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Safety evaluation of the food enzyme xylanase from Bacillus pumilus (strain BLXSC)

Abstract The food enzyme 1,4‐β‐d‐xylan xylanohydrolase (EC 3.2.1.8) is produced with the non‐genetically modified strain Bacillus pumilus (strain BLXSC) by Advanced Enzyme Technologies Ltd. The food enzyme is intended to be used in baking processes, grain treatment for the production of starch and gluten fractions, and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation and during grain treatment, dietary exposure was only calculated for baking processes. Based on the maximum recommended use levels for baking processes, and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme–Total Organic Solids (TOS) was estimated to be up to 0.138 mg TOS/kg body weight (bw) per day. As the production strain of B. pumilus meets the requirements for a Qualified Presumption of Safety (QPS) approach, no toxicological data are required. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that under the intended conditions of use (other than distilled alcohol production), the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but is considered to be low. Based on the QPS status of the production strain and the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

'Food enzyme' means a product obtained from plants, animals or micro-organisms or products thereof including a product obtained by a fermentation process using micro-organisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.
'Food enzyme preparation' means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.
Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 1 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 2 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that: i) it does not pose a safety concern to the health of the consumer at the level of use proposed; ii) there is a reasonable technological need; iii) its use does not mislead the consumer.
All food enzymes currently on the European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.
The 'Guidance on submission of a dossier on food enzymes for safety evaluation' (EFSA CEF Panel, 2009) lays down the administrative, technical and toxicological data required.

1.1.
Background and Terms of Reference as provided by the requestor 1.1.

Background as provided by the European Commission
Only food enzymes included in the European Union (EU) Community list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 1 on food enzymes.
Three applications have been submitted by the company 'Advanced Enzyme Technologies Ltd.' and 'Novozymes A/S' of the food enzymes pectinase, polygalacturonase, pectinesterase, pectin lyase, and arabanase from Aspergillus niger (strain ASNSC), xylanase from Bacillus pumilus (strain BLXSC) and asparaginase from a genetically modified strain of Bacillus subtilis (strain NZYM-CK).
Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 3 implementing Regulation (EC) No 1331/2008 2 , the Commission has verified that the three applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference
The European Commission (EC) requests the European Food Safety Authority (EFSA) to carry out safety assessments on the food enzymes pectinase, polygalacturonase, pectinesterase, pectin lyase,

Interpretation of the Terms of Reference
The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme xylanase from a non-genetically modified microorganism B. pumilus (strain BLXSC).

2.
Data and methodologies

Data
The applicant has submitted a dossier in support of the application for authorisation of the food enzyme xylanase from non-genetically modified B. pumilus (strain BLXSC).
Additional information was requested from the applicant during the assessment process on 19 April 2018 and on 16 October 2018 and was consequently provided (see 'Documentation provided to EFSA').

Methodologies
The assessment was conducted in line with the principles described in the EFSA 'Guidance on transparency in the scientific aspects of risk assessment' (EFSA, 2009) and following the relevant existing guidance from the EFSA Scientific Committee.
The current 'Guidance on the submission of a dossier for safety evaluation of a food enzyme' (EFSA CEF Panel, 2009) has been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016).

3.1.
Source of the food enzyme 5 The xylanase is produced with the non-genetically modified strain of B. pumilus designated BLXSC, 6 which is deposited at with number 7 The production strain was identified as B. The species B. pumilus is included in the 'Qualified Presumption of Safety' (QPS) list with the qualifications of absence of cytotoxic activity and antimicrobial resistance activity (EFSA, 2007;EFSA BIOHAZ Panel, 2017). The absence of cytotoxicity against Vero cells 10 and of haemolytic activity on blood agar plates 11 was demonstrated for B. pumilus BLXSC. Antimicrobial resistance of B. pumilus BLXSC was tested as recommended by EFSA FEEDAP Panel (2018). 10 of the strain BLXSC indicates that it does not contain any acquired antimicrobial gene. 9

3.2.
Production of the food enzyme 12 The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 13 , with food safety procedures based on Hazard Analysis and Critical Control Points (HACCP 14 ), and in accordance with current Good Manufacturing Practice (GMP).
The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration leaving a supernatant containing the food enzyme. The filtrate containing the enzyme is then further purified and concentrated, including ultrafiltration steps in which enzyme protein is retained while most of the low molecular weight material passes the filtration membrane and is discarded. The applicant provided information on the identity and analysis of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.
The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern. 3.3.
Characteristics of the food enzyme

