertained. The sorption capacity of the EPS for Zn(II), Cd(II), Pb(II), and Cu(II) was investigated. Sorption was maximal (q = 481 mg g-1) for Cu(II) ions. In model experiments, treatment of wheat seeds with EPS solution significantly increased the length of seedling roots and shoots.
... Natural polysaccharides are highly susceptible to biodegradation and are less harmful than synthetic polymers (Liang and Wang 2015). In recent years, substantial progress has been made in searching for microbial exopolysaccharides (EPSs) that possess novel and highly functional properties (Shih 2010). The surface localization of the EPSs makes them mediators of intercellular interactions with micro-and macroorganisms. ...
Bacterial polysaccharides are promising stimulants of protective functions in humans and animals. We investigated the ability of exopolysaccharide from the rhizobacterium Paenibacillus polymyxa CCM 1465 to induce nonspecific resistance factors in the macroorganism. We examined in vitro the effect of the exopolysaccharide, produced with different carbon sources, on the phagocytic activity of murine macrophages, on the generation of reactive oxygen species and of enzymes (acid phosphatase and myeloperoxidase), on the proliferation of murine splenocytes, and on the synthesis of proinflammatory cytokines [interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)] by human mononuclear cells. The exopolysaccharide promoted the phagocytosis of bacterial cells, activated metabolic processes in human and animal leukocytes, and moderately affected the production of TNF-α and IL-1β. The exopolysaccharides produced on media with glucose and sucrose differed in their effect on the immune cells, possibly owing to their different compositions, structures, and properties. The results validly indicate that the exopolysaccharide of P. polymyxa CCM 1465 promotes nonspecific immunity. Therefore, it can find application as a biologically active immunomodulatory substance.
... Currently, the microbial polysaccharides are widely used in the various fields of the human activity, such as medicinal 1 , pharmaceutical 2 , food, textile and chemical industries, oil production, and hydrometallurgy, to obtain the composite materials 3,4 . Despite advances in biotechnology, the number of commercially available microbial polysaccharides is extremely limited. ...
Using the classic biotechnological methods, the dependence of A. vinelandii D-05 culture alginate production from the media carbon and nitrogen content was investigated. The maximal alginate production was observed during cultivation bacterium in the medium with 2 to 4% of sucrose, but the maximal growth was found in the medium with 4% glucose. It was found that for the alginate production the optimal nitrogen contents could take from 0.05% yeast extract (carbon: nitrogen ratio 168:1). For the first time we demonstrated possibility the A. vinelandii growth during the cultivation in a medium with molasses (a by-product of sugar production) and the significant polysaccharide production (16.6 g/l) was obtained. It was established, that A. vinelandii culture broth could be used as a biological binder for obtaining the biocomposite materials.
... Microbial extracellular polysaccharides (EPS) are normally soluble, biodegradable and non-toxic biopolymers that have special properties, such as antitumor, antioxidant, anti-inflammatory, and antibacterial activities [1][2][3]. Currently, EPS are being used as the active ingredients of some biological medicines, functional foods, food additives and polysaccharide vaccines, and function as microbial flocculants, film-forming agents, antistaling agents, emulsifiers and antioxidants as well as anti-aging, anti-radiation and anti-cancer agents [4][5][6][7]. ...
Industrial grade soluble corn starch was used directly and effectively as the fermentation substrate for microbial exopolysaccharides production. Bacillus subtilis mutant strain NJ308 grew with untreated starch raw material as the sole carbon source. The real-time PCR results demonstrated that up-regulated genes encoding N-acetylglucosaminyltransferase, mannosyltransferase, and N-acetylglucosamine-1-phosphate uridyltransferase were the key elements of B. subtilis mutant strain NJ308 for exopolysaccharides production from industrial grade starch. Subsequently, the culture conditions for B. subtilis NJ308 were optimized using Plackett–Burman design and central composite design methods, and the related key genes in the synthesis pathway of exopolysaccharides from the starch raw material were analyzed by real-time PCR. The maximum exopolysaccharides titration (3.41 g/L) was obtained when the initial starch concentration was 45 g/L. This corresponds to volumetric productivity values of 71.04 mg/L h.
... Compared with plant polysaccharides, EPS has the advantage of efficient production duration, convenient extraction and the absence of geographical restrictions (17). Investigations on the various types of biological activity of EPS have revealed antioxidant effects (18) and enhanced immunity (19). The EPS produced by Cordycept militaris has an antihyperglycemic effect in diabetic mice induced by streptozotocoin injection (20), and EPS separated from Morchella conica markedly prolongs the life-span of fruit flies (21). ...
Marasmius androsaceus, a well‑known medical fungus, possesses antihypertensive, analgesic and antioxidant effects. Exopolysaccharide (EPS), produced by microorganism secretion, exerts various types of biological activities. The present study aimed to investigate the antidepressant‑like effect of the EPS produced during Marasmius androsaceus submerge fermentation (MEPS). Based on the assessment of acute toxicity and behavior, a forced swimming test (FST), tail suspension test (TST), 5‑hydroxytryptophan‑induced head‑twitch assessment and reserpine‑induced hypothermia assessment were performed. The administration of MEPS for 7 days enhanced mouse locomotor and balance ability in the mice. Similar to the results following treatment with fluoxetine, which was used as positive control drug, MEPS significantly decreased the duration of immobility in the FST and TST, increased head twitches in the 5‑HTP‑induced head‑twitch test and enhanced rectal temperature in resperpine‑induced hypothermia. MEPS altered the abnormal concentrations of 5‑hydroxytryptamine, 5‑hydroxyindoleacetic acid, dopamine and norepinephrine in the hypothalamus in the resperine‑induced mouse model. Additionally, an increase in the expression of tyrosine hydroxylase and a reduction in the level of dopamine transporter in the hypothalamus were noted following 7 days of MEPS administration. Taken together, the EPS produced during MEPS exhibited antidepressant‑like effects, which may be associated with its regulation on the dopaminergic system. The results of the present study provide experimental evidence supporting the clinical use of MEPS as an effective agent against depression.
... Pullulan and dextran are naturally derived polysaccharides that are widely used in the pharmaceutical and biomedical industries due to their high abundance, low cost, biocompatibility and biodegradability. 10,11 Being biochemically similar to the natural ECM, these polysaccharides are also used as biodegradable scaffolds for tissue engineering. [12][13][14][15] Tubular-shaped P/D hydrogels can recapitulate the mechanical properties of a blood vessel. ...
