Safety of pea and rice protein fermented by Shiitake (Lentinula edodes) mycelia as a Novel food pursuant to Regulation (EU) 2015/2283
2022; Wiley; Volume: 20; Issue: 4 Linguagem: Inglês
10.2903/j.efsa.2022.7205
ISSN1831-4732
AutoresDominique Turck, Torsten Bohn, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch‐Ernst, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska, Carmen Peláez, Kristina Pentieva, Alfonso Siani, Frank Thiès, Sophia Tsabouri, Marco Vinceti, Francesco Cubadda, Tim Frenzel, Marina Heinonen, Rosangela Marchelli, Monika Neuhäuser‐Berthold, Morten Poulsen, Miguel Prieto Maradona, Josef Rudolf Schlatter, Henk van Loveren, Ruth Roldán‐Torres, Helle Katrine Knutsen,
Tópico(s)Food Allergy and Anaphylaxis Research
ResumoEFSA JournalVolume 20, Issue 4 e07205 Scientific OpinionOpen Access Safety of pea and rice protein fermented by Shiitake (Lentinula edodes) mycelia as a Novel food pursuant to Regulation (EU) 2015/2283 EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Corresponding Author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) nif@efsa.europa.eu Correspondence:nif@efsa.europa.euSearch for more papers by this authorDominique Turck, Dominique TurckSearch for more papers by this authorTorsten Bohn, Torsten BohnSearch for more papers by this authorJacqueline Castenmiller, Jacqueline CastenmillerSearch for more papers by this authorStefaan De Henauw, Stefaan De HenauwSearch for more papers by this authorKaren Ildico Hirsch-Ernst, Karen Ildico Hirsch-ErnstSearch for more papers by this authorAlexandre Maciuk, Alexandre MaciukSearch for more papers by this authorInge Mangelsdorf, Inge MangelsdorfSearch for more papers by this authorHarry J McArdle, Harry J McArdleSearch for more papers by this authorAndroniki Naska, Androniki NaskaSearch for more papers by this authorCarmen Pelaez, Carmen PelaezSearch for more papers by this authorKristina Pentieva, Kristina PentievaSearch for more papers by this authorAlfonso Siani, Alfonso SianiSearch for more papers by this authorFrank Thies, Frank ThiesSearch for more papers by this authorSophia Tsabouri, Sophia TsabouriSearch for more papers by this authorMarco Vinceti, Marco VincetiSearch for more papers by this authorFrancesco Cubadda, Francesco CubaddaSearch for more papers by this authorThomas Frenzel, Thomas FrenzelSearch for more papers by this authorMarina Heinonen, Marina HeinonenSearch for more papers by this authorRosangela Marchelli, Rosangela MarchelliSearch for more papers by this authorMonika Neuhäuser-Berthold, Monika Neuhäuser-BertholdSearch for more papers by this authorMorten Poulsen, Morten PoulsenSearch for more papers by this authorMiguel Prieto Maradona, Miguel Prieto MaradonaSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorHenk van Loveren, Henk van LoverenSearch for more papers by this authorRuth Roldán-Torres, Ruth Roldán-TorresSearch for more papers by this authorHelle Katrine Knutsen, Helle Katrine KnutsenSearch for more papers by this author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Corresponding Author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) nif@efsa.europa.eu Correspondence:nif@efsa.europa.euSearch for more papers by this authorDominique Turck, Dominique TurckSearch for more papers by this authorTorsten Bohn, Torsten BohnSearch for more papers by this authorJacqueline Castenmiller, Jacqueline CastenmillerSearch for more papers by this authorStefaan De Henauw, Stefaan De HenauwSearch for more papers by this authorKaren Ildico Hirsch-Ernst, Karen Ildico Hirsch-ErnstSearch for more papers by this authorAlexandre Maciuk, Alexandre MaciukSearch for more papers by this authorInge Mangelsdorf, Inge MangelsdorfSearch for more papers by this authorHarry J McArdle, Harry J McArdleSearch for more papers by this authorAndroniki Naska, Androniki NaskaSearch for more papers by this authorCarmen Pelaez, Carmen PelaezSearch for more papers by this authorKristina Pentieva, Kristina PentievaSearch for