Botryosphaeran

The ascomycete and filamentous fungus, Botryosphaeria rhodina (strain MAMB-05), was isolated from a canker on the trunk of a eucalypt tree, and was molecularly characterized by sequencing the Internal Transcribed Spacer (ITS) region of rDNA.{{Cite journal|date=2004-04-29|title=Botryosphaeria rhodina isolate MAMB 05 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence|url=http://www.ncbi.nlm.nih.gov/nuccore/AY612337.1}}{{Cite journal|last1=Saldanha|first1=Roze L.|last2=Garcia|first2=José E.|last3=Dekker|first3=Robert F. H.|last4=Vilas-Boas|first4=Laurival A.|last5=Barbosa|first5=Aneli M.|date=June 2007|title=Genetic diversity among Botryosphaeria isolates and their correlation with cell wall-lytic enzyme production|journal=Brazilian Journal of Microbiology|volume=38|issue=2|pages=259–264|doi=10.1590/s1517-83822007000200013|issn=1517-8382|doi-access=free}}

The β-glucan, botryosphaeran, was discovered accidentally in 1994 while cultivating Botryosphaeria rhodina MAMB-05 on nutrient media containing glucose to produce the enzyme, laccase. This fungal isolate produces a constitutive laccase that could be induced to higher enzyme titers by various lignin-like aromatic compounds, and especially veratryl alcohol.{{Cite journal|last1=Barbosa|first1=A.M.|last2=Dekker|first2=R.F.H.|last3=Hardy|first3=G.E. St|date=August 1996|title=Veratryl alcohol as an inducer of laccase by an ascomycete, Botryosphaeria sp., when screened on the polymeric dye Poly R-478|journal=Letters in Applied Microbiology|volume=23|issue=2|pages=93–96|doi=10.1111/j.1472-765x.1996.tb00038.x|s2cid=84782439|issn=0266-8254}} The fungus was found to be ligninolytic.{{Cite journal|last=BARBOSA|first=A. M.|date=1995|title=In-vivo decolorisation of Poly R-478 as a method for screening ligninolytic microorganisms for use in bioremediation|url=https://ci.nii.ac.jp/naid/10017443476/|journal=4th Pacific Rim Biotechnol. Conf., Melbourne, Australia, Feb 6-9, 1995, Pp. 88-90.}}{{Cite journal|last1=Dekker|first1=Robert F.H.|last2=Barbosa|first2=Aneli M.|last3=Sargent|first3=Keith|date=March 2002|title=The effect of lignin-related compounds on the growth and production of laccases by the ascomycete, Botryosphaeria sp.|journal=Enzyme and Microbial Technology|volume=30|issue=3|pages=374–380|doi=10.1016/s0141-0229(01)00503-8|issn=0141-0229}}

Botryosphaeran is secreted by the fungus during growth and appears in the fermentation broth where its presence causes an increase in the broth's viscosity. It can easily be extracted from the broth by precipitation methods. Veratryl alcohol, however, suppresses the formation of botryosphaeran.{{Cite journal|last1=Dekker|first1=Robert F.H.|last2=Vasconcelos|first2=Ana-Flora D.|last3=Barbosa|first3=Aneli M.|last4=Giese|first4=Ellen C.|last5=Paccola-Meirelles|first5=Luzia|date=2001-12-01|title=A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source|journal=Biotechnology Letters|volume=23|issue=24|pages=1987–1993|doi=10.1023/A:1013742527550|s2cid=11468117|issn=1573-6776}}

Botryosphaeran is produced under submerged fermentation conditions when Botryosphaeria rhodina MAMB-05 is grown on nutrient media containing glucose and mineral salts. Extracting the fermentation broth with alcohol causes the EPS (botryosphaeran) to precipitate from solution, and this can be separated by centrifugation or filtration.

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 through chemical derivatization with various functional groups.

