EFSA Journal 211;9(6):2275 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Lactobacillus plantarum (NCIMB 3236) as a silage additive for all species 1,2 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 3, 4 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT Lactobacillus plantarum is a technological additive intended to improve the ensiling process at a proposed dose of 2.4 x 1 8 CFU/kg fresh material. The bacterial species L. plantarum is considered by EFSA 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. However, the proteinaceous nature of the active agents means that they have the potential to be a skin/respiratory sensitizer and risks for the user cannot be fully excluded. Seven studies with laboratory-scale silos are described, each lasting at least 1 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 2 ºC. The additive containing L. plantarum (NCIMB 3236) consistently showed the potential to improve the production of silage from easy, moderately difficult and difficult to ensile forage species by reducing the ph, increasing the preservation of dry matter and protein. European Food Safety Authority, 211 KEY WORDS Technological additive, silage additive, Lactobacillus plantarum, QPS, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q-211-62, adopted on 14 June 211. 2 This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/23. 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 plantarum (NCIMB 3236) as a silage additive for all species. EFSA Journal 211;9(6):2275. [12 pp.] doi:1.293/j.efsa.211.2275. Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 211
SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FFEDAP) 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 plantarum, when used as a technological additive intended to improve the ensiling process at a proposed dose of 2.4 x 1 8 CFU/Kg fresh material. The bacterial species L. plantarum is considered by EFSA to be suitable for the Qualified Presumption of Safety approach. 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. 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. The proteinaceous nature of the active agent means that they have the potential to be a skin/respiratory sensitizer and risks for the user cannot be fully excluded. Seven studies with laboratory-scale silos are described, each lasting at least 1 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 2 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 and protein. EFSA Journal 211;9(6):2275 2
TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment... 7 1. Introduction... 7 2. Characterisation... 7 2.1. Identity and properties of the active agent... 7 2.2. Production and characteristics of the additive... 7 2.3. Stability... 8 2.4. Conditions of use... 8 2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 8 3. Safety... 8 4. Efficacy... 9 Conclusions... 1 Documentation provided to EFSA... 1 References... 11 Appendix... 12 EFSA Journal 211;9(6):2275 3
BACKGROUND Regulation (EC) No 1831/23 5 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 Bio-Competence Centre of Healthy Dairy Products LLC 6 for authorisation of the product Lactobacillus plantarum (NCIMB 3236), 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 1831/23, 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 2 March 211. The additive Lactobacillus plantarum (NCIMB 3236) has not been previously authorised in the Community. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/23, 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 plantarum (NCIMB 3236), when used under the conditions described in Table 1. 5 6 7 OJ L 268, 18.1.23, p. 29. Bio-Competence Centre of Healthy Dairy Products LLC, Kreutzwaldi 1, 5114 Tartu, Estonia. EFSA Dossier reference: FAD-211-4. EFSA Journal 211;9(6):2275 4
Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Lactobacillus plantarum NCIMB 3236 Registration number/ec No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive - 1.Technological additive K) Silage additive Composition, description Lactobacillus plantarum - NCIMB 3236 Activity: >1,2E + 11 CFU/g Description Chemical Purity criteria formula (if appropriate) - - Method of analysis (if appropriate) Quantification of lactic acid bacteria according to EN 15787:29 Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Not appropiate - Conditions of use Species or category of animal All animal species Maximum Age n.a. Minimum content Maximum content Withdrawal mg or Units of activity or CFU/kg of complete feedingstuffs (select what applicable) period (if appropriate) The minimum dose is 24 CFU/g fresh n.a. n.a. matter 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) It is recommended to user wear goggles and protection mask. The directions for handling must indicate storage temperature, shelf life. n.a. EFSA Journal 211;9(6):2275 5
