Scientific Opinion on the safety and efficacy of Pediococcus pentosaceus (DSM 12834) as a silage additive for all species 1

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Pediococcus pentosaceus (DSM 12834) as a silage additive for all species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Pediococcus pentosaceus is a technological additive intended to improve the ensiling process at a proposed dose of 1.0 x 10 8 CFU/kg fresh material. The bacterial species P. pentosaceus is considered by the European Food Safety Authority to be suitable for the Qualified Presumption of Safety approach. As the identity of the strain has been clearly established and as no antibiotic resistance was detected, the use of the strain in the production of silage is considered safe for livestock species, consumers of products from animals fed the treated silage and for the environment. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that considering the nature of the alternative food grade excipients, different results would be obtained for other formulations containing P. pentosaceus DSM However, due to the lack of information and its proteinaceous nature, the active agent has the potential to be a skin/respiratory sensitizer. Three studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of grass forage of differing water-soluble carbohydrate content and representing material easy to ensile, moderately difficult to ensile and difficult to ensile. In each case, replicate silos containing treated forage were compared to identical silos containing the same untreated forage at 20 ºC. The additive consistently showed the potential to improve the production of silage from all forages by a reduction of ph and increased preservation of dry matter. European Food Safety Authority, 2011 KEY WORDS Technological additive, silage additive, Pediococcus pentosaceus, QPS, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q , adopted on 6 September Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino Lopez Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Silage for the preparatory work on this scientific opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the safety and efficacy of Pediococcus pentosaceus (DSM 12834) as a silage additive for all species. EFSA Journal 2011;9(9):2369. [11 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2011

2 SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety for the target animals, consumer, user and for the environment and on the efficacy of a product based on a specific strain of Pediococcus pentosaceus, when used as a technological additive intended to improve the ensiling process at a proposed dose of 1.0 x 10 8 CFU/kg fresh material. The bacterial species P. pentosaceus is considered by EFSA to be suitable for the Qualified Presumption of Safety approach. Therefore, the strain does not require any specific demonstration of safety other than confirming the absence of any determinants of resistance to antibiotics of human and veterinary clinical significance. As the identity of the strain has been clearly established and as no antibiotic resistance was detected, the use of the strain in the production of silage is considered safe for livestock species, consumers of products from animals fed the treated silage and for the environment. Once an active agent has been authorised as a silage additive, different formulations can be placed on the market with reference to that authorisation. The applicant listed several cryoprotectants and carriers which would allow multiple formulations of the additive to be produced and consequently, not all forms can be directly tested for user safety. However, for assessing the safety for the user of the additive, the active agent is the principal concern provided that other components do not introduce concerns. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that considering the nature of the alternative food grade excipients, different results would be obtained for other formulations containing P. pentosaceus DSM Given the lack of information and its proteinaceous nature, the active agent has the potential to be a skin/respiratory sensitizer. Three studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of grass forage of differing water-soluble carbohydrate content and representing material easy to ensile, moderately difficult to ensile and difficult to ensile. In each case, replicate silos containing treated forage were compared to identical silos containing the same untreated forage at 20 ºC. The additive consistently showed the potential to improve the production of silage from all forages by a reduction of ph and increased preservation of dry matter. 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Identity and properties of the active agent Production and characteristics of the additive Stability Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)7 3. Safety Efficacy... 8 Conclusions... 9 Documentation provided to EFSA... 9 References Appendix

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular Article 10(2)/(7) of that Regulation specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, within a maximum of seven years after the entry into force of this Regulation. The European Commission received a request from the company Lactosan GmbH&Co.KG 5 for reevaluation of the product Pediococcus pentosaceus DSM 12834, to be used as a feed additive for all animal species (category: technological additive; functional group: silage additive) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 10(2)/(7) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 1 June This product was included in the European Union Register of Feed Additives following the provisions of Article 10(1) of Regulation (EC) No 1831/2003. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and the efficacy of the product Pediococcus pentosaceus DSM 12834, when used under the conditions described in Table 1. 4 OJ L 268, , p.29 5 Lactosan GmbH&Co.KG. Industriestrasse West 5, 8605 Kapfenberg, Austria 6 EFSA Dossier reference: FAD

