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 on food enzymes.

Five applications have been introduced by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP), and by the companies “DSM Food Specialties B.V" and “Novozymes A/S" for the authorisation of the food enzymes Pectinase, Poly-galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A2 from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo-1,4-B-xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM-SO), respectively.

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, 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 Pectinase, Poly-galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A2 from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo-1,4-B-xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM-SO) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

3.3.1 Properties of the food enzyme

The food enzyme contains ■■■■■ endo-polygalacturonases of ■■■■■ amino acids and a pectin lyase of ■■■■■ amino acids.12 The molecular masses of the mature proteins, calculated from the amino acid sequences, are ■■■■■ kDa, respectively.12 The food enzyme was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.13 A consistent protein pattern was observed across all batches. The gel showed protein bands consistent with the expected masses of the enzymes, together with other bands of lesser staining intensity. The food enzyme was tested for α-amylase, lipase and protease activities.14 only α-amylase activity was detected. No other enzymatic activities were reported.

The in-house determination of endo-polygalacturonase activity is based on the hydrolysis of polygalacturonic acid with a consequent release of reducing groups determined using a colorimetric assay (reaction conditions: pH 4.5, 50°C, 5 min). Enzyme activity is expressed as polygalacturonase units (PGNU)/g. One PGNU is defined as the amount of enzyme producing reducing groups equivalent to 1 mg of galacturonic acid under the conditions of the assay.15

The in-house determination of pectin lyase activity is based on the cleavage of pectin (reaction conditions: pH 5.5, 45°C, 10 min). The enzymatic activity is determined by measuring the release of unsaturated Δ-4,5 polygalacturonides, which are determined spectrophotometrically at 235 nm. Enzyme activity is expressed in PECtin Transeliminase Units (PN)/g (PECTU(PN)/g). One PECTU(PN) is defined as the quantity of the enzyme releasing 1 μmol of unsaturated Δ-4,5 polygalacturonides per minute under the conditions of the assay.16

The optimum temperature is around 60°C (pH 4.0) for the endo-polygalacturonase and around 40°C (pH 4.0) for pectin lyase activities. The optimum pH is around pH 4.0 (37°C) for endo-polygalacturonase and around pH 4.0 (37°C) for pectin lyase activities. Thermostability was tested after a pre-incubation of the food enzyme for 30 min at different temperatures (pH 4.0). Both activities decreased at temperatures greater than 70°C, with no residual activity detected at 80°C.17

3.3.2 Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation, among which batch 1 was used for the genotoxicity tests (Table 1). In addition, batch 4 was produced for the 90-day toxicity study.18 The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 14% and the mean enzyme activity/TOS ratio was 261.6 PGNU(PE)/mg TOS for the endo-polygalacturonase and 94.7 PECTU(PN)/mg TOS for the pectin lyase, respectively.

3.3.3 Purity

The lead content in all batches was below 0.5 mg/kg,19 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, arsenic, mercury and cadmium contents were below the limits of detection (LoD) of the employed methods.19, 20

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).21 No antimicrobial activity was detected in any of the tested batches.22

Strains of Aspergillus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The applicant did not provide information on the potential secondary metabolites produced under the conditions of fermentation which might contribute to the food enzyme-TOS. This issue was addressed by the toxicological examination of the food enzyme-TOS.

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

3.3.4 Viable cells 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. A positive control was included.23

3.4.1 Genotoxicity

3.4.2 Repeated dose 90-day oral toxicity study in rodents

The repeated dose 90-day oral toxicity study was performed following GLP26 and in accordance with OECD Test Guideline 408 (OECD, 1981) with the following deviations: serum ornithine decarboxylase (ODC) was not measured and microscopic examination of lungs at low and intermediate doses was not performed. The Panel considered that these deviations are minor and do not impact the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received by gavage the food enzyme in doses of 14.3, 143 or 1,430 mg TOS/kg bw per day. Controls received the vehicle (purified water obtained by reverse osmosis).

One high-dose male died following blood sampling procedures on day 87 (week 13). The Panel considered the death as incidental based on its isolated occurrence and lack of test article-related microscopic findings in this, or any other rat, in the study.

Salivation after dosing increased from day 1 throughout the study duration in high-dose males and females, reaching 8/10 males and 5/10 females on day 78. The Panel considered this change test item-related, possibly associated with the organoleptic properties of the food enzyme and/or with the gavage procedure. However, it was not regarded as adverse based on the transient presentation and the lack of effect on the overall health status.

