Scientific Opinion on the safety and efficacy of Lactobacillus brevis (DSMZ 21982) as a silage additive for all species 1,2

Transcription

1 EFSA Journal 2012;10(3):2617 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Lactobacillus brevis (DSMZ 21982) as a silage additive for all species 1,2 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 3,4 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy The strain of Lactobacillus brevis under application is intended for use as a technological additive to improve the ensiling process at a proposed dose of 1x10 8 to 1x10 9 CFU/kg fresh material. The bacterial species L. brevis 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. Given the lack of information and its proteinaceous nature, the active agent should be considered as a potential skin and respiratory sensitiser. Five studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of forage covering a range of dry matter content (19 to 52 %) with differing water-soluble carbohydrate content. In each case, replicate silos containing treated forage were compared to silos containing the same untreated forage. The results showed that the additive has the potential to improve the production of silage from forages by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. European Food Safety Authority, 2012 KEY WORDS Technological additive, silage additive, Lactobacillus brevis, QPS, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q , adopted on 6 March This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. The modified sections are indicated in the text. 3 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 4 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 Lactobacillus brevis (DSMZ 21982) as a silage additive for all species. EFSA Journal EFSA Journal 2012;10(3):2617. [11 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2012

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 single strain of Lactobacillus brevis, when used as a technological additive intended to improve the ensiling process at a proposed dose of 1x10 8 to 1x10 9 CFU/Kg fresh material. The bacterial species L. brevis 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. However, for assessing the safety for the user of the additive, the active agent is the principal issue provided that other components do not introduce concerns. 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. Given the lack of information and its proteinaceous nature, the active agent should be considered as a potential skin and respiratory sensitiser. Five studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of forage covering a range of dry matter content (19 to 52 %) with differing water-soluble carbohydrate content. In each case, replicate silos containing treated forage were compared to identical silos containing the same untreated forage. The results showed that this strain of L. brevis has the potential to improve the production of silage from forages by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. EFSA Journal 2012;10(3):2617 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... 7 Conclusions... 9 Documentation provided to EFSA... 9 References Appendix EFSA Journal 2012;10(3):2617 3

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from the company Microferm Limited 6 for authorisation of the product Lactobacillus brevis DSMZ 21982, when 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 4(1) (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 7 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 23 May The additive Lactobacillus brevis DSMZ has not been previously authorised in the European Union. 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 Lactobacillus brevis DSMZ 21982, when used under the conditions described in Table Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, , p. 29. Microferm Limited, Spring Lane North, Malvern Link, WR14 1BU, Worcester, United Kingdom. EFSA Dossier reference: FAD EFSA Journal 2012;10(3):2617 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Lactobacillus brevis DSMZ Registration number/ec No/No (if appropriate) Category(-ies) of additive - Technological feed additive Functional group(s) of additive Silage additive Composition, description A preparation of Lactobacillus brevis DSMZ Chemical formula - Description Purity criteria (if appropriate) E. coli <100 CFU/g Salmonella nil in 25g. Yeast/Mold<100 CFU/g Method of analysis (if appropriate) BS EN:15787:2009 Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Conditions of use Species or category of animal Maximum Age Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs (if appropriate) All animal species - - Not relevant Other provisions and additional requirements for the labelling 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) - Respiratory sensitizer, wear appropriate PPE including dust masks and gloves, wash hands after use. - - Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues Not relevant Not relevant Not relevant Not relevant EFSA Journal 2012;10(3):2617 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, Lactobacillus brevis, and is intended to be added to forages to promote ensiling (technological additive, functional group: silage additives) for eventual use of the silage in any animal species. It is a bacterial species considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 2007, 2011). This approach requires the identity of the strain to be conclusively established and evidence that the strain does not show resistance to antibiotics of human and veterinary importance. 2. Characterisation 2.1. Identity and properties of the active agent The strain of L. brevis is deposited with the Deutsche Sammlung von Mikroorganisms und Zellkulturen (DSMZ) with the accession number DSMZ It has not been genetically modified. Strain identity was established by its phenotypic properties (API) and by the partial 16S rrna gene sequence which, by comparison with sequences recorded in databases, was unambiguously identified as L. brevis. Strain-specific detection is based on a Multi Locus Sequence Typing (MLST), an approach which analyses allelic variation in house keeping genes. Data on genetic stability, comparing the master culture with working cultures used to inoculate fermentation batches were not provided. The strain was tested for antibiotic susceptibility using two-fold broth dilutions. The battery of antibiotics tested was that recommended by EFSA (EFSA, 2008). As all minimum inhibitory concentration values for the L. brevis 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 10 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. The production process and the details are fully described in the dossier. Material safety data sheets are provided for all cryoprotectants and all are of food grade and do not introduce safety concerns. 11 Data on five production batches showed that the minimum specification (8 x CFU/g) was exceeded in all cases (mean 6.6 x CFU/g additive). 12 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 84.4 µm with approximately 12 % by weight of the additive consisting of particles with diameters below 50 µm and ~3 % below 5 µm. However, the dusting potential of 0.78 g/m 3 is considered moderate to low. 13 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. 8 Technical dossier/section II. 9 Technical dossier/section II/ Annex II This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/ Technical dossier/section III/Annex MSDS. 12 Technical dossier/section II. 13 Technical dossier/section II/Annex II.3. EFSA Journal 2012;10(3):2617 6

