Safety evaluation of the food enzyme glucan‐1,4‐α‐maltohydrolase from the genetically modified Bacillus subtilis strain AR‐453

Abstract

The food enzyme glucan‐1,4‐α‐maltohydrolase (4‐α‐d‐glucan α‐maltohydrolase; EC 3.2.1.133) is produced with the genetically modified Bacillus subtilis strain AR‐453 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in baking processes. Dietary exposure was estimated to be up to 0.262 mg TOS/kg body weight per day in European populations. As the production strain of B. subtilis strain AR‐453 qualifies for the qualified presumption of safety (QPS) approach to safety assessment and no issue of concern arose from the production process, no toxicological data were required. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and six matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

1. Introduction

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (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 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:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;

• there is a reasonable technological need;

• 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, 2009a) lays down the administrative, technical and toxicological data required.

1.1. Background and terms of reference as provided by the requestor

1.1.1. 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.

An application has been introduced by the applicant “AB Enzymes GmbH” for the authorization of the food enzyme maltogenic amylase from a genetically modified strain of Bacillus subtilis (strain AR‐453).

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 application falls within the scope of the food enzyme Regulation and contains all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

In accordance with Article 29(1)(a) of Regulation (EC) No 178/2002 ⁴ , the European Commission requests the European Food Safety Authority to carry out the safety assessment on the following food enzyme: Maltogenic amylase from a genetically modified strain of Bacillus subtilis (strain AR‐453), in accordance with Regulation (EC) No 1331/2008 ² establishing a common authorization procedure for food additives, food enzymes and food flavourings.

2. Data and methodologies

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme maltogenic amylase from a genetically modified Bacillus subtilis strain AR‐453. The dossier was updated on 13 October 2021.

Additional information was requested from the applicant during the assessment process on 26 January 2022 and 15 December 2022 and received on 26 October 2022 and 15 March 2023 (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 15 December 2022, the applicant requested a clarification teleconference on 09 January 2023, after which the applicant provided additional data on 15 March 2023.

2.2. 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, 2009b) and following the relevant existing guidance documents of EFSA Scientific Committee.

The current ‘Scientific Guidance for the submission of dossiers on Food Enzymes’ (EFSA CEP Panel, 2021a) has been followed for the evaluation of the application.

3. Assessment

IUBMB nomenclature	glucan 1,4‐α‐maltohydrolase
Systematic name	4‐α‐d‐glucan α‐maltohydrolase
Synonyms	maltogenic α‐amylase
IUBMB No	3.2.1.133
CAS No	160611‐47‐2
EINECS No	630‐523‐5

Glucan‐1,4‐α‐maltohydrolases catalyse the hydrolysis of (1,4)‐α‐d‐glucosidic linkages in starch polysaccharides, to successively remove maltose residues from the non‐reducing ends of the chains. The enzyme under assessment is intended to be used only in baking processes.

3.1. Source of the food enzyme

The glucan 1,4‐α‐maltohydrolase is produced with the genetically modified bacterium Bacillus subtilis strain AR‐453 (■■■■■), which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (the Netherlands), with the deposit number ■■■■■ ⁵ The production strain was identified as B. subtilis by ■■■■■■■■■■■■■■■ ⁷

The species B. subtilis is included in the list of organisms for which the qualified presumption of safety (QPS) may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022). The production strain B. subtilis AR‐453 was found not to be cytotoxic to VERO cells. ⁸ The WGS of the production strain was interrogated for the presence of antimicrobial resistance genes using two databases with thresholds of > 80% identity and > 70% coverage. ■■■■■ ⁹ ■■■■■.

Therefore, the production strain is considered to qualify for the QPS approach.

3.1.1. Characteristics of the parental microorganism

The parental microorganism is B. subtilis strain ■■■■■ ¹⁰

■■■■■ ¹¹

3.1.2. Characteristics of introduced sequences

The sequence encoding the glucan 1,4‐α‐maltohydrolase ■■■■■

■■■■■ ¹²

3.1.3. Description of the genetic modification process

The aim of the genetic modification was to enable the production strain to synthesise the glucan 1,4‐α‐maltohydrolase ■■■■■

■■■■■

■■■■■ ¹³ ■■■■■ ¹⁴

3.1.4. Safety aspects of the genetic modification

The technical dossier contains all necessary information on the parental microorganism, the donor organism and the genetic modification process.