Specific conditions for use in complementary feedingstuffs (if appropriate) n.a. Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues n.a. n.a. n.a. n.a. EFSA Journal 211;9(6):2275 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 plantarum, and is intended to be added to forages to promote ensiling (technological additive, functional group: silage additive) for eventual use of the silage in any animal species. The species L. plantarum is considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 27, 21). 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 L. plantarum was isolated from silage and is deposited with the National Collection of Industrial and Marine Bacteria (NCIMB) with the accession number NCIMB 3236. 8 It has not been genetically modified within the meaning of GM legislation. Strain identity was established by the full 16S rdna gene sequence which by comparison with sequences recorded in databases was unambiguously identified as L. plantarum. Strain-specific detection is based on the use of pulsed field gel electrophoresis after cleavage with a number of restriction enzymes. The strain was tested for antibiotic susceptibility using two-fold broth dilutions. The battery of antibiotics tested was that recommended by EFSA (EFSA, 28) excluding vancomycin which is not required for this species. As all MIC values for the L. plantarum strain fell below the corresponding breakpoints defined by the FEEDAP Panel no further investigation is required. 2.2. Production and characteristics of the additive 9 Cells were grown in a sterilised medium typical of those used for lactic acid bacteria and then separated from the growth medium. The production process and the details are fully described in the dossier. Material safety datasheets are provided for all cryoprotectants and carrier materials and all are of food grade and do not introduce safety concerns. 1 Data on five production batches showed that the minimum specification (1.2 x 1 11 CFU/g additive) was exceeded in all cases (mean 1.6 x 1 11 CFU/g additive). 11 A single batch of the additive (lactose carrier) was examined for particle size distribution by sieving and for dusting potential using a Stauber-Heubach dustometer. The additive consisting of particles with diameters below 1 µm 22.2 %, and 16.5 % below 63 µm. 12 The dusting potential of 6.2 g/m 3 found is considered high. 13 8 Technical dossier/section II/Annexes II.1. 9 This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/23. 1 Technical dossier/section II/Annexes II.13 to II.19. 11 Technical dossier/section II/Annex II.3. 12 Technical dossier/section II/Annex II.6. 13 Supplementary Information/Annex I. EFSA Journal 211;9(6):2275 7
The additive is routinely monitored for microbial contamination at various points in the manufacturing process and in the final product. Limits are set for Enterobacteriace and yeasts and filamentous fungi (<1 3 CFU/g additive), Escherichia coli (<1 CFU/g additive) and Salmonella (absence in 25 g additive). Data from a single batch confirmed compliance with the value for yeasts and fungi and Enterobacteriace, absence of Salmonella and E.coli. 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. Analysis of one batch of additive confirmed this position. 2.3. Stability The additive is described as hydroscopic and must be stored in packaging which protects against moisture. When stored in the original packaging the additive met the minimum specification after four days storage at 4 ºC, three months at 25 ºC and six months at 4 ºC (two batches). 15 As the additive is intended to be distributed by the spraying of an aqueous suspension, the short-term stability in water was measured. 16 This showed that bacterial numbers were maintained for at least six hours at 2 ºC. 2.4. Conditions of use The additive is intended for use with forages at a proposed minimum dose of 2.4 x 1 8 CFU/kg fresh matter and to be applied as an aqueous suspension. 2.5. 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, L. plantarum, 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. Given its proteinaceous nature, the active agent has the potential to be a skin/respiratory sensitizer, although users at the farm level are exposed to the additive only for a short period of time when preparing the aqueous suspension. 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 14 Technical dossier/section II/Annex II.5. 15 Technical dossier/section II/Annex II.2. 16 Technical dossier/section II/Annex II.21. EFSA Journal 211;9(6):2275 8
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 Seven laboratory studies are described with durations of at least 1 days. All of the experiments were made using 3. L capacity laboratory silos. All laboratory silos had the capacity to vent gas. The contents of seven replicate silos were sprayed with the additive at 1. x 1 9 CFU/kg forage (experiments 1 and 2), 2.6 x 1 8 CFU/kg forage (experiments 3 and 4), 2. x 1 8 CFU/kg forage (experiments 5 and 6) and 1. x 1 8 CFU/kg forage (experiment 7) dissolved in 1 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 2 ± 2 ºC. The seven studies involved forages of different water-soluble carbohydrate (WSC) content, representing material easy to ensile (experiments 2, 4 and 6), moderately difficult to ensile (experiments 1, 3 and 5) and difficult to ensile (experiment 7) as defined in Regulation (EC) No 429/28 (see Table 2). Table 2: Characteristics of the forage samples used in the ensiling experiments Study Test material Dry matter content (% fresh material) WSC content 1 (% fresh material) 1 17 Timothy 1 st cut 21.4 1.7 2 18 Timothy 1 st cut (wilted) 3.9 3.2 3 19 Red clover 1 st cut 18. 2.2 4 2 Perennial ryegrass 1 st cut (wilted) 31.9 7.2 5 21 Timothy 2 nd cut 23.7 2. 6 22 Perennial ryegrass 2 nd cut (wilted) 38.4 4.2 7 23 Red clover-timothy mixture 2 nd cut 14.2.6 1 WSC: water-soluble carbohydrate Replicate silos were opened at the end of the experiment and contents were analysed for dry matter, ph, lactic acid, acetic acid, ethanol, ammonia and total nitrogen. Data were examined by non-parametric Wilcoxon and Kruskal-Wallis test. 24 17 Technical dossier/section IV/Annex IV.1. 18 Technical dossier/section IV/Annex IV.2. 19 Technical dossier/section IV/Annex IV.3. 2 Technical dossier/section IV/Annex IV.4. 21 Technical dossier/section IV/Annex IV.5. 22 Technical dossier/section IV/Annex IV.6. 23 Technical dossier/section IV/Annex IV.7. 24 Supplementary Information/Annex II. EFSA Journal 211;9(6):2275 9
