Safety evaluation of the food enzyme pullulanase from the non‐genetically modified Klebsiella pneumoniae strain AE‐PUL

Abstract

The food enzyme pullulanase (pullulan 6‐α‐glucanohydrolase, EC 3.2.1.41) is produced by Amano Enzymes Inc. with the non‐genetically modified Klebsiella pneumoniae strain AE‐PUL, a known human pathogen. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in 10 food manufacturing processes. Since residual amounts of food enzyme—total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the remaining eight food manufacturing processes. It was estimated to be up to 0.075 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 0 124 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 1653. A search for the homology of the amino acid sequence of the pullulanase to known allergens was made and matches with two food allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded. 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 EU 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.

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 Union 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 Article7(2) of Regulation (EC) No 1332/2008 on food enzymes.

On 20 March 2023, a new application has been introduced by the applicant ‘Amano Enzyme Inc.’ for the authorisation of the food enzyme pullulanase from a non‐genetically modified strain of Klebsiella pneumoniae (strain AE‐PUL).

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests for the following food enzyme: pullulanase from a non‐genetically modified strain of Klebsiella pneumoniae (strain AE‐PUL), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. ³

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme pullulanase from Klebsiella pneumoniae AE‐PUL.

Additional information was requested from the applicant during the assessment process on 16 April 2024 and received on 7 November 2024. In addition, the applicant provided spontaneous data on 5 December 2024 (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 guidance documents of the EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation of the application.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁴ and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 24 January to 14 February 2025. ⁵ No comments were received.

3. ASSESSMENT

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

Pullulanases catalyse the hydrolysis of (1–6)‐α‐D‐glucosidic linkages in pullulan, amylopectin and glycogen, and in the α‐ and β‐limit dextrins of amylopectin and glycogen, releasing debranched dextrins and/or maltotriose. The food enzyme under assessment is intended to be used in 10 food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) baked products, (2) cereal‐based products other than baked, (3) brewed products, (4) glucose syrups and other starch hydrolysates, (5) distilled alcohol and (6) non‐wine vinegars; processing of fruits and vegetables for the production of (7) juices, (8) fruit and vegetable products other than juices and (9) alcoholic beverages other than grape wine; (10) processing of plant‐ and fungal‐derived products for the production of plant‐based analogues of milk and milk products.

3.1. Source of the food enzyme

The enzyme is produced with the bacterium Klebsiella pneumoniae strain AE‐PUL, which is deposited at the ■■■■■ with the deposition number ■■■■■ ■■■■■. ⁶ The production strain was identified as K. pneumoniae by ■■■■■, with an ■■■■■ compared to the ■■■■■ K. pneumoniae ■■■■■ and by ■■■■■. ⁷

K. pneumoniae is a known human pathogen. The WGS of the production strain was interrogated for the presence of virulence factors and antimicrobial resistance genes using two maintained databases with thresholds of > 80% identity and > 70% coverage. Matches were found with ■■■■■ virulence factors, including those typical for Klebsiella, and with genes conferring resistance to ■■■■■ and ■■■■■. ⁸ Taken together, this is considered to be a hazard.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ⁹ with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practices. 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 and treatment with ■■■■■, ¹¹ the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is 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. ¹² 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. ¹³

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 pullulanase is a ■■■■■ of ■■■■■. ¹⁴ The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■. ¹⁵ The food enzyme was analysed by ■■■■■. ¹⁶ A consistent ■■■■■ was observed across all batches. ■■■■■ showed a ■■■■■ corresponding to an ■■■■■, consistent with the expected molecular mass of the enzyme.

No other enzyme activities were reported.