The production strain B. subtilis strain AR‐453 differs from the parental strain in its capacity to synthesise the glucan 1,4‐α‐maltohydrolase ■■■■■

No issues of concern arising from the genetic modifications were identified by the Panel.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 ¹⁵ , with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice.

The production strain is grown as a pure culture using a typical industrial medium in a submerged, ■■■■■ 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 an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. ¹⁶ The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme. ¹⁷

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

3.3.1. Properties of the food enzyme

The glucan 1,4‐α‐maltohydrolase is a single polypeptide chain of ■■■■■ amino acids. ¹⁸ The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁹ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gels showed a major protein band migrating slightly above the ■■■■■ kDa marker band, consistent with the expected molecular mass of the enzyme, and a number of bands of lesser staining intensity. ¹⁹ The food enzyme was tested for amylase, protease and ■■■■■ activities and only amylase activity was detected. No other enzymatic activities were reported. ²⁰

The in‐house determination of glucan 1,4‐α‐maltohydrolase activity is based on hydrolysis of maltotriose (reaction conditions: ■■■■■. Released glucose is quantified by means of a coupled reaction that forms NADPH, which is then detected spectrophotometrically at 340 nm. The enzyme activity is expressed in Maltogenic Amylase Units (MAZ)/g. One MAZ is defined as the amount of enzyme which catalyses the hydrolysis of one μmol of maltotriose per minute under the assay conditions. ²¹

The food enzyme has a temperature optimum around ■■■■■ and a pH optimum around pH ■■■■■ ²² Thermostability was tested by pre‐incubation of the food enzyme at 90°C for different time periods (■■■■■). No glucan 1,4‐α‐maltohydrolase activity remained after 5 min of pre‐incubation. ²²

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation (Table 1). The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 10.4% and the mean enzyme activity/TOS ratio was 19.1 MAZ/mg TOS. ²³

Table 1.

Composition of the food enzyme

Parameters	Unit	Batches
		1	2	3
Glucan 1,4‐α‐maltohydrolase activity	MAZ/g (a)	2,130	2,350	1,480
Protein	%	2.4	2.8	2.4
Ash	%	0.2	0.2	1.0
Water	%	88.0	88.0	91.3
Total organic solids (TOS) (b)	%	11.8	11.8	7.7
Activity/TOS	MAZ/mg TOS	18.1	19.9	19.2

^(a)MAZ: Maltogenic Amylase Unit (see Section 3.3.1).

^(b)TOS calculated as 100%–% water–% ash.

3.3.3. Purity

The lead content in the three commercial batches was below 5 mg/kg ²⁴ which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ²⁵ In addition, arsenic, cadmium and mercury were below the limits of detection (LoD) of the employed methods. ^{24 , 26}

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ^{24 , 25} No antimicrobial activity was detected in any of the tested batches. ²⁴

The Panel considered that the information provided on the purity of the food enzyme is sufficient.

3.3.4. Viable cells and DNA of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■ No colonies of the production strain were produced. A positive control was included. ²⁷

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify ■■■■■ ²⁸

3.4. Toxicological data

As the production strain qualifies for the QPS approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified (see Sections 3.1, 3.2 and 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021a).

3.4.1. Allergenicity

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 glucan 1,4‐α‐maltohydrolase produced with the genetically modified B. subtilis strain AR‐453 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, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, six matches were found. The matching allergens were Asp o 21 and Asp o 21.0101 (TAKA amylase A and α‐amylase A type‐1/2 from Aspergillus oryzae); Sch c 1.0101 (glycoside hydrolase family 15 from Schizophyllum commune (Split Gill fungus)); Asp f 13.0101 (uncleaved alkaline protease from Aspergillus fumigatus) and Asp f 13 (partial alkaline protease from Aspergillus fumigatus), all known as respiratory allergens and Aed a 4.0101 (glycosyl hydrolase from Aedes aegypti (yellow fever mosquito)). ²⁹

No information is available on oral and respiratory sensitisation or elicitation reactions of this glucan 1,4‐α‐maltohydrolase.