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Pediococcus pentosaceus DSM Registration number/ec No/No (if appropriate) Category(ies) of additive Functional group(s) of additive - Technological Silage additive Composition, description Pediococcus pentosaceus DSM Activity: min. 4 x10 11 CFU/g Description Chemical Purity criteria formula (if appropriate) Impurities: Fungi < 100 CFU/g Clostridia < 10 CFU/g Enterobacteria <10 CFU/g Salmonella not detectable in 25 g Method of analysis (if appropriate) Quantification of lactic acid bacteria (according ISO 15786:2009) Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Not applicable Not applicable Species or category of animal All species and animal categories Maximum Age Conditions of use Minimum content Maximum content mg or Units of activity or CFU kg -1 of complete feedingstuffs (select what applicable) The min. dose is CFU/g fresh matter, corresponding to 0.2 g of the additive Pediococcus pentosaceus DSM per ton forage. Withdrawal period (if appropriate) Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) Other provisions and additional requirements for the labelling The directions for use must indicate storage temperature, shelf-life Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues 5

6 ASSESSMENT 1. Introduction Six genera of lactic acid producing bacteria are commonly associated with forage species and collectively contribute to the natural ensiling process. The present additive is based on a preparation of a single strain of one of those six genera, Pediococcus pentosaceus, and is intended to be added to forages to promote ensiling (technological additive, functional group: silage additive) for the eventual use of the silage in any animal species. The species P. pentosaceus is considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 2007, 2010). This approach requires the identity of the strain to be conclusively established and evidence that the strain does not show acquired resistance to antibiotics of human and veterinary importance. 2. Characterisation 2.1. Identity and properties of the active agent The strain of P. pentosaceus was isolated from silage and is deposited with the Deutsche Sammlung von Mikroorganisms und Zellkulturen (DSMZ) with the accession number DSM It has not been genetically modified. Strain identity was established by its phenotypic properties and by partial 16S rrna gene sequence which by comparison with sequences recorded in databases was unambiguously identified as P. pentosaceus. Strain-specific detection is based on the use of pulsed field gel electrophoresis after cleavage with a number of restriction enzymes used individually (ApaI, SfiI, and SmaI). Genetic stability was established using amplification products of a finger-printing method based on random amplification of polymorphic DNA polymerase chain reaction (RAPD- PCR). Using this method the master culture is routinely compared with working cultures used to inoculate fermentation batches. No differences in the resultant patterns have been observed to date. The strain was tested for antibiotic susceptibility using two-fold broth dilutions. The battery of antibiotics tested was that recommended by EFSA (EFSA, 2008) excluding vancomycin which is not required for this species. As all minimum inhibitory concentration values for the P. pentosaceus strain were equal to or fell below the corresponding breakpoints defined by the FEEDAP Panel no further investigation is required Production and characteristics of the additive The active agent is grown in a sterilised medium typical of those used for lactic acid bacteria and then separated from the growth medium by centrifugation. Cryoprotectants are added and the cell mix is freeze-dried and ground. The ground powder is then blended with sufficient carrier to meet the minimum specified concentration of 5 x CFU/g additive. The resultant additive consists of % cells and solids from the fermentation and % carriers. Material safety data sheets are provided for all cryoprotectants (ascorbic acid, lactose, mannitol, monosodium glutamate, sodium citrate or whey powder) and carrier materials (glucose. maltodextrin or whey powder) and all are of food grade and do not introduce safety concerns. 9 Data on five production batches showed that the minimum specification was exceeded in all cases (mean 6.4 x CFU/g additive). 10 A single batch of the additive (excipients unknown) was examined for particle size distribution by laser diffraction and for dusting potential using a Heubach dustometer. The mean particle size was ~160 µm with approximately 10 % by weight of the additive consisting of particles with diameters 7 Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annexes II.3-4 to II Technical dossier/section II/Annex II.1-2 6