The applicant's in‐house determination of pullulanase activity is based ■■■■■ (reaction conditions: ■■■■■). The enzyme activity is expressed in units (U)/g. One Unit is defined as the amount of enzyme that ■■■■■ under the conditions of the assay. ¹⁷

The food enzyme has a temperature optimum around 55°C (pH 6.0) and a pH optimum around pH 6.0 (40°C). ¹⁸ Thermostability was tested after a pre‐incubation of the food enzyme for 30 min at different temperatures (pH 6.0). Enzyme activity decreased above 40°C, showing no residual activity after pre‐incubation at 65°C. ¹⁹

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation and two batches produced for the toxicological tests (Table 1). ²⁰ The mean total organic solids (TOS) of the three batches used for commercialisation was 7.1% and the mean enzyme activity/TOS ratio was 73.4 U/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a	5 b
Pullulanase activity	U/mL c	4550	5450	5580	5300	4220
Protein	%	5.1	5.6	5.6	5.9	4.4
Ash	%	7.3	7.7	7.7	1.2	4.7
Water	%	86.0	85.3	84.8	92.6	87.9
Total organic solids (TOS) d	%	6.7	7.0	7.5	6.2	7.4
Activity/TOS ratio	U/mg TOS	67.9	77.9	74.4	85.5	57.0

^a Batch used for the repeated dose 90‐day oral toxicity study in rats.

^b Batch used for the Ames test and the in vitro mammalian cell micronucleus test.

^c U: Pullulanase Unit (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three commercial batches and in the two batches used for toxicological studies was below 0.01 mg/kg, ^{21 , 22} which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

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). ²³ 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 was 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 ■■■■■ analysed ■■■■■. Samples of ■■■■■. No colonies were produced. A positive control was included. ²⁵

The absence of 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 a ■■■■■ fragment corresponding to the gene encoding ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme. ^{26 , 27}

3.4. Toxicological data

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian cell micronucleus test and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batches 4 and 5 (Table 1) used in these studies had a similar composition as the batches used for commercialisation, and thus were considered suitable as test items.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020) and following good laboratory practice (GLP). ²⁸ ■■■■■.

■■■■■.

■■■■■.

■■■■■.

The study was considered reliable without restrictions and the results were of high relevance.

The Panel concluded that the food enzyme pullulanase did not induce gene mutations in bacteria under the conditions applied in this study.

3.4.1.2. In vitro mammalian cell micronucleus test

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP. ²⁹ A dose range‐finding and two main experiments were performed with duplicate cultures of the TK6 human lymphoblastoid cell line. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

Based on the results from the dose range‐finding test, cells were exposed to the food enzyme and scored for the frequency of mononucleated cells with micronuclei at concentrations of 3000, 4000 and 5000 μg TOS/mL either in a short‐term treatment (3‐hours exposure and 21‐hours recovery period) with and without S9‐mix or in a long‐term treatment (24‐hours exposure without recovery period) without S9‐mix.

No cytotoxicity was seen in the short‐term treatment either with or without S9‐mix. In the long‐term treatment, cytotoxicity of 50% (based on relative population doubling) was observed at 5000 μg TOS/mL.

The frequency of micronucleated cells was not statistically significantly different from the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results were of high relevance.

The Panel concluded that the food enzyme pullulanase did not induce an increase in the frequency of micronucleated cells under the test conditions applied in this study.

3.4.2. Repeated dose 90‐day oral toxicity study in rodents

The repeated dose 90‐day oral toxicity study was performed following GLP and in accordance with guidelines of the Japanese Ministry of Health and Welfare (1999). ³⁰ The study is in accordance with OECD Test Guideline 408 (OECD, 1998) with the following deviations: only two sections of the brain and one section of spinal cord (thoracic level) were examined, urea was not determined. The Panel considered that these deviations are minor and do not impact on the evaluation of the study.

Groups of 12 male and 12 female Sprague–Dawley (Crl:CD(SD)) rats received by gavage the food enzyme in doses of 500, 1000 or 2000 mg/kg body weight (bw) per day, corresponding to 31, 62 or 124 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

Haematological investigations revealed a statistically significant increase in haemoglobin (Hb) and a decrease in large unstained cells (LUC) in high‐dose males (+3% and −20%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex, the change was small (Hb), there were no changes in other relevant parameters (other blood or white cells' parameters) and the changes were within the historical control values.

Statistically significant changes detected in organ weights were an increase in absolute thyroid weight in high‐dose males (+18%), in relative liver to body weight in mid‐dose males (+8%) and in absolute thymus weight in mid‐dose females (+28%); a decrease in relative adrenals to body weight was observed in mid‐dose females (−13%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), there was no dose–response relationship (liver, thymus and adrenals) and there were no histopathological changes in thyroid, liver, thymus or adrenals.

