Botryosphaeran

Identifiers
CAS Number	610312-16-8
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). Infobox references

Botryosphaeran is anexopolysaccharide (EPS) produced by theascomyceteous fungusBotryosphaeria rhodina.^[1][2] Characterization of the chemical structure of botryosphaeran showed this EPS to be a (1→3)(1→6)-β-D-glucan.^[3] This particularβ-glucan can be produced by several strains ofBotryosphaeria rhodina that include: MAMB-05,^[1] DABAC-P82,^[4] and RCYU 30101.^[5] Botryosphaeran exhibits interestingrheological properties and novel biological functions includinghypoglycaemia,hypocholesterolaemia, anti-atheroslerosis and anti-cancer activity, with potential commercial applications. Threecosmetic products formulated with botryosphaeran have been developed to promote skin health and treatskin conditions for future intended commercialization purposes.

The ascomycete andfilamentous fungus,Botryosphaeria rhodina (strain MAMB-05), was isolated from acanker on the trunk of aeucalypt tree, and was molecularly characterized by sequencing theInternal Transcribed Spacer (ITS) region ofrDNA.^[6][7]

The β-glucan, botryosphaeran, was discovered accidentally in 1994^[1] while cultivatingBotryosphaeria rhodina MAMB-05 on nutrient media containing glucose to produce theenzyme,laccase. This fungal isolate produces a constitutive laccase that could be induced to higherenzyme titers by variouslignin-likearomatic compounds, and especiallyveratryl alcohol.^[1][8] The fungus was found to be ligninolytic.^[9][10]

Botryosphaeran is secreted by the fungus during growth and appears in the fermentation broth where its presence causes an increase in the broth'sviscosity. It can easily be extracted from the broth byprecipitation methods.^[1]Veratryl alcohol, however, suppresses the formation of botryosphaeran.^[11]

Botryosphaeran is produced under submerged fermentation conditions whenBotryosphaeria rhodina MAMB-05 is grown onnutrient media containing glucose and mineral salts.^[1] Extracting the fermentation broth withalcohol causes the EPS (botryosphaeran) to precipitate from solution, and this can be separated bycentrifugation orfiltration.

The precipitate recovered can be lyophilized to a white fibrous material that is sparingly soluble in water. Alternatively, the recovered precipitate is resolubilized in water (gentle heating with stirring) to form a viscous solution that forms a firm gel when cooled to 5 °C. Solubilization of botryosphaeran can be enhanced throughchemical derivatization with variousfunctional groups.

The influence of the composition of the nutrient medium,^[12] including nitrogen,^[13] phosphate,^[13] minerals, supplements (soybean oil,Tween 80),^[14] and thecarbon source (carbohydrates),^[12][15] is important in enhancing the production of botryosphaeran andbiomass during fermentation byBotryosphaeria rhodina MAMB-05.

Catabolite repression,^[16] and the presence of β-glucan-hydrolyzing enzymes that attack botryosphaeran^[17] during the fermentation process are critical and limit the production of botryosphaeran.

Statisticalfactorial design methods, such as theresponse surface methodology (RSM),^[18][19] are effective in investigating complex fermentation parameters and their interactions to optimize metabolite production bymicroorganisms. RSM assists in defining the effects and interactions of thephysiological factors playing a role in biotechnological processes in the production of microbialmetabolites including exopolysaccharides such as β-glucans. Statistical methodologies reduce the number of experiments to provide sufficient information for statistically acceptable results.

The validation of the fermentation parameters by statistical factorial design improved botryosphaeran production byBotryosphaeria rhodina MAMB-05^[13][14] over unoptimized conditions.^[12]

Botryosphaeria rhodina MAMB-05 when grown on nutrient media containing different carbohydrate substrates produces afamily of botryosphaerans.^[12][15] These β-glucans differ only in the extent and frequency ofside-chain substituents.