Vascularization of tissue-engineered constructs is critical for proper cell and graft survival. In order to achieve this, pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), are often incorporated into scaffolds by methods that either involve multiple steps or risk compromising protein bioactivity. In this study, we demonstrate a simple approach to incorporate VEGF into polysaccharide electrospun fibers by taking advantage of the interactions between VEGF and sulfated polysaccharide, fucoidan. Pullulan/dextran (P/D) electrospun fibers (diameter 500 nm) incorporating fucoidan were fabricated by a one-step electrospinning process. Thereafter, VEGF was loaded onto the scaffolds. By varying the content of the chemical crosslinker, trisodium trimetaphosphate (STMP), from 10 to 12 and 16 wt% (denoted as STMP10, 12 and 16 respectively), the extent of fucoidan incorporation was significantly enhanced (<2.5 mg g−1 for STMP10 vs. 5 mg g−1 for STMP12 and 16). In addition, increased fucoidan content resulted in prolonged retention of VEGF bioactivity (≥14 days for STMP12 and 16 vs. 3 days for STMP10 and 1 day for VEGF by bolus delivery). Subcutaneous implantation of P/D scaffolds in mice demonstrated enhanced angiogenic response towards fucoidan and VEGF loaded scaffolds at 14 days post-implantation. In addition, P/D constructs supported rapid cellular infiltration and complete biodegradation of the scaffolds was observed at 7 days post-implantation. Taken together, the results demonstrate the potential of P/D electrospun fibers endowed with fucoidan as tunable reservoirs for the effective delivery of VEGF to control vascularization of tissue-engineered constructs.
... Recently, submerged fermentation of mushrooms has provided a scalable production technology to afford relative higher yields of various biomaterials with lower contamination. Especially, the resulting fungal polysaccharides such as scheroglucan, schizophyllum, lentinan and pullulan offer a potential new source of functional biopolymers for food, medical and industrial applications due to their unique physico-chemical properties [4]. Some physiologically active polysaccharides, to date, are successfully produced from commercially cultivated mushrooms such as Grifola frondosa, Phellinus gilvus, Sarcodon aspratus, etc [5]. ...
The parasitic fungus, Paecilomyces hepiali, is used to produce Cordyceps materials as succedaneum of natural Cordyceps sinensis in China. The purpose of this research was to investigate the effects of glucose, mannose, sucrose, lactose as solo carbon source and sucrose + lactose or mannose + sucrose as synthetic carbon source on the growth of mycelium and production, chemical composition, molecular weight distribution and monosaccharide composition of exopolysaccharides from P. hepiali HN1 (PHEPS). The maximum mycelium biomass of 12.16 kg m−3 and PHEPS yield of 4.57 kg m−3 were achieved from the culture with sucrose (50 kg m−3) as carbon source. The resulting PHEPS was characterized by analyses of chemical composition, size-exclusion chromatography and high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization. It was found that the chemical compositions and monosaccharide ratios in PHEPS were significantly affected by the carbon sources used. Glucose or mannose as carbon source enhanced the biosynthesis of PHEPS with higher-molecular weight (>1000 kD), but solo carbon source of lactose or synthetic carbon source of mannose + lactose did not increase the ratio of galactose in PHEPS. The metabolism kinetics of carbon sources demonstrated the correlation between PHEPS synthesis and the utilization of carbon sources. These findings will be useful for further works on the production, structure and function of PHEPS.
... Bacterial cellulose (BC) is an extracellular polysaccharide composed of glucose molecules connected together by b-1,4glycosidic bonds (Klemm et al., 2005;Shih, 2010). In contrast to plant cellulose, BC exhibits high chemical purity and high crystallinity as it is not associated lignin and hemicellulose (Klemm et al., 2005;Nishiyama et al., 2002Nishiyama et al., , 2003. ...
The effect of ultrafiltration on the cheesemaking properties of donkey milk was assessed. Milk was coagulated by rennet with or without modification of some technological parameters, i.e., pre-acidification with lactic acid or EPS-producing starter, addition of small amount of bovine milk. After assessing the gross composition, the milk samples were processed, and coagulation was monitored with a viscosimeter. The obtained cheeses were subjected to chemical analyses, calculation of the yield and electrophoretic characterization of the protein profile. The results indicated that the milk protein concentration was the main limiting factor for coagulation and that pre-acidification played a minor role. The most satisfactory results were obtained for milk with added EPS-producing starter, since the cheese showed the highest yield (about 11%) and the firmest texture. The outcomes of the study could be easily transferred to the dairy level, after suitable economic evaluation.
Marine polysaccharides (MPs) are an eco-friendly and renewable resource with a distinctive set of biological functions and are regarded as biological materials that can be in contact with tissues and body fluids for an extended time and promote tissue or organ regeneration. Skin tissue is easily invaded by the external environment due to its softness and large surface area. However, the body's natural physiological healing process is often too slow or suffers from the incomplete restoration of skin structure and function. Functional wound dressings are crucial for skin tissue engineering. Herein, popular MPs from different sources are summarized systematically. In particular, the structure-effectiveness of MP-based wound dressings and the physiological remodeling process of different wounds are reviewed in detail. Finally, the prospect of MP-based smart wound dressings is stated in conjunction with the wound microenvironment and provides new opportunities for high-value biomedical applications of MPs.
Bacterial exopolysaccharides (EPS) are water-soluble polymers consisting of repeating sugar moieties that serve a wide range of functions for the bacterial species that produce them. Their functions include biofilm matrix constituent, nutrient retention, protection from environmental threats and even pathogenicity. EPS have also been exploited for use in various applications in the biomedical field: most notably as viscosupplements, drug delivery vehicles and in tissue engineering constructs. The use of EPS in bone tissue engineering has increased in recent years due to the wide range of compounds available, low cost, and ease of production on an industrial scale. This review discusses the extraction and purification methods employed to produce bacterial EPS. A particular focus is on bone-related tissue engineering applications where EPS is the primary active agent, or as a scaffold matrix, as well as a carrier for osteopromotive agents.
One of the most important challenges in drug delivery is the permeability of drugs as carriers to target cells. Depending on the medication interaction with target cell cause efficiency and high uptake of them. Hence drug loading happens in each cell of the body without the various side effects of the drugs. One example of various side effects of the drugs is enzyme interference in oral drug delivery. New drug delivery systems are designed to reduce the side effects of the drugs, optimize their therapeutic efficacy, and increase patient satisfaction. With modern drug delivery systems, we will be able to control and determine speed, time and place of the drug release. In recent years, coating a class of drugs with biodegradable polymeric compounds as controllers for drug delivery systems has become increasingly important in biomedical research, drug delivery, and pharmacy. Microbial exopolysaccharides (EPS), the most significant group of polymeric materials, are renewable and their structural and physicochemical diversity enables them to play diverse roles in various fields. Based on the recent findings in glycobiotechnologies, EPS will have wide applications in future notably in medicine and pharmacy as drug delivery systems.
