Safety and efficacy of a feed additive consisting of Lacticaseibacillus huelsenbergensis DSM 115424 for all animal species (Lactosan GmbH & Co. KG)

TL;DR

This study evaluates the safety and effectiveness of a bacterial additive for improving silage in animal feed, finding it safe for animals, consumers, and the environment.

Contribution

The study confirms the safety and efficacy of Lacticaseibacillus huelsenbergensis DSM 115424 as a silage additive for all animal species.

Findings

The strain is safe for animals, consumers, and the environment when used as a silage additive.

It can improve silage production at a minimum concentration of 1 × 108 CFU/kg fresh plant material.

One preparation of the additive is not irritating to skin or eyes, but others remain unassessed.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on Lacticaseibacillus huelsenbergensis DSM 115424 when used as a technological additive to improve ensiling of all fresh plant material for all animal species at a proposed minimum concentration of 1 × 108 colony forming units (CFU)/kg fresh plant material. The bacterial species L. huelsenbergensis is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. The identity of the strain was established, and no acquired antimicrobial resistance genes of concern were detected. Therefore, the FEEDAP Panel concluded that the use of the strain as a silage additive is considered safe for all animal species, for consumers of products from animals fed with the treated silage and for the environment. Regarding user safety, inhalation and dermal exposure are considered a risk. One preparation of the additive containing whey powder as a carrier was shown not to be irritant to skin or eyes. However, the Panel cannot assess the irritation potential of other possible preparations. The FEEDAP Panel concluded that L. huelsenbergensis DSM 115424 has the potential to improve the production of silage prepared from all fresh plant materials at a minimum concentration of 1 × 108 CFU/kg fresh material.

Tables

TABLE 1. Data on the batch‐to‐batch variation, impurities and physical properties of a preparation containing L. huelsenbergensis DSM 115424.

Parameter	Specification	Analysis
		Average	Range	# batches
Batch‐to‐batch variation
CFU/g	4.0 × 1011	5.4 × 1011	4.8–6.1 × 1011	5
Impurities
Lead (mg/kg)			< 0.1	3
Mercury (mg/kg)			< 0.1	3
Cadmium (mg/kg)			< 0.1	3
Arsenic (mg/kg)			< 0.1–0.11	3
Mycotoxins (μg/kg)
Aflatoxins B1, B2, G1, G2			< 0.03	3
Deoxynivalenol			< 10	3
Zearalenone			< 5	3
Microbial contamination
Salmonella spp. (per 25g)			Not detected	3
Enterobacteriaceae (CFU/g)			< 1000	3
Yeast and moulds (CFU/g)			< 1000	3
Physical properties
Physical form		Solid
Bulk density (kg/m3)		645.7	642–649	3
Dusting potential (Stauber Heubach) (mg/m3)		713	695–743	3
Particle size distribution (laser diffraction) (% of particles below, v/v)
100 μm		57.6	57.3–57.8	3
50 μm		31.8	31.5–32.2
10 μm		8.5	8.4–8.5
Shelf‐life (losses)
20°C up to 12 months		< 0.5 log		3
Stability in water (losses)
48 h 20°C		< 0.5 log		3
7 days 4°C		< 0.5 log		3

TABLE 2. Characteristics of the fresh material samples used in the four ensiling experiments.

Study	Test material	Dry matter content (%)	Water‐soluble carbohydrate content (% fresh matter)
1	Grass	41.7	6
2	Corn cob mix	70.2	1.14
3	Maize whole plant	39.5	1.82
4	Grass	26.6	1.22

TABLE 3. Effects of L. huelsenbergensis DSM 115424 on the characteristics of ensiled material recovered at the end of the ensiling period (90 days).

Study	Application rate (CFU/kg forage)	Dry matter loss 1 (%)	pH	Lactic acid (% fresh matter)	Acetic acid (% fresh matter)	Ammonia‐N (% of total N)
1	0	3.10	4.54	1.63	0.35	11.19
	1 × 108	6.83	3.73*	4.14*	0.21*	7.36*
2	0	1.43	4.04	0.91	0.21	3.65
	1 × 108	0.88	3.83*	1.38*	0.36*	3.00*
3	0	8.13	4.05	1.07	0.62	7.59
	1 × 108	7.61	3.98*	1.17*	0.99*	4.94*
4	0	1.24	4.98	1.48	0.98	17.23
	1 × 108	0.30*	4.28*	3.34*	0.81*	9.26*

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 Lactosan GmbH & Co. KG2 for the authorisation of the additive consisting of Lacticaseibacillus huelsenbergensis DSM 115424, when used as a feed additive for all animal 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 01 August 2024 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00510. The particulars and documents in support of the application were considered valid by EFSA as of 04 November 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 L. huelsenbergensis DSM 115424, when used under the proposed conditions of use (see Section 3.1.3).