Botryosphaeran production can be enhanced whenBotryosphaeria rhodina MAMB-05 is cultivated on glucose media containing soybean oil and thesurfactant, Tween 80.^[14]

The most attractive feature for the commercialization of botryosphaeran is the ease by which it can be produced by simple fermentation processes on low-cost nutrient media, and its subsequent isolation throughprecipitation with ethanol,^[3][12][13] which all takes place on atime-scale of days compared to other commercial β-glucans available on the market. The latter are extracted fromfungal fruiting bodies (mushrooms, fungal brackets), spentBrewers yeast, andcereal grains (barley, oat) that can takeweeks-to-months to prepare.

Themycelium ofBotryosphaeria rhodina MAMB-05 is a rich source of β-glucans.^[20]

The chemical structure of botryosphaeran was first described^[3] in 2003, and was determined using the methods:methylation analysis, Smith degradation, Gas Chromatography-Mass Spectroscopy (GC-MS) and¹³C NMR.

Totalacid hydrolysis of botryosphaeran produces onlyD-glucose,^[1][3] while partial acid hydrolysis^[3] andenzymatic hydrolysis^[21][22][23] produces a series ofhomologous gluco-oligosaccharides of differentdegrees of polymerization, which can be analyzed byHigh Performance Liquid Chromatography (HPLC).

Enzymatic digestion of botryosphaeran under controlled conditions employing the enzymes: β-(1→3)-glucanases and β-(1→6)-glucanases fromBotryosphaeria rhodina MAMB-05,^[24]Trichoderma harzianum Rifai,^[24] andAureobasidium pullulans 1WA1,^[23] produces a mixed series of β-(1→3)- and β-(1→6)- linked gluco-oligosaccharides^[21][22][23] that can serve asprebiotics.

Enzymes hydrolyzing botryosphaeran can be obtained by cultivatingBotryosphaeria rhodina MAMB-05,Trichoderma harzianum Rifai andAureobasidium pullulans 1WA1 on nutrient media containing either botryosphaeran,^[23][24] or the biomass^[17][24] derived fromBotryosphaeria rhodina MAMB-05, which is a rich source of β-glucans.^[20]

Prebiotics such as the (1→3)-linked gluco-oligosaccharides are emerging asnutraceuticals for inclusion in foods. Botryosphaeran can serve as a source of conveniently generating these oligosaccharides through enzymatic hydrolysis.^[21]

Methylation and Smith degradation analysis revealed that botryosphaeran constituted abackbone chain made up of (1→3)-β-linked glucose residues (i.e., it is a (1→3)-β-D-glucan) with β-(1→6)-linked glucose and di-glucose (gentiobiose) side-branches located at the C-6 position of glucose along the (1→3)-linked backbone chain.^[3][4] The chemical structure of botryosphaeran is a (1→3)(1→6)-β-D-glucan.^[3]13C NMR spectroscopy confirmed its structure.

The degree of branching of the family of botryosphaerans varies from 21 to 31%.^[3][15] depending upon the carbohydrate source in the nutrient media during fermentation by the fungus, and this also affects themolecular weight (MW) of the botryosphaerans produced, which can be large (order of >1 x 10⁶ daltons)^[4][15]

Botryosphaeran exists in atriple helixconformation,^[25] an important structural feature in manifestingbiological response modifying activities.^[26][27][28]

Derivatization of botryosphaeran by carboxymethylation and sulfonylation^[29][30] results in improved solubility in water, and diminishes its viscous nature in solution.

In the case of sulfonated botryosphaeran, the chemically modified polysaccharide containingsulfonate groups exhibited new biological functions:anticoagulation,^[29][30] andantiviral activity againstenveloped viruses such ashuman herpes simplex andDengue,^{[citation needed]} The latter is amosquito-borne virus.

