80th JECFA - Chemical and Technical Assessment (CTA), 2015 © FAO 2015

MIXED -GLUCANASE AND XYLANASE FROM DISPOROTRICHUM DIMORPHOSPORUM

Chemical and Technical Assessment (CTA)

Prepared by Jannavi R. Srinivasan, Ph. D., and reviewed by Inge Meyland

1. Summary

This Chemical and Technical Assessment summarizes data and information on the mixed - glucanase and xylanase enzyme preparation submitted to JECFA by DSM Food Specialties (DSM Food Specialties, P. O. Box 1, 2600 MA Deft, The Netherlands). This document also discusses published information relevant to -glucanase, xylanase, and D. dimorphosporum, the production organism. This document uses the following expressions: “-glucanase” and “xylanase” to refer to the enzymes, and the expression “mixed -glucanase and xylanase enzyme preparation” to refer to the preparation formulated for commercial use.

-glucanase is an enzyme that catalyses the hydrolysis of 1,4-bonds in -D-glucans that contain 1,3- bonds. Xylanase is an enzyme that catalyses the hydrolysis of 1,4--D-xylosidic bonds in xylans. The mixed -glucanase and xylanase enzyme preparation is used as a processing aid in brewing, grain processing and in the production of potable alcohol up to 36.5 milligrams of Total Organic Solids per kilogram of raw material (mg TOS/kg RM). The mixed -glucanase and xylanase enzyme preparation is manufactured by a submerged fed- batch pure culture fermentation of a pure culture of D. dimorphosporum carrying the -glucanase and xylanase genes. D. dimorphosporum is considered to be non-pathogenic with a history of use in commercial food enzyme production. Tests have been performed to demonstrate that the D. dimorphosporum production strain is nontoxigenic; it does not produce toxins or antibiotics under conditions used for enzyme production. Authorities in the U. S. (NIH, 1998), Germany (Germany, 2013) and Switzerland (Switzerland, 2004) consider D. dimorphosporum to be a Risk Group 1 microorganism. It is not listed as a pathogen in Belgium (Belgium, 2010) and the Netherlands (Dutch Commission on Genetic Modification, 2011). The production strain, D. dimorphosporum DXL1, is obtained from a parent wild-type (WT) strain D. dimorphosporum (DSMZ) obtained from the ATCC collection as Sporotrichum dimorphosporum ATCC24562. The strain is also known as Basidiomycete QM-806. This WT strain is available in other culture collections as well, such as CBS484.76 and IMI155711. According to the culture collection catalogues the strain was isolated by E.G. Simmons from a blanket in New Guinea. The production strain was derived by re-isolation and subculturing of the wild-type D. dimorphosporum. The -glucanase and xylanase enzymes are secreted simultaneously to the fermentation broth and are subsequently purified and concentrated. The final enzyme product is standardized with glycerol, stabilized with sodium benzoate, and diluted with water to desired activity. The -glucanase and

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (1) of (6) © FAO 2015 xylanase enzyme preparation complies with the General Specifications and Considerations for Enzyme Preparations Used in Food Processing (FAO/WHO, 2006). β-Glucanase and xylanase from D. dimorphosporum have commonly been present in food, and there are no indications for allergic reactions due to the ingestion of β-glucanase and xylanase from D. dimorphosporum.  Description Light brown to dark brown liquid.

3. Method of Manufacture

3.1 D. dimorphosporum

D. dimorphosporum has been taxonomically identified to be from the genus Sporotrichum by the Dutch culture collection, the Centraalbureau voor Schimmelcultures. D. dimorphosporum is a saprophyte, and a basidiomycete fungus; it is capable of growing at pH 3.5-5.5, and at 28-32 °C. D. dimorphosporum is a non-pathogenic microbe with a history of use in food. The taxonomy of D. dimorphosporum is as follows:

Kingdom: Fungi Division: Eumycota Sub-division: Deuteromycetes Class: Hyphomycetes Order: Moniliales Family: Dematiaceae Genus: Disporotrichum Species: Disporotrichum dimorphosporum