The influence of the composition of the nutrient medium,{{Cite journal|last1=Steluti|first1=Rosangela M.|last2=Giese|first2=Ellen C.|last3=Piggato|first3=Mariane M.|last4=Sumiya|first4=Andressa F. G.|last5=Covizzi|first5=Luiz G.|last6=Job|first6=Aldo E.|last7=Cardoso|first7=Marilsa S.|last8=De Lourdes Corradi Da Silva|first8=Maria|last9=Dekker|first9=Robert F. H.|date=December 2004|title=Comparison of Botryosphaeran production by the ascomyceteous fungusBotryosphaeria sp., grown on different carbohydrate carbon sources, and their partial structural features|journal=Journal of Basic Microbiology|volume=44|issue=6|pages=480–486|doi=10.1002/jobm.200410415|pmid=15558819|s2cid=37841100|issn=0233-111X}} including nitrogen,{{Cite journal|last1=Giese|first1=Ellen Cristine|last2=Sumiya|first2=Andressa|last3=Borsato|first3=Dioníso|last4=Dekker|first4=Robert|last5=Barbosa|first5=Aneli|date=2015-04-05|title=Evaluation of Fermentative Parameters for the Production of Botryosphaeran (a(1-->3;1-->6)-β-D-glucan) and Mycelial Biomass by Botryosphaeria rhodina MAMB-05|journal=Orbital - the Electronic Journal of Chemistry|volume=7|issue=1|doi=10.17807/orbital.v7i1.637|issn=1984-6428|doi-access=free}} phosphate, minerals, supplements (soybean oil, Tween 80),{{Cite journal|last1=Silva|first1=Cassiano C.|last2=Dekker|first2=Robert F.H.|last3=Silva|first3=Rui Sérgio S.F.|last4=Silva|first4=Maria de Lourdes Corradi da|last5=Barbosa|first5=Aneli M.|date=August 2007|title=Effect of soybean oil and Tween 80 on the production of botryosphaeran by Botryosphaeria rhodina MAMB-05|journal=Process Biochemistry|volume=42|issue=8|pages=1254–1258|doi=10.1016/j.procbio.2007.05.009|issn=1359-5113}} and the carbon source (carbohydrates),{{Cite journal|last1=DELOURDESCORRADIDASILVA|first1=M|last2=IZELI|first2=N|last3=MARTINEZ|first3=P|last4=SILVA|first4=I|last5=CONSTANTINO|first5=C|last6=CARDOSO|first6=M|last7=BARBOSA|first7=A|last8=DEKKER|first8=R|last9=DASILVA|first9=G|date=2005-07-04|title=Purification and structural characterisation of (1→3;1→6)-β--glucans (botryosphaerans) from grown on sucrose and fructose as carbon sources: a comparative study|journal=Carbohydrate Polymers|volume=61|issue=1|pages=10–17|doi=10.1016/j.carbpol.2005.01.002|issn=0144-8617}} is important in enhancing the production of botryosphaeran and biomass during fermentation by Botryosphaeria rhodina MAMB-05.

Catabolite repression,{{Cite journal|last1=Crognale|first1=Silvia|last2=Bruno|first2=Maria|last3=Moresi|first3=Mauro|last4=Petruccioli|first4=Maurizio|date=July 2007|title=Enhanced production of β-glucan from Botryosphaeria rhodina using emulsified media or fan impellers|journal=Enzyme and Microbial Technology|volume=41|issue=1–2|pages=111–120|doi=10.1016/j.enzmictec.2006.12.008|issn=0141-0229}} and the presence of β-glucan-hydrolyzing enzymes that attack botryosphaeran{{Cite journal|last1=Giese|first1=Ellen C.|last2=Dekker|first2=Robert F.H.|last3=Scarminio|first3=Ieda S.|last4=Barbosa|first4=Aneli M.|last5=da Silva|first5=Roberto|date=January 2011|title=Comparison of β-1,3-glucanase production by Botryosphaeria rhodina MAMB-05 and Trichoderma harzianum Rifai and its optimization using a statistical mixture-design|journal=Biochemical Engineering Journal|volume=53|issue=2|pages=239–243|doi=10.1016/j.bej.2010.10.013|bibcode=2011BioEJ..53..239G |issn=1369-703X|hdl=11449/72299|hdl-access=free}} during the fermentation process are critical and limit the production of botryosphaeran.

Statistical factorial design methods, such as the response surface methodology (RSM),{{Cite book|title=Statistics for experimenters : design, innovation, and discovery|last=P.|first=Box, George E.|date=2005|publisher=Wiley-Interscience|others=Hunter, J. Stuart, 1923-, Hunter, William Gordon, 1937-|isbn=978-0471718130|edition=2nd|location=Hoboken, N.J.|oclc=57286064}}{{Cite book|title=Statistical design--chemometrics|last=E.|first=Bruns, R.|date=2006|publisher=Elsevier|others=Scarminio, I. S., Barros Neto, B. de.|isbn=9780444521811|edition=1st|location=Amsterdam|oclc=162587290}} are effective in investigating complex fermentation parameters and their interactions to optimize metabolite production by microorganisms. RSM assists in defining the effects and interactions of the physiological factors playing a role in biotechnological processes in the production of microbial metabolites 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 by Botryosphaeria rhodina MAMB-05 over unoptimized conditions.

Botryosphaeria rhodina MAMB-05 when grown on nutrient media containing different carbohydrate substrates produces a family of botryosphaerans. These β-glucans differ only in the extent and frequency of side-chain substituents.

Botryosphaeran production can be enhanced when Botryosphaeria rhodina MAMB-05 is cultivated on glucose media containing soybean oil and the surfactant, Tween 80.