Related exopolysaccharides (β-glucans) from several strains ofBotryosphaeria rhodina (theteleomorphLasiodiplodiatheobromae^[7]) isolated from rotting tropical fruits have been described,^[31][32][33] The chemical structures of three β-glucans produced were characterized; a (1→3)(1→6)-β-D-glucan with a single glucose repeat substituent (frequency of 20%),^[31] an unbranched (1→6)-β-glucan namedlasiodiplodan,^[31][32][33] and a new (1→3)(1→6)-β-glucan with unique branches comprising gentiobiose andgentiotriose residues, but not glucose.^[33]

Structural characterization of thecell wall (mycelium) ofBotryosphaeria rhodina MAMB-05 revealed the presence of three different D-glucans; a linear (1→6)-β-glucan, a branched (1→3)(1→6)-β-glucan with single glucose repeat branches (frequency of 18%), and aglycogen-like (1→4)(1→6)-α-glucan.^[20]

Botryosphaeran was demonstrated in extensive studies on mice^[34][35] and mammaliancell-lines (hamster, rat, human)^[36][37][38] that it was notmutagenic (assessed by themicronucleus test),^[34][35] nor was itgenotoxic as assessed by theAmes test andComet assay.^[36][38]

When administered orally to mice by gavage, botryosphaeran reduced theclastogenic effect ofcyclophosphamide-inducedmicronucleus formation inbone marrow (polychromatic erythrocytes)^[34] andperipheral blood (reticulocytes) cells.^[34][35]

Using mammalian cell lines: lungfibroblasts (Chinese hamster) andhepatocarcinoma cells (rat), botryosphaeran was confirmed not to bemutagenic nor genotoxic by the micronucleus test and Comet assay procedures.^[38] Botryosphaeran exhibited nomutagenicity, and protected cultured human whole bloodlymphocytes againstDNA damage andcell death induced bybleomycin throughout thecell cycle stage.^{[citation needed]} Botryosphaeran exhibited antigenotoxic activity against damage induced bymethyl methanesulfonate, in normal andtumorigenic (Jurkat) human lymphocytes.^[36]

The absence of mutagenicity and genotoxicity assessed by the micronucleus, Ames andMTT tests, and the Comet assay, established that botryosphaeran hasGRAS status (Generally Recommended As Safe), and is safe for use by humans and animals.

Therheological properties of botryosphaeran has been described.^[39][40]

Botryosphaeran forms a viscous solution when dissolved in water that is stable to heat as occurs duringautoclaving (steamsterilization). When an aqueous solution of botryosphaeran is cooled to 5 °C, it forms a stronggel that is firm and transparent.

Botryosphaeran possessesin-vitrofree-radicalscavenging properties andantioxidant activities.^[41]

Botryosphaeran exhibits anin-vivo antioxidant role in theβ-cell line INS-1E derived from ratinsulinoma (tumor of thepancreas derived from β-cells).^[42]Oxidative stress was induced byhydrogen peroxide (H₂O₂) in the INS-1E cells under highglucose, and botryosphaeran decreased this condition by reducing the production ofreactive oxygen species (ROS).^[42]Apoptosis increased in the INS-1E cells treated with H₂O₂ in high glucose conditions, and treatment with botryosphaeran attenuated apoptosis.

Botryosphaeran exerts achemoprotective effect exhibiting strongantimutagenic (anticlastogenic) activity against thein-vivo DNA-damaging effect of cyclophosphamide in mice.,^[35][43] and genotoxic damage bydoxorubicin infibroblasts and hepatocarcinoma cells,^[38] bleomycin in human lymphocytes,^{[citation needed]} and methyl methanesulfonate in Jurkat cells.^[36]

Botryosphaeran exhibits hypoglycaemic activity (lowering of blood glucose levels) in rats in which diabetes was induced byintramuscular injection ofstreptozotocin, which selectively damages the pancreaticinsulin-secreting β-cells resulting intype-1 diabetes condition.^[43]

