Safety evaluation of the food enzyme with 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase activities from the Gryllotalpicola ginsengisoli strain S34

Abstract

The food enzyme with 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase ((4‐α‐d ‐[(1,4‐α‐d ‐glucano]trehalose glucanohydrolase (trehalose‐producing)), EC 3.2.1.141) and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase ((1,4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase, EC 5.4.99.15) activities is produced with a non‐genetically modified microorganism Gryllotalpicola ginsengisoli (formally Arthrobacter ramosus ) by Hayashibara Co., Ltd. The applicant states that the food enzyme will not be placed on the open market, but will only be used in‐house in starch processing for trehalose production. Since residual amounts of total organic solids are removed by the purification steps applied during the production of trehalose, toxicological studies were considered not necessary and no dietary exposure was calculated. Similarities of the amino acid sequences to those of known allergens were searched and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

1 Introduction

Article 3 of the Regulation (EC) No 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘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 1332/2008¹ 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 1331/200822 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
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:

1. it does not pose a safety concern to the health of the consumer at the level of use proposed;
2. there is a reasonable technological need;
3. 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

1.1.1 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 1332/2008¹ on food enzymes.

Five applications have been introduced by the companies ‘Nagase (Europa) GmbH’ for the authorisation of the food enzyme phospholipase A₂ from a genetically modified strain of Streptomyces violaceoruber (strain AS‐10), ‘Novozymes A/S’ for the authorisation of the food enzyme glucose oxidase from Aspergillus niger (strain NZYM‐KA), ‘Hayashibara Co., Ltd.’ for the authorisation of the food enzymes 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase from Arthrobacter ramosus and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase from Arthrobacter ramosus, and ‘the Association of Manufacturers and Formulators of Enzyme Products (AMFEP)’ for the authorisation of the food enzyme alpha‐amylase from Bacillus subtilis .

Following the requirements of Article 12.1 of Regulation (EC) No 234/201133 Commission Regulation (EU) No 234/2011 of 10 March 2011 implementing Regulation (EC) No 1331/2008 of the European Parliament and of the Council establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 64, 11.3.2011, pp. 15–24.
implementing Regulation (EC) No 1331/2008², the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2 Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes phospholipase A₂ from a genetically modified strain of Streptomyces violaceoruber (strain AS‐10), glucose oxidase from Aspergillus niger (strain NZYM‐KA), 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase from Arthrobacter ramosus, (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase from Arthrobacter ramosus and alpha‐amylase from Bacillus subtilis in accordance with Article 17.3 of Regulation (EC) No 1332/2008¹ on food enzymes.

1.2 Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzymes 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase from Gryllotalpicola ginsengisoli strain S34, initially identified as A. ramosus .

2 Data and methodologies

2.1 Data

The applicant has submitted dossiers in support of the applications for authorisation of the food enzyme with 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase activities from a non‐genetically modified microorganism G. ginsengisoli .

Additional information was requested from the applicant during the assessment process on 14 June 2019 and was consequently provided (see ˋDocumentation provided to EFSAˊ).

During pre‐notification sent on 2 March 2020 the applicant provided additional revised data (see ‘Documentation provided to EFSA’).

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, 2009) and following the relevant existing guidances of EFSA Scientific Committee.

The current ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (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 Assessment

The food enzyme contains two declared activities:

IUBMB nomenclature/systematic name: (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase

Synonyms: maltooligosyl trehalose synthase; maltodextrin α‐d ‐glucosyltransferase; trehalosyl dextrin‐forming enzyme

IUBMB No.: EC 5.4.99.15

CAS No.: Not available.

The (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase catalyses the transglucosylation of the reducing terminal linkages in α‐glucans containing a minimum of three glucose residues resulting in the formation of a trehalose residue.

IUBMB nomenclature: 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase

Systematic name: 4‐α‐d ‐(1,4‐α‐d ‐glucano)trehalose glucanohydrolase (trehalose producing)

Synonyms: maltooligosyl trehalose trehalohydrolase; glycosyltrehalose trehalohydrolase; maltooligosyltrehalose trehalosidase

IUBMB No.: EC 3.2.1.141

CAS No.: Not available.