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 through precipitation with ethanol, which all takes place on a time-scale of days compared to other commercial β-glucans available on the market. The latter are extracted from fungal fruiting bodies (mushrooms, fungal brackets), spent Brewers yeast, and cereal grains (barley, oat) that can take weeks-to-months to prepare.

The mycelium of Botryosphaeria rhodina MAMB-05 is a rich source of β-glucans.{{Cite journal|last1=Corradi da Silva|first1=Maria de Lourdes|last2=Fukuda|first2=Eliane K.|last3=Vasconcelos|first3=Ana Flora D.|last4=Dekker|first4=Robert F.H.|last5=Matias|first5=Andreza C.|last6=Monteiro|first6=Nilson K.|last7=Cardoso|first7=Marilsa S.|last8=Barbosa|first8=Aneli M.|last9=Silveira|first9=Joana L.M.|date=March 2008|title=Structural characterization of the cell wall d-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05|journal=Carbohydrate Research|volume=343|issue=4|pages=793–798|doi=10.1016/j.carres.2007.12.021|pmid=18237722|issn=0008-6215}}

The chemical structure of botryosphaeran was first described in 2003, and was determined using the methods: methylation analysis, Smith degradation, Gas Chromatography-Mass Spectroscopy (GC-MS) and ¹³C NMR.

Total acid hydrolysis of botryosphaeran produces only D-glucose, while partial acid hydrolysis and enzymatic hydrolysis{{Cite journal|last1=Giese|first1=Ellen C.|last2=Covizzi|first2=Luiz G.|last3=Dekker|first3=Robert F.H.|last4=Monteiro|first4=Nilson K.|last5=Corradi da Silva|first5=Maria de Lourdes|last6=Barbosa|first6=Aneli M.|date=June 2006|title=Enzymatic hydrolysis of botryosphaeran and laminarin by β-1,3-glucanases produced by Botryosphaeria rhodina and Trichoderma harzianum Rifai|journal=Process Biochemistry|volume=41|issue=6|pages=1265–1271|doi=10.1016/j.procbio.2005.12.023|issn=1359-5113}}{{Cite book|last1=Giese|first1=Ellen Cristine|last2=Monteiro|first2=Alexandre C.|last3=de Melo Barbosa|first3=Aneli|last4=Dekker|first4=Robert F. H.|last5=dos Santos|first5=Osvaldo|last6=de Lourdes Corradi da Silva|first6=Maria|last7=Gomes|first7=Eleni|last8=da Silva|first8=Roberto|date=January 2009|title=Evaluation of the β-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-β-D-glucans|journal=Current Research Topics in Applied Microbiology and Microbial Biotechnology|publisher=WORLD SCIENTIFIC|doi=10.1142/9789812837554_0091|isbn=9789812837547}}{{Cite journal|last1=Bauermeister|first1=Anelize|last2=Amador|first2=Ismael R.|last3=Pretti|first3=Carla P.|last4=Giese|first4=Ellen C.|last5=Oliveira|first5=André L. M.|last6=Alves da Cunha|first6=Mário A.|last7=Rezende|first7=Maria Inês|last8=Dekker|first8=Robert F. H.|last9=Barbosa|first9=Aneli M.|date=2015-01-05|title=β-(1 → 3)-Glucanolytic Yeasts from Brazilian Grape Microbiota: Production and Characterization of β-Glucanolytic Enzymes by Aureobasidium pullulans 1WA1 Cultivated on Fungal Mycelium|journal=Journal of Agricultural and Food Chemistry|volume=63|issue=1|pages=269–278|doi=10.1021/jf504333h|pmid=25559084|bibcode=2015JAFC...63..269B |issn=0021-8561}} produces a series of homologous gluco-oligosaccharides of different degrees of polymerization, which can be analyzed by High Performance Liquid Chromatography (HPLC).

Enzymatic digestion of botryosphaeran under controlled conditions employing the enzymes: β-(1→3)-glucanases and β-(1→6)-glucanases from Botryosphaeria rhodina MAMB-05,{{Cite journal|last1=Giese|first1=Ellen C.|last2=Covizzi|first2=Luiz G.|last3=Borsato|first3=Dionísio|last4=Dekker|first4=Robert F.H.|last5=de Lourdes Corradi da Silva|first5=Maria|last6=Barbosa|first6=Aneli M.|date=December 2005|title=Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai|journal=Process Biochemistry|volume=40|issue=12|pages=3783–3788|doi=10.1016/j.procbio.2005.04.004|issn=1359-5113}}Trichoderma harzianum Rifai, and Aureobasidium pullulans 1WA1, produces a mixed series of β-(1→3)- and β-(1→6)- linked gluco-oligosaccharides that can serve as prebiotics.

Enzymes hydrolyzing botryosphaeran can be obtained by cultivating Botryosphaeria rhodina MAMB-05, Trichoderma harzianum Rifai and Aureobasidium pullulans 1WA1 on nutrient media containing either botryosphaeran, or the biomass derived from Botryosphaeria rhodina MAMB-05, which is a rich source of β-glucans.