Dextran hydrogels (Dex-SS) containing both disulfide and Schiff base bonds were developed via facile method based on the dextran oxidation and subsequent formation of Schiff base linkages between polyaldehyde dextran and cystamine, denoted as the disulfide-containing Schiff base reactions. Results of rheology, swelling and ¹³C CP/MAS NMR study indicated that cross-linking degree of Dex-SS hydrogels depended strongly on the molar ratio of -CHO/-NH2. Acidic and reductive (GSH) environment sensitive degradation behaviors of Dex-SS hydrogels were then evidenced by SEM, rheology study and Ellman’s assay. Moreover, doxorubicin (DOX) was loaded into the hydrogel matrix and pH/GSH-responsive release behaviors were demonstrated. Cytocompatibility of Dex-SS hydrogel and effective cell uptake of released DOX was finally proved by transwell assay with HepG2 cells. Take advantages of the abundance of vicinal hydroxyl on a variety of polysaccharides, the disulfide-containing Schiff base reactions is considered as versatile method to develop stimuli-sensitive hydrogels for local drug delivery.
This work aims to enhance the bioproduction of xanthan gum by screening a hyper-yield producer from the wild-type Xanthomonas campestris during a long-term continuous subculture. We reported a cell-wall deficient mutant, which performed a shift of cell morphology from rod-shaped to round-shaped. Both the yield of xanthan gum and the conversion rate of feedstock were assessed using sucrose as a carbon source with the supplement of yeast extract powder, l-glutamic acid, and other raw materials. After 96 h aerobic fermentation, the yield of xanthan gum of the mutant reached up to 32 g/L, which was 3.4 times of that of the wild-type strain. The conversion rate of feedstock in the mutant was up to 92.1%, which was 3 times of that of the wild-type (31.2%). Furthermore, pigments generated were determined and compared. As a result, the fermentation broth of the wild-type performed an OD560nm of 0.296, which was 5.8 times of that (OD560nm = 0.051) of the mutant. Microscopy analysis showed that the percentage of free-living cells in broth affected the color of the final product. Moreover, the robustness of the fermentation performance of the cell-wall deficient mutant at a pilot scale showed potential for industrial application.
In this study, a bacterial cellulose (BC) producing strain was isolated from Kombucha tea and identified as Komagataeibacter hansenii JR‐02 by morphological, physiological, biochemical characterization and 16S rRNA sequence. Then, the media components and culture conditions for BC production were optimized. Result showed that the highest BC yield was 3.14 ± 0.22 g/L and 8.36 ± 0.19 g/L after fermentation for 7 days under shaking and static cultivation, respectively. Moreover, it was interesting that JR‐02 could produce BC in nitrogen‐free medium with the highest yield of 0.76 ± 0.06 g/L/7days, and the possible nitrogen fixation gene nifH was cloned from its genomic DNA. The BC produced by JR‐02 was type‐I cellulose with high crystallinity and thermodynamic stability which was revealed from fourier transform infrared spectroscopy (FTIR), X‐ray diffraction (XRD) and thermogravimetric analysis (TGA) methods. The crystallinity of static and shaking cultured BC were 91.76% and 90.69%, respectively. The maximum rate of weight loss of static and shaking BC occurred at temperature of approximately 373.1°C and 369.1°C, respectively. Overall, these results indicated that K. hansenii JR‐02 had great potential to produce high crystallinity type‐I BC in manufacture. This article is protected by copyright. All rights reserved
Microfluidic methods are frequently used to produce cell-laden microgels for various biomedical purposes. Such microfluidic methods generally employ oil-water systems. The poor distribution of crosslinking reagents in the oil phase limits the available gelation strategies. Extracting the microgel from the oil-phase also reduces its production efficiency. In this study, an aqueous two-phase system (ATPS) involving dextran (DEX) and polyethylene glycol (PEG) was used to prepare cell-laden microgel. This avoided the problems associated with an oil phase. The microgel precursor polymers and crosslinking reagents were dispersed in the DEX and PEG phases, respectively. The ultra-low interfacial tension of the ATPS hindered droplet formation. A co-flow microfluidic device was fabricated to overcome this problem. The device incorporated a square-wave-changing injection force, to improve the efficiency of droplet formation. The microgel precursor (including alginate and carboxymethyl cellulose derivatives posse
In recent years, significant progress has been done to discover a novel microbial exopolysaccharides that possess novel and highly functional properties. During our screening program for curdlan producing bacteria, 35 bacterial strains were isolated from 15 sandy soils collected from Rafha governorate, Northern Border region, Saudi Arabia. Among them isolate, NBR-10 was selected for its promising ability for curdlan production. The selected isolate was identified based on methods or results mentioned in this paper as Paenibacillus sp. For enhancing the curdlan yield produced by Paenibacillus sp. NBR-10, different culture conditions and medium compositions were optimized. It was found that, the maximum yield was obtained at 35 °C, initial pH 7 after 48 h of incubation. Also different carbon and nitrogen sources were used to improve the curdlan yield, it was indicated that sucrose and yeast extract were the best carbon and nitrogen sources respectively. Generally, optimization of the different parameters was approximately duplicated the curdlan yield from 2.34 g/l to 4.82 g/l. The precipitated curdlan dissolved in 2M NaOH exhibited high affinity to gel formation. Analysis of FT-IR, ¹H NMR and ¹³C NMR spectra proved that the produced polymer by Paenibacillus sp. NBR-10 has β-(1-3)-D-glucan (curdlan) structure.
Microporous bacterial cellulose/potato starch (BC/PS) composites composed of a compact upper surface and transparent lower surface were fabricated by an in situ method by adding PS into the culture medium. The special structure formation mechanism was explored. Compared with original BC, a locally oriented surface morphology was observed when the concentration of PS in the culture media was above 1.0 %. Many more free spaces were made after modification with pore size reaching 40 mu m. An obvious cell ingrowth tendency was observed on the porous surface of BC/PS composites as the starch content increased, while most of muscle-derived cells could only proliferate on the surface of original BCs. In vivo implantation showed the transparent fibrous lower side of BC/PS composites was much easier for neovascularization, and no obvious sign of inflammation was observed.
