Safety evaluation of the food enzyme papain, a cysteine endopeptidase complex from the latex of Carica papaya L.

Abstract

The food enzyme is a cysteine endopeptidase complex, containing papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30) and glycyl endopeptidase (EC 3.4.22.25), obtained from the latex of unripe Carica papaya L. by Enzyme Development Corporation. It is intended to be used in 13 food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in one process, dietary exposure was calculated for the remaining 12 food manufacturing processes. It was estimated to be up to 20.963 mg TOS/kg body weight per day. This exposure is up to one order of magnitude higher than the intake of the corresponding fraction from unripe C. papaya L. latex. Considering the overestimation of the exposure to the food enzyme–TOS, in a realistic exposure scenario, both values can be expected to be within the same order of magnitude. Toxicological studies were not required according to the current guidance. Among the four proteins in the cysteine endopeptidase complex, papain and chymopapain are known food allergens. Homology searches with the amino acid sequences of the four proteins in the complex to known allergens identified matches with six food and eight respiratory allergens. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the origin of the food enzyme being an edible plant source and the estimated dietary exposure, 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 1332/2008 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 Article 7(2) of Regulation (EC) No 1332/2008 on food enzymes.

On 5 April 2023, a new application has been introduced by the applicant “Enzyme Development Corporation” for the authorisation of the food enzyme Papain from Carica papaya.

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 of the following food enzyme: papain from Carica papaya, 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 papain from Carica papaya L. as a new food enzyme.

Additional information was requested four times during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00441?search=EFSA‐Q‐2023‐00441.

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 current ‘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 to evaluate this 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 26 June 2024 to 17 July 2024. ⁵ No comments were received.

3. ASSESSMENT

The food enzyme under application is a cysteine endopeptidase complex, containing four proteolytic activities: papain, chymopapain, caricain and glycyl endopeptidase. Papain is the generally accepted name of this complex, and therefore the term ‘papain’ is used throughout the text of the scientific opinion.

IUBMB nomenclature	Papain
Systematic name	–
Synonyms	Papayotin; Papaya peptidase I
IUBMB No	EC 3.4.22.2
CAS No	9001‐73‐4
EINECS No	232‐627‐2

IUBMB nomenclature	Chymopapain
Systematic name	–
Synonyms	Chymopapain A; chymopapain B; chymopapain S
IUBMB No	EC 3.4.22.6
CAS No	9001‐09‐6
EINECS No	232‐580‐8

IUBMB nomenclature	Caricain
Systematic name	–
Synonyms	Papaya peptidase A; papaya peptidase II; chymopapain S
IUBMB No	EC 3.4.22.30
CAS No	39307‐22‐7
EINECS No	–

IUBMB nomenclature	Glycyl endopeptidase
Systematic name	–
Synonyms	Papaya peptidase B; papaya proteinase IV; chymopapain M
IUBMB No	EC 3.4.22.25
CAS No	149719‐24‐4
EINECS No	–

Papains catalyse the hydrolysis of proteins with broad specificity for peptide bonds, with preference for amino acids with large hydrophobic side chains at the P2 position, resulting in the generation of peptides and amino acids.

The food enzyme is intended to be used in 13 food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of dairy products for the production of (1) cheese, (2) flavouring preparations from dairy products and (3) modified milk proteins; (4) processing of eggs and egg products; processing of meat and fish products for the production of (5) modified meat and fish products and (6) protein hydrolysates from meat and fish proteins; processing of cereals and other grains for the production of (7) baked products, (8) cereal‐based products other than baked; (9) brewed products and (10) starch and gluten fractions; (11) processing of fruits and vegetables for the production of juices; (12) processing of plant‐ and fungal‐derived products for the production of protein hydrolysates from plants and fungi and (13) processing of yeast and yeast products.

3.1. Source of the food enzyme

The food enzyme papain is obtained from the latex harvested from the unripe fruit of non‐genetically modified Carica papaya L., ⁶ a species belonging to the Caricaceae family (OECD, 2010) and a perennial tree native to Mexico and South America (Garrett, 1995; Siar et al., 1998).

The milky latex is stored in secretory structures known as laticifers, which are distributed throughout various parts of the papaya plant. The highest concentration of latex is present in the peels of unripe papaya fruit. During the ripening of the papaya fruit, the production of latex by the laticifers gradually declines. Mechanical injury, such as longitudinal incisions on the unripe fruit induce latex exudation (El Moussaoui et al., 2001).