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

Methylation and Smith degradation analysis revealed that botryosphaeran constituted a backbone 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. The chemical structure of botryosphaeran is a (1→3)(1→6)-β-D-glucan. ¹³C NMR spectroscopy confirmed its structure.

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

Botryosphaeran exists in a triple helix conformation,{{Cite journal|last1=Giese|first1=Ellen C.|last2=Dekker|first2=Robert F.H.|last3=Barbosa|first3=Aneli M.|last4=da Silva|first4=Roberto|date=November 2008|title=Triple helix conformation of botryosphaeran, a (1→3;1→6)-β-d-glucan produced by Botryosphaeria rhodina MAMB-05|journal=Carbohydrate Polymers|volume=74|issue=4|pages=953–956|doi=10.1016/j.carbpol.2008.04.038|issn=0144-8617}} an important structural feature in manifesting biological response modifying activities.{{Cite journal|last1=Bohn|first1=John A.|last2=BeMiller|first2=James N.|date=January 1995|title=(1→3)-β-d-Glucans as biological response modifiers: a review of structure-functional activity relationships|journal=Carbohydrate Polymers|volume=28|issue=1|pages=3–14|doi=10.1016/0144-8617(95)00076-3|issn=0144-8617}}{{Cite journal|last1=Leung|first1=M.Y.K.|last2=Liu|first2=C.|last3=Koon|first3=J.C.M.|last4=Fung|first4=K.P.|date=June 2006|title=Polysaccharide biological response modifiers|journal=Immunology Letters|volume=105|issue=2|pages=101–114|doi=10.1016/j.imlet.2006.01.009|pmid=16554097|issn=0165-2478}}{{Cite journal|last1=Wang|first1=Qiang|last2=Sheng|first2=Xiaojing|last3=Shi|first3=Aimin|last4=Hu|first4=Hui|last5=Yang|first5=Ying|last6=Liu|first6=Li|last7=Fei|first7=Ling|last8=Liu|first8=Hongzhi|date=2017-02-09|title=β-Glucans: Relationships between Modification, Conformation and Functional Activities|journal=Molecules|volume=22|issue=2|pages=257|doi=10.3390/molecules22020257|pmid=28208790|pmc=6155770|issn=1420-3049|doi-access=free}}

Derivatization of botryosphaeran by carboxymethylation and sulfonylation{{Cite journal|last1=Mendes|first1=Simone Ferreira|last2=Santos|first2=Osvaldo dos|last3=Barbosa|first3=Aneli M.|last4=Vasconcelos|first4=Ana Flora D.|last5=Aranda-Selverio|first5=Gabriel|last6=Monteiro|first6=Nilson K.|last7=Dekker|first7=Robert F.H.|last8=Pereira|first8=Mariana Sá|last9=Tovar|first9=Ana Maria F.|date=October 2009|title=Sulfonation and anticoagulant activity of botryosphaeran from Botryosphaeria rhodina MAMB-05 grown on fructose|journal=International Journal of Biological Macromolecules|volume=45|issue=3|pages=305–309|doi=10.1016/j.ijbiomac.2009.06.004|pmid=19549543|issn=0141-8130}}{{Cite journal|last=Brandi|first=Jamile|date=2011-10-28|title=Chemical Modification of Botryosphaeran: Structural Characterization and Anticoagulant Activity of a Water-Soluble Sulfonated (1→3)(1→6)-β-D-Glucan|journal=Journal of Microbiology and Biotechnology|volume=21|issue=10|pages=1036–1042|doi=10.4014/jmb.1105.05020|pmid=22031027|issn=1017-7825}} results in improved solubility in water, and diminishes its viscous nature in solution.

In the case of sulfonated botryosphaeran, the chemically modified polysaccharide containing sulfonate groups exhibited new biological functions: anticoagulation, and antiviral activity against enveloped viruses such as human herpes simplex and Dengue,{{citation needed|date=October 2019}} The latter is a mosquito-borne virus.

