Safety and efficacy of a feed additive consisting of Enterococcus lactis NCIMB 10415 for all animal species (Agri‐King, Inc.)

TL;DR

This study evaluates the safety and effectiveness of using Enterococcus lactis NCIMB 10415 as a feed additive to improve silage quality for all animal species.

Contribution

The study confirms the safety of Enterococcus lactis NCIMB 10415 for animals, consumers, and the environment, and its potential to improve silage fermentation.

Findings

E. lactis NCIMB 10415 is safe for all animal species and does not pose a risk to consumers or the environment.

The additive has the potential to improve silage production and fermentation quality when used at a minimum level of 1 × 108 CFU/kg.

The additive is irritant to eyes and a skin and respiratory sensitiser, posing a risk to users upon exposure.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on Enterococcus lactis NCIMB 10415 when used as a technological additive to improve ensiling of all types of fresh plant material for all animal species at a proposed minimum level of 1 × 108 colony forming units (CFU)/kg fresh plant material. The strain NCIMB 10415 was unambiguously identified as Enterococcus lactis, found not to belong to the hospital‐associated clade of Enterococcus faecium and acquired antimicrobial resistance genes were not detected. Consequently, the FEEDAP Panel concluded that E. lactis NCIMB 10415 is safe for all animal species, for consumers of products from animals fed with the treated silage and for the environment. Regarding the safety for the user, the additive is considered irritant to eyes and a skin and respiratory sensitiser. Therefore, any exposure is considered a risk. The FEEDAP Panel concluded that the addition of E. lactis NCIMB 10415 at a minimum level of 1 × 108 CFU/kg to fresh plant material has the potential to improve the production and fermentation quality of silage made from all types of plant materials.

Tables

TABLE 1. Data on the batch‐to‐batch variation, impurities and physical properties of the additive containing E. lactis NCIMB 10415.

Parameter	Specification	Analysis
		Average	Range	No. batches
Batch‐to‐batch variation (CFU/g)
E. lactis NCIMB 10415	≥ 1.0 × 1010	5.6 × 1010	4.5–6.6 × 1010	5
Impurities (mg/kg)
Arsenic			< 0.0067	3
Mercury			< 0.0017	3
Cadmium			< 0.0017 to < 0.0050	3
Lead			<0.0050	3
Mycotoxins (μg/kg)
Aflatoxin B1			< 0.5	3
Zearalenone			< 10	3
Deoxynivalenol			< 20	3
Ochratoxin A			< 0.5	3
Fumonisin B1 + B2			< 20	3
Microbial contamination
Total aerobic counts (CFU/g)			< 1000	5
Escherichia coli (per 1 g)			Not detected	5
Yeast and moulds (CFU/g)			< 100	5
Salmonella spp. (per 25 g)			Not detected	5
Pseudomonas aeruginosa (per 1 g)			Not detected	5
Staphylococcus aureus (per 0.1 g)			Not detected	5
Enterobacteriaceae (CFU/g)			< 10	3
Physical properties
Bulk density (kg/m3)			930–940	5
Dusting potential (Stauber Heubach) (mg/m3)			1.25–17.50	3
Particle size distribution (laser diffraction) (% of particles below, v/v)
100 μm			0.09–0.13	3
50 μm			0.04–0.05
10 μm			0.02–0.03
1 μm			0.01–0.02
Shelf‐life (losses)
2–8°C 24 months		< 0.5 log		3
Stability in water (losses)
20°C 48 h		< 0.5 log		3
Homogeneity in water (coefficient of variation %)		7–13		3

TABLE 2. Characteristics of the fresh materials used in the three ensiling experiments.

Study	Test material	Dry matter content (%)	Water‐soluble carbohydrate content (% fresh matter)
1	High‐moisture maize cobs	63.5	4.1
2	Whole crop maize	33.7	2.7
3	Alfalfa silage	37.4	1.4

TABLE 3. Effects of E. lactis NCIMB 10415 on the characteristics of ensiled material recovered at the end of the ensiling period.

Study	CFU/kg forage	Dry matter loss 1 (% dry matter)	Ammonia‐N (as % of total N)	pH	Lactic acid (% dry matter)	Acetic acid (% dry matter)	Ethanol (% dry matter)
1	0	4.09	9.40	3.65	2.99	0.57	2.07
	1 × 108	3.09	8.66*	3.67*	3.82*	0.57	2.01
2	0	4.07	16.76	3.60	6.91	1.68	1.38
	1 × 108	2.10*	14.86	3.62	8.63*	1.61	1.54*
3	0	2.77	19.79	4.50	8.42	3.55	0.74
	1 × 108	1.83	16.90*	4.39*	8.63*	2.63*	0.49*

Full text

INTRODUCTION

1

Background and Terms of Reference

1.1

Regulation (EC) No 1831/20031 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 Agri‐King, Inc. (represented in the EU by Agri‐King Limited)2 for the authorisation of the additive consisting of Enterococcus lactis NCIMB 10415, when used as a feed additive for target species (category: technological additives; functional group: silage additives).