Two odorless, water-soluble exopolysaccharide (EPS) fractions, EPS-1 and EPS-2, were isolated from a newly isolated Bacillus amyloliquefaciens strain C-1 and purified by ion exchange and gel chromatography. The purified EPS-1 contained glucose/mannose/galactose/arabinose in a relative proportion of 15:4:2:1, and possessed a molecular weight of 79.6 kDa, while EPS-2 contained only glucose and mannose in a 3:1 ratio, with the molecular weights of 19.8 kDa. The antioxidant activity results showed that EPS-1 exhibited strong reducing power, superoxide radicals (O(2-)·), and hydroxyl free radicals (OH·) scavenging activities. For the H2O2-induced injury in HepG2 cells, EPS-1 significantly decreased the formation of reactive oxygen species, intracellular malondialdehyde levels, and restored intracellular superoxide dismutase activity. For EPS-2, there had no detectable antioxidant activities. And all these results collectively showed that as a natural antioxidant, only EPS-1 produced by C-1 had considerable potential to be used as medical compounds or functional additives.
A novel exopolysaccharide (EPS), namely B4-EPS, is produced byArthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett-Burman design (PBD), glucose, yeast extract and KH2PO4 were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH2PO4. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 h batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.
Significance:
Inflammatory diseases (such as arthritis) of the extracellular matrix (ECM) are of considerable socioeconomic significance. There is clear evidence that reactive oxygen species (ROS) and nitrogen species released by, for instance, neutrophils contribute to the degradation of the ECM. Here we will focus on the ROS-induced degradation of the glycosaminoglycans, one important component of the ECM.
Recent advances:
The recently developed "anti-TNF-α" therapy is primarily directed against neutrophilic granulocytes that are powerful sources of ROS. Therefore, a more detailed look into the mechanisms of the reactions of these ROS is reasonable.
Critical issues:
Since both enzymes and ROS contribute to the pathogenesis of inflammatory diseases, it is very difficult to estimate the contributions of the individual species in a complex biological environment. This particularly applies as many products are not stable but only transient products that decompose in a time-dependent manner. Thus, the development of suitable analytical methods as well as the establishment of useful biomarkers is a challenging aspect.
Future directions:
If the mechanisms of ECM destruction are understood in more detail, then the development of suitable drugs to treat inflammatory diseases will be hopefully much more successful.
Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It is composed of a pure cellulose nanofiber mesh spun by bacteria. It is remarkable for its strength and its ability to be engineered structurally and chemically at nano-, micro-, and macroscales. Its high water content and purity make the material biocompatible for multiple medical applications. Its biocompatibility, mechanical strength, chemical and morphologic controllability make it a natural choice for use in the body in biomedical devices with broader application than has yet been utilized. This paper reviews the current state of understanding of bacterial cellulose, known methods for controlling its physical and chemical structure (e.g., porosity, fiber alignment, etc.), biomedical applications for which it is currently being used, or investigated for use, challenges yet to be overcome, and future possibilities for BC.
Seventeen wild-type Xanthomonas isolates were screened in terms of broth viscosity in shake-flasks. As culture conditions affect polymer characteristics, a fair comparison among isolates required their cultivation in a fermenter under controlled dissolved oxygen tension. Three isolates and a reference strain were studied. The mean molecular weights and molecular weight distributions of their xanthans were determined. Products showed different pyruvate (0.2-7%), acetate (5-10%) and proteinaceous nitrogen (1-3%) contents. The selected isolates exhibit properties which could improve xanthan gum production and some could be used to produce polymers with specific characteristics.
Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.
Different isolated strains of Leuconostoc mesenteroides produce dextran compared with that of L. mesenteroides NRRL B-512 F. Among nine newly isolated strains, strains PCSIR-4 and PCSIR-9 produced dextran of different quality. Using these two newly isolated strains, the development of dextran coincided closely with the disappearance of sucrose. The yield of dextran increased during growth and maxi- mum yield was obtained at the end of exponential phase. Dextran yield was higher in media containing nitrogen source supplemented with different salts. Medium 2 was found to be best for the dextran production as compared with other media. Initial sucrose concentration in the media plays an important role in dextran pro- duction. The higher the initial concentration of sucrose in the medium, the higher is the yield of dextran produced per unit volume; however, the percentage conversion of sucrose into dextran decreases after reaching maxima. A 10% sucrose concentra- tion in the medium was the optimal level of dextran production. Molecular mass distribution of dextran from PCSIR-4 is near to 2 million Dalton whereas the dex- tran produced by PCSIR-9 is quiet higher than the dextran from PCSIR-4.
Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.
Levan is a non-toxic, biologically active, extra cellular polysaccharide composed solely by fructose units. Optimization of levan production by Zymomonas mobilis strain ZAG-12 employing a 24-1 fractional factorial design was performed to analyze the influence of the temperature (20, 25 e 30ºC) agitation (50, 75 e 100 rpm), and the initial concentrations of both sucrose (150, 200 e 250 g.L-1) and yeast extract (2.0, 3.5 e 5.0g.L-1) on final levan concentration. Aerobic fermentation was performed batchwise in 500mL Pyrex flasks for 72 hours. Biomass, ethanol, levan and sucrose were determined at beginning and also at end of the fermentations. The experiments showed that the final levan concentration depended on initial sucrose concentration, temperature and agitation velocity and that the initial concentration of yeast extract did not influence levan production. However, when the production of ethanol and biomass were considered, it became evident that yeast extract was a significant variable. The best conditions for levan production occurred at 100 rpm agitation, 20ºC and 250g.L-1 of initial sucrose resulting in 14.67g.L-1 of levan.
Seventeen wild-type Xanthomonas isolates were screened in terms of broth viscosity in shake-flasks. As culture conditions affect polymer characteristics, a fair comparison among isolates required their cultivation in a fermenter under controlled dissolved oxygen tension. Three isolates and a reference strain were studied. The mean molecular weights and molecular weight distributions of their xanthans were determined. Products showed different pyruvate (0.2–7%), acetate (5–10%) and proteinaceous nitrogen (1–3%) contents. The selected isolates exhibit properties which could improve xanthan gum production and some could be used to produce polymers with specific characteristics.
A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration,
the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative
to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity
regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production
rate were found to be less favourable in the chemically defined media.