Related exopolysaccharides (β-glucans) from several strains of Botryosphaeria rhodina (the teleomorph Lasiodiplodia theobromae) isolated from rotting tropical fruits have been described,{{Cite journal|last1=Vasconcelos|first1=Ana Flora D.|last2=Monteiro|first2=Nilson K.|last3=Dekker|first3=Robert F.H.|last4=Barbosa|first4=Aneli M.|last5=Carbonero|first5=Elaine R.|last6=Silveira|first6=Joana L.M.|last7=Sassaki|first7=Guilherme L.|last8=da Silva|first8=Roberto|last9=de Lourdes Corradi da Silva|first9=Maria|date=September 2008|title=Three exopolysaccharides of the β-(1→6)-d-glucan type and a β-(1→3;1→6)-d-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit|journal=Carbohydrate Research|volume=343|issue=14|pages=2481–2485|doi=10.1016/j.carres.2008.06.013|pmid=18639868|issn=0008-6215}}{{Cite journal|last1=Alves da Cunha|first1=Mário A.|last2=Turmina|first2=Janaína A.|last3=Ivanov|first3=Raphael C.|last4=Barroso|first4=Roney R.|last5=Marques|first5=Patrícia T.|last6=Fonseca|first6=Eveline A. I.|last7=Fortes|first7=Zuleica B.|last8=Dekker|first8=Robert F. H.|last9=Khaper|first9=Neelam|date=2012-03-08|title=Lasiodiplodan, an exocellular (1→6)-β-d-glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity|journal=Journal of Industrial Microbiology & Biotechnology|volume=39|issue=8|pages=1179–1188|doi=10.1007/s10295-012-1112-2|pmid=22399240|s2cid=15375855|issn=1367-5435}}{{Cite journal|last1=Oliveira|first1=Kassandra S.M.|last2=Di Bastiani|first2=Mirela|last3=Cordeiro|first3=Lucimara M.C.|last4=Costa|first4=Mírian F.|last5=Toledo|first5=Karina A.|last6=Iacomini|first6=Marcello|last7=Babosa|first7=Aneli M.|last8=Dekker|first8=Robert F.H.|last9=Nascimento|first9=Valéria M.G.|date=November 2015|title=(1→6)- and (1→3)(1→6)-β-glucans from Lasiodiplodia theobromae MMBJ: Structural characterization and pro-inflammatory activity|journal=Carbohydrate Polymers|volume=133|pages=539–546|doi=10.1016/j.carbpol.2015.07.060|pmid=26344312|issn=0144-8617}} 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%), an unbranched (1→6)-β-glucan named [https://www.ncbi.nlm.nih.gov/pubmed/22399240 lasiodiplodan], and a new (1→3)(1→6)-β-glucan with unique branches comprising gentiobiose and [https://pubchem.ncbi.nlm.nih.gov/compound/4632877#section=Top gentiotriose] residues, but not glucose.

Structural characterization of the cell wall (mycelium) of Botryosphaeria 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 a glycogen-like (1→4)(1→6)-α-glucan.

Botryosphaeran was demonstrated in extensive studies on mice{{Cite journal|last1=Miranda|first1=Carolina C.B.O.|last2=Dekker|first2=Robert F.H.|last3=Serpeloni|first3=Juliana M.|last4=Fonseca|first4=Eveline A.I.|last5=Cólus|first5=Ilce M.S.|last6=Barbosa|first6=Aneli M.|date=March 2008|title=Anticlastogenic activity exhibited by botryosphaeran, a new exopolysaccharide produced by Botryosphaeria rhodina MAMB-05|journal=International Journal of Biological Macromolecules|volume=42|issue=2|pages=172–177|doi=10.1016/j.ijbiomac.2007.10.010|pmid=18022685|issn=0141-8130}}{{Cite journal|last1=Silva-Sena|first1=Geralda Gillian|last2=Malini|first2=Maressa|last3=Delarmelina|first3=Juliana Macedo|last4=Dutra|first4=Jean Carlos Vencioneck|last5=Gervásio|first5=Suiany Vitorino|last6=Leal|first6=Marcos André Soares|last7=Costa Pereira|first7=Thiago de Melo|last8=Barbosa-Dekker|first8=Aneli M.|last9=Dekker|first9=Robert F.H.|date=February 2018|title=In vivo antimutagenic and antiatherogenic effects of the (1 → 3)(1 → 6)-β-d-glucan botryosphaeran|journal=Mutation Research/Genetic Toxicology and Environmental Mutagenesis|volume=826|pages=6–14|doi=10.1016/j.mrgentox.2017.12.008|pmid=29412871|issn=1383-5718|doi-access=free|bibcode=2018MRGTE.826....6S }} and mammalian cell-lines (hamster, rat, human){{Cite journal|last1=Kerche-Silva|first1=Leandra E.|last2=Cólus|first2=Ilce M.S.|last3=Malini|first3=Maressa|last4=Mori|first4=Mateus Prates|last5=Dekker|first5=Robert F.H.|last6=Barbosa-Dekker|first6=Aneli M.|date=February 2017|title=In vitro protective effects of botryosphaeran, a (1 → 3;1 → 6)-β- d -glucan, against mutagens in normal and tumor rodent cells|journal=Mutation Research/Genetic Toxicology and Environmental Mutagenesis|volume=814|pages=29–36|doi=10.1016/j.mrgentox.2016.12.003|pmid=28137365|issn=1383-5718|doi-access=free|bibcode=2017MRGTE.814...29K }}{{Cite journal|last1=Malini|first1=M.|last2=Camargo|first2=M.S.|last3=Hernandes|first3=L.C.|last4=Vargas-Rechia|first4=C.G.|last5=Varanda|first5=E.A.|last6=Barbosa|first6=A.M.|last7=Dekker|first7=R.F.H.|last8=Matsumoto|first8=S.T.|last9=Antunes|first9=L.M.G.|date=October 2016|title=Chemopreventive effect and lack of genotoxicity and mutagenicity of the exopolysaccharide botryosphaeran on human lymphocytes|journal=Toxicology in Vitro|volume=36|pages=18–25|doi=10.1016/j.tiv.2016.06.008|pmid=27387458|bibcode=2016ToxVi..36...18M |issn=0887-2333|hdl=11449/161952|hdl-access=free}}{{Cite journal|last1=Malini|first1=Maressa|last2=Souza|first2=Marilesia Ferreira de|last3=Oliveira|first3=Marcelo Tempesta de|last4=Antunes|first4=Lusânia Maria Greggi|last5=Figueiredo|first5=Suely Gomes de|last6=Barbosa|first6=Aneli M.|last7=Dekker|first7=Robert F.H.|last8=Cólus|first8=Ilce Mara de Syllos|date=June 2015|title=Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes|journal=International Journal of Biological Macromolecules|volume=77|pages=214–221|doi=10.1016/j.ijbiomac.2015.03.010|pmid=25795388|issn=0141-8130}} that it was not mutagenic (assessed by the micronucleus test), nor was it genotoxic as assessed by the Ames test and Comet assay.