more papers by this authorAlfonso Siani, Alfonso SianiSearch for more papers by this authorFrank Thies, Frank ThiesSearch for more papers by this authorSophia Tsabouri, Sophia TsabouriSearch for more papers by this authorMarco Vinceti, Marco VincetiSearch for more papers by this authorFrancesco Cubadda, Francesco CubaddaSearch for more papers by this authorThomas Frenzel, Thomas FrenzelSearch for more papers by this authorMarina Heinonen, Marina HeinonenSearch for more papers by this authorRosangela Marchelli, Rosangela MarchelliSearch for more papers by this authorMonika Neuhäuser-Berthold, Monika Neuhäuser-BertholdSearch for more papers by this authorMorten Poulsen, Morten PoulsenSearch for more papers by this authorMiguel Prieto Maradona, Miguel Prieto MaradonaSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorHenk van Loveren, Henk van LoverenSearch for more papers by this authorRuth Roldán-Torres, Ruth Roldán-TorresSearch for more papers by this authorHelle Katrine Knutsen, Helle Katrine KnutsenSearch for more papers by this author First published: 06 April 2022 https://doi.org/10.2903/j.efsa.2022.7205 Requestor: European Commission Question number: EFSA-Q-2020-00040 Panel members: Dominique Turck, Torsten Bohn, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch-Ernst, Helle Katrine Knutsen, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska, Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri and Marco Vinceti. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgments: The Panel wishes to thank the EFSA Staff Petra Gergelova and EFSA trainee and interim staff Gabriela Precup for the support and contributions provided to the preparation of this scientific output. Adopted: 28 February 2022 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on pea and rice protein fermented by Shiitake mushroom (Lentinula edodes) mycelia as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is a mixture of fermented pea and rice protein concentrates (65% and 35%, respectively). The NF is proposed to be used as a food ingredient in specific food categories. The target population is the general population. The major constituent of this NF is protein (≥ 75% dry weight), which is well digestible and provides sufficient amounts of essential amino acids. Although a tolerable upper intake level (UL) has not been derived for protein, the protein intake from the NF may nevertheless further contribute to an already high dietary protein intake in Europe. The Panel notes that the cumulative exposure to the nutrients and contaminants analysed does not raise concern. The reported values for the levels of antinutritional factors in the NF are comparable to those in other foodstuffs. The Panel considers that taking into account the composition of the NF and the proposed conditions of use, consumption of the NF is not nutritionally disadvantageous. No toxicological studies with the NF were provided by the applicant; however, the Panel considers that no toxicological studies are required for this NF. The NF has the potential capacity to sensitise individuals or to induce allergic reactions in individuals allergic to pea, rice and Shiitake mushroom. However, this risk is expected not to be higher than that resulting from the normal consumption of pea, rice or the fruiting body of the Shiitake mushroom. The Panel considers that the NF is safe at the proposed conditions of use. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor On 12 December 2019, the company MycoTechnology, Inc., submitted a request to the Commission in accordance with Article 10 of Regulation (EU) No 2015/2283 to place on the EU market pea and rice protein fermented by Shiitake mushroom (Lentinula edodes) mycelia as a novel food. Pea and rice protein fermented by Shiitake mushroom (Lentinula edodes) mycelia is intended to be used in a number of foods. The applicant has requested data protection according to the provisions of Article 26 of Regulation (EU) 2015/2283. In accordance with Article 10(3) of Regulation (EU) 2015/2283, the European Commission asks the European Food Safety Authority to provide a scientific opinion on pea and rice protein fermented by Shiitake mushroom (Lentinula edodes) mycelia as a novel food. 2 Data and methodologies 2.1 Data The safety assessment of this NF is based on data supplied in the application and information submitted by the applicant following EFSA's requests for supplementary information. During the assessment, the Panel identified additional data which were not included in the application. Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in the Commission Implementing Regulation (EU) 2017/246911 Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientific requirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. OJ L 351, 30.12.2017, pp. 64-71.. A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of an NF application (EFSA NDA Panel, 2016). As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data (both in favour and not in favour) that are pertinent to the safety of the NF. This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The data requested by the applicant to be protected comprise the NF production process and information concerning the composition, recipe and analysis of the novel food. 2.2 Methodologies The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2016) and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of the Commission Implementing Regulation (EU) 2017/2469. This assessment concerns only the risks that might be associated with consumption of the NF under the proposed conditions of use and is not an assessment of the efficacy of the NF with regard to any claimed benefit. 3 Assessment 3.1 Introduction The NF which is the subject of the application is pea and rice protein fermented by Shiitake (Lentinula edodes) mycelia. The NF falls under category (ii), i.e. food consisting of, isolated from or produced from microorganisms, fungi or algae according to Article 3(2)(a) of Regulation (EU) No 2015/2283. The NF is produced by the fermentation with Shiitake mycelia of pea and rice protein and consists of ≥ 75% protein on a dry basis and an estimated level of Shiitake mycelia biomass of < 0.1 weight (wt) %. The NF is proposed to be used as food ingredient in specific food categories. The target population proposed by the applicant is the general population. 3.2 Identity of the NF The NF is the heat-treated mixture of pea and rice protein concentrates (65% pea protein and 35% rice protein) obtained after fermentation by Shiitake mycelia (Lentinula edodes), in a powder form. The fermentation is performed to improve organoleptic properties of the pea and rice protein starting materials. The fungus Lentinula edodes (basidiomycete family) is listed in the Index fungorum,22 http://www.indexfungorum.org/Names/Names.asp and it is commonly known by its Japanese name 'Shiitake'. Under the conditions of use in aqueous culture, the mushrooms Lentinula edodes grow as vegetative form. According to the applicant, the organism used in the production process of the NF is Lentinula edodes vegetative form (also identified as Shiitake mycelia). The strain of Lentinula edodes used to produce the NF was originally obtained from Pennsylvania State University, ID No. WC 1008 (https://plantpath.psu.edu/facilities/mushroom/cultures/spawn). The taxonomic identification of the strain as Lentinula edodes was established by comparing the highly conserved Internal Transcribed Spacer (ITS) region of L. edodes to the genetic sequences in the NCBI public database (GenBank: AB366150.1) and confirmed as 100% identity to L. edodes. 