D. dimorphosporum has been classified as a Risk Group 1 microorganism (i.e., not associated with disease in healthy adult humans) by the U.S. National Institutes of Health. It has also been classified as a Risk Group 1 microorganism in the German Federal Office of Consumer Protection and Food Safety (BVL), and in the Swiss Agency for the Environment, Forests and Landscape, SAEF) lists. D. dimorphosporum is not on the list of pathogens on the Belgian Biosafety Server and on the Dutch Commission on Genetic Modification. Strains of D. dimorphosporum that have been deposited in ATCC the American Type Culture Collection (ATCC, 2014) and DSMZ (Germany, 2014) Culture Collections are classified as Biosafety Level 1. Additionally, according to EU Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work. D. dimorphosporum does not appear on the list of pathogens in Annex III of the Directive (EU, 2000). D. dimorphosporum has been described in the literature as a safe source of food-processing enzymes (Belitz, et. al., 2008, Fraatz, M. A., et al, 2014). In 2014, the United States Food and Drug Administration did not question the conclusion that the mixed -glucanase and xylanase enzyme preparation from D. dimorphosporum strain is Generally Recognized As Safe (GRAS) under the intended conditions of use (GRAS Notice (GRN) 482) (US FDA, 2014).

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (2) of (6) © FAO 2015 3.2 D. dimorphosporum Production Strain

The D. dimorphosporum production strain is identified as DXL1. This production strain has been tested to be genetically stable under laboratory conditions with no significant degeneration in yield or appearance of morphological variants. -glucanase and xylanase preparation from DXL1 produced at large scale since 1999 have been on the market since. Unpublished data indicate that the production strain does not produce mycotoxins under large-scale fermentation conditions.

3.3 Fermentation, Recovery, and Formulation

-glucanase and xylanase are produced by a controlled aerobic submerged fed-batch pure culture fermentation of a pure culture of D. dimorphosporum. The equipment to produce -glucanase and xylanase from D. dimorphosporum is carefully designed, constructed, operated, cleaned, and maintained to prevent contamination by foreign microorganisms. Measures are in place during the various fermentation steps for physical and chemical quality control; microbiological analyses are also routinely done to ensure absence of foreign microorganisms. Periodic samples are removed to test for enzyme generating ability. The fermentation broth carrying the liquid enzyme is separated from the biomass that contains the production organism and spent fermentation media, by several filtration steps (polish and germ reduction). The liquid filtrate containing the enzyme is then concentrated by ultrafiltration, to remove low molecular weight compounds. 40-45% glycerol is added to the polished liquid enzyme concentrate, to standardize to desired activity. Sodium benzoate is added as a stabilizing agent, and the liquid enzyme preparation is filtered again prior to packaging. The mixed -glucanase and xylanase enzyme preparation conforms to the General specifications for enzyme preparations used in food processing, and the enzyme preparation is tested to be free from the production organism.

4. Characterization

4.1 Beta-glucanase

-glucanase catalyses the hydrolysis of (1,3)- or (1,4)-linkages in β-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3. The Chemical Abstract Service Registry Number (CAS No.) of -glucanase is CAS No. 62213-14- 3. -glucanase is classified by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB, online edition) as follows:

Accepted name: endo-1,3(4)-β-glucanase

Other names(s): endo-1,3-β-D-glucanase; laminarinase; laminaranase; β-1,3-glucanase; β-1,3-1,4-glucanase; endo-1,3-β-glucanase; endo-β-1,3(4)-glucanase; endo-β-1,3-1,4-glucanase; endo-β-(1→3)-D-glucanase; endo-1,3-1,4-β- D-glucanase; endo-β-(1-3)-D-glucanase; endo-β-1,3-glucanase IV; endo-1,3-β-D-glucanase; 1,3-(1,3;1,4)-β-D-glucan 3(4)- glucanohydrolase

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (3) of (6) © FAO 2015 Reaction: Endohydrolysis of (1→3)- or (1→4)-linkages in β-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3

Systematic name: 3-(1,3-1,4)-β-D-glucan 3(4) glucanohydrolase

EC No.: 3.2.1.6

Enzyme activity is measured by a method based on the reduction of viscosity of a β-glucan substrate at pH 5.60 and 45 °C, in the presence of -glucanase. This is measured using a calibrated Ubbelhode viscosimeter. -glucanase activity is expressed in Beta-Glucanase Fungique (BGF) units, as defined in the specific assay that measures a change in viscosity of a β-glucan substrate solution in the presence of -glucanase.