7 (absence in 25 g additive). Data from three batches confirmed compliance with the set microbiological values. 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. Several batches of the additive were, however, sent for analysis to confirm this position. Aflatoxins B 1, B 2, G 1 and G 2, zearalenone and deoxynivalenol, Pb, Hg and Cd and As could not be detected Stability The product is hygroscopic and is therefore distributed in packaging that protects from moisture and light. Data was provided on the stability of three batches of the additive stored in the original packaging at ambient temperature with maltodextrin or dextrose carrier material. Both formulations of the product were stable at the tested temperature over 15 months. As the additive is intended to be distributed by the spraying of an aqueous suspension, the short-term stability in water was measured. This showed that bacterial numbers were maintained for at least two days under ambient conditions Conditions of use The additive is intended for use with forages at a proposed minimum dose of 1.0 x 10 8 CFU/kg and maximum dose of 1.0 x 10 9 CFU/kg fresh matter, and applied as an aqueous suspension 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 substance in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 3. Safety The species L. brevis is considered by EFSA to be suitable for the QPS approach to safety. Therefore, it 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 and the safety for the user. 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. Dermal, eye irritation and skin sensitisation were not tested. 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 and respiratory sensitizer. 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. 4. Efficacy A total of five laboratory experiments are described made with five different forage samples. In all of the studies 1 kg capacity plastic drain pipe silos were used with the capacity to vent gas. In each case, 14 Technical dossier/section II. EFSA Journal 2012;10(3):2617 7

8 the contents of four replicate silos were sprayed with the additive at 1 x 10 8 CFU/kg forage or 1 x 10 9 CFU/kg forage (dose confirmed by analysis of the applied suspension). Forage for the four control silos were sprayed with an equal volume of water without the additive. The four studies involved a range of forages of different botanical origin, dry matter content and and water-soluble carbohydrate (WSC) content, representing material easy to ensile (experiments 1 and 3) and difficult to ensile (experiments 2, 4 and 5) as defined in Regulation (EC) No 429/2008 (see Table 2). Replicate silos were opened at the end of the experiments (364 days in experiment 1, 359 days in experiment 2, 213 days in experiment 3, 153 days in experiment 4 and 90 days in experiment 5) and the contents were analysed for dry matter content, ph, lactic acid, acetic acid, ethanol and ammonianitrogen concentration and aerobic stability. Aerobic stability was determined by the period taken to increase the temperature of the silage by 2 o C (experiments 1, 2, 3 and 4) and 3 o C (experiment 5) above the ambient temperature. Data of four replicates in each treatment were examined by non-parametric Kruskal-Wallis and Mann- Whitney tests (studies 1, 2, 3 and 5). Table 2: Characteristics of the forage samples used in the ensiling experiments Study No Test material Dry matter content Water-soluble carbohydrate content (% fresh matter) (% fresh matter) 1 15 Whole crop maize 34.0 N.D Whole crop wheat Grass Grass/clover Grass The data from five experiments are summarised in Table 3. In four of these five experiments consistent results were obtained, with a significant increase in acetic acid and a significant improvement of the aerobic stability of the ensiled material. 15 Technical dossier/section IV/Annex IV Technical dossier/section IV/Annex IV Technical dossier/section IV/Annex IV Technical dossier/section IV/Annex IV Supplementary Information February EFSA Journal 2012;10(3):2617 8

9 Table 3: Summary of the analysis of ensiled material recovered at the end of experiments Study Treatment (CFU/kg) 0 1x10 8 1x x10 8 1x x10 8 1x10 9 Dry matter loss (%) ph * * Lactic acid (% dry matter) * * 2.14 * Acetic acid (% dry matter) * 0.41 * * 2.37 * * 4.77 * NH3-N (% total N) * Ethanol (% dry matter) * 0.66 * * 0.34 * Aerobic stability (hours) ** * 211 * * 237 * 155 >240 > x >168 > x10 8 1x10 9 n.d. n.d. n.d * * 2.03 * * 8.40 * * 8.30 * * 2.65 * * 192 * * Significantly different from control value at P < 0.05 n.d.: not determined ** Period to reach 2ºC or 3ºC rise over the ambient temperature (hours) CONCLUSIONS The species Lactobacillus brevis is considered by EFSA to be suitable for the QPS approach to safety assessment. 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 and the safety for the user. As the identity of the strain DSMZ has been established and no antibiotic resistance detected, 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. Given its proteinaceous nature, the active agent should be considered to have the potential to be skin and respiratory sensitisers. The additive containing L. brevis DSMZ has the potential to improve the production of silage by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. DOCUMENTATION PROVIDED TO EFSA 1. Lactobacillus brevis DSMZ November Submitted by Microferm Limited 2. Lactobacillus brevis DSMZ Supplementary information. August Submitted by Microferm Limited 3. Lactobacillus brevis DSMZ Supplementary information. February Submitted by Microferm Limited 4. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Lactobacillus brevis DSMZ Comments from Member States received through the ScienceNet. EFSA Journal 2012;10(3):2617 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, 587, 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, 732, EFSA Panel on Biological Hazards (BIOHAZ) Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2011 update). EFSA Journal.9(12):2497. EFSA Journal 2012;10(3):

11 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method of Analysis for Lactobacillus brevis DSMZ for all animal species. 20 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. 20 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. 21 Full list provided in EURL evaluation report, available on the EURL website: EFSA Journal 2012;10(3):