3.3 Production process According to the information provided, the NF is produced following good manufacturing practice (GMP) and hazard analysis critical control points (HACCP) principles. Flow charts of the manufacturing process with detailed description of the methods involved at each step, a complete list of the culture media constituents and processing aids and the respective certificates of analysis were provided by the applicant. The starting materials include pea protein concentrates and rice protein concentrates. Further information on the origin, specifications, certificates of analysis and process flow-charts from the protein concentrates suppliers were provided by the applicant. Additional starting materials include also processing aids (e.g. maltodextrin, carrot powder, anti-foam agent) and the mycelium from Shiitake. The production process refers to a closed system that includes subsequent fermentation steps. In short, an inoculum of L. edodes (Shiitake mycelia) is made by successive fermentation steps in liquid media at a certain temperature and pH in order to prepare for the inoculation of the main fermenter. The main fermentation is conducted at specific conditions, where the L. edodes biomass is mixed with 65% pea protein and 35% rice protein concentrates, while slowly stirring for up to 40 h. The growth of the shiitake biomass between fermentations is quantified by pH monitoring and visual appearance. The production process for the fermented pea and rice protein concentrates by L. edodes consists of fermentation followed by heat treatment steps and spray-drying. The powder is packaged into poly-lined kraft bags, heat-sealed and stored at ambient conditions. The Panel considers that the production process is sufficiently described and does not raise safety concerns. 3.4 Compositional data The NF is a heat-treated mixture of pea and rice protein concentrates fermented by L. edodes, and the major constituents are proteins ≥ 75% (dry weight), produced from a mixture which consists of a combination of 65% pea protein concentrate and 35% rice protein concentrate and remaining processing aids. The NF also contains carbohydrates, dietary fibre, fat, ash and water. In order to confirm that the production process is reproducible and adequate to produce the NF on a commercial scale, the applicant provided analytical information for five batches of the NF (Table 1). Table 1. Batch-to-batch analysis of the NF Parameter (unit) Batch number Method of analysis #1 #2 #3 #4 #5 Crude protein (% DW)** < 4 IU/100 g Converted from International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the European Food Safety Authority Technical Report on Dietary Reference Values for nutrients (EFSA, 2017). (N-protein conversion factor 6.25) 78.11 78.48 78.47 79.71 78.45 Combustion-Dumas; AOAC 990.03; AOAC 992.15 Moisture (%) 2.70 3.00 1.80 2.70 2.80 Gravimetric method AOAC 925.09; 985.14 Vacuum Oven Ash (% DW) 3.25 3.08 3.44 6.21 6.33 Gravimetric method AOAC 942.05 Total fat (% DW) 8.19 5.33 9.68 6.62 6.84 GC-FID AOAC 996.06 internal adaption Carbohydrates (%DW) 10.45 13.13 8.40 7.46 8.38 By calculation Total dietary fibre (%) 12.1 13 12.5 11.6 12.1 AOAC 991.43 Mod. Total sugars (%) 1.06 1.02 0.65 0.94 0.62 AOAC 982.14, Water activity (aw) 0.120 0.462 0.103 0.127 N.A. AOAC 978.18 mod. Potassium (%) 0.06 0.07 0.06 0.06 0.06 ICP, AOAC 984.27 mod.927.02 Sodium (%) 0.66 0.63 0.68 0.69 0.58 Calcium (%) 0.03 0.04 0.03 0.03 0.03 Iron (%) 0.02 0.01 0.02 0.01 0.02 Vitamin D2 (µg/100 g)** < 4 IU/100 g Converted from International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the European Food Safety Authority Technical Report on Dietary Reference Values for nutrients (EFSA, 2017). < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 LC-MS/MS Vitamin D3 ** < 4 IU/100 g Converted from International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the European Food Safety Authority Technical Report on Dietary Reference Values for nutrients (EFSA, 2017). (µg/100 g) < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 DW: Dry weight; AOAC: Association of Official Analytical Chemists; GC-FID: Gas Chromatography with Flame Ionisation Detection; LC-MS/MS: Liquid chromatography with tandem mass spectrometry; ICP: Inductively