Haematological investigations revealed a statistically significant decrease in neutrophils (NEU) in high-dose males (−39%), in monocytes (MONO) in low- and mid-dose males (−33% in both cases) and in large unstained cells (LUC) in low-dose males (−50%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all), there was no dose–response relationship (MONO and LUC), there were no changes in other relevant parameters (other white blood cell parameters) and there were no histopathological changes in lymphohematopoietic tissues or organs.

Clinical chemistry investigations revealed statistically significant changes in alanine aminotransferase (ALT), with increase in low-dose males (+16%) and decrease in high-dose males and females (−11% and − 35%, respectively), decrease in aspartate aminotransferase (AST) in mid- and high-dose females (−19% and − 26%, respectively), decrease in total proteins (TP) in high-dose males (−6%), decrease in albumin (ALB) in mid-dose females (−8%), decrease in α-2-globulin in low-dose females (−20%), decrease in γ-globulin in mid-dose males (−33%), decrease in albumin/globulin ratio in low-dose males (−11%), changes in sodium (Na), with decrease in high-dose males and mid-dose females (−0.7% and − 1%, respectively) and increase in low-dose females (+0.7%), decrease in potassium (K) in low-dose females (−9%), increase in chloride (Cl) in mid-dose males (+2%) and decrease in mid-dose females (−1%), decrease in calcium (Ca) in mid- and high-dose males (−4% and −3%, respectively) and in females at all doses (−4%, −5% and − 3%, respectively) and decrease in phosphorus (Phos) in high-dose males (−20%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (AST, TP, ALB, alpha-2-globulin, gamma-globulin, albumin/globulin, K), there was no consistency between the changes in males and females (ALT), there was no dose–response relationship (ALT, ALB, alpha-2-globulin, gamma-globulin, albumin/globulin, all electrolytes) and there were no histopathological changes in the liver (ALT, AST and proteins).

Statistically significant changes in organ weights included increase in absolute kidney weight in high-dose males and females (+9% and +13%, respectively), and relative testes weight in mid- and high-dose males (+19% and +33%, respectively). The Panel considered increase in kidney weight as not toxicologically relevant, as the changes were small, they were within the historical control values and did not correlate with histopathological findings (males).

The microscopic examination revealed increased mineralisation in the kidneys from females at mid- and high dose compared with controls; the finding was dose-related in terms of incidence, only reaching statistical significance at the high dose and it was localised in the corticomedullary junction (1/10 rats affected in control and low-dose groups, 4/10 in mid-dose and 10/10 in high-dose) and the medulla (1/10 in controls, 2/10 in mid-dose and 9/10 in high-dose). The Panel noted that renal mineralisation is frequently found along the corticomedullary junction in rats, particularly in females, as a background finding and is regarded as of no clinical consequence (Ritskes-Hoitinga and Beynen, 1992; Frazier et al., 2012). In this study, considering the dose relationship observed, an effect of the test item could not be ruled out and the Panel considered the increased incidence of renal mineralisation observed in females a test item exacerbation of a background change. However, the Panel did not regard microscopic renal changes as adverse taking into consideration that: (i) there was no evidence of associated degenerative changes in the kidneys, (ii) there was no concurrent alteration in serum biomarkers of renal function (i.e. urea and creatinine), (iii) there was no correlated increase in serum Ca, while the minimal decrease in serum Ca observed was regarded as incidental and of no toxicological relevance.

In addition, in the testes from the control group, an unusual high incidence of degeneration of the germinal epithelium was observed in the seminiferous tubules not seen in the high-dose group. Degenerative changes in the control group were variably associated with interstitial cell hyperplasia (observed in 2/10 rats), reduced sperm content in the epididymides (in 3/10) and accounted for the flaccidity observed macroscopically in one male, and for the lower mean weight of testes in control group when compared to historical control data.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1,430 mg TOS/kg bw per day, the highest dose tested.

3.4.3 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 endo-polygalacturonases and pectin lyase produced from the non-genetically modified A. tubingensis strain NZYM-PE was assessed by comparing its amino acid sequences 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, no match was found for the pectin lyase, but 13 matches were found for the two endo-polygalacturonases. Twelve of them were with pollen allergens: Sor h 13.0101 from Sorghum halepense (Johnson grass), Pla or 2.0101 from Platanus orientalis (oriental plane tree), Cry j 2.0101 from Cryptomeria japonica (Japanese cedar), Pla a 2.0101 from Platanus acerifolia (London plane tree), Cha o 2.0101 from Chamaecyparis obtusa (Japanese cypress), Jun a 2.0101 from Juniperus ashei (mountain cedar), Phl p 13.0101 from Phleum pratense (thimothy), Zea m 13 from Zea mays (maize), gi|338930674 from Paspalum notatum (bahiagrass), Sal k 6.01, partial (gi|589912883) from Salsola kali (prickly saltwort), gi|73913442 from Lilium longiflorum (trumpet lily) and Ole e 14.0101 from Olea europaea (olive tree). The remaining match was with the food allergen Cari p 1.0101 from Carica papaya (papaya).

