Safety evaluation of the food enzyme pullulanase from the genetically modified Bacillus licheniformis strain NZYM-LU.

The food enzyme pullulanase (pullulan 6-α-glucanohydrolase, EC 3.2.1.41) is produced with the genetically modified Bacillus licheniformis strain NZYM-LU by Novozyme A/S. The genetic modifications did not give rise to safety concerns. The production strain has been shown to qualify for the qualified presumption of safety (QPS) status. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in brewing processes and in starch processing for production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was calculated only for the brewing processes. It was estimated to be up to 0.59 mg TOS/kg body weight (bw) per day in European populations. Given the QPS status of the production strain and the lack of hazards resulting from the food enzyme manufacturing process, toxicological studies were not considered necessary. The 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, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme pullulanase produced with the genetically modified B. licheniformis strain NZYM-LU does not give rise to safety concerns under the intended conditions of use.

Introduction

Article 3 of the Regulation (EC) No 1332/2008 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 1332/2008 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 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 CEF Panel, 2009) lays down the administrative, technical and toxicological data required.

Background and Terms of Reference as provided by the requestor

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 on food enzymes.

An application has been introduced by the applicant “Novozymes A/S” for the authorization of food enzyme pullulanase from a genetically modified Bacillus licheniformis (strain NZYM‐LU).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 implementing Regulation (EC) No 1331/2008, the Commission has verified that the application falls within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

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

Data and methodologies

Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme pullulanase produced by a genetically modified strain of B. licheniformis (strain NZYM‐LU).

Additional information was requested from the applicant during the assessment process on 15 September 2021 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 guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA CEF Panel, 2009) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021a).

Assessment

Pullulanases catalyse the hydrolysis of (1→6)‐α‐d‐glucosidic linkages in pullulan, amylopectin and glycogen as well as in α‐ and β‐limit dextrins of amylopectin and glycogen. The enzyme under this assessment is intended to be used in brewing processes and in starch processing for production of glucose syrups and other starch hydrolysates.

Source of the food enzyme

The pullulanase is produced with the genetically modified bacterium B. licheniformis strain NZYM‐LU, which is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), with deposit number ■■■■■

The production strain was identified as B. licheniformis ■■■■■

The species B. licheniformis 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 absence of cytotoxic activity of the production strain was confirmed ■■■■■ ■■■■■.

Characteristics of the parental and recipient microorganisms

The parental strain is B. licheniformis ■■■■■

The recipient strain was derived from ■■■■■

■■■■■

Characteristics of introduced sequences

The sequence encoding the pullulanase ■■■■■ ■■■■■

■■■■■

■■■■■

Description of the genetic modification process

The purpose of the genetic modification was to enable the production strain to synthesise pullulanase. ■■■■■

The genetic modifications were confirmed ■■■■■ ■■■■■.

Safety aspects of the genetic modification

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

The production strain B. licheniformis strain NZYM‐LU differs from the recipient strain in its capacity to produce the pullulanase ■■■■■.

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

Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 , 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 ■■■■■ 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 the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. Then, the concentrated food enzyme is stabilised. The applicant provided information on the identity of the substances used to control the fermentation and 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.

Characteristics of the food enzyme

Properties of the food enzyme

The pullulanase is a single polypeptide chain of ■■■■■ amino acids. The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.18 The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). A consistent protein pattern was observed across all batches. The gels showed a single major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme. The food enzyme was tested for α‐amylase, glucoamylase, lipase and protease activities. Only lipase activity was detected in two of three batches. No other enzymatic activities were reported.

The in‐house determination of pullulanase activity is based on the hydrolysis of borohydride‐reduced pullulan (reaction conditions: pH 5.0, 50°C, 20 min). The released maltotriose reacts with 4‐hydroxybenzoic acid hydrazide and generates a yellow complex. The enzymatic activity is determined spectrophotometrically at 405 nm. It is expressed in Pullulanase Unit Novozymes (G pullulanase)/gram (PUN‐G/g). One PUN‐G is defined as the amount of enzyme that releases 1 µmol of maltotriose under the reaction conditions per minute.

The food enzyme has a temperature optimum around 60°C (pH 5.0) and a pH optimum around pH 4.0 (55°C). Thermostability was tested after a pre‐incubation of the food enzyme for 30 min at different temperatures (pH 5.0). The pullulanase activity was stable up to 60°C but decreased sharply at higher temperatures. No residual activity was detected at 70°C.

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 was 10.4% and the mean enzyme activity/TOS ratio 77.6 PUN(G)/mg TOS.

Table 1

Composition of the food enzyme

Parameters	Unit	Batches
		1	2	3
Pullulanase activity	PUN(G)/g batch (a)	8,640	7,420	8,140
Protein	%	10.0	9.4	9.5
Ash	%	0.6	0.8	0.7
Water	%	88.7	88.7	89.3
Total organic solids (TOS) (b)	%	10.7	10.5	10.0
Activity/mg TOS	PUN(G)/mg TOS	80.7	70.7	81.4

PUN(G): Pullulanase Unit Novozymes (G Pullulanase) (see Section 3.3.1).

TOS calculated as 100% – % water – % ash.