coupled plasma. * < 4 IU/100 g Converted from International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the European Food Safety Authority Technical Report on Dietary Reference Values for nutrients (EFSA, 2017). The applicant also provided a detailed description of the amino acid composition (See Section '3.9 Nutritional information'). The appearance of the batches was that of a light-tan powder, as confirmed by colorimetric analysis. The level of contaminants was also monitored in the NF. Analytical information on chemical and microbiological parameters for five batches of the NF was also provided (Table 2). Table 2. Batch-to-batch analysis of chemical contaminants and microbiological parameters in the NF Parameter (unit) Batch number Method of analysis #1 #2 #3 #4 #5 Mycotoxins Aflatoxin B1 (µg/kg) < 0.5 < 0.5 < 0.5 0.619 < 0.5 AOAC 999.07 Modified (UHPLC-MS/MS) Aflatoxin B2 (µg/kg) < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 Aflatoxin G1 (µg/kg) < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 Aflatoxin G2 (µg/kg) < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 Aflatoxin total (B1 + B2 + G1 + G2) (µg/kg) < 2 < 2 < 2 2.119 < 2 Deoxynivalenol (µg/kg) < 100 < 100 < 100 < 100 < 100 Internal Method (UHPLC-MS/MS) Fumonisin B1 (µg/kg) < 25 < 25 < 25 < 25 < 25 Anal. Bioanal. Chem (2012) 402:2675-2686 (UHPLC-MS/MS) Fumonisin B2 (µg/kg) < 25 < 25 < 25 < 25 < 25 HT-2 toxin (µg/kg) < 100 < 100 < 100 < 100 < 100 Ochratoxin A (µg/kg) 2.17 < 1 < 1 < 1 < 1 T-2 toxin (µg/kg) < 10 < 10 < 10 < 10 < 10 Zearalenone (µg/kg) < 30 < 30 < 30 < 30 < 30 Heavy metals Arsenic (mg/kg) 0.02 0.02 0.02 0.03 0.02 ICP-MS1 Lead (mg/kg) 0.04 0.04 0.04 0.02 0.04 Cadmium (mg/kg) 0.03 0.02 0.03 0.03 0.02 Mercury (mg/kg) 0.01 < 0.005 < 0.005 0.01 0.01 Microbiological Aerobic Plate Count (CFU/g) < 10 < 10 < 10 < 10 290 AOAC 966.23 Yeast (CFU/g) < 10 < 10 < 10 < 10 < 10 FDA-BAM, Ch 18; AOAC 997.02 Mould (CFU/g) < 10 < 10 200 < 10 < 10 FDA-BAM, Ch 18; AOAC 997.02 Coliforms (CFU/g) < 10 < 10 < 10 < 10 < 10 AOAC 991.14 E. coli (CFU/g) < 10 < 10 < 10 < 10 < 10 AOAC 991.14 S. aureus (CFU/g) < 10 < 10 < 10 < 10 N.A. AOAC 2003.07 B. cereus (CFU/g) / 20 10 < 10 N.A. FDA BAM chapter 14 Salmonella spp. (in 25 g) Not detected Not detected Not detected Not detected Not detected AOAC 2016.01 Listeria spp. (in 25 g) Not detected Not detected Not detected Not detected Not detected AOAC 2004.06 AOAC 2016.07 AOAC: Association of Official Analytical Chemists; FDA BAM: FDA's Bacteriological Analytical Manual; N.A: Not available; /: not provided; ICP-MS: inductively coupled plasma-mass-spectrometry; UHPLC-MS/MS: Ultra-high-performance liquid chromatography tandem mass spectrometry; 1: Method reference: J. AOAC (2007) v. 90: 844-856 (modified). Pesticides residues were below the limit of detection and glyphosate was reported at levels between 0.01 and 0.05 mg/kg which is under the lowest MRL (0.05 mg/kg). Information was provided on the accreditation of the laboratories that conducted the analyses presented in the application. The Panel considers that the information provided on the composition is sufficient for characterising the NF. 3.4.1 Stability The applicant performed two separate stability studies with two independently produced batches of the NF. The tests were carried out under accelerated conditions at 40°C and at 75% RH for a period of 24–32 weeks (168–224 days). In the first study, the samples of the NF were taken every 2 weeks for 168 days and were analysed for pH, colour, moisture, protein and nitrogen solubility at a pH ranging from 3 to 7. In the second study, samples of a second batch of the NF were taken on days 0, 112, 166 and 224, and analysed for pH, colour, moisture, protein, proximates, amino acid profile, microbiological contaminants and nitrogen solubility at pH 7. The NF was found to be stable under accelerated storage conditions for 168–224 days. The applicant provided certificates of analysis of both studies as well as