This chapter discusses the synthesis of levansucrase from Bacillus subtilis. Levansucrase is an enzyme produced by many bacteria and is extensively studied in Bacillus subtilis, where it is inducible and exocellular, and in Aerobacter levanicum, where it is constitutive and endocellular. It catalyses mainly the reversible reaction described in the chapter. However in presence of sucrose, its action leads always to a formation of free fructose and of oligosaccharides. The assay procedure is based on the determination of free glucose under conditions where the velocity of the glucose formation depends only on the amount of enzyme. The reaction is stopped by dilution in boiling buffer and the free glucose is determined by a colorimetric procedure using glucose oxidase. The addition of levan primer to the reaction mixture gives more constant conditions for the assay during the purification procedure. Supernatant from cultures of sucrose grown B. subtilis serves as the source of levansucrase. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter.
The bacterial polymers reviewed here include the extracellular polysaccharides (EPS) alginate, emulsan, gellan, hyaluronic acid, xanthan, Erwinia-EPS, cellulose, curdlan, succinoglucan, dextran, alternan and levan; as well as the intracellular polyesters poly-β-hydroxybutyrate/ poly-β-hydroxyvalerate and the bacterial capsular materialpoly-D-glutamate. Several other bacterial EPS-molecules contain unusual sugars as constituents and, apart from their use based on their polymer characteristics, they are increasingly considered as an easy source of such specialty sugars upon chemical or enzymatic hydrolysis. Bacterial formation of cyclodextrins is also included. As to the fungal extracellular polysaccharides, pullulan, elsinan and scleroglucan are discussed as well as the fungal cell wall components chitosan and the immunostimulatory β-1,3-D-glucans. In addition, the microbial synthesis of metabolites, suitable as building blocks for biocompatible polymers such as poly-(D/L)-lactate, nylon, polyacrylamide and polyphenylene is reviewed.
Die Bildung eines kompakten, gleichmäßigen Vlieses von Bakteriencellulose ist begünstigt, wenn sich die syntheseaktiven Zellen von Acetobacter xylinum an der Grenzfläche Gas/Flüssigkeit der Nährlösung oder an einer festen Oberfläche akkumulieren und an sie anlagern können. Es wurde ein Walzenfermentor konstruiert. In diesem lagern sich die Zellen an die speziell bearbeitete, aufgerauhte Oberfläche einer Glaswalze an und bilden ein Vlies aus Bakteriencellulose. Im Vergleich zur stationären Emersfermentation wird auf diese Weise die Nährstoffversorgung des wachsenden Vlieses wesentlich verbessert. Die Wachstumsrate nimmt zu, die Celluloseausbeute steigt und die Produktivität der Cellulosebildung wird nahezu verdoppelt.
CellulonTM fiber, a cellulose produced by a bacterial fermentation process employing a strain of Acetobacter aceti subsp. xylinum, was tested for genotoxicity in four assays: (1) microbial reverse mutation assay in Salmonella typhimurium (Ames assay), (2) an assay for chromosomal aberrations in Chinese hamster ovary (CHO) cells, (3) an assay for induction of unscheduled DNA synthesis (UDS) in rat primary hepatocytes, and (4) the CHO/HGPRT forward mutation assay. Each assay was conducted at a wide range of dose levels, both with and without metabolic activation (assay 1, 2, and 4). Test results gave no indication that Cellulon fiber possessed any genotoxic potential. The pyrogenicity of five batches of Cellulon fiber was tested in the Limulus Amebocyte Lysate assay, gel-clot method. Test results were negative for the presence of gram-negative bacterial endotoxin. The primary eye and dermal irritation potential of Cellulon fiber were examined in New Zealand White rabbits. The Draize method was employed to evaluate and grade ocular and dermal irritation as a result of test material administration. Test results indicated that Cellulon fiber is a minor ocular irritant up to 1 hour postadministration. However, the resultant irritation was considered to be mechanical and related to the dry, granular form of the test material. In addition, test results indicated that Cellulon fiber is not a dermal irritant in the rabbit. The acute oral toxicity of Cellulon fiber was determined in Sprague-Dawley rats, and the LD50 was found to be greater than 2000 mg/kg of body weight. The subchronic toxicity of Cellulon fiber was examined in Sprague-Dawley rats fed diets containing 0, 5, and 10% Cellulon fiber or microcrystalline cellulose for 13 weeks. No dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or pathologic lesions were observed. The results of these studies indicate that Cellulon fiber and microcrystalline cellulose are toxicologically equivalent and that Cellulon fiber does not possess genotoxic potential, is nonpyrogenic, and that animals are not adversely affected by acute or subchronic exposure to Cellulon fiber.
The use of microbial polysaccharides in the food, pharmaceutical and chemical industries has increased steadily during the past decade. The biopolymer gellan is a more recent addition to the family of microbial polysaccharides that is gaining much importance due to its novel property of forming thermo-reversible gels when heated and cooled. It is produced and marketed by some companies of Europe, USA, etc under trade names such as ‘Gelrite’, ‘Phytagel’ and ‘Kelcogel’. It has applications in diverse fields in the food, pharmaceutical and many other industries. Further research and development in biopolymer technology is expected to expand its use. This article presents a critical review of the available published information on the gellan exopolysaccharide synthesized by Pseudomonas species. In particular information on its structure, physico-chemical properties and the rheology of its solutions etc. is critically assessed. Emphasis has also been paid to characterization of gellan. A brief historical background of the polymer and the biochemical and physiological characteristics of several different existing bacterial isolates which secrete gellan and related polysaccharides are discussed. An attempt has also been made to review the potential and future prospects, highlighting some novel techniques adopted to overcome the mass transfer problems associated with the fermentative production of gellan gum. The efficient downstream processes used for obtaining purified gellan are also highlighted. Attention has also been drawn to the problem associated with the fermentation processes due to the highly viscous nature of gellan gum and effect of different impeller systems on gellan fermentation kinetics and rheological properties.
Recently, an environmental criteria was required for every product and process. The great amount of pollution caused by the paper and cellulose industry effluents, especially those produced from nonwood fibrous materials, e.g., rice straw and bagasse. Using new technologies or redesigning products to meet new environments criteria is necessary. The production of cellulose by microorganisms is considered as one of the most beneficial sources for environment requirements. Microbial polysaccharides are valuable commodities, since they display many useful properties and can be produced in large quantities. This article reviews previous research on the biosynthesis of bacterial cellulose, structural features, properties, as well as potential applications as new industrial materials, i.e., paper industry, wastes treatment, sewage purification, food, medicine, etc. Suggestions for an economic production process for bacterial cellulose and cellulose derivatives also were reported.