The data from the experiments (summarised in Table 3) produced consistent results with the significantly lower dry matter loss and higher lactic acid content of the ensiled material, which resulted significantly lower ph, except in experiment 7 (difficult to ensiled forage). These significant differences were also seen in lower ammonia-nitrogen and ethanol content of the ensiled material. Table 3: Summary of the analysis of ensiled material recovered at the end of experiments Study Dose (CFU/Kg forage) Dry matter loss (%) ph Lactic acid (% d.m.) Acetic acid (% d.m.) Ethanol (% d.m.) Ammonia-N (% total N) 1 13.2 1. x 1 9 5.6* 6.5 4.18*. 9.8* 1.9 1.6 3.6 2.* 17.62 4.4* 2 9.6 1. x 1 9 5.5* 5.52 3.9* 2. 11.7*.5.7 2.7 1.5* 3.64 1.76* 3 13.3 2.6 x 1 8 4.9* 5.3 3.95* 4.9 15.1*.9 1.3* 4.2 1.2* 8.54 3.82* 4 11.9 2.6 x 1 8 6.6* 5.13 4.4* 2.4 9.4* 1.3 1.2 2.8 1.* 4.72 3.16* 5 4.5 2. x 1 8 3.1* 4.35 3.97* 5.2 8.1* 1.4 2.4* 1.7 1.4* 8.88 4.52* 6 6. 2. x 1 8 2.5* 5.32 4.11* 3.3 7.4* 1.2 1.5* 1.5.7* 9.76 5.28* 7 4.3 1. x 1 8 3.7 4.23 4.18 11.5 1.4 3.6 3.5.9.6* 5.62 4.* *Significantly different from control value at P <.5 CONCLUSIONS As the identity of the strain Lactobacillus plantarum (NCIMB 3236) 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. The proteinaceous nature of the active agent means that they have the potential to be a skin/respiratory sensitizer and risks for the user cannot be fully excluded. The additive containing L. plantarum (NCIMB 3236) has the potential to improve the production of silage from all forages by reducing the ph, increasing the preservation of dry matter and protein. DOCUMENTATION PROVIDED TO EFSA 1. Lactobacillus plantarum E98 NCIMB 3236. January 211. Submitted by Bio-Competence Centre of Healthy Dairy Products LLC. EFSA Journal 211;9(6):2275 1
2. Lactobacillus plantarum E98 NCIMB 3236. Supplementary information. April 211. Submitted by Bio-Competence of Healthy Dairy Products LLC. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods of analysis for Lactobacillus plantarum (NCIMB 3236). 4. Comments from Member States received through the ScienceNet. REFERENCES EFSA (European Food Safety Authority), 27. 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 (27) 587. 1-16. EFSA (European Food Safety Authority), 28. 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 (28) 732. 1-15. EFSA (European Food Safety Authority), 21. EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (21 update). EFSA Journal 21;8(12):1944. EFSA Journal 211;9(6):2275 11
APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for Lactobacillus plantarum (NCIMB 3236) for all animal species 25 In the current application authorisation is sought for the feed additive Lactobacillus plantarum NCIMB 3236 under the category 'technological additives', functional group 'silage additives' according to Annex I of Regulation (EC) No 1831/23. Specifically, authorisation is sought for the feed additive to be placed on the market in the form of powder, containing a minimum concentration of 1.2 x 1 11 CFU/g of Lactobacillus plantarum NCIMB 3236. The intended use of the current application is for all animal species. It is proposed to be mixed into silage providing a minimum concentration of 2.4 x 1 5 CFU/g fresh matter ensilage. For enumeration of Lactobacillus plantarum NCIMB 3236 in feed additive, the Applicant proposes internationally recognised, ring trial validated spread plate method developed by CEN (EN 15787). The performance characteristics reported after logarithmic transformation of measured values (CFU) are: - a standard deviation for repeatability (S r ) of.24 log 1 CFU/g; - a standard deviation for reproducibility (S R ) ranging from.29 to.38 log 1 CFU/g; and - a limit of detection (LOD) of 1 5 CFU/kg feedingstuffs. Based on the performance characteristics of the method the EURL recommends for official control the EN 15787 method for the determination of Lactobacillus plantarum NCIMB 3236 in the feed additive per se. The Applicant did not provide any experimental method or data for the determination of Lactobacillus plantarum NCIMB 3236 in silage. Furthermore, the unambiguous determination of the content of Lactobacillus plantarum NCIMB 3236 added to silage is not achievable by analysis. Therefore the EURL cannot evaluate nor recommend any method for official control to determine Lactobacillus plantarum NCIMB 3236 in silage. Molecular methods were used by the Applicant for identification of the active agent in the feed additive. The EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification. Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by article 1 (Commission Regulation (EC) No 378/25) is not considered necessary. 25 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/sitecollectiondocuments/finrep-fad- 211-4.pdf EFSA Journal 211;9(6):2275 12