When administered orally to mice by gavage, botryosphaeran reduced the clastogenic effect of cyclophosphamide-induced micronucleus formation in bone marrow (polychromatic erythrocytes) and peripheral blood (reticulocytes) cells.

Using mammalian cell lines: lung fibroblasts (Chinese hamster) and hepatocarcinoma cells (rat), botryosphaeran was confirmed not to be mutagenic nor genotoxic by the micronucleus test and Comet assay procedures. Botryosphaeran exhibited no mutagenicity, and protected cultured human whole blood lymphocytes against DNA damage and cell death induced by bleomycin throughout the cell cycle stage.{{citation needed|date=October 2019}} Botryosphaeran exhibited antigenotoxic activity against damage induced by methyl methanesulfonate, in normal and tumorigenic (Jurkat) human lymphocytes.

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

The rheological properties of botryosphaeran has been described.{{Cite journal|last=BONGIOVANI|first=Raphael A. M.|date=2009-04-09|title=Nota Científica: Caracterização reológica dos botriosferanas produzidos pelo Botryosphaeria rhodina MAMB-05 em glucose, sacarose e frutose como fontes de carbono|journal=Brazilian Journal of Food Technology|volume=12|issue=1|pages=53–59|doi=10.4260/bjft2009800900008|doi-broken-date=1 November 2024 |issn=1981-6723}}{{Cite journal|last1=Fonseca|first1=Paulo R.M.S.|last2=Dekker|first2=Robert F.H.|last3=Barbosa|first3=Aneli M.|last4=Silveira|first4=Joana L.M.|last5=Vasconcelos|first5=Ana F.D.|last6=Monteiro|first6=Nilson K.|last7=Aranda-Selverio|first7=Gabriel|last8=Da Silva|first8=Maria de Lourdes Corradi|date=2011-09-02|title=Thermal and Rheological Properties of a Family of Botryosphaerans Produced by Botryosphaeria rhodina MAMB-05|journal=Molecules|volume=16|issue=9|pages=7488–7501|doi=10.3390/molecules16097488|pmid=21892127|pmc=6264532|issn=1420-3049|doi-access=free}}

Botryosphaeran forms a viscous solution when dissolved in water that is stable to heat as occurs during autoclaving (steam sterilization). When an aqueous solution of botryosphaeran is cooled to 5 °C, it forms a strong gel that is firm and transparent.

Botryosphaeran possesses in-vitro free-radical scavenging properties and antioxidant activities.{{Cite journal|last1=Giese|first1=Ellen C.|last2=Gascon|first2=Jacob|last3=Anzelmo|first3=Gianluca|last4=Barbosa|first4=Aneli M.|last5=da Cunha|first5=Mário A. Alves|last6=Dekker|first6=Robert F.H.|date=January 2015|title=Free-radical scavenging properties and antioxidant activities of botryosphaeran and some other β-D-glucans|journal=International Journal of Biological Macromolecules|volume=72|pages=125–130|doi=10.1016/j.ijbiomac.2014.07.046|pmid=25128096|issn=0141-8130}}