Long-chain, high-molecular-weight polymers that dissolve or disperse in water to give thickening and sometimes gelling and/or emulsifying effects are indispensable tools in food product formulation. Most of these polymers are polysaccharides, and their functional properties in foods are determined by quite subtle structural characteristics. Biotechnology offers the means for the modification of biopolymer structure to achieve desirable changes in functional properties. In this article, the impact of recent advances in traditional biotechnology (fermentation and enzymology), as well as in the newer sciences of molecular biology (genetic manipulation and protein engineering), on the development of food biopolymers is critically assessed.
We found that Acetobacter xylinum KU-1 produced cellulose from D-arabitol. The maximum cellulose production was obtained when it was grown in a medium containing 2.0% D-arabitol, 1.0% tryptone, and 1.0% yeast extract (pH 5) at 30°C for 96h statically. The productivity was more than 6 times as much as that of D-glucose [productivity (mg/ml-medium): from D-arabitol, 12.4; from D-glucose, 2.0].
The mixing properties of bacterial cellulose (BC) in an aerated and agitated vessel are described. To characterize the mixing of BC culture broth, which can affect the productivity of BC, non-Newtonian behavior during mixing of a 1% BC suspension was studied using an image processor capable of detecting decoloration of a pH indicator and was compared with that of a 2% carboxy methyl cellulose (CMC) solution. CMC solution was mixed homogeneously within the measured range of agitation speed, but the BC suspension was not mixed homogeneously at agitation speeds lower than 15 rps because mixing was delayed in some areas of the vessel. A possible reason for the inhomogeneity of the BC suspension at low agitation speeds is the non-Newtonian behavior which increases viscosity at low shear rates.
To improve the bacterial cellulose (BC) productivity in aerated and agitated culture, the agitator configuration was improved. Agitators were selected by measuring mixing delay time and KLa of a 1% BC suspension and cultivating Acetobacter xylinum subsp. sucrofermentans BPR3001A. We concluded that (i) the impellers such as Maxblend and gate with turbine were suitable for BC fermentation because they mixed culture broth well and had large KLa, (ii) the production rate and yield of BC were dependent on KLa and the oxygen consumption rate, and (iii) the static gassing-out method to measure KLa was useful for characterization of the agitation conditions and the agitator configuration.
A potent cellulose producer, Acetobacter xylinum BRC5, was cultivated in shaking flasks and jar fermentors with glucose, fructose, sucrose and mixtures of these as carbon sources. It was confirmed that corn steep liquor (CSL) was cheap and a suitable organic nitrogen source for cellulose production by strain BRC5. When glucose was used as a sole carbon source, strain BRC5 oxidized almost all the glucose to gluconic acid, thereafter it biosynthesized cellulose by utilizing the gluconic acid accumulated in the broth under limited glucose conditions. Since strain BRC5 did not metabolize fructose to acid, the fermentation pattern of fructose was found to be typically growth associated with cellulose production. However, when glucose and fructose coexisted in the medium, strain BRC5 preferentially metabolized all the glucose to gluconic acid. Thereafter, it produced cellulose mainly by utilizing fructose. Overall cellulose productivity in a jar fermentor ranged from 0.071 to 0.086 g/l/h.
Polymers such as hydroxypropylcellulose, hydroxyethylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, pectin, carrageenan and guar gum have wide application in the pharmaceutical industry, and many techniques in polymer characterization are performed with the polymer molecules in aqueous solution; this is because the thermodynamic properties of polymer solutions can be readily measured and the results interpreted in terms of the size and structure of the macromolecules, thus enabling characterization of the polymer. The authors address some important properties and practical applications of water-soluble polymers.
The hydrogel nanoparticle that was formed by self-assembly of cholesterol-bearing pullulan complexed with 5 - 10 insulin monomers in water. Biocolloidal and thermal stability of insulin drastically increased upon the supramolecular assembly with the hydrophobized polysaccharide.
Depolymerization of high-molecular-weight sodium hyaluronan by superoxide anions and hydroxyl free radicals was studied. The changes in number-average and weight-average molecular weight, molecular-weight distribution, polydispersity, and concentration were measured over time by high-performance liquid chromatography on a TSK 6000 PW size-exclusion column coupled to multiangle laser light scattering and refractive index detection. There was no significant change in the elution profile and in the peak height of the refractive index chromatogram for either of the radical species at different reaction times. Furthermore, the weight-average molecular weight of hyaluronan decreased from 1.3 × 106 to 3.0 × 105 g/mol; however, the polydispersity did not significantly increase. These experimental results indicate that superoxide anions and hydroxyl free radicals do not have different modes of action on hyaluronan. Also, both radical species seem to attack hyaluronan randomly from the end of the molecule. Depolymerization studies on sodium hyaluronate by superoxide anions and hydroxyl free radicals indicate similar modes of action for both species.
Hydrolysis of pullulan, derived from strain P50 of Aureobasidium pullulans, by amyloglucosidase leaves a major portion of the glucan untouched, suggesting that the polysaccharide does not contain the simple linear sequence of α-(1→6)-linked maltotriosyl residues hitherto ascribed.
The process of cellulose formation in a nata-de-coco culture system has been investigated. The medium was prepared with coconut-water by adding sugar and N-compounds and the culture was conducted in static conditions. The growth of gel thickness, wet weight and dry weight was almost independent of the concentrations of N-compound and sugar at least when they were above 0.1 and 1%, respectively, suggesting that the process was controlled by oxygen supply. Glucose was the only saccharide found in the coconut water stored for 3days. While the concentration of sugar dropped quickly to a certain level and decreased monotonically with time, a part of glucose turned to something other than cellulose. No fructose was found after the initial stage, presumably being consumed by side-reactions. Computer simulation showed that, after the induction time, the process of cellulose formation or the consumption of glucose is controlled by the diffusion of atmospheric oxygen.
Continuous filamentation of bacterial cellulose (BC) was successfully achieved by using shallow pan for the incubation to regulate thickness of the BC gel produced by Acetobacter xylinum. The BC filament was harvested and prepared directly by picking up BC pellicles, the thin BC gel, and winding slowly from the surface of the culture medium passed through a preliminary bactericidal washing bath. The X-ray diffraction analysis and scanning electron microscopic observation of the BC filament thus obtained showed that the filament was smooth and the fairly good orientation of BC molecules.The average tensile strength was 4.4 g denier−1 for the filament prepared by hot alkaline treatment and subsequent washing with distilled water and dried under tension (Filament W): 3.4 g denier−1 for washing with 10% aqueous ethylene glycol after alkaline treatment followed by drying under tension (Filament E) and 2.4 g denier−1 for the treatment with 10% ethylene glycol after normal water-washing followed by drying under tension.