Ripe papaya fruit is commonly consumed worldwide. Unripe papaya is also used as vegetable in south Asian countries as Thailand, where it is cooked in soup or consumed raw as a salad ingredient (Sone et al., 1998), or canned in sugar syrup in Puerto Rico (Morton, 1987).

A literature search was made by EFSA to identify compounds in papaya peels or latex or extracts thereof that could be hazardous to human health upon oral exposure. The Panel did not identify any studies both reliable and relevant to the source of the food enzyme, except for allergenicity.

No issues of concern were identified by the Panel from the source material.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ⁷ with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice. ⁸

The food enzyme is obtained by aqueous extraction from papaya latex provided by a third‐party supplier. ⁹ After the dried latex is rehydrated, the liquid containing the enzyme is filtrated and further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. Finally, the food enzyme is spray‐dried. Around 5 kg of dry latex is needed to produce 1 kg of papain. ¹⁰ Taken into account the weight loss during drying (about 7 times), this corresponds to a yield factor of 0.03 (w/w, papain/fresh latex). The applicant provided information on the identity of the substances used in the extraction and in the subsequent downstream processing of the food enzyme. ¹¹

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 papain activity of the food enzyme is attributed to four proteolytic enzymes of 345, 352, 348 and 348 amino acids, which include the signal peptide, propeptide and the mature protein. ¹² The molecular masses of the mature proteins, calculated from the amino acid sequences, are 23.3, 23.3, 23.4 and 23.7 kDa. The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about 25 kDa, consistent with the expected mass of the enzyme. ¹³

No other enzyme activities were reported. ¹⁴

The applicant's in‐house determination of papain activity is based on the hydrolysis of casein (reaction conditions: pH 6, 40°C, 60 min). The release of peptides containing l‐tyrosine is measured spectrophotometrically at 280 nm. The enzyme activity is expressed in Tyrosine Units (TU)/g. One TU is defined as the amount of enzyme which will release 1 μg of l‐tyrosine equivalents from casein per minute under the conditions of the assay. ¹⁵

The food enzyme has a temperature optimum around 80°C (pH 6) and a pH optimum around pH 9 (40°C), the maximum pH tested. ¹⁶ Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 6). Enzyme activity decreased above 60°C showing no residual activity at 100°C. ¹⁷

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches (Table 1). ¹⁸ The mean total organic solids (TOS) was 91.3% and the mean enzyme activity/TOS ratio was 904.7 TU/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Proteolytic activity	TU/mg a	887	820	772
Protein	%	94.9	90.6	93.1
Ash	%	0.4	2.9	1.6
Water	%	7.8	6.4	6.9
Total organic solids (TOS) b	%	91.8	90.7	91.5
Activity/TOS ratio	TU/mg TOS	966.2	904.1	843.7

^a TU: Tyrosine Unit (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three batches was below 0.3 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, the mercury content was below the limit of quantification (LoQ) of the employed method. ^{20 , 21 , 22} For arsenic and cadmium, the average concentrations determined in the three batches were 0.31 and 0.06 mg/kg, respectively. ²³ The Panel considered these concentrations as not of concern.

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Enterobacteriaceae as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ²⁴ The counts of osmotolerant yeasts and filamentous fungi were ≤ 100 CFU/g in three food enzyme batches tested, meeting EFSA's requirements (EFSA CEP Panel, 2021). ²⁵ In addition, the counts of Bacillus cereus were below the limit of detection (100 CFU/g) in three tested batches. ²⁶

The presence of aflatoxin B1, B2, G1, G2, deoxynivalenol, zearalenone and ochratoxin A was examined in three food enzyme batches. All were below the LoQ of the applied analytical methods. ^{27 , 28 , 29}

More than 600 pesticides were tested in three food enzyme batches, and no pesticide residues were detected, except for 2‐phenylphenol, which was found in one batch (0.012 mg/kg) and for anthraquinone, which was detected in two batches (0.016 mg/kg and 0.014 mg/kg). ³⁰

Using the highest estimated dietary exposure to the food enzyme–TOS of 20.963 mg/kg body weight (bw) per day (see Section 3.5.2), this results in an exposure (0.27 × 10⁻⁶ mg/kg bw per day), which is below the acceptable daily intake (ADI) of 0.4 mg/kg bw per day set by EFSA for 2‐phenylphenol (EFSA, 2008). Therefore, the Panel considers that the amount detected in the food enzyme batch is of no safety concern.