Botryosphaeran exhibits an in-vivo antioxidant role in the β-cell line INS-1E derived from rat insulinoma (tumor of the pancreas derived from β-cells).{{Cite book|title=Biological activities of bacterial levan, and three fungal β-glucans, botryosphaeran and lasiodiplodan, under high glucose condition in the pancreatic β-cell line INS-1E.|last=Kazak|first=Hande|publisher=Narosa Publishing House, New Delhi, India|year=2014|isbn=978-81-8487-214-9|doi= 10.13140/2.1.1799.2649|url=http://www.narosa.com/books_display.asp?catgcode=978-81-8487-214-9|pages=Vol. 7, Ch. 8, p. 105–115}} Oxidative stress was induced by hydrogen peroxide (H₂O₂) in the INS-1E cells under high glucose, and botryosphaeran decreased this condition by reducing the production of reactive oxygen species (ROS). Apoptosis increased in the INS-1E cells treated with H₂O₂ in high glucose conditions, and treatment with botryosphaeran attenuated apoptosis.

Botryosphaeran exerts a chemoprotective effect exhibiting strong antimutagenic (anticlastogenic) activity against the in-vivo DNA-damaging effect of cyclophosphamide in mice.,{{Cite journal|last1=Miranda-Nantes|first1=Carolina C. B. O.|last2=Fonseca|first2=Eveline A. I.|last3=Zaia|first3=Cassia T. B. V.|last4=Dekker|first4=Robert F. H.|last5=Khaper|first5=Neelam|last6=Castro|first6=Inar A.|last7=Barbosa|first7=Aneli M.|date=September 2011|title=Hypoglycemic and Hypocholesterolemic Effects of Botryosphaeran from Botryosphaeria rhodina MAMB-05 in Diabetes-Induced and Hyperlipidemia Conditions in Rats|journal=Mycobiology|volume=39|issue=3|pages=187–193|doi=10.5941/myco.2011.39.3.187|pmid=22783102|pmc=3385115|issn=1229-8093}} and genotoxic damage by doxorubicin in fibroblasts and hepatocarcinoma cells, bleomycin in human lymphocytes,{{citation needed|date=October 2019}} and methyl methanesulfonate in Jurkat cells.

Botryosphaeran exhibits hypoglycaemic activity (lowering of blood glucose levels) in rats in which diabetes was induced by intramuscular injection of streptozotocin, which selectively damages the pancreatic insulin-secreting β-cells resulting in type-1 diabetes condition.

The cholesterol-lowering effect (hypocholesterolaemia) of β-glucans derived from oat and barley (β-(1→3)(1→4)-linked D-glucans) is well established.{{Cite journal|last1=Daou|first1=Cheickna|last2=Zhang|first2=Hui|date=2012-06-12|title=Oat Beta-Glucan: Its Role in Health Promotion and Prevention of Diseases|journal=Comprehensive Reviews in Food Science and Food Safety|volume=11|issue=4|pages=355–365|doi=10.1111/j.1541-4337.2012.00189.x|issn=1541-4337|doi-access=}} Botryosphaeran exhibits hypocholesterolaemic activity lowering total cholesterol and Low Density Lipoprotein (LDL)-cholesterol blood levels in rats preconditioned on hyperlipidaemic diets.{{Cite journal|last1=Silva|first1=Amadeu Z.|last2=Costa|first2=Felipe P.L.|last3=Souza|first3=Ingrid L.|last4=Ribeiro|first4=Mariana C.|last5=Giordani|first5=Morenna Alana|last6=Queiroz|first6=Diogo A.|last7=Luvizotto|first7=Renata A.M.|last8=Nascimento|first8=André F.|last9=Bomfim|first9=Gisele F.|date=October 2018|title=Botryosphaeran reduces obesity, hepatic steatosis, dyslipidaemia, insulin resistance and glucose intolerance in diet-induced obese rats|journal=Life Sciences|volume=211|pages=147–156|doi=10.1016/j.lfs.2018.09.027|pmid=30227131|s2cid=52293952|issn=0024-3205}}

In experiments with elderly male knockout LDLr^-/-mice (LDLreceptor-deficient mice that show elevated plasma cholesterol levels and develop atherosclerosis), botryosphaeran reduced the plasma glucose levels, improved the lipidic profiles, reduced LDL-cholesterol, and decreased aortic lipid deposition that lowers cardiovascular risks of atherosclerosis.

Treatment of obese rats with botryosphaeran by gavage was effective in ameliorating the comorbidities (diabetes, dyslipidaemia, hepatic steatosis) associated with obesity. Botryosphaeran reduces hepatic steatosis and dyslipidaemia, and glucose intolerance in diet-induced obese rats through activation of AMP-activated protein-kinase (AMPK) and the expression of the Forkhead transcription factor, FOXO3a, in adipose tissue.