Thecholesterol-lowering effect (hypocholesterolaemia) of β-glucans derived from oat and barley (β-(1→3)(1→4)-linked D-glucans) is well established.^[44] Botryosphaeran exhibits hypocholesterolaemic activity lowering totalcholesterol andLow Density Lipoprotein (LDL)-cholesterol blood levels in rats preconditioned onhyperlipidaemic diets.^[35][45]

In experiments with elderly maleknockout LDLr^-/-mice (LDLreceptor-deficient mice that show elevatedplasma cholesterol levels and developatherosclerosis), botryosphaeran reduced theplasma glucose levels, improved thelipidic profiles, reducedLDL-cholesterol, and decreasedaortic lipid deposition that lowerscardiovascular risks ofatherosclerosis.^[35]

Treatment ofobese rats with botryosphaeran by gavage was effective in ameliorating thecomorbidities (diabetes,dyslipidaemia,hepatic steatosis) associated with obesity. Botryosphaeran reduces hepatic steatosis and dyslipidaemia, andglucose intolerance in diet-induced obese rats through activation ofAMP-activated protein-kinase (AMPK) and the expression of theForkhead transcription factor, FOXO3a, inadipose tissue.^[45]

Obese rats showed significant increases inweight gain, adipose tissue mass, andadiposity andatherogenic indices, and presented glucose intolerance,insulin resistance, dyslipidaemia, and hepatic steatosis. Botryosphaeran significantly reduces feed intake, weight gains, periepididymal andmesenteric fat, and improvesglucose tolerance in obese rats. Botryosphaeran, furthermore, reduces theserum levels oftriglyceride andVLDL-cholesterol, and increasedHDL-cholesterol andglycogen in liver, and reduces the atherogenic index.^[45]

The above data demonstrated the beneficial effects of botryosphaeran in reducing the stimulatory effect of obesity on dyslipidaemia and hepatic steatosis, and can play a potential role in the management of obesity comorbidities.

Studies on human carcinoma cell-lines: Jurkat (lymphocytes)^[37] and breast (MCF-7)^[46] demonstrated that botryosphaeran manifests anti-cancer activity.

The action by whichanticancer activity occurs is still not well understood, but a mechanistic insight on how this may occur inbreast cancerMCF-7 cells was advanced in 2015, and involves cell-signaling pathways that suppresstumourigenesis (cellantiproliferation) through apoptosis,necrosis andoxidative stress. Botryosphaeran-induced apoptosis was mediated by AMPK and FOXO3a.^[46]

In tumorigenic human lymphocytes (Jurkat cells), botryosphaeran modulates gene expression and regulatescell cycle and thecell cycle checkpoint.^[37]

Encapsulation ofprobioticbacteria (Lactobacillus casei) inalginatemicrospheres together with botryosphaeran^[47] andmucilages fromlinseed andokra increases the encapsulation efficiency, and improves the stability of the encapsulated probiotic bacteria during prolonged storage at 5 °C.Gastrointestinal simulation of themicroencapsulatedLactobacillus casei cells demonstrated preservation of theviability of the probiotic bacteria against lowpH andbile salts.^[47] The use of botryosphaeran in microencapsulating probiotic bacteria appears to be another promising application for this β-glucan.

New applications for botryosphaeran have included:biological response modifying activities of derivatized botryosphaerans; treatment ofskin conditions (eczema,psoriasis,wound healing);antimicrobial activities;antinociceptive activity; as a matrix for enzyme (laccase)immobilization;^{[citation needed]} as a platform forelectrochemicalsensors;^[48] and as afood additive (nutraceutical).

Thebiomass resulting fromBotryosphaeria rhodina MAMB-05 on producing botryosphaeran bysubmerged fermentation has been successfully used as abiosorbent to extractmetallic species;rare earth elements (lanthanides;La,Sm),^[49] andheavy metals (lead; Pb)^{[citation needed]} fromindustrial effluents.

• Biomolecules
• Rheology
• Probiotics
• Carbohydrates
• Fermentation
• Biomass
• Polysaccharides
• Prebiotic chemistry
• Botryosphaeriales

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