Anthraquinone is not permitted for use as a pesticide in the EU; ³¹ therefore, EFSA did not derive an ADI value. ³² Using the highest estimated dietary exposure of 20.963 mg TOS/kg bw per day, this results in an exposure (0.34 × 10⁻⁶ mg/kg bw per day), which is below the subchronic provisional reference dose of 0.01 mg/kg bw per day set by the US EPA for anthraquinone (EPA, 2011). The Panel considers that the amount detected in the food enzyme batches is of no safety concern.

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

3.4. Toxicological data

According to the EFSA ‘Scientific Guidance for the submission of dossiers on Food Enzymes’, for food enzymes derived from plants and animals that are consumed by the European population, three criteria must be met to waive toxicological studies: (i) the food enzyme is obtained from an edible plant or animal source (ii) no hazard is introduced through the manufacturing process and (iii) when it can be demonstrated that the dietary exposure to the food enzyme–TOS is within the same order of magnitude as the dietary intake of the fraction of the plant or animal material comparable to the food enzyme–TOS (EFSA CEP Panel, 2021).

The Panel considered that sufficient information has been provided on the plant source, its history of consumption as well as the food enzyme manufacturing process. Therefore, the need for toxicological data is waived.

3.4.1. 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 papain, chymopapain, caricain and glycyl endopeptidase was assessed by the Panel by separately comparing each amino acid sequence with those of known allergens as described in the EFSA GMO Scientific opinion (EFSA GMO Panel, 2010). The amino acid sequences of these four proteins present a high degree of homology (62%–81% sequence identity). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, matches with 6 food and 8 respiratory allergens were found using the AllergenOnline and COMPARE databases. ³³

Papain and chymopapain (Cari p 2) are food allergens from papaya (Carica papaya L.). Cari p 2 was detected in pollen and pulp of raw and ripe papaya fruits (Bhowmik et al., 2021). Several studies reported occupational rhinitis and asthma in workers of industries where papain is handled (Baur et al., 1982; Baur & Fruhmann, 1979; Niinimaki et al., 1993; Soto‐Mera et al., 2000; Van Kampen et al., 2005). Administration of chymopapain for chemonucleolysis resulted in sensitisation in some patients (Garcia‐Ortega et al., 1991). Severe systemic reactions mediated by papain‐specific Immunoglobulin E (IgE) were observed in some individuals that ingested papain‐containing meat tenderiser (Mansfield & Bowers, 1983). Caricain and glycyl endopeptidase showed 82.5%–87.5% sequence identity to papain and chymopapain, and IgE binding to caricain and glycyl endopeptidase was reported (Dando et al., 1995).

Matching food allergens were Ana c 2 (52.5%–62.5% sequence identity), a bromelain from pineapple (Ananas comosus); actinidins (56.3%–61.3% sequence identity) from kiwi fruits (Actinidia deliciosa, A. chinensis) and a cysteine protease (47.5%–50% sequence identity) from soybean (Glycine max).

Bromelain from pineapple was described as occupational allergen eliciting allergic reactions after inhalation or dietary exposure (Gailhofer et al., 1988; Nettis et al., 2001).

Actinidins are major kiwi allergens (Grozdanovic et al., 2014). Kiwi allergic individuals also demonstrated IgE reactivity to bromelain and papain. In addition, the presence of cross‐reactive allergens in papaya and fig was shown by cross‐inhibition experiments (Hemmer et al., 2004).

IgE binding to the soybean cysteine protease was described in soybean allergic individuals with skin‐related symptoms (Morita et al., 2012; Ogawa et al., 1993).

The matching pollen allergen was Amb a 11 (53.8%–58.8% sequence identity), a cysteine protease from ragweed (Ambrosia artemisiifolia). Ragweed is associated with the pollen‐food allergy syndrome. Reactions within this syndrome are usually restricted to the buccal cavity and seldomly lead to anaphylaxis (Sarkar et al., 2018).

The matching respiratory allergens were group 1 mite allergens (36.3%–48.8% sequence identity), cysteine proteases from Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Euroglyphus maynei, Tyrophagus putrescentiae and Sarcoptes scabiei. Der p 1 and Der f 1 are major mite allergens associated with rhinitis and asthma (Xu et al., 2025). No evidence of papain‐related allergic reactions upon dietary exposure in individuals sensitised to mites is available (Giangrieco et al., 2023).

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for papaya, ananas, kiwi, soy, fig and pollen‐allergic individuals upon dietary exposure to the papain under assessment.