7 below 50 µm and ~2 % below 5 µm. 11 However, the dusting potential of g/m 3 is considered low. 12 The additive is routinely monitored for microbial contamination at various points in the manufacturing process and in the final product. Limits are set for Enterobacteriaceae and yeasts and filamentous fungi (<10 3 CFU/g additive), Escherichia coli (<10 CFU/g additive) and Salmonella spp. (absence in 25 g additive). Data from five batches confirmed compliance with the value for yeasts and fungi and Enterobacteriaceae 13 but absence of Salmonella and E. coli was shown only for a single batch. 14 Given the nature of the fermentation medium and the food grade excipients, the probability of contamination with heavy metals or mycotoxins is considered to be low and consequently not included in routine monitoring. One batch of the additive was, however, sent for analysis to confirm this position. Aflatoxins B 1, B 2, G 1, and G 2, zearalenone and deoxynivalenol, the metals Pb, Hg and Cd and arsenic could not be detected Stability The additive is described as hygroscopic and must be stored in packaging which protects against moisture. When stored in the original packaging, although losses were recorded, the additive met the minimum specification after two months storage at 40 ºC, one year at 25 ºC and two years at 4 ºC. 16 This data is based on three batches of the additive. Although the precise formulation of the additive used for these studies was not given, the stability data is likely to apply to any combination of the excipients listed in Section 2.2. As the additive is intended to be distributed by the spraying of an aqueous suspension, the short-term stability in water was measured. 17 This showed that bacterial numbers were maintained for at least two days at 20 ºC and seven days if refrigerated (4 ºC) Conditions of use The additive is intended for use with easy to ensile forage (> 3 % soluble carbohydrates in fresh material), moderately difficult to ensile forage (1.5 % - 3 % soluble carbohydrates in fresh material) and difficult to ensile forage (< 1.5 % soluble carbohydrates in fresh material) at ensiling at a proposed minimum dose of 1.0 x 10 8 CFU/kg fresh materials Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report as it relates to the methods used for the control of the active agent in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 3. Safety The species, P. pentosaceus, is considered by EFSA to be suitable for the QPS approach to safety. Therefore, the strain does not require any specific demonstration of safety other than confirming the absence of any determinants of resistance to antibiotics of human and veterinary clinical significance. In the view of the FEEDAP Panel, the antibiotic resistance qualification has been met and the identity of the production strain established. Accordingly, no further assessment of safety for the target species, consumers of products from animals fed treated silage or the environment is required. Safety for the user is considered bellow. 11 Technical dossier/section III/Annex III Technical dossier/section III/Annex III Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II.4-2 7

8 Dermal and eye irritation studies were made in rabbits following the relevant OECD protocols. 18 No cutaneous reactions and only mild and reversible effects on the conjunctiva were observed. Consequently, the form of the additive tested can be considered as non-irritant to skin and eyes. Skin sensitisation was not tested but its absence was assumed by the applicant as no adverse effects have been reported for workers exposed to the product in the manufacturing plant. Although users at the farm level are exposed to the additive only for a short period of time when preparing the aqueous suspension, given the lack of specific information and its proteinaceous nature, the active agent should be considered to have the potential to be a skin/respiratory sensitizer. Thus precautions should be taken. Once an active agent has been authorised as a silage additive, different formulations can be placed on the market with reference to that authorisation. The applicant listed several cryoprotectants and carriers which would allow multiple formulations of the additive to be produced and consequently, not all forms can be directly tested for user safety. However, for assessing the safety for the user of the additive, the active agent is the principal concern provided that other components do not introduce safety issues. For this specific product, all excipients used are food grade, and their use in the additive would not introduce an additional risk to their conventional use. 4. Efficacy Three laboratory studies are described with durations of at least 90 days. Study 1 was made using 1.5 L capacity laboratory silos. Study 2 and study 3 used 6.5 L laboratory silos. All laboratory silos had the capacity to vent gas. In each case, the contents of three replicate silos were sprayed with the additive at 1 x 10 8 CFU/kg forage dissolved in 10 ml water (not confirmed by analysis of the applied suspension). Forage for the control silos were sprayed with an equal volume of water without the additive. Ambient temperature was controlled at 20±2 ºC. The three studies involved forages of different water-soluble carbohydrate (WSC) content, representing material easy to ensile (study 1), moderately difficult to ensile (study 2) and difficult to ensile (study 3) as defined in Regulation (EC) No 429/2008 (see Table 2). Table 2: Characteristics of the grass samples used in the four ensiling experiments Study No Test material Dry matter content (%) WSC 1 content (% fresh matter) 1 19 Perennial rye grass, 1 st cut Permanent grass, 2 nd cut Permanent grass, 2 nd cut WSC: water-soluble carbohydrate Replicate silos were opened at the end of the experiment and contents were analysed for dry matter, ph, lactic acid, volatile fatty acids (acetic, butyric, propionic acid), ethanol, ammonia and total nitrogen. In addition intermediate samples were taken in study 1 after 13 days and in study 3 after three days. Intermediate samples were assayed only for ph and lactic acid concentration. Data were examined by the one-sided non-parametric Wilcoxon Kruskal-Wallis test (rank sum followed by chi-square approximation). 18 Technical dossier/section III/Annexes III.3-3 and III Technical Dossier Section IV. Annex IV.1_easy 20 Technical Dossier Section IV. Annex IV.2_mod difficult 21 Technical Dossier Section IV. Annex IV.3_difficult 8