Purity

The lead content in the three commercial batches was below 0.5 mg/kg, which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). The levels of arsenic, cadmium and mercury were below the limits of detection (LoDs) of the employed methodologies. ,

The food enzyme complies with the criteria for total coliforms, Escherichia coli and Salmonella,24 as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). No antimicrobial activity was detected in any of the tested batches (FAO/WHO, 2006).26

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

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 were produced.

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 ■■■■■

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).

Allergenicity

The allergenicity assessment considered only the food enzyme and not carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the pullulanase with the genetically modified B. licheniformis strain NZYM‐LU 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.

No information is available on oral and respiratory sensitisation or elicitation reactions to this pullulanase. In addition, no allergic reactions upon dietary exposure to any pullulanase have been reported in the literature.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ) is used as protein source. However, during the fermentation process, it 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 are present in the food enzyme.

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

Dietary exposure

Intended use of the food enzyme

The food enzyme is intended to be used in two food processes at the recommended use levels summarised in Table 2.

Table 2

Intended uses and recommended use levels of the food enzyme as provided by the applicant

Food manufacturing process (a)	Raw material	Recommended use level (b) , (c)
Brewing processes	Cereals (malted or not)	6.4–128.9 mg TOS/kg cereals
Starch processing for production of glucose syrup and other starch hydrolysates	Starch	2.5–257.7 mg TOS/kg starch

TOS: total organic solids.

The description has been harmonised according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.

Based on 77.6 PUN(G)/mg TOS.

The number in bold was used for calculations.

In brewing processes, the food enzyme is added to the raw materials during mashing. It acts together with other amylolytic enzymes to degrade starch into fermentable sugars. It may also be applied during fermentation to debranch starch and aids the release of glucose. The use of the pullulanase reduces the mashing time and improves the alcohol yield. The food enzyme–TOS remains in the beer.

Based on the data provided on thermostability (see Section 3.3.1), it is expected that the enzyme is inactivated during brewing processes.

In starch processing, the food enzyme is added during the saccharification step. It degrades gelatinised starch into polysaccharides or shorter dextrins. The food enzyme–TOS is removed from the syrups and other hydrolysates by treatment with activated charcoal or similar and with ion‐exchange resins (EFSA CEP Panel, 2021b).

Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021a), a dietary exposure was calculated only for food manufacturing processes where the food enzyme–TOS remains in the final foods, namely brewing processes.

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 one 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 3 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 41 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure to the food enzyme–TOS was estimated to be about 0.591 mg TOS/kg bw per day in adults at the 95th percentile.

Table 3

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)	0–0.010 (11)	0–0.024 (15)	0–0.019 (19)	0–0.024 (21)	0.010–0.131 (22)	0.003–0.065 (22)
Min–max 95th percentile (number of surveys)	0–0 (9)	0–0.154 (13)	0–0.148 (19)	0–0.119 (20)	0.073–0.591 (22)	0.016–0.270 (21)

TOS: total organic solids.

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.

Table 4

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	+/–
Exclusion of one processes from the exposure assessment: starch processing for production of glucose syrups and other starch hydrolysates	–

TOS: total organic solids.

+: 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.

The exclusion of one food manufacturing process (starch processing for the production of glucose syrups and other hydrolysates) from the exposure assessment was based on > 99% TOS removal. This is not expected to have an impact on the overall estimate derived.

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 are considered unnecessary by the Panel and the margin of exposure was not calculated.

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, the Panel concluded that the food enzyme pullulanase produced with the genetically modified B. licheniformis strain NZYM‐LU does not give rise to safety concerns under the intended conditions of use.

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

Documentation as provided to EFSA

1) Application for authorisation pullulanase produced by a genetically modified strain of Bacillus licheniformis (strain NZYM LU). July 2021. Submitted by Novozymes A/S.

2) Additional information. March 2022. Submitted by Novozymes A/S.

Abbreviations

average nucleotide identity

Pullulanase Unit Novozymes (G pullulanase)/gram

body weight

Chemical Abstracts Service

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

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

German Collection of Microorganisms and Cell Cultures

Deutsche Sammlung von Mikroorganismen und Zellkulturen

European Inventory of Existing Commercial Chemical Substances

Food and Agricultural Organization of the United Nations

genetically modified organism

International Union of Biochemistry and Molecular Biology

Joint FAO/WHO Expert Committee on Food Additives

limit of detection

polymerase chain reaction

Pullulanase Unit Novozymes (G Pullulanase)

qualified presumption of safety

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

total organic solids

whole genome sequence

World Health Organization

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

Information provided in this appendix is shown in an excel file (downloadable https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2022.7359#support‐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

Dietary exposure estimates to the food enzyme–TOS in details

Click here for additional data file.

IUBMB nomenclature	Pullulanase
Systematic name	Pullulan 6‐α‐glucanohydrolase
Synonyms	α‐Dextrin endo‐1,6‐alpha‐glucosidase
IUBMB No	EC 3.2.1.41
CAS No	9075‐68‐7
EINECS No	232‐983‐9

Population	Age range	Countries with food consumption surveys covering more than one 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
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
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
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
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
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

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).