A great deal of recent interest has been shown in the ability of some microbes to synthesize exopolysaccharides. Most attention has been directed toward the prokaryote producers, yet many filamentous fungi also produce exopolysaccharides that have chemical and physical properties of considerable commercial potential. Surprisingly little is known about how and why fungi overproduce these metabolites and how yields are affected by both the physical and chemical environments. This review attempts to critically appraise the current literature on fungal exopolysaccharides, considers their chemical diversity, and examines factors that seem to affect their production. Although much of the published work has been carried out with the α-glucan pullulan, there is considerable literature on the β-glucans and, hence, both of these are discussed.
Summary The optimum pH for both the rate of production and yield of hyaluronic acid (HA) byStreptococcus zooepidemicus from glucose medium was 6.7±0.2 under anaerobic conditions. High agitation rates (600 rpm) gave superior results compared to 300 rpm. Aeration of the culture (0.3 VVM) improved the HA yield, but not the rate of production and lead to some acetate and CO2 being formed, in addition to lactate and HA.
Microbial extracellular heteropolysaccharides are mainly linear molecules to which side chains of varying length and complexity are attached at regular intervals. Examination of 'families' of microbial exopolysaccharide with closely related structures enables us to determine the effect of minor (or major) changes to structure on the physical properties of these macromolecules. Changes may also be seen in some of the biological properties of the polysaccharides, such as susceptibility to enzymes; interaction with antibodies or lectins; and capacity and specificity of ion binding. Acetyl groups frequently assert very marked effects on the properties of microbial polysaccharides. The presence or absence of an O-acetyl or pyruvate (ketal) group on each oligosaccharide repeat unit can greatly alter the properties of a number of exopolysaccharides.
Acetobacter xylinum produces both cellulase and bacterial cellulose, but some report believed that this cellulase activity does not decrease the d.p. (DP) of bacterial cellulose during cultivation. A. xylinum subsp. sucrofermentans BPR2001 produces two enzymes that hydrolyze CM-cellulose and cellotriose, resp. We examd. the effect of the two cellulase activities on the DP of bacterial cellulose when bacterial cells were cultured with agitation at pH 4, where little cellulase is produced, and at pH 5, where much cellulase is produced. The wt.-av. d.p. (DPw) of bacterial cellulose remained in the range of 14,000 to 16,000 during cultivation at pH 4, but at pH 5, the DPw decreased from 16,800 to 11,000. The mech. strength of a sheet prepd. from the bacterial cellulose produced at pH 4 was higher than those of BC produced at pH 5. These results suggest that the two cellulase activities cause the decrease in DP and deterioration of phys. properties of bacterial cellulose seen during cultivation.
A levan-producing bacterium was isolated from soil. Cultural and physiological characteristics of the isolate identified the organism as a strain of Bacillus polymyxa. The organism's characteristics for polysaccharide synthesis were studied. The bacterium produced polysaccharide in high yield when grown on sucrose solution. Hydrolysis and subsequent analysis showed the product to consist entirely of D-fructose. 13C NMR and methylation analyses indicated the product to be a β(2→6)-linked polymer of fructose, with 12% branching. The polysaccharide has a molecular weight of about 2 million and is readily soluble in water.
Macromolecular complexation between bovine serum albumin (BSA) and self-assembled hydrogel nanoparticle formed by the self-aggregation of cholesterol-bearing pullulan (CHP) was studied by high performance size exclusion column chromatography (HPSEC) and circular dichroism (CD). The CHP self-aggregates complexed with one BSA molecule to give colloidally stable nanoparticles (RG = 17 nm) at pH 7.0 and 25 °C. This was almost irrespective of the substitution degree of the cholesterol group of CHP. The helical content of BSA decreased upon complexation. Unfolding of BSA by either heating or a denaturant such as urea was largely suppressed upon complexation. BSA would be incorporated inside into the hydrogel matrix of the CHP nanoparticle. Kinetic analysis of the complexation suggested a two-step process: namely, the fast pre-equilibrium of looser binding of BSA to the CHP self-aggregate followed by the slower process of tighter inclusion into the hydrogel network. The substitution degree of the cholesterol group in CHP significantly affected the complexation kinetics.
Hyalobiuronic acid, a glucuronido glucosamine earlier isolated from hydrolysates of hyaluronic acid from umbilical cord, has been converted to its heptaacetyl methyl ester and its N-acetyl derivative. Esterification of the disaccharide, oxidation of the glucosamine residue to glucosaminic acid, and reduction of the uronic ester residue to a glucose residue yields a crystalline glucosidoglucosaminic acid. This is oxidatively deaminated to give a glucosidoarabinose, isolated as its crystalline heptaacetate, identical with the heptaacetate VII obtained by Zemplén degradation of laminaribiose (VIII). Hyalobiuronic acid is thus 3-O-(β-D-glucopyranosyluronic acid)-2-amino-2-deoxy-D-glucose (I). That its N-acetyl derivative II is the basic repeating unit of hyaluronic acid linked linearly in the polymer, probably by 3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl) linkages (Fig. 2), follows from earlier hydrolytic and enzymatic experiments, and from periodate oxidation data in the literature. A modification of the hydroxamic acid test suitable for sugar esters is described.
Summary The effects of pH (2, 7 and 10), NaCl concentrations (0, 1, 3 and 5% w/w), and temperatures (−20, 25 and 100 °C) on the physical properties of nata, a bacteria cellulose produced by Acetobacter aceti ssp. xylinum, were evaluated in this study. The addition of NaCl was found to increase the hardness of nata but to decrease the water-holding capacity; however, no effect of NaCl on the hardness of nata was found when nata was acidified or alkalized. Compared to the texture properties of nata at 25 °C, freezing at −20 °C for 24 h gave a more elastic texture, but heating at 100 °C for 3 h had no significant effect on the textural properties. The results of this study indicated that acidification or alkalization was necessary to prepare salted nata products without increasing their hardness.
Zymomonas mobilis strain 113 “S” produces levan – an extracellular, viscous, biologically active, non-toxic fructose polymer with a unique structure and extraordinary properties. This polysaccharide was isolated at two different degrees of purity by alcohol precipitation from aqueous solutions and was characterized with respect to some rheological properties and stability of viscous solutions.