The food enzyme is obtained from the latex of unripe papaya. Papaya also contains other food allergens, for example endo‐polygalacturonase (Cari p 1) and endochitinase, involved in the latex‐fruit syndrome (Rojas‐Mandujano et al., 2018). Cari p 1 was detected in papaya peel, pulp, and pollen (Sarkar et al., 2018). The Panel considered that these allergenic proteins could also be present in the food enzyme.

In conclusion, the Panel considered that under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme, particularly for papaya, ananas, kiwi, soy, fig and pollen‐allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after consumption of papaya, ananas, kiwi, soy and fig.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in 13 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)	Recommended use level (mg TOS/kg RM) b
Processing of dairy products
Production of cheese	Milk	25–100
Production of flavouring preparations from dairy products	Cheese	5–15
Production of modified milk proteins c	Milk protein	500–750
Processing of eggs and egg products	Whole egg/egg white	500–750
Processing of meat and fish products
Production of modified meat and fish products	Fish and shellfish	0.05–0.3
	Meat	0.8–3.0
Production of protein hydrolysates from meat and fish proteins	Fish and shellfish protein	50–250
	Meat protein	50–250
Processing of cereals and other grains
Production of baked products	Flour	0.3–5
Production of cereal‐based products other than baked 35	Grains	2–15
	Rice bran	100–150
	Corn	75–125
	Barley	100–125
Production of brewed products	Beer	1–5
Production of starch and gluten fractions	Grains	2–15
	Rice bran	100–150
	Corn	75–125
	Barley	100–125
Processing of fruits and vegetables
Production of juices	Fruit and vegetable juice	150–300
Processing of plant‐ and fungal‐derived products
Production of protein hydrolysates from plants and fungi	Vegetable protein	100–600
Processing of yeast and yeast products	Yeast biomass	250–2000

Abbreviation: TOS, total organic solids.

^a The name has been harmonised by EFSA in accordance with (EFSA CEP Panel, 2023).

^b The numbers in bold were used for calculation.

^c This Papain is intended to be used only in the production of infant formulae and follow‐on formulae. ³⁶

In the production of cheese, the food enzyme is added to milk during the coagulation step ³⁷ to hydrolyse κ‐casein. ³⁸ After coagulation, the food enzyme–TOS partition into the whey and the curd.

In the production of flavouring preparations from dairy products, the food enzyme is added to cheese before heat treatment ³⁹ to improve the sensory properties of the final foods. The food enzyme–TOS remain in these enzyme‐modified dairy ingredients.

In the production of modified milk proteins, the food enzyme is added to hydrolyse milk proteins ⁴⁰ to improve the solubility and taste. The subsequent downstream processing steps applied, that include an ultrafiltration step, could theoretically remove the food enzyme–TOS from the final food ingredients. However, in the absence of analytical data demonstrating the extent of such removal, ⁴¹ the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS are transferred into the modified milk proteins.

In the processing of eggs and eggs products, the food enzyme is added to treat the whole liquid egg or the egg white ⁴² to improve the sensory and technological properties of these products. The food enzyme–TOS remain in these enzyme‐modified egg products.

In the production of modified meat and fish products, the food enzyme is added to meat and fish ⁴³ to hydrolyse the fibrous proteins for tenderising purposes. ⁴⁴ The food enzyme–TOS remain in the final processed foods.

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. meat, fish, gelatine, collagen) or plant sources (e.g. wheat, corn). ⁴⁵ The food enzyme–TOS remain in these protein hydrolysates.

In the production of baked products, the food enzyme is added to flour during the preparation of the dough ⁴⁶ to hydrolyse gluten proteins, facilitating the handling of the dough. ⁴⁷ The food enzyme–TOS remain in the baked foods.

In the production of cereal‐based products other than baked, the food enzyme is added to a mixture of cereals and water during soaking to produce cooked cereals or stabilised rice bran. ⁴⁸ Papain catalyses the protein hydrolysis on the surface of the cereals to degrade naturally occurring enzymes that could cause rancidity. ⁴⁹ The food enzyme–TOS remain in the final foods.

In the production of brewed products, the food enzyme is added to beer during ageing ⁵⁰ to prevent haze formation. ⁵¹ The food enzyme–TOS remain in the brewed products.