The 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase catalyses the hydrolysis of (1‐>4)‐α‐d ‐glucosidic linkage in 4‐α‐d ‐[(1‐>4)‐α‐d ‐glucanosyl](n) trehalose to yield trehalose and (1‐>4)‐α‐d ‐glucan.

The food enzyme is used exclusively for the production of trehalose from starch.

3.1 Source of the food enzyme

The food enzyme with the two activities, 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase, is produced with a non‐genetically modified microorganism G. ginsengisoli S34.44 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 12 and Annex 12; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 12 and Annex 12; Technical dossier/Additional information, 25 November 2019/Annex 7, Annex 8 and Annex 9.
This strain is deposited under the name of Arthrobacter sp. S34 at the National Institute of Bioscience and Human‐Technology Agency of Industrial Science and Technology (Japan) with accession number FERM BP 6450.55 Technical dossier/Additional information, 25 November 2019/Annex 8.

The production strain was been identified by ■■■■■.66 Technical dossier/Additional information, 25 November 2019/Annex 7.
From the results of these analyses the food enzyme producing bacterium was identified as G. ginsengisoli (■■■■■).⁶ The production strain S34 was obtained by chemical mutagenesis from a wild type isolate.77 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 12; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 12.

The absence of antimicrobial resistance genes was confirmed by ■■■■■ of the production strain.88 Technical dossier/Additional information, 25 November 2019/Annex 10.

3.2 Production of the food enzyme99 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 41–49; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 41–49; Technical dossier/Additional information, 25 November 2019.

The food enzyme is manufactured according the Food Sanitation Act in Japan. In addition, the Japan Quality Assurance Organization certifies that the plant and its production process follow a Quality Management System, which complies with the requirements of ISO 9001:2008 and is in accordance with current Good Manufacturing Practice (GMP).1010 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 10; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 10.

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, lysozyme is added to the medium to lyse the bacterial cells and a ■■■■■1111 Technical dossier/Additional information, 25 November 2019.
added to aid downstream processing. The solid biomass is removed from the fermentation broth by filtration leaving a supernatant containing the bulk of the food enzyme. The filtrate containing the food enzyme is then further purified and concentrated, including ■■■■■ 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.1212 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 3; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 3; Technical dossier/Additional information, 25 November 2019/Annex 6.

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 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase is a single polypeptide chain of 575 amino acids1313 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 27 and Annex 9.
and the (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase is a single polypeptide chain of 757 amino acids.1414 Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 27 and Annex 9.
The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).1515 Technical dossier/Additional information, 25 November 2019/Annex 4.
The SDS–PAGE gels showed two protein bands, one corresponding to an apparent molecular mass of around 65 kDa as expected for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and a second band corresponding to an apparent molecular mass of around 83 kDa and attributed to the (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase.¹⁵ The food enzyme was examined for neutral protease and lipase activities, which were found to be below their respective limits of detection (LODs).1616 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 7; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 7; Technical dossier/Additional information, 25 November 2019/Annex 2.

The in‐house determination of 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase is based on hydrolysis of the substrate maltotetraosyl glucoside (reaction conditions: pH 5.5, 50°C, 20 min) with a colourimetric detection of reducing groups. One unit of 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase activity is defined as that the amount of enzyme which releases 1 μmol glucose per minute at pH 5.5 and 50.0°C.1717 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 6.

The determination of (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase is based on the hydrolysis of the substrate amylose (reaction conditions: pH 5.5, 50°C, 30 min, in the presence of 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase). After acidification to stop the reaction and the addition of an iodine solution (45 min, 25°C) the absorbance is measured spectrophotometrically at 660 nm. One (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase unit (U) is defined as the quantity of enzyme that causes the iodine–starch reaction of 25 mg of amylose to completely disappear under the conditions of the assay (at pH 5.5 and 50°C).1818 Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 6.

The temperature and pH profiles of the two food enzyme activities have been described by Yamamoto et al. (2001).1919 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 14; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 14.
The 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase both show maximum activity around 50°C (pH 6.0) and around pH 6.0 (50°C). Thermostability was tested after a pre‐incubation of the food enzyme for 60 min at different temperatures (pH 7.0). Under these conditions, both activities were stable up to 55°C.