laboratory accreditations. From the results of the stability studies, the applicant proposed a shelf-life for the NF of 2 years at 25°C. Upon EFSA's request to prove the shelf-life of 2 years, the applicant performed tests under accelerated conditions at 40°C and at 75% RH and used a lot which was 659 days old as starting material to simulate a total real-time storage of 3.5 years. The samples were taken at baseline (5 days), 9.6 weeks, 18.1 weeks, 27.1 weeks and were analysed for proximates, colour, pH, lipid oxidation (thiobarbituric acid, hexanal), microbiological parameters (APC, mould, yeast, E. coli, Coliforms, S. aureus, B. cereus), sensorial analysis (Table 3). Table 3. Stability of the NF Parameters NF Time points (days) Baseline 5 days Baseline 659 Days Baseline + Accelerated for 8 Weeks (877 days) Baseline + Accelerated for 16 Weeks (1072 days) Baseline + Accelerated for 24 Weeks (1277 days) Microbiological (cfu/g) Aerobic Plate Count (APC) < 10 < 10 < 10 < 10 < 10 Yeasts < 10 < 10 < 10 < 10 < 10 Moulds < 10 < 10 < 10 < 10 < 10 S. aureus N.A. N.A. < 10 < 10 < 10 Coliforms < 10 < 10 < 10 < 10 < 10 E. coli < 10 < 10 < 10 < 10 < 10 L. monocytogenes (in 25 g) ND ND ND ND ND B. cereus N.A. N.A. < 10 < 10 < 10 Salmonella (in 25 g) ND ND ND ND ND Physical/Chemical parameters Moisture (% m/m) 2.59 2.80 4.20 4.69 5.50 pH 6.10 6.03 5.96 5.82 5.78 Protein (measured) % 76.81 76.94 76.19 73.63 75.19 Carbohydrates (%) N.A. 4.07 8.45 4.69 7.29 Fat (%) N.A. 8.59 6.86 9.00 8.52 Ash (%) N.A. 7.60 4.30 8.08 3.83 Calories (kcal/100 g) N.A. 401 400 394 404 TBA – Rancidity (mg/kg) N.A. 2.46 N.A. 2.26 N.A. Hexanal (ppm) N.A. 38.8 N.A. 19.8 N.A. N.A: Not analysed – not provided, ND: Non-detected. The applicant explained that for calculating the simulated age of the product, they used a factor of 3.25 based on the principle of the Arrhenius equation (Arrhenius Equation, 2021), to the days the product was stored at accelerated conditions. The Panel considers that the data provided sufficient information with respect to the stability of the NF for 2 years. Upon EFSA's request of information on stability in intended food matrix, the applicant provided a stability study of the NF as an ingredient in plant-based burger meat analogue, plant-based whole milk analogue (unflavoured) and chocolate chip muffin. The burger meat and whole milk analogues with the NF were refrigerated at 5°C (% RH not recorded but expected to be standard for commercial refrigerated coolers) whereas the chocolate chip muffin with the NF was stored at (25°C/60%RH). Samples were collected at baseline (0 days), 5/7 days and 10 days and analysed for the following: visual appearance, acrylamide, proximates (carbohydrates, protein content, fatty acids profile, moisture, ash), heavy metals, microbiological parameters and amino acid concentrations and showed no changes. Microbiological stability in the matrix was also assessed (milk analogue, plant-based burger and baked chocolate chip muffins). The results showed an increase in microbiological parameters compatible with microbial growth during storage of highly perishable food matrices. The Panel notes that the NF itself has very low total microbial counts, and low water activity and hence, the microbiological levels in the representative foods are more related to the quality level of the food itself (matrix) rather than that of the NF. Provided that the specifications are met also at the end of shelf-life and that products containing the NF are compliant with respective legislative limits, the stability data do not raise safety concerns. 