Several studies have shown that adults sensitised to respiratory allergens can ingest the allergens without acquiring clinical symptoms of food allergy (Cullinan et al., 1997; Poulsen, 2004; Armentia et al., 2009). Taking into account the wide use of α‐amylase as a food enzyme, only a low number of case reports has been described in literature that focused on allergic reactions upon oral exposure to α‐amylase in individuals respiratory sensitised to α‐amylase (Losada et al., 1992; Quirce et al., 1992; Baur and Czuppon, 1995; Kanny and Moneret‐Vautrin, 1995; Moreno‐Ancillo et al., 2004). Such information has not been reported for glucoamylase and serine protease. α‐Glucosidase is associated with allergic reactions to insect bites, but allergic reactions after oral exposure have not been reported. In addition, no allergic reactions upon dietary exposure to any glucan 1,4‐α‐maltohydrolase have been reported in the literature.

■■■■■, a known source of allergens, is present in the media fed to the microorganisms. However, during the fermentation process, this product 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 no potentially allergenic residues from this source are present in the food enzyme.

The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in baking processes at a maximum recommended use level of 22 mg TOS/kg raw material. ³⁰

In baking processes, the food enzyme performs its technological function during dough or batter handling, contributing to an improved and consistent baking process. The conversion of starch lowers the rate of retrogradation, thereby reducing staling, and improves crumb structure. The food enzyme‐TOS remains in the dough and batter.

Based on data provided on thermostability (see Section 3.3.1), it is expected that the glucan 1,4‐α‐maltohydrolase is inactivated during baking processes.

3.5.2. Dietary exposure estimation

Chronic exposure to the food enzyme‐TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021a). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2021b). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. 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 1 day per subject were excluded and high‐level 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 2 provides an overview of the derived exposure estimates across all surveys. Detailed mean 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 43 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 23 European countries (Appendix B). The highest dietary exposure to the food enzyme‐TOS was estimated to be 0.262 mg TOS/kg bw per day in infant at the 95th percentile.

Table 2.

Summary of estimated dietary exposure to food enzyme–TOS in six population groups

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.004–0.061 (12)	0.047–0.132 (16)	0.053–0.127 (19)	0.029–0.078 (20)	0.022–0.048 (22)	0.022–0.045 (21)
Min–max 95th percentile (number of surveys)	0.024–0.262 (10)	0.117–0.224 (14)	0.104–0.239 (19)	0.064–0.165 (19)	0.048–0.093 (22)	0.043–0.078 (21)

3.5.3. 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 3.

Table 3.

Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
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	+
Use of recipe fractions in disaggregation FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

+: uncertainty with potential to cause overestimation of exposure.

–: uncertainty with potential to cause underestimation of exposure.

The conservative approach applied to the exposure estimate to food enzyme‐TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to overestimation of the exposure.

3.6. Margin of exposure

Given the QPS status of the production strain and the lack of hazards resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. Consequently, the margin of exposure was not calculated.

4. Conclusions

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process of the food enzyme, the Panel concluded that the food enzyme glucan 1,4‐α‐maltohydrolase produced with the genetically modified Bacillus subtilis strain AR‐453 does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism and its recombinant DNA.

5. Documentation as provided to EFSA

Application for authorisation of a maltogenic amylase from a genetically modified strain of Bacillus subtilis in accordance with Regulation (EC) NO 1331/2008. March 2021. Submitted by AB Enzymes GmbH.

Additional information. October 2022 and March 2023. Submitted by AB Enzymes GmbH.

Abbreviations

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LoD

limit of detection

PCR

polymerase chain reaction

TOS

total organic solids

WGS

Whole genome sequence

WHO

World Health Organization

Appendix A – Dietary exposure estimates to the food enzyme–TOS in details

1.

Appendix A can be found in the online version of this output (in the ‘Supporting information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

Appendix B – Population groups considered for the exposure assessment

1.

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, United Kingdom
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, Slovenia, Spain, United Kingdom
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Spain, Sweden, United Kingdom
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden, United Kingdom
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden, United Kingdom
The elderly (a)	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden, United Kingdom

^(a)The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

Suggested citation: EFSA CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré C, Barat Baviera JM, Bolognesi C, Cocconcelli PS, Crebelli R, Gott DM, Grob K, Lampi E, Mengelers M, Mortensen A, Rivière G, Steffensen I‐L, Tlustos C, Van Loveren H, Vernis L, Zorn H, Herman L, Roos Y, Andryszkiewicz M, Liu Y, Lunardi S and Chesson A, 2023. Scientific Opinion on the safety evaluation of the food enzyme glucan‐1,4‐α‐maltohydrolase from the genetically modified Bacillus subtilis strain AR‐453. EFSA Journal 2023;21(7):8089, 14 pp. 10.2903/j.efsa.2023.8089