9 The data from the experiments (summarised in Table 3) produced consistent results with the lactic acid content of the ensiled material being significantly increased in all three cases resulting in a significantly lower ph at the end of the experiments. These significant differences were also seen in the intermediate samples (of studies 1 and 3 after 14 days) indicating that the additive promoted a more rapid increase in lactic acid concentration and reduction in ph than was seen in the control samples. Table 3: Summary of the analysis of ensiled material recovered at the end of the experiment Study No (Duration) 1 (90 days) 2 (90 days) 3 (92 days) Dose (CFU/kg forage) Dry matter loss (%) ph Lactic acid (% Fresh material) Volatile fatty acids (% Fresh material) Ammonia-N (% total N) x * 2.7 * 0.2 * 9.2 * x * 4.20 * 3.0 * 0.4 * 4.7 * , x * 4.21 * 1.7 * 0.5 * 7.4 * : Significantly different from the control value at P<0.05 Dry matter loss and volatile fatty acid production decreased in the treated material compared to controls in all three studies. Ammonia nitrogen production was significantly decreased in easy and moderately difficult ensiled treated materials compared to controls, but not in the more difficult to ensile material. CONCLUSIONS As the identity of the strain Pediococcus pentosaceus (DSM 12834), has been established and no antibiotic resistance detected, following the QPS approach the use of this strain in the production of silage is considered safe for target species, consumers of products from animals fed treated silage and for the environment. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that considering the nature of the alternative food grade excipients, different results would be obtained for other formulations containing P. pentosaceus DSM Given the lack of specific information and its proteinaceous nature, the active agent should be considered to have the potential to be a skin/respiratory sensitizer. The additive containing P. pentosaceus DSM has the potential to improve the production of silage from all forages by reducing the ph and increasing the preservation of dry matter. DOCUMENTATION PROVIDED TO EFSA 1. Pediococcus pentosaceus DSM October Submitted by Lactosan GmbH & Co. KG. 2. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for Pediococcus pentosaceus (DSM 12834) 3. Comments from Member States received through the ScienceNet. 9

10 REFERENCES EFSA (European Food Safety Authority), Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal (2007) 587, 1-16 EFSA (European Food Safety Authority), Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. The EFSA Journal (2008) 732, EFSA (European Food Safety Authority), EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2010 update). EFSA Journal 2010;8(12):

11 Appendix Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for Pediococcus pentosaceus (DSM 12834), for all animal species. 22 This report is on the evaluation of feed additives "micro-organisms used as silage agents", which is related to the application of (1) forty two micro-organisms for which authorisation is sought under Article 10(2) and (2) three additional micro-organisms for which authorisation is sought under Article 4(1). Authorisation is sought for all the above mentioned micro-organisms under category/functional group 1(k), technological additives/silage additives, according to Annex I of Regulation (EC) No 1831/2003. The list of micro-organisms of interest and the minimum activities in the feed additives and in silage, as sought in the authorisation, are presented in Table The intended use of the current applications is for all animal species, except for FAD , for which pigs, bovines, sheep, goats and horses are specified. For identification and characterisation of Saccharomyces cerevisiae the EURL recommends for official control Polymerase Chain Reaction (PCR), a generally recognised standard methodology for identification of yeasts. For identification and characterisation of all the other micro-organisms of concern (i.e. lactococci, lactobacilli, pediococci and bacilli) the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification. The EURL recommends for enumeration in the feed additives the following ring trial validated methods: - Pour plate method using MRS agar (ISO 15214) for Lactococci; - Spread plate method using MRS agar (EN 15787) for Lactobacilli; - Spread plate method using MRS agar (EN 15786) for Pediococci; - Spread plate method using tryptone soya agar (EN 15784) for Bacilli; and - Pour plate method using CGYE agar (EN 15789) for Saccharomyces. None of the Applicants provide experimental data for the determination of micro-organisms in silage. Furthermore, the unambiguous determination of the content of micro-organisms added to silage is not achievable by analysis. Therefore the EURL cannot evaluate nor recommend any method for official control to determine any of the forty five micro-organisms of concern in silage. Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary. 22 The EURL produced a combined report for the L. lactis, L. plantarum, L. buchneri, L. paracasei, L. rhamnosus, L. Salivarius,L. casei, L. brevis, L. pentosus, P. acidilactici, P. pentosaceus, Bacillus, Saccharomyces cerevisiae and lactococcus lactis. 23 Full list provided in EURL evaluation report, available from the EURL website. 11