Properties of the food enzyme
The xylanase is a single polypeptide chain of 228 amino acids. 15 The molecular mass of the mature protein, based on the amino acid sequence, was calculated to be 24 kDa. 16 The homogeneity of the food enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The gels presented for all batches are comparable and consistently showed one major protein band with an apparent molecular weight close to 24 kDa. 17 No other side enzyme activities were reported by the applicant. 18 The in-house determination of xylanase activity 19 is based on hydrolysis of beechwood xylan to reducing carbohydrates, which reduce dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, producing a colour (reaction conditions: pH 6.0, temperature 50°C, incubation time 20 min). One bacterial xylanase unit (BXU) is defined as the amount of enzyme required that liberates 1 micromole of reducing sugars (measured as xylose equivalents) from beechwood xylan in 1 min under the standard assay conditions.
The food enzyme has a temperature optimum of around 55°C (pH 6.0) and a pH optimum of around 6.5 (T = 50°C). 20 Thermostability 21 was tested after pre-incubation of the food enzyme for 2 h at different temperatures. Under the conditions (pH 6.0) of the applied temperature stability assay, the xylanase showed no residual activity above 70°C.

Chemical parameters 22
Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation (Table 1). The average Total Organic Solids (TOS) of the three commercial batches was 87.45%. The three food enzyme batches presented in Table 1 are spray dried concentrates.
The average enzyme activity/mg TOS ratio of the three food enzyme batches for commercialisation is 51.18 BXU/mg TOS.

Purity
The lead content in the three commercial batches was below 0.5 mg/kg which complies with the specification for lead (≤5 mg/kg) 23 as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). In addition, the levels of heavy metals (arsenic, mercury, cadmium) were below the limits of detection (LODs) of the employed methodologies. 24 The food enzyme complies with the microbiological criteria as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006), which stipulate that Escherichia coli and Salmonella species are absent in 25 g of sample, and total coliforms should not exceed 30 colony forming units (CFU) per gram. 25 No antimicrobial activity was detected in any of these batches (FAO/WHO, 2006). 26 The presence of mycotoxins (aflatoxin B1, B2, G1, G2, zearalenone, ochratoxin A, deoxynivalenol, T-2 toxin, HT-2 toxin, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine and ergotamine 27 ) was examined in three food enzyme batches and were below LOD of the applied analytical methods. 28

Toxicological data
No toxicological tests were provided by the applicant. The Panel considers no toxicological studies other than assessment of allergenicity necessary. This is based on the QPS status of the production strain (see Section 3.1) and the absence of any hazards arising from the manufacturing of the food enzyme.

Allergenicity 29
The allergenicity assessment considers only the food enzyme and not any carrier or other excipient which may be used in the final formulation.
The potential allergenicity of the xylanase produced with B. pumilus (strain BLXSC) was assessed by comparing its amino acid sequence with those of known allergens according to the scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms (EFSA GMO Panel, 2017). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found. 30 No information is available on oral sensitisation or elicitation reactions of this xylanase. However, respiratory allergy, e.g. baker's asthma, following occupational exposure to xylanase has been described in some epidemiological studies (Elms et al., 2003;Harris-Roberts et al., 2009;Martel et al., 2010;Budnik et al., 2017) and case reports (Tarvainen et al., 1991;Baur et al., 1998;Merget et al., 2001;Lipi nska-Ojrzanowska et al., 2016). However, several studies have shown that adults with occupational asthma to an enzyme may be able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Brisman, 2002;Poulsen, 2004;Armentia et al., 2009). Overall, allergic reactions upon oral ingestion of this xylanase, produced with the non-genetically modified B. pumilus strain BLXSC in individuals respiratory sensitised to xylanase cannot be excluded, but the likelihood of such a reaction to occur is low.
According to the information provided, substances or products that may cause allergies or intolerances (Regulation (EU) No 1169/2011 31 ) are used as raw materials ( 32 ) in the media fed to the microorganisms. However, during the fermentation process, these products will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the microbial biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that potentially allergenic residues of these foods employed as protein sources are not expected to be present.
Quantifying the risk for allergenicity is not possible in view of the individual susceptibility to food allergens. Allergenicity can be ruled out only if the proteins are removed, as is the case for distilled alcohol production.
The Panel considered that, under the intended conditions of use (other than distilled alcohol production), the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded but the likelihood of such reactions occurring is considered to be low.