3.5 Specifications The specifications of the NF are indicated in Table 4. Table 4. Specifications of the NF Description: powder from pea and rice protein fermented by Shiitake (Lentinula edodes) mycelia Parameter Specification Protein, % dry weight (DW) basis ≥ 75.0% Moisture ≤ 7% Total Fat ≤ 10% Ash ≤ 10% Carbohydrates (by calculation) ≤ 15% Mycotoxins Aflatoxin B1 < 1 µg/kg Aflatoxin B2 < 1 µg/kg Aflatoxin G1 < 1 µg/kg Aflatoxin G2 < 1 µg/kg Aflatoxin total (B1 + B2 + G1 + G2) < 3 µg/kg Heavy metals Arsenic < 0.1 µg/g Lead < 0.3 µg/g Cadmium < 0.1 µg/g Mercury < 0.1 µg/g Microbiological TAMC < 1,000 CFU/g TYMC < 100 CFU/g Coliforms < 10 CFU/g Salmonella spp. Not detected in 25 g E. coli < 10 CFU/g Listeria monocytogenes Not detected in 25 g TAMC: total aerobic microbial count; TYMC: total yeast and mould count; CFU: colony forming units. The applicant proposes a specification limit for TAMC as < 5,000 CFU/g. The Panel notes that considering the NF production process (thermal processing) and compositional analyses of five batches (below 10 CFU/g with only one batch showing a maximum value of 290 CFU/g), a lower specification limit could be met. The Panel also notes that TAMC is considered an indicator of hygiene and that this parameter could affect the safety of the NF. Therefore, the Panel proposes a criterion for this parameter of ≤ 1,000 CFU/g. The Panel considers that the information provided on the specifications of the NF is sufficient and does not raise safety concerns. 3.6 History of use of the NF and/or of its source 3.6.1 History of use of the source The NF is composed of pea and rice protein fermented with the mycelium of Shiitake mushroom (L. edodes). Pea, rice and its protein concentrates are consumed in the EU. Pea (Pisum sativum) is a legume in the family Fabaceae, grown from its edible seeds. Pea is a crop cultivated extensively around the world and is rich in protein (23–31%). Pea proteins have a good capacity for fat/water binding, allowing adequate texture, emulsification and gelation and are being proposed as a possible alternative protein in different food products (Bessada et al., 2019). Rice is the seed of the grass species Oryza sativa (plant family Poaceae (Gramineae)) which has its origin in Asia. Rice is a cereal widely consumed in the EU and around the world. Rice protein content is 6–10% depending on cultivation conditions and rice variety. According to the applicant, the starting materials rice and pea protein concentrates used to produce the NF contain > 80% protein and have been recognised by the U.S. FDA as GRAS for use in food (GRN 60933 https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=609; GRN 60844 https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=608). The mycelium of the fermentation organism (L. edodes) used for the production of the NF was not used for food production within the Union prior to 15 May 1997, although the fruiting body of L. edodes has been consumed all over Europe long before. L. edodes is cultivated and consumed in many Asian countries, as well as being dried and exported to many countries around the world. It is a feature of many Asian cuisines including Chinese, Japanese, Korean and Thai in both fresh and dried forms (EFSA NDA Panel, 2010). 3.6.2 History of use of the NF According to the applicant, the NF is authorised as a food ingredient in the United States.55 https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=794 3.7 Proposed uses and use levels and anticipated intake 3.7.1 Target population The target population proposed by the applicant is the general population. 3.7.2 Proposed uses and use levels The NF is proposed by the applicant to be used as an ingredient in a number of food products. These food products are defined using the FoodEx2 hierarchy (EFSA, 2015), and the maximum use levels are reported in Table 5. Table 5. Food categories and maximum use levels intended by the applicant Proposed food category & use FoodEx2 level FoodEx2 code Food category (Foodex2) Max use level (g NF/100 g) Baked goods- bread & rolls 4 A005R Gluten-free bread 5 3 A005Z Extruded, pressed or puffed bread 5 3 A04KZ Unleavened or flat bread and similar 5 3 A0BY0 Leavened bread and similar 5 4 A007A Breadcrumbs 5 4 A007B Croutons(a(a) Recipe – only the proportion of food
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