No other statistically significant or biologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 124 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

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

The potential allergenicity of the pullulanase produced with the Klebsiella pneumoniae strain AE‐PUL was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, matches with two allergens were found using the AllergenOnline database and Allergen Database for Food Safety. The matching food allergens were Sal s 6 (62.3%–69.0% sequence identity), a collagen alpha from Atlantic salmon (Salmo salar) and a collagen alpha (95.3% sequence identity) from cow (Bos taurus). ³¹

No reports on oral or respiratory sensitisation or elicitation reactions of the pullulanase under assessment have been published. ³²

Sal s 6, a collagen alpha from Atlantic salmon (Salmo salar) is known as a food allergen (Ruethers et al., 2021). Sensitisation to bovine collagen has been observed, mostly in association with vaccines containing gelatin or collagen‐derived therapeutic devices (Hori et al., 2002; Mullins et al., 1996). Food allergies involving IgE reactivity to the peptide backbone of beef proteins are rare, but cases of allergic reactions after consuming bovine collagen have been described (Sakaguchi et al., 1996).

No allergic reactions upon dietary exposure to any pullulanase have been reported in the literature. ³³

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for salmon‐allergic individuals upon dietary exposure to the pullulanase under assessment.

■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly for salmon‐allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after salmon consumption.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in 10 food manufacturing 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. ³⁴

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of baked products	Flour	1.5
Production of cereal‐based products other than baked	Cereals	2.9
Production of brewed products	Cereals	0.3
Production of glucose syrups and other starch hydrolysates	Starch	2.9
Production of distilled alcohol	Cereals	116.2
Production of non‐wine vinegar	Cereals	2.9
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	1.5
Production of fruit and vegetable products other than juices	Fruit and vegetables	1.5
Production of alcoholic beverages other than grape wine	Fruit	1.5
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Plant‐based beverages	2.9

^a The name has been harmonised by EFSA in accordance with the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

^b The numbers in bold were used for calculation.

In the production of baked products, the food enzyme is added to flour during dough preparation. ³⁵ This pullulanase is used in conjunction with other enzymes to hydrolyse starch, which retards starch retrogradation. ³⁶ The food enzyme–TOS remain in the baked products.

In the production of cereal‐based products other than baked, the food enzyme is added in the production of cooked rice, or to cereals such as corn, wheat and barley for the production of processed cereal products. ³⁷ The pullulanase hydrolyses starch within the raw materials to retard starch retrogradation and reduce product viscosity. ³⁸ The food enzyme–TOS remain in the cereal‐based products.

In the production of brewed products, the food enzyme is added during mashing and liquefaction in the production of beer. ³⁹ In the production of fermented beverages such as sake or rice wine, it may be added during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps. ⁴⁰ This reduces the mashing time and improves the alcohol yield. The food enzyme‐TOS remain in the brewed products.

In the production of glucose syrups and other starch hydrolysates, the food enzyme is added together with other enzymes during the saccharification step. ⁴¹ It degrades gelatinised starch into polysaccharides or shorter dextrins. The food enzyme–TOS are removed from the syrups and other hydrolysates by treatment with activated charcoal or similar and with ion‐exchange resins (EFSA CEP Panel, 2023).

In the production of distilled alcohol, the food enzyme could be added to cereals during slurry mixing, liquefaction, pre‐saccharification and fermentation steps. ⁴² In combination with other starch‐degrading enzymes, it improves the hydrolysis of starch, which increases alcohol yield and improves performance in the pre‐saccharification process. ⁴³ The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of non‐wine vinegar, the food enzyme could be added to cereals during slurry mixing, liquefaction, saccharification or fermentation steps. ⁴⁴ The pullulanase acts on the starch present in different grains and cereals to improve the yield of the process. ⁴⁵ The food enzyme–TOS remain in the non‐wine vinegars.

In the production of juices, the food enzyme is added to fruits and vegetables during both the mash treatment and the depectinisation step. ⁴⁶ The pullulanase degrades starch in the juices, improving the filtration rate and preventing haze. ⁴⁷ The food enzyme–TOS remain in the juices.