In the production of starch and gluten fractions, the food enzyme is added to raw materials (e.g. grains, rice, corn and barley) before separation of starch and proteins. ⁵² The food enzyme cleaves the peptide bonds in the gluten network, improving the rheology of the dough. Repeated washing steps applied in the downstream processes remove the food enzyme–TOS in the final starch or gluten (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme is added to the raw juices before the filtration step ⁵³ to reduce cloudiness and turbidity. ⁵⁴ The food enzyme–TOS remain in the juices.

In the processing of yeast and yeast products, the food enzyme is added to autolysed yeast ⁵⁵ to enrich the savoury taste of the yeast products, in which the food enzyme–TOS remain.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that the food enzyme may remain in its active form in all food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain, depending on the processing conditions.

3.5.2. Dietary exposure estimation

Following the EFSA Guidance Document on food enzymes (EFSA CEP Panel, 2021), a comparison was made between the chronic exposures:

• dietary exposure to the food enzyme–TOS, resulting from the intended uses as proposed by the applicant (herein referred as ‘FE–TOS’) and

• dietary exposure to a fraction of C. papaya L. unripe fruit comparable to the food enzyme–TOS, resulting from the consumption of raw or cooked unripe C. papaya L. (herein referred to as source material TOS equivalent, ‘SMT–Equivalent’)

3.5.2.1. Estimated dietary exposure to the food enzyme–TOS

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the 12 food manufacturing processes where the food enzyme–TOS remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool ⁵⁶ 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).

Table 3 provides an overview of the derived exposure estimates across all surveys from actual consumers of the relevant food categories. 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 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix B). The highest dietary exposure was estimated to be 20.963 mg TOS/kg bw per day in toddlers at the 95th percentile. As the food categories relevant to the intended uses of this food enzyme are commonly consumed by Europeans, the estimates calculated for survey respondents are the same as those calculated for actual consumers.

TABLE 3.

Summary of the estimated dietary exposure to the food enzyme–TOS in six consumer groups.

Consumer group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Actual consumers of the relevant food categories
Min–max mean (number of surveys)	0.880–2.926 (14)	1.330–8.680 (17)	0.671–4.683 (21)	0.415–3.002 (23)	0.222–1.677 (23)	0.206–1.112 (25)
Min–max 95th percentile (number of surveys)	2.107–8.695 (13)	4.491–20.963 (16)	1.642–13.310 (21)	0.942–8.089 (22)	0.543–5.991 (23)	0.468–4.063 (24)

3.5.2.2. Estimated dietary exposure to the SMT–Equivalent

Unripe papaya (also known as green papaya) is rich of papain, especially in the latex. The food enzyme is obtained from the papaya latex. In Europe, papaya is consumed mainly as ripe fruit, while the consumption of unripe papaya is limited and consumption data are not available. In Southeast Asia, unripe papaya is commonly eaten raw as salad or cooked in soup. ⁵⁷ For the estimation of the SMT–Equivalent, consumption data from actual consumers of green papaya salad or soup in the Philippines ⁵⁸ and Thailand ⁵⁹ were used.

The chronic dietary exposure to the SMT–Equivalent was calculated in two steps: firstly, the intake of unripe papaya was converted to the intake of latex, using a factor of 0.016, ⁶⁰ as provided by the applicant to account for the amount of latex in unripe C. papaya L. Secondly, the intake of latex was converted into a fraction comparable to the food enzyme–TOS, by applying a yield factor (0.03) to take into account the yield of the food enzyme from fresh latex (Section 3.2).

Table 4 provides an overview of the estimated exposure to the SMT–Equivalent for the actual consumers of raw and cooked unripe papaya. The age ranges are presented as provided in the surveys from the Philippines and Thailand. The highest dietary exposure was estimated to be 2.527 mg/kg bw per day in children at the 95th percentile.

TABLE 4.

Summary of estimated dietary exposure to the SMT–Equivalent resulting from eating raw and cooked unripe papaya.

Consumer group		Estimated exposure (mg/kg body weight per day)
		Infants	Toddlers	Children		Adolescents			Adults				The elderly
	EFSA age range	4–11 months	12–35 months	3–9 years		10–17 years			18–64 years				≥ 65 years
Philippines	Age range	6–11 months	1–2 years	3–5 years	6–9 years	10–12 years	13–15 years	16–18 years	19–29 years	30–49 years	50–59 years	60–69 years	≥ 70 years
	Mean	0.503	1.011	0.876	0.828	0.603	0.584	0.587	0.541	0.518	0.584	0.555	0.575
	95th Percentile	0.652	2.432	2.306	2.527	1.867	1.670	2.207	1.297	1.213	1.418	1.306	1.447
Thailand	Age range			3–5.9 years	6–12.9 years		13–17.9 years		18–34.9 years	35–64.9 years			≥ 65 years
	Mean			1.917	1.782		0.973		0.859	0.761			0.707
	95th Percentile	Not available

3.5.2.3. Comparison of the two exposure estimates

The intakes of the SMT–Equivalent by consumers from the Philippines and Thailand (Table 4) are 3–14 folds lower than the dietary exposure to the food enzyme–TOS for European consumers (Table 3).