3.5.
Dietary exposure 3.5.1. Intended use of the food enzyme This food enzyme is intended to be used in three food manufacturing processes at the recommended use levels summarised in Table 2. In baking processes, 33 the xylanase is added during the preparation of the dough. It hydrolyses (arabino)xylans, which interact with gluten and bind water, thus reducing the dough viscosity and shortening the processing time. The decrease in viscosity facilitates the handling of the dough, results in more uniform products with better properties (increased firmness, reduced oil absorption and less stockiness).
The food enzyme remains in dough. Based on data provided on thermostability (see Section 3.3.1), it is anticipated that the xylanase is inactivated during baking processes.
In grain treatment for the production of starch and gluten fractions, 34 the xylanase is added during dough preparation. It is used to reduce technical difficulties (e.g. high viscosity), to give higher yield due to efficient hydrolysis of xylans, improving starch purity due to greater extraction yield of the high value fraction and efficient removal of fibres and proteins, resulting in more efficient processes and more consistent product quality.
Analytical data have been provided to investigate the transfer of food enzyme-TOS (several activities from different sources) into the starch and gluten fractions during grain treatment (Documentation provided to EFSA No. 5). The data provided includes a theoretical calculation of enzyme transfer based on measured amounts of intermediate and final fractions; and provision of measured enzymatic activities in the weighed intermediate and final products. Based on both data sets, a removal of > 99% of the amount of enzyme added to the raw material (e.g. grain, flour) during production of the final fractions (starch and gluten) can be estimated. The Panel considered the evidence as sufficient to conclude that TOS (including substances other than proteins) are efficiently removed by mechanical separation and repeated washing, resulting in negligible amount of residual TOS remaining in the starch and gluten fractions.
In distilled alcohol production, the xylanase food enzyme is applied during liquefaction and fermentation, and may also be added during slurry mixing and pre-saccharification. Endo-1,4-bxylanase catalyses the hydrolysis of 1,4-b-D-xylosidic linkages in xylan. The reaction products are (1?4)b-D-xylan oligosaccharides of different lengths.
Experimental data have been provided on the removal (> 99%) of protein in the course of distilled alcohol production (Documentation provided to EFSA No. 6). The Panel considered the evidence as sufficient to conclude that residual amounts of TOS (including substances other than proteins) are removed by distillation.

Dietary exposure estimation
As residual amounts of TOS are removed during grain treatment for the fractionation of starch and gluten and by distillation (see Section 3.5.1), dietary exposure to the food enzyme resulting from these two processes was not calculated.
For baking processes, chronic exposure was calculated using the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016). The assessment involved selection of relevant food categories from the EFSA Comprehensive European Food Consumption Database and application of process and technical conversion factors (Annex B in EFSA CEF Panel, 2016). Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant (see Section 3.5.1/Table 2) with the relevant FoodEx categories (Annex B in EFSA CEF Panel, 2016), based on individual consumption data. Exposure from individual FoodEx categories was subsequently summed up, averaged over the total survey period and normalised for bodyweight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only one day per subject were excluded and highlevel exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011). Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed average and 95th percentile exposure to the food enzyme-TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A -Tables 1 and 2. For the present assessment, food consumption data were available from 35 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B).
Based on the maximum use levels recommended for baking processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-total organic solids was estimated to be up to 0.138 mg TOS/kg bw per day.

Uncertainty analysis
In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.  Use of data from food consumption surveys of a few days to estimate long-term (chronic) exposure for high percentiles (95th percentile) + Possible national differences in categorisation and classification of food +/-

Model assumptions and factors
FoodEx categories included in the exposure assessment were assumed to always contain the food enzyme-TOS + Exposure to food enzyme-TOS was always calculated based on the recommended maximum use level + Selection of broad FoodEx categories for the exposure assessment + Safety evaluation of the food enzyme xylanase from B. pumilus (strain BLXSC) www.efsa.europa.eu/efsajournal