For the production of fruit and vegetable products other than juices, the food enzyme is added to crushed fruit and vegetables during the mash treatment or before the straining. ⁴⁸ The hydrolysis of starch reduces viscosity and improves the yield. ⁴⁹ The food enzyme–TOS remain in the final foods.

In the production of alcoholic beverages other than grape wine, the food enzyme is added to the fruit during mashing or directly to the juice before fermentation. ⁵⁰ The hydrolysis of starch reduces viscosity and improves the yield. ⁵¹ The food enzyme–TOS remain in the final alcoholic beverages.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to aqueous homogenates ⁵² to hydrolyse the gelatinised starch to produce malto‐oligosaccharides. ⁵³ The food enzyme–TOS remain in these food products.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied, the Panel considered that this pullulanase will be inactivated or removed in most of the food manufacturing processes listed in Table 1. However, it may remain in its active form in juices, brewed and baked products, and non‐wine vinegars, depending on the processing conditions.

3.5.2. Dietary exposure estimation

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

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023). 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 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.075 mg TOS/kg bw per day in toddlers and children at the 95th percentile.

TABLE 3.

Summary of the 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)	0.004–0.023 (12)	0.013–0.054 (15)	0.009–0.029 (19)	0.006–0.020 (21)	0.004–0.011 (22)	0.003–0.008 (23)
Min–max 95th percentile (number of surveys)	0.011–0.055 (11)	0.034–0.075 (14)	0.019–0.075 (19)	0.013–0.046 (20)	0.009–0.033 (22)	0.006–0.023 (22)

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 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
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of two processes from the exposure estimation: –Production of glucose syrups and other starch hydrolysates –Production of distilled alcohol	−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

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

The exclusion of two food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

3.6. Margin of exposure

A comparison of the NOAEL (124 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.003–0.054 mg TOS/kg bw per day at the mean and from 0.006 to 0.075 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 1653.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme pullulanase produced with the non‐genetically modified Klebsiella pneumoniae strain AE‐PUL does not give rise to safety concerns under the intended conditions of use.

The production strain of the food enzyme is a known human pathogen and contains genes encoding for antimicrobial resistance. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, the Panel did not consider this to be a risk.

5. DOCUMENTATION AS PROVIDED TO EFSA

Dossier for pullulanase from Klebsiella pneumoniae AE‐PUL. December 2023. Submitted by Amano Enzyme Inc.

Additional information. November 2024. Submitted by Amano Enzyme Inc.

Additional information. December 2024. Submitted by Amano Enzyme Inc.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMM

genetically modified microorganism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LOD

limit of detection

MOE

margin of exposure

OECD

Organisation for Economic Cooperation and Development

PCR

polymerase chain reaction

QPS

qualified presumption of safety

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00567

PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Monika Sramkova, Henk Van Loveren, Laurence Vernis, Holger Zorn

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

NOTE

This scientific opinion was first adopted by the Panel on Food Enzymes on 12 March 2025. After this date the opinion was revised in order to incorporate a new Ames test, which was unavailable when this opinion was originally adopted due to administrative error. The scientific opinion was re‐adopted by the Panel on Food Enzymes on 10 April 2025. The original version is available upon request.

Supporting information

APPENDIX A: Dietary exposure estimates to the food enzyme–TOS in detail

APPENDIX A. Dietary exposure estimates to the food enzyme–TOS in detail

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

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, Spain
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, Republic of North Macedonia*, Serbia*, 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, Republic of North Macedonia*, Serbia*, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina*, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina*, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, 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, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden

^*Consumption data from these pre‐accession countries are not reported in Table 3 of this opinion; however, they are included in Appendix B for testing purposes.

^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).

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Peluso, S. , Andryszkiewicz, M. , Cavanna, D. , Criado, A. , Fernàndez‐Fraguas, C. , & Liu, Y. (2025). Safety evaluation of the food enzyme pullulanase from the non‐genetically modified Klebsiella pneumoniae strain AE‐PUL . EFSA Journal, 23(5), e9332. 10.2903/j.efsa.2025.9332