4.2 Xylanase

Xylanase catalyses the hydrolysis of (1→4)-β-D-xylosidic linkages in xylans. The CAS No. of xylanase is 9025-57-4. Xylanase is classified by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB, online edition) as follows:

Accepted name: endo-1,4-β-xylanase

Other names(s): endo-(1→4)-β-xylan 4-xylanohydrolase; endo-1,4-xylanase; xylanase; β-1,4-xylanase; endo-1,4-xylanase; endo-β-1,4-xylanase; endo-1,4-β-D- xylanase; 1,4-β-xylan xylanohydrolase; β-xylanase; β-1,4-xylan xylanohydrolase; endo-1,4-β-xylanase; β-D-xylanase

Reaction: Endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans

Systematic name: 4-β-D-xylan xylanohydrolase

EC No.: 3.2.1.8

Enzyme activity is measured by a method based on the reduction in viscosity of a xylan (from rye) substrate in the presence of xylanase. The activity is measured in a calibrated viscometer, as defined in the specific assay that measures a change in viscosity of a xylan substrate solution in the presence of xylanase.

4.2 Mixed -glucanase and Xylanase Enzyme Preparation

The mixed glucanase and xylanase enzyme preparation will be marketed as a liquid product under the trade name Filtrase BR-X L. A representative composition of a commercial liquid formulation of the mixed -glucanase and xylanase enzyme preparation is provided below:

Total Organic Solids (TOS): 10.8-16.65% w/w Water: 38-43.6% w/w Protein: 5.5-11 % w/w

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (4) of (6) © FAO 2015 Glycerol: up to 45% w/w Sodium Benzoate: 0.1-0.3% w/w

The TOS content is calculated according to the following equation:

TOS (%) = 100 – (A + W + D)

where:

A = % ash, W = % water and D = % diluents and/or other formulation ingredients (NAS/NRC, 1981; FAO/WHO, 2006). TOS consists of the enzyme of interest and residues of organic materials, such as proteins, peptides, and carbohydrates, derived from the production organism and the manufacturing process.

β-Glucanase and xylanase from D. dimorphosporum have commonly been present in food, and there are no indications for allergic reactions due to their ingestion. Also, since these enzymes are not genetically modified, an assessment of their potential allergenicity is not required.

5. Functional uses

The mixed -glucanase and xylanase enzyme preparation is used as a processing aid in brewing, grain processing and in the production of potable alcohol. The action of these enzymes allow for the hydrolysis of endo (1→3)- or (1→4)-bonds in β-D-glucans, to make smaller oligosaccharide units, and the hydrolysis of the (1,4)-β-D-xylosidic bonds that are present in xylans, including arabinoxylan resulting in mono- and oligosaccharide, respectively. Degradation of β-D-glucans and (arabino)xylan result in decreased viscosity, which in turn improves filterability, yield and consistency of product quality. The mixed-glucanase and xylanase enzyme preparation is used in brewing at a maximum level of 36.5 milligram TOS per kilogram raw material (mg TOS/kg RM), in grain processing at a maximum level of 25.5 mg TOS/kg RM, and in manufacture of potable alcohol at a maximum level of 25.5 mg TOS/kg RM.

6. Reactions and fate in food

The reaction products of the enzymatic conversion of -D-glucans in the presence of -glucanase are (glucose and di-,) tri-, tetra- and oligosaccharides of glucose residues. The reaction products of the hydrolysis of (arabino)xylans in the presence of xylanase are oligomers of 1,4-β-xylan and 1,4-β- arabinoxylan. Brewing, grain processing and production of potable alcohol include steps that would inactivate and/or remove -glucanase and xylanase. Thus, the carry-over of the active enzymes to food is expected to be negligible.

7. References http://www.dsmz.de/catalogues/details/culture/DSM- 8230.html?tx_dsmzresources_pi5%5BreturnPid%5D=304

FAO/WHO, 2006. General specifications and considerations for enzyme preparations used in food processing. In: Compendium of food additive specifications, volume 4. Analytical methods, test

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (5) of (6) © FAO 2015 procedures and laboratory solutions used by and referenced in the food additive specifications. FAO/JECFA Monographs 1. Rome, Italy, pp. xxi-xxv. www.atcc.org/~/ps/24562.ashx https://osha.europa.eu/en/legislation/directives/exposure-to-biological-agents/77

Food Chemistry; H.-D. Belitz, W. Grosch, P. Schieberle, May 2008, pp. 146-47.

M. Fraatz, M. Ruhl, H. Zorn, in Advances in Biochemical Engineering/Biotechnology, Vol 143, 2014, Ed. H. Zorn, P. Czermak, 2014, pp. 229.

US FDA, 2014; http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm425584.htm

Mixed β-Glucanase And Xylanase From Disporotrichum Dimorphosporum (CTA) 2015 - Page (6) of (6) © FAO 2015