Additional information

1.2

The additive is a preparation containing viable cells of L. huelsenbergensis DSM 115424. It is not currently authorised in the European Union.

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of L. huelsenbergensis DSM 115424 as a feed additive.

In accordance with Article 38 of the Regulation (EC) No 178/20024 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,5 a non‐confidential version of the dossier has been published on Open.EFSA.6

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 13 March to 03 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 08 November 2024 to 08 February 2025, for which the received comments were considered for the assessment.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the L. huelsenbergensis DSM 115424 in animal feed.7

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of L. huelsenbergensis DSM 115424 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); 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); EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2024); Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023).

ASSESSMENT

3

The additive under assessment is based on viable cells of L. huelsenbergensis DSM 115424 to be added to fresh plant material to promote ensiling (technological additive, functional group: silage additives) with the eventual use of the resulting silage for all animal species.

Characterisation

3.1

Characterisation of the active agent

3.1.1

The active agent was originally isolated from corn silage9 and is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the deposition number DSM 115424.10 The applicant declared that it has not been genetically modified.11

The taxonomical identification of the strain was confirmed by average nucleotide identity (ANI) and digital DNA–DNA hybridisation (dDDH) based on the whole genome sequence (WGS) data.12 ■■■■■

The antimicrobial susceptibility of the strain DSM 115424 was tested against the set of antimicrobials recommended by FEEDAP Panel (EFSA FEEDAP Panel, 2018).13 All the minimum inhibitory concentration values were equal to or fell below the corresponding EFSA cut‐off values of L. casei/paracasei, which is the closest relative. Therefore, the strain is considered to be phenotypically susceptible to all the relevant antimicrobials.

The WGS of the strain was interrogated for the presence of antimicrobial resistance (AMR) genes by a search against the ResFinder and NCBI databases.14 No hits were identified above the thresholds recommended by EFSA (2024). Therefore, the FEEDAP Panel concludes that the strain harbours no acquired AMR genes and raises no safety concerns.

Characterisation of the additive

3.1.2

After growing the active agent in appropriate media by a fermentation process, the biomass is concentrated by centrifugation and blended with cryoprotectants.15 The freeze‐dried cell concentrate is then mixed with other carriers to the final standardised concentration. The applicant provided examples of different carriers that could be used in the formulation of the final preparation, which are all feed materials (whey powder, dextrose anhydrous, maltodextrin), except PEG 4000. The safety of PEG 4000 is assessed in Section 3.2. The applicant states that no antimicrobial substances are used during the manufacturing process.16

The additive is specified to contain a minimum concentration of the active agent of 4 × 10^11^ colony forming units (CFU)/g of additive.17 The applicant provided data on the characterisation of the additive as a preparation containing whey powder as carrier.18

The data provided by the applicant on the batch‐to‐batch variation,19 impurities20 and physico‐chemical properties21 of the additive are reported in Table 1.

The Panel considers that the microbial contamination and the levels of the impurities analysed in these preparations are of no concern.

Conditions of use

3.1.3

The additive is intended for use with all fresh plant material and for all animal species at a proposed minimum concentration of 1 × 10^8^ CFU/kg fresh material. It is to be applied as an aqueous suspension.22

Safety

3.2

The species Lacticaseibacillus huelsenbergensis is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA BIOHAZ Panel, 2025). This approach requires the identity of the strain to be conclusively established and evidence that it does not carry acquired resistance to antibiotics of human and veterinary importance. In the view of the FEEDAP Panel, the identity of the strain has been established as L. huelsenbergensis and the antibiotic resistance qualification met. Consequently, the active agent L. huelsenbergensis DSM 115424 is considered safe for the target species, consumers and the environment.