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). The dossier was received on 06 June 2024 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00329. The particulars and documents in support of the application were considered valid by EFSA as of 12 August 2024.

According to Article 8 of Regulation (EC) No 1831/2003, 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the feed additive consisting of Enterococcus lactis NCIMB 10415 when used under the proposed conditions of use (see Section 3.1.3).

Additional information

1.2

The additive is a preparation containing Enterococcus lactis NCIMB 10415. EFSA issued an opinion on the safety and efficacy of E. lactis NCIMB 10415 (originally identified as Enterococcus faecium) as a silage additive for all animal species (FEEDAP Panel, 2013). Enterococcus lactis NCIMB 10415 was authorised as a technological additive (functional group: silage additives) for all animal species (1k20601).3 This authorisation expired on 15 April 2024.

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier4 in support of the authorisation request for the use of Enterococcus lactis NCIMB 10415 as a feed additive.

In accordance with Article 38 of the Regulation (EC) No 178/20025 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 and 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,6 a non‐confidential version of the dossier has been published on Open.EFSA.

According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 31 March to 21 April 2025, for which no comments were received.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 16 August to 16 November 2024; the comments received were considered for the assessment.

The European Union Reference Laboratory (EURL) considered that the conclusions and recommendations reached in the previous assessment regarding the methods used for the control of Enterococcus lactis NCIMB 10415 (originally identified as Enterococcus faecium) in animal feed are valid and applicable for the current application.7

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and efficacy of E. lactis NCIMB 10415 is in line with the principles laid down in Regulation (EC) No 429/20088 and the relevant guidance documents: Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024a, 2024b), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2024).

ASSESSMENT

3

The additive is a preparation containing viable cells of E. lactis NCIMB 10415 as an active agent. It is intended to be used as a technological additive (functional group: silage additives) in fresh plant material for all animal species.

Characterisation

3.1

Characterisation of the active agent

3.1.1

The active agent was originally isolated from faeces of a healthy infant and is deposited in the National Collections of Industrial and Marine Bacteria (NCIMB) with the accession number NCIMB 10415.9 The applicant claimed that it has not been genetically modified.10

The active agent, originally assigned to the Enterococcus faecium species, was identified as Enterococcus lactis, based on a bioinformatic analysis of the complete whole genome sequence (WGS) data.11 The taxonomic assignment was based on an average nucleotide identity (ANI) value of 98.61% with the type strain E. lactis LMG 25958^T^. Moreover, the comparison showed an ANI value of 94.58% with the type strain E. faecium DSM 20477^T^. The strain is predicted to harbour one plasmid based on the WGS data.

The susceptibility of the active agent to antimicrobials was tested using a broth microdilution method and including the set of antimicrobials recommended by EFSA (EFSA FEEDAP Panel, 2018). The minimum inhibitory concentration (MIC) values were compared with the defined EFSA cut‐off values for the closest related species E. faecium.12 All MIC values were equal to or fell below the cut‐off values, with the exception of clindamycin, erythromycin and kanamycin. The MIC values for these antibiotics were one dilution above the corresponding cut‐off values. Exceeding the cut‐off value by one dilution is considered to be within the normal range of variation and, thus, not a matter of concern. Therefore, the strain is considered to be phenotypically susceptible to all the relevant antibiotics.

The WGS of the strain, including the plasmid, was interrogated for the presence of antimicrobial resistance (AMR) genes by a search against the CARD and ResFinder databases.13 A total of eight hits were found exceeding the thresholds recommended by EFSA (EFSA, 2024). Further analysis of these hits following EFSA's criteria (EFSA BIOHAZ Panel, 2023) did not reveal any acquired AMR genes.14 Therefore, the FEEDAP Panel concludes that the active agent harbours no acquired AMR genes, and therefore, raises no safety concerns.

According to the FEEDAP guidance on characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018), the safety of E. faecium should be established by the susceptibility to ampicillin and the lack of genetic markers typical of the E. faecium clade A clinical isolates (IS16, esp, hylEfm). In view of the allocation of clade B strains to the E. lactis species, the FEEDAP Panel considers these criteria are also applicable to E. lactis strains. The WGS of the strain, including the plasmid, was interrogated for the presence of the virulence markers by a search against the VFDB database.15 No sequences of concern were identified. Additionally, the strain was shown to be susceptible to ampicillin (MIC < 2 mg/L). Therefore, the safety of the strain is established.

Characterisation of the additive

3.1.2

■■■■■ The final additive is a micro‐encapsulated preparation consisting of approximately 2% active agent, 88% saccharose and 10% hydroxypropyl methyl cellulose.16 The carriers proposed by the applicant are feed materials or authorised feed additives. The applicant states that no antimicrobial substances are used during the manufacturing process.17

The additive is specified to contain a minimum concentration of active agent of 1 × 10^10^ colony forming units (CFU)/g of additive.

The data provided by the applicant on the batch‐to‐batch variation,18 impurities19 and physical properties20 of the additive are reported in Table 1.