3.3.2 Chemical parameters2020 Technical dossier/Additional information, 25 November 2019/Annex 1, Annex 2, and Annex 3.

Data on the chemical parameters of the food enzyme were provided for three food enzyme batches used for commercialisation (Table 1). The average Total Organic Solids (TOS) of the three food enzyme batches for commercialisation was 2.8%. The mean enzyme activity/mg TOS ratio of the three food enzyme batches for commercialisation are 24.7 U/mg TOS for both declared activities. The mean enzyme activity of (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/mg TOS ratio of the three food enzyme batches for commercialisation is 70.8 U/mg TOS.

Table 1. Compositional data of the food enzyme preparation

Parameter	Unit	Batch
		1	2	3
4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase activity + (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase activity	U/ga^a U: Unit (see Section 3.3.1).	717	673	686
(1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase activity	U/ga^a U: Unit (see Section 3.3.1).	1,983	1,963	1,998
Proteinb^b Measured by the method of Lowry et al. (1951).	%	3.3	3.4	3.3
Ash	%	6.1	6.1	6.2
Water	%	83.0	83.1	83.1
Excipients	%	8.0	8.0	8.0
Total Organic Solids (TOS)c^c TOS calculated as 100% – % water – % ash – additives.	%	2.9	2.8	2.7
4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase activity + (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase activity/mg TOS	U/mg TOS	24.7	24.0	25.4
(1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/mg TOS	U/mg TOS	68.4	70.1	74.0

• ^a U: Unit (see Section 3.3.1).
• ^b Measured by the method of Lowry et al. (1951).
• ^c TOS calculated as 100% – % water – % ash – additives.

3.3.3 Purity

The lead content in the three commercial batches was below 5 mg/kg which complies with the specification for lead (≤ 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006).2121 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 2 and Annex 11; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 2 and Annex 11; Technical dossier/Additional information, 25 November 2019/Annex 3; LOD for lead: 5 mg/kg.

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 Salmonella species are absent in 25 g of sample and that the count of total coliforms does not exceed 30 colony forming units (CFU) per gram.2222 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 2 and Annex 11; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 2 and Annex 11; Technical dossier/Additional information, 25 November 2019/Annex 2 and Annex 3.
Escherichia coli was not detected by a method using 10 g of starting material.2323 Technical dossier/Additional data during pre‐notification, 6 March 2020/Hayashibara Co., Ltd. Statement, 6 March 2020; Revised technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Revised Annex 2 and revised Annex 11; Revised technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Revised Annex 2 and revised Annex 11.
No antimicrobial activity was detected in any of three batches tested (FAO/WHO, 2006).2424 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 2 and Annex 5; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 2 and Annex 5.

The presence of mycotoxins/secondary metabolites (total aflatoxins, aflatoxin B1, B2, G1 and G2) was examined in the three food enzyme preparation batches and were below the LODs of the applied analytical methods and of no concern.2525 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 4; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 4; Technical dossier/Additional information, 25 November 2019/Annex 2; LOD for aflatoxin B1, B2, G1 and G2: 1.0 μg/kg.

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

3.3.4 Viable cells of the production strain

■■■■■ during the production of the food enzyme means that the presence of the viable cells of the production strain cannot be excluded.

3.4 Toxicological data2626 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 16; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 16.

No toxicological tests were provided by the applicant or requested by EFSA.

The Panel considers that in the course of the production of trehalose, the food enzyme is removed by the applied purification steps (see Section 3.5) and so exposure can be considered negligible. Toxicological tests, except allergenicity, are not needed for the assessment of the food enzyme.

3.4.1 Allergenicity2727 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 14 and Annex 9; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 14 and Annex 9.

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 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase produced with G. ginsengisoli S34 was assessed by comparing their amino acid sequences with those of known allergens according to the scientific opinion on the assessment of allergenicity of genetically modified 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.2828 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 9; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 9.

No information is available on oral and respiratory sensitisation or elicitation reactions of either 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase or (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase under evaluation. The applicant claimed that no single case of allergic or adverse reaction in workers exposed to these food enzymes had been reported.2929 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/Annex 21; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/Annex 21.
Therefore, it can be concluded that the likelihood of an allergic reaction upon oral ingestion of this 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase, produced with G. ginsengisoli, cannot be excluded, but the likelihood of such a reaction to occur is considered to be low.