Obese rats showed significant increases in weight gain, adipose tissue mass, and adiposity and atherogenic indices, and presented glucose intolerance, insulin resistance, dyslipidaemia, and hepatic steatosis. Botryosphaeran significantly reduces feed intake, weight gains, periepididymal and mesenteric fat, and improves glucose tolerance in obese rats. Botryosphaeran, furthermore, reduces the serum levels of triglyceride and VLDL-cholesterol, and increased HDL-cholesterol and glycogen in liver, and reduces the atherogenic index.

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) and breast (MCF-7){{Cite journal|last1=Queiroz|first1=Eveline A.I.F.|last2=Fortes|first2=Zuleica B.|last3=da Cunha|first3=Mário A.A.|last4=Barbosa|first4=Aneli M.|last5=Khaper|first5=Neelam|last6=Dekker|first6=Robert F.H.|date=October 2015|title=Antiproliferative and pro-apoptotic effects of three fungal exocellular β-glucans in MCF-7 breast cancer cells is mediated by oxidative stress, AMP-activated protein kinase (AMPK) and the Forkhead transcription factor, FOXO3a|journal=The International Journal of Biochemistry & Cell Biology|volume=67|pages=14–24|doi=10.1016/j.biocel.2015.08.003|pmid=26255117|issn=1357-2725}} demonstrated that botryosphaeran manifests anti-cancer activity.

The action by which anticancer activity occurs is still not well understood, but a mechanistic insight on how this may occur in breast cancer MCF-7 cells was advanced in 2015, and involves cell-signaling pathways that suppress tumourigenesis (cell antiproliferation) through apoptosis, necrosis and oxidative stress. Botryosphaeran-induced apoptosis was mediated by AMPK and FOXO3a.

In tumorigenic human lymphocytes (Jurkat cells), botryosphaeran modulates gene expression and regulates cell cycle and the cell cycle checkpoint.

Encapsulation of probiotic bacteria (Lactobacillus casei) in alginate microspheres together with botryosphaeran{{Cite journal|last1=Rodrigues|first1=Fábio J.|last2=Omura|first2=Michele H.|last3=Cedran|first3=Marina F.|last4=Dekker|first4=Robert F. H.|last5=Barbosa-Dekker|first5=Aneli M.|last6=Garcia|first6=Sandra|date=2017-07-04|title=Effect of natural polymers on the survival of Lactobacillus casei encapsulated in alginate microspheres|journal=Journal of Microencapsulation|volume=34|issue=5|pages=431–439|doi=10.1080/02652048.2017.1343872|pmid=28618877|s2cid=41482357|issn=0265-2048}} and mucilages from linseed and okra increases the encapsulation efficiency, and improves the stability of the encapsulated probiotic bacteria during prolonged storage at 5 °C. Gastrointestinal simulation of the microencapsulated Lactobacillus casei cells demonstrated preservation of the viability of the probiotic bacteria against low pH and bile salts. 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 of skin conditions (eczema, psoriasis, wound healing); antimicrobial activities; antinociceptive activity; as a matrix for enzyme (laccase) immobilization;{{citation needed|date=October 2019}} as a platform for electrochemical sensors;{{Cite journal|last1=Eisele|first1=Ana Paula Pires|last2=Valezi|first2=Camila Farinha|last3=Mazziero|first3=Tatiane|last4=Dekker|first4=Robert F.H.|last5=Barbosa-Dekker|first5=Aneli M.|last6=Sartori|first6=Elen Romão|date=May 2019|title=Layering of a film of carboxymethyl-botryosphaeran onto carbon black as a novel sensitive electrochemical platform on glassy carbon electrodes for the improvement in the simultaneous determination of phenolic compounds|journal=Sensors and Actuators B: Chemical|volume=287|pages=18–26|doi=10.1016/j.snb.2019.02.004|bibcode=2019SeAcB.287...18E |s2cid=104322350|issn=0925-4005}} and as a food additive (nutraceutical).

The biomass resulting from Botryosphaeria rhodina MAMB-05 on producing botryosphaeran by submerged fermentation has been successfully used as a biosorbent to extract metallic species; rare earth elements (lanthanides; La, Sm),{{Cite journal|last1=Giese|first1=Ellen C.|last2=Dekker|first2=Robert F.H.|last3=Barbosa-Dekker|first3=Aneli M.|date=2019-02-09|title=Biosorption of lanthanum and samarium by viable and autoclaved mycelium of Botryosphaeria rhodina MAMB-05|journal=Biotechnology Progress|volume=35|issue=3|pages=e2783|doi=10.1002/btpr.2783|pmid=30738002|s2cid=73427692|issn=8756-7938}} and heavy metals (lead; Pb){{citation needed|date=October 2019}} from industrial effluents.

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Category:Biomolecules

Category:Rheology

Category:Probiotics

Category:Carbohydrates

Category:Fermentation

Category:Biomass

Category:Polysaccharides

Category:Prebiotic chemistry

Category:Botryosphaeriales