The data provided showed compliance with the specifications set by the applicant. The FEEDAP Panel considers that the microbial contamination and the amounts of the detected impurities do not raise safety concerns.

Conditions of use

3.1.3

The additive is intended for use in all types of fresh plant material and for all animal species at a proposed minimum inclusion level of 1 × 10^8^ CFU/kg fresh material. It is to be applied as an aqueous suspension or applied directly to the silage.21

Safety

3.2

The additive under assessment has previously been authorised for use in the European Union (1k20601).22 The applicant states that no adverse effects on target animals, consumers, users or the environment have been reported during the period of the previous authorisation of the additive (2014–2024).23

In the context of the current application, the identity of strain NCIMB 10415 was reassigned to E. lactis and was found not to belong to the hospital‐associated clade of Enterococcus faecium and not to express resistance to clinically relevant antibiotics. Consequently, the FEEDAP Panel concludes that E. lactis NCIMB 10415 is safe for the target species, consumers and the environment. All the proposed carriers are feed materials or authorised feed additives; therefore, the preparation under assessment is considered safe.

With regard to user safety, based on the highest dusting potential measured (see Section 3.1.2), the FEEDAP Panel considers that the exposure of users through inhalation is likely.

The eye irritation potential of the additive (one batch) was investigated in vitro according to OECD TG 492 (2023). The test was conducted in full compliance with the guideline; however, the control data were outside the normal range. The results indicated that the additive is an eye irritant, but a false positive could not be ruled out. The FEEDAP Panel concludes that the test material should be considered to be an eye irritant.24

The additive is a preparation containing a microorganism and therefore should be considered as a skin and respiratory sensitiser.

Conclusions on safety

3.2.1

The FEEDAP Panel concludes that E. lactis NCIMB 10415 is safe for the target species, consumers and the environment. Regarding user safety, the additive is considered an irritant to the eyes and a skin and respiratory sensitiser. Any exposure is considered a risk.

Efficacy

3.3

The additive is intended to improve silage production and quality, as well as preserve the nutrients of all types of fresh plant material, at a proposed minimum inclusion rate of 1 × 10^8^ CFU/kg of fresh matter. To support this claim, the applicant provided three laboratory experiments performed with easy (trial 125), moderately difficult (trial 226) and difficult to ensile (trial 327) fresh plant materials as specified by Regulation (EC) No 429/2008. The characteristics of the fresh plant materials are shown in Table 2.

In all trials, the fresh materials were sprayed with a fixed amount of water, which was either not inoculated (control) or inoculated with E. lactis NCIMB 10415 at a level of 1 × 10^8^ CFU/kg fresh matter. Samples of the fresh material were randomly distributed in 20 2.9 L mini‐silos (10 replicates per group) and allowed to ensile for a minimum of 90 days at room temperature (23 ± 2°C). Silos had gas release valves and devices for evacuating liquid effluents.28

In all trials, after the fermentation period, the silos were opened, weighed and the ensiled material was analysed for the content of dry matter, pH, lactic acid and other volatile fatty acids (acetic, propionic, butyric), ethanol and ammonia nitrogen. The silage dry matter content was corrected for the loss of volatiles during drying, and the dry matter loss was calculated (Krueger et al., 2023).29

The experimental data were analysed with Student's t‐test, with the additive supplementation as the fixed effect and the mini‐silo as the experimental unit. The significance level was set at 0.05. Results are shown in Table 3.

The addition of E. lactis NCIMB 10415 to the fresh plant materials at 1 × 10^8^ CFU/kg resulted in a significantly lower ammonia‐N production in trials 1 and 3, and lower dry matter loss in trial 2 compared to the control materials. In all trials, these effects were accompanied by a higher lactic acid concentration of the supplemented silages. In trials 1 and 2, a significant but minimal increase in pH or ethanol, respectively, was observed in the supplemented silage compared to the control. In trial 3, the supplemented silage exhibited lower pH, acetic acid and ethanol concentrations than the control.

Conclusions on efficacy

3.3.1

The addition of E. lactis NCIMB 10415 at a minimum level of 1 × 10^8^ CFU/kg to fresh plant material has the potential to improve the production and fermentation quality of silage made from all types of plant materials.

CONCLUSIONS

4

The FEEDAP Panel concludes that E. lactis NCIMB 10415 is safe for all animal species, consumers and the environment.

Regarding user safety, the additive is considered an irritant to eyes and a skin and respiratory sensitiser. Any exposure is considered a risk.

The addition of E. lactis NCIMB 10415 at a minimum level of 1 × 10^8^ CFU/kg to fresh plant material has the potential to improve the production and fermentation quality of silage made from all types of plant materials.

ABBREVIATIONSAMRantimicrobial resistanceANIAverage Nucleotide IdentityCFUcolony‐forming unitEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedMICminimum inhibitory concentrationWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00329

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PANEL MEMBERS

Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Ilen Röhe, and Katerina Theodoridou.

LEGAL NOTICE

The scientific output published implements EFSA&s decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA, they are consequently withheld from public disclosure by redaction.