According to the information provided, a product that may cause allergies or intolerances (Regulation (EU) No 1169/20113030 Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004.
) are used as a raw material (■■■■■) 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 potentially allergenic residues of ■■■■■ employed as a nitrogen source is not expected to be present.

Hen egg white lysozyme, a recognised allergen, is added after completion of the fermentation. Since the downstream processing of the food enzyme is designed to retain and maintain enzyme activity, it is probable that lysozyme will be found in the food enzyme and any food enzyme preparations.

The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to the two declared activities in the food enzyme cannot be excluded but the likelihood of such reactions occurring is considered to be low. The Panel notes, however, that the food enzyme may contain hen egg white lysozyme, a known allergen.

3.5 Dietary exposure

3.5.1 Intended use of the food enzyme

The applicant states that the food enzyme will not be placed on the open market, but only will be used in‐house in starch processing for the production of trehalose at a recommended use level of 1–300 mg TOS/kg starch.3131 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 14; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 14.

A flow chart depicting the manufacturing of starch‐derived trehalose used by the applicant was provided.3232 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 50; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 50.

During production of trehalose from starch, and after the saccharification stages the activities found in the food enzyme are inactivated by ■■■■■ and any residues are reduced or removed during the follow‐up purification procedures. These include ■■■■■ followed by ■■■■■.3333 Technical dossier for 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase/p. 49; Technical dossier for (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glycosylmutase/p. 49.

Taking into consideration the process used for the manufacturing of trehalose described by the applicant, the Panel considers the presence of viable cells of the production strain in the trehalose is highly unlikely.

The Panel considered the evidence as sufficient to conclude that residual amounts of TOS (including substances other than proteins) are removed by production of trehalose from starch.

3.5.2 Dietary exposure estimation

The technical information and experimental data provided on the removal of food enzyme‐TOS during starch processing for the production of glucose syrup were considered by the Panel as sufficient to exclude this process from the exposure assessment. As trehalose is obtained from starch and the purification steps applied during its production are virtually the same as for glucose syrup, for which TOS removal has been demonstrated in food enzyme dossiers evaluated, the Panel decided to exclude also these types of starch hydrolysates from the exposure calculation (Annex B in EFSA CEF Panel, 2016). Consequently, a dietary exposure was not calculated.

4 Conclusions

The Panel notes that the food enzyme may contain hen egg white lysozyme, a known allergen.

Based on the data provided, and the removal of TOS during the intended food production process, the Panel concluded that the food enzyme with 4‐α‐d ‐{(1‐>4)‐α‐d ‐glucano}trehalose trehalohydrolase and (1‐>4)‐α‐d ‐glucan 1‐α‐d ‐glucosylmutase activities produced with non‐genetically modified G. ginsengisoli strain S34 does not give rise to safety concerns under the intended conditions of use.

Documentation provided to EFSA

1. Technical dossier ˋApplication for authorisation of 4‐α‐D‐{(1‐>4)‐α‐D‐glucano}trehalose trehalohydrolase preparation from Arthrobacter ramosus′ . 5 March 2015. Submitted by Hayashibara Co., Ltd., Japan.
2. Technical dossier ˋApplication for authorisation of (1‐>4)‐α‐D‐glucan 1‐α‐D‐glucosylmutase preparation from Arthrobacter ramosus′ . 5 March 2015 (revised on November 10, 2015). Submitted by Hayashibara Co., Ltd., Japan.
3. Additional information. 25 November 2019. Submitted by Hayashibara Co., Ltd., Japan.
4. Additional data received during pre‐notification. 6 March 2020. Submitted by Hayashibara Co., Ltd., Japan.

Abbreviations

AMFEP

Association of Manufacturers and Formulators of Enzyme Products

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CAS

Chemical Abstracts Service

CEF

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

CFU

colony forming units

EC

Enzyme Commission

FAO

Food and Agricultural Organization of the United Nations

GMO

genetically modified organism

GMP

Good Manufacturing Practice

IUBMB

International Union of Biochemistry and Molecular Biology

LOD

limit of detection

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SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TOS

total organic solids

U

Unit

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WHO

World Health Organization