No information is available on oral and respiratory sensitisation or elicitation reactions of these endo-polygalacturonase and pectin lyase enzymes.

Endo-polygalacturonases are allergens often present in grass and tree pollen. The oral allergy syndrome, i.e. allergic reactions mainly in the mouth, is associated with sensitisation to pollen such as from cedar trees (Midoro-Horiuti et al., 2003) and grasses. Such reactions are seldomly leading to severe systemic anaphylaxis.

Cari p 1 (Cari p 1.0101) is the main allergen present in C. papaya, described as both a food and a respiratory allergen (Sarkar et al., 2018). Several studies reported occupational rhinitis and asthma in workers of industries where papain is handled (Baur and Fruhmann, 1979; Baur et al., 1982; Niinimaki et al., 1993; Soto-Mera et al., 2000; Van Kampen et al., 2005). In other studies, allergy to papaya-derived products unrelated to occupational exposure has also been described. Garcia-Ortega et al. (1991) showed that administration of chymopapain for chemonucleolysis resulted in sensitisation in some patients. Mansfield and Bowers (1983) reported severe systemic allergic reactions mediated by papain-specific IgE in some individuals that ingested papain-containing meat tenderiser. Sensitisation to papaya does not typically occur from eating papaya fruit. However, once sensitised, individuals may suffer allergic reactions following any type of exposure to papaya or papaya-derived products (Morton, 1987).

■■■■■, a product that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201127), is used as raw material. 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 fungal 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, in particular for individuals sensitised to papaya, but that the risk will not exceed that of consumption of papaya. In addition, oral allergy reactions cannot be excluded in pollen-sensitised individuals.

3.5.1 Intended use of the food enzyme

The food enzyme is intended to be used in four food processes at the recommended use levels summarised in Table 2.28

In fruit and vegetable processing for juice production, the food enzyme is added to fruits or vegetables during mash treatment and to the pomace during the second mash treatment. It can also be added to the raw juice during depectinisation.29 The endo-polygalacturonase degrades galacturonans in the cell wall, improving the processability, resulting in increased yield and avoidance of haziness.30 The food enzyme-TOS remains in the juices.

In fruit and vegetable processing for products other than juices, the food enzyme is added to fruits or vegetables during the maceration step, where it catalyses the breakdown of pectins,31 improving processability. The food enzyme-TOS remains in the final products.

In refined olive oil production, the food enzyme is added to the olive paste during the malaxation step. The food enzyme catalyses the breakdown of pectins in the cell wall, facilitating the release of oil from vacuoles and thus increasing the yield.32

The term ‘olive oil’ is defined in the Regulation (EU) No 1308/201333 as ‘composed of refined olive oils and virgin olive oils’. The term ‘virgin olive oils’ means ‘oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alterations in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oils obtained using solvents or using adjuvants having a chemical or biochemical action, or by re-esterification process and any mixture with oils of other kinds’.

In accordance with the law, the use of enzymes is not permitted in the production of virgin olive oils in the European Union. Therefore, this assessment is limited to the use of this food enzyme in the production of refined olive oil only. The food enzyme-TOS is removed from the refined olive oil by repeated washing during the refinement process (EFSA CEP Panel, 2021b).

The production processes of olive oil and palm oil are very similar. The applicant provided a theoretical calculation, showing that > 99.96% of the food enzyme-TOS could be removed in olive oil.34 The residual amounts of enzyme in crude palm oil were below the LoD of the method.35,36 Although equivalent analytical data were not available for olive oil, the Panel considered that only negligible amounts of enzyme TOS (< 1%) would remain in refined olive oils.

In wine and wine vinegar production, the food enzyme is added during the crushing and maceration, the clarification, and before the ageing and filtration steps.37 It is used to improve processability, to increase yield and to improve clarification.28 The food enzyme-TOS remains in the final products.

based on data provided on thermostability (see Section 3.3.1), the food enzyme is expected to be inactivated by heat in fruit and vegetable processing for products other than juices, but may remain active in wine and wine vinegar, and in juices, depending on the pasteurisation conditions.

3.5.2 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: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices and wine and wine vinegar production.

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 43 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries. The highest dietary exposure was estimated to be about 0.132 mg TOS/kg bw per day in toddlers and children at the 95th percentile.

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.

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 overestimation of the exposure.

The exclusion of one food manufacturing process from the exposure assessment was based on > 99% of TOS removal. This is not expected to have an impact on the overall estimate derived.