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 5.

TABLE 5.

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

Sources of uncertainties	Direction of impact
	Exposure to FE–TOS	Exposure to SMT–Equivalent
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)	+	NA
Possible national differences in categorisation and classification of food	+/−	+/−
Use of the consumption data of unripe C. papaya L. from Thailand and Philippines to calculate the exposure to the SMT–Equivalent	NA	+/−
Model assumptions and factors
Selection of broad FoodEx categories to calculate the exposure to FE–TOS	+	NA
Only green papaya salad and soup are included to calculate the intake of SMT–Equivalent	NA	−
Use of recipe fractions to disaggregate FoodEx categories	+/−	NA
Use of technical factors in the exposure model	+/−	NA
Assumption that the food enzyme–TOS are fully transferred in the modified milk proteins	+	NA
Whenever two or more use levels are applied to different raw materials for the same food manufacturing process, the higher use level was used in the calculation.	+	NA
For the production of cereal‐based products other than baked, the food categories chosen for calculation are not only those containing cooked cereals, but also those containing other types of cereal‐based products	+	NA
Exclusion of one process from the exposure estimation: – production of starch and gluten fractions	−	NA
The applied food enzyme yield factor was the mean value	NA	+/−

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

Abbreviations: FE, food enzyme; NA, not applicable; SMT, source material–TOS; TOS, total organic solids.

The exclusion of one food manufacturing process from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimates derived for the food enzyme–TOS.

The estimation of the SMT–Equivalent is based on a realistic scenario, while the estimation of the dietary exposure to the food enzyme–TOS is based on a conservative approach. In particular, assumptions made on the occurrence and use levels of this specific enzyme, have likely led to an overestimation of the dietary exposure to the food enzyme–TOS. According to the Panel, undesirable changes in the sensory properties of some of the final foods or food ingredients are likely, should the maximum use levels proposed by the applicant be used. Considering this overestimation of the exposure to the food enzyme–TOS, in a realistic exposure scenario, both values can be expected to be within the same magnitude.

3.6. Margin of exposure

Since toxicological tests were not required for this food enzyme (see Section 3.4), the margin of exposure was not calculated.

4. CONCLUSIONS

Based on the data provided, the origin of the food enzyme being an edible plant source and the estimated dietary exposure, the Panel concluded that the food enzyme endopeptidase complex (papain) extracted from the latex of the unripe Carica papaya L. does not give rise to safety concerns under the intended conditions of use.

5. REMARK

Anthraquinone was detected in two of the three food enzyme batches tested. The Panel notes that anthraquinone is not permitted for use as a pesticide in the EU. ^{61 , 62}

6. DOCUMENTATION AS PROVIDED TO EFSA

Dossier and additional information can be accessed at https://open.efsa.europa.eu/dossier/FEN‐2023‐14690.

ABBREVIATIONS

ADI

acceptable daily intake

bw

body weight

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

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FEIM

Food Enzyme Intake Model

FEZ

EFSA Panel on Food Enzymes

GMO

genetically modified organism

IgE

Immunoglobulin E

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LOQ

limit of quantification

OECD

Organisation for Economic Cooperation and Development

RM

raw material

TOS

total organic solids

TU

Tyrosine Unit

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00441

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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, Henk Van Loveren, Laurence Vernis, Holger Zorn.

Supporting information

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

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

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, obtained considering the actual consumers of the relevant food categories

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 16 weeks on up to and including 11 months of age	Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Poland, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, Portugal, Republic of North Macedonia*, Serbia*, Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, 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*, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, 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, Poland, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Poland, 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 purpose.

^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. , de Marzo Solano, M. , Van Loveren, H. , Vernis, L. , Fraguas, C. F. , Cavanna, D. , & Liu, Y. (2026). Safety evaluation of the food enzyme papain, a cysteine endopeptidase complex from the latex of Carica papaya L. EFSA Journal, 24(3), e9950. 10.2903/j.efsa.2026.9950