The effects of temperature, pH and salt concentration on the viscosity of 1–3% levan solutions were examined. The viscosity of levan solutions was found to be quite stable and reversible at room temperature over a wide range of pH from 4 to 11. The viscosity was slightly affected by increased salt concentration. Levan solutions were rather stable at high temperatures (up to 70°C, 1 h, pH 6), where the viscosity could be almost completerly restored (up to 80–100%). Therefore, the degradation of the polymer structure under these conditions is probably insignificant. Temperatures of 70–100°C with a pH of less than 3.5 caused irreversible degradation of the levan structure.
The above-mentioned properties of levan, obtained from Zymomonas mobilis 113 “S”, demonstrated the potential for the development of various therapeutic forms of pharmacologically-active levan and their application in medicine as well as in the food and other industries.
B. MANNA, A. GAMBHIR AND P. GHOSH. 1996. Sphingomonas paucimobilis was used for the synthesis of the microbial polysaccharide gellan. In a 60 h fermentation, polysaccharide yield and productivity obtained were 0.45 g gellan per g glucose consumed and 0.21 g I−1 h−1 respectively. The broth showed pseudoplastic behaviour with yield stress. The requirement of the solvent propanol to precipitate gellan from the broth depended on the volume of the broth rather than on gellan concentration. The addition of salt to the broth reduced the propanol requirement. Attempts to separate cells from the highly viscous broth by microfiltration were not successful. Ultrafiltration reduced the propanol requirement but appreciable membrane fouling and loss of gellan was observed.
IntroductionIsolation of Polysaccharide-Producing MicroorganismsThe Potential for Strain DevelopmentLaboratory and Commercial Preparation of Microbial PolysaccharidesUses of Microbial PolysaccharidesAcceptability of Microbial PolysaccharidesCommercialized Microbial Polysaccharides and Potential New ProductsThe FutureReferences
Thermosensitive hydrogels were prepared by free radical polymerization starting from a methacrylated pullulan derivative (acting as the cross-linker) and using N-isopropylacrylamide (NIPAAM) as the monomer. Several hydrogels were obtained by changing the monomer to cross-linker ratio. A significant thermosensitivity was observed only when the molar amount of NIPAAM incorporated in the network was at least eight times higher that of methacrylate groups on pullulan. The hydrogel with high amount of NIPAAM deswells more than 80% after the T-jump. The lower critical solution temperature of thermosensitive hydrogels decreases with increasing amount of NIPAAM. The mechanical properties of the hydrogels are strongly affected by the percentage of incorporated NIPAAM and by the temperature.
Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.
The structure and some properties of bacterial cellulose produced in agitated culture were studied. Scanning electron microscopy revealed that there was almost no difference between reticulated structures of bacterial cellulose fibrils produced in agitated culture and in static culture. Nevertheless, bacterial cellulose produced in agitated culture exhibited microstuctural changes, namely, a low degree of polymerization and a low crystallinity index. A CP/MAS 13C NMR analysis revealed that the cellulose I content of the cellulose produced in agitated culture was lower than that of the cellulose produced in static culture. The bacterial cellulose produced in agitated culture had a lower Young''s modulus of sheet, a higher water holding capacity and a higher suspension viscosity in the disintegrated form than that produced in static culture.
The biosynthesis of xanthan by Xanthomonas campestris was found to be affected by the addition of citric acid in fed batch mode. Under oxygen-limiting conditions, the addition of up to 2.6 g citric acid per litre improved cell viability as well as increasing xanthan yield by up to 80%. Comparative xanthan formation profiles at different operating conditions indicate that at higher aeration (when there was no oxygen limitation), citric acid addition did not improve xanthan production.
A levan-producing bacterium was isolated from soils and its characteristics for polysaccharide synthesis were studied. A series of enrichment and plating techniques enabled the isolation of a levan-producing bacterium from closely related contaminants. Cultural and physiological characteristics of the isolate identified the organism an a strain ofBacillus polymyxa. The organism produced about 40 g extracellular polysaccharide per liter of sucrose medium, which was about three times more yield than levan obtained from known levan producers. The highest amount of polysaccharide was on a 8% sucrose medium. Hydrolysis of the product showed that the polysaccharide consisted entirely ofd-fructose, and13C.n.m.r. spectra confirmed that the product was levan, a fructose polymer linked by B-(26) fructofuranosyl linkage.
Raw sewage and activated sludge have been shown to be good sources for isolating a levan-synthesising bacterium. The morphological,
cultural and physiological characteristics of the bacterium have been described and the designation.Corynebacterium laevaniformans, nov. spec. has been proposed for it. Some of the factors which influence levan synthesis are reported.
Bacterial cellulose (BC) was produced from fructose by Acetobacter xylinum subsp. sucrofermentans BPR3001A was performed in continuous culture and the effect of lactate on the production was investigated. In continuous culture with feeding of CSL-Fru medium containing 30 g·l−1 fructose at a dilution rate of 0.07 h−1, a higher production rate (0.62 g·l−1·h−1) was obtained than that (0.40 g·l−1·h−1) in a batch culture using CSL-Fru medium with 70 g·l−1 initial fructose. However, when the dilution rate or fructose concentration in the feed medium were increased, the total yield of BC declined because the residual fructose concentration in the drawn broth increased. Supplementing 12.5 g·l−1 lactate to the feed medium increased the cell concentration and fructose consumption at a steady state, resulting in a production rate of 0.90 g·l−1·h−1 and a BC yield of 36% at a dilution rate of 0.1 h−1. The ATP content of viable cells was maintained at a higher level by feeding a lactate-supplemented medium rather than the unsupplemented CSL-Fru medium. In a batch culture using lactate as the main carbon source, 77% of the lactate consumed was oxidized to CO2 and only 6.9% was converted to BC. These findings indicated that lactate functioned as an energy source, not as a substrate for BC biosynthesis. Increased intracellular ATP resulting from lactate oxidation may have improved the fructose consumption and BC production in the continuous culture.
The production of pigment-free pullulan by Aureobasidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l−1), polysaccharide productivity (4.5 g l−1 per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l−1) and pullulan productivity (3.5 g l−1 per day) were obtained in culture grown with feeding substrate containing 50 g l−1 sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly α-(1→4) (∼66%) and α-(1→6) (∼31%) glucosidic linkages; however, a small amount (<3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by 13C-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.
Industrial polymers are, in the main, petrochemical based. The textiles, gums, films, adhesives and coatings which comprise all or part of manufactured articles in daily use have been developed over many years of research. They are produced cheaply and in very large volume, two features which go hand in hand.In what form is the challenge from biopolymers? Many biological polymers undoubtedly have useful properties but is it possible to produce them efficiently enough to create volume markets? This article will concentrate largely on the production of poly-β-hydroxybutyrate where some of the most advanced technology has been applied.