All the proposed cryoprotectants and carriers are feed materials or authorised feed additives, with the exception of PEG 4000. ■■■■■.23 The safety of PEG 4000 used at the same concentration in the formulation of another silage additive intended for use under the same conditions of use was already assessed by the FEEDAP Panel in a previous opinion (EFSA FEEDAP Panel, 2024b). The FEEDAP Panel concluded that, at this concentration, PEG 4000 is safe for target animals, consumers, users and the environment.

With regard to user safety, the applicant provided data on a preparation based on whey powder.

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 skin irritation potential of L. huelsenbergensis DSM 115424 was tested in a study performed according to OECD TG 439, which showed that this preparation of the additive is not a skin irritant (UN GHS ‘No Category’).24

The eye irritation potential of L. huelsenbergensis DSM 115424 was tested in a study performed according to OECD TG 405, which showed that this preparation of the additive is not an eye irritant (UN GHS ‘No Category’).25

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 the active agent and the preparations proposed by the applicant are safe for the target species, consumers and the environment. Regarding user safety, one preparation containing whey powder as a carrier was shown not to be irritant to skin and eyes. The FEEDAP Panel cannot assess the irritation potential of other preparations. The additive should be considered as a skin and respiratory sensitiser. Inhalation and dermal exposure are considered a risk. These latter conclusions would apply, in principle, to any preparations containing the active agent.

Efficacy

3.3

Four laboratory experiments were made with fresh plant material samples representing materials easy to ensile (study 1),26 moderately difficult to ensile (study 3)27 and difficult to ensile (studies 2 and 4)28 as specified by Regulation (EC) No 429/2008, the characteristics of which are shown in Table 2. All the studies included a control and a group inoculated with L. huelsenbergensis DSM 115424. The additive was dissolved in water and sprayed on the fresh plant material at an intended inclusion rate of 1 × 10^8^ CFU/kg fresh matter. The fresh material for the control was sprayed with an equal volume of water but without the additive. Samples of the fresh material were ensiled for 90 days in 1.5 L mini‐silos (studies 1, 3 and 4) or 6.5 L mini‐silos (study 2) (three replicates per group). All experiments were conducted at 20 ± 2°C.

After 90 days, the silos were opened, and their contents were analysed for total dry matter (DM) content, pH, lactic, acetic, butyric and propionic acids, ethanol and ammonia‐N content. The DM loss (correcting silage DM content for the loss of volatiles) during ensiling was calculated.

The experimental data were analysed with a non‐parametric Mann‐Whitney U‐test, with the additive supplementation as the fixed effect and the silo as the experimental unit. The significance level was set at p = 0.05. Results are shown in Table 3.

In all the studies, the addition of L. huelsenbergensis DSM 115424 in the fresh plant material resulted in lower ammonia‐N production and higher lactic acid concentration in the silage compared to the control. In studies 1 and 4, the treated silage showed a significantly lower concentration of acetic acid compared to the control. Similarly, in study 4, the treated silage showed a significantly lower dry matter loss compared to the control.

Conclusions on efficacy

3.3.1

The use of L. huelsenbergensis DSM 115424 at the proposed inclusion level has the potential to improve the production of silages from all fresh plant materials by enhancing the preservation of nutrients.

CONCLUSIONS

4

The FEEDAP Panel concludes that the preparations containing L. huelsenbergensis DSM 115424 are safe for all animal species, consumers and the environment.

Regarding user safety, one preparation containing whey powder as carrier was shown not to be irritant to skin and eyes. No conclusions can be reached on the potential of other preparations to be irritant. The additive should be considered as a skin and respiratory sensitiser. Inhalation and dermal exposure are considered a risk. This conclusion would apply, in principle, to any preparations containing the active agent.

The addition of L. huelsenbergensis DSM 115424 at a minimum level of 1 × 10^8^ CFU/kg fresh plant material has the potential to improve the production of silages from all fresh plant materials by enhancing the preservation of nutrients.

ABBREVIATIONSAMRantimicrobial resistanceANIAverage Nucleotide IdentityCFUcolony forming unitdDDHdigital DNA–DNA hybridisationEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedMICminimum inhibitory concentrationOECDOrganisation for Economic Co‐operation and DevelopmentPEG 4000polyethylene glycol 4000QPSQualified Presumption of SafetyWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00510

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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.