Safety and efficacy of a feed additive consisting of Loigolactobacillus coryniformis DSM 34345 as a silage additive for all animal species (Lactosan GmbH & Co.KG)

TL;DR

This paper evaluates the safety and effectiveness of a bacterial additive for improving silage production in animal feed.

Contribution

The study confirms the safety and efficacy of Loigolactobacillus coryniformis DSM 34345 as a silage additive for all animal species.

Findings

The additive is safe for animals, consumers, and the environment.

The strain can improve silage production at a minimum concentration of 1 × 108 CFU/kg.

The additive may pose risks as a skin and respiratory sensitiser.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on Loigolactobacillus coryniformis DSM 34345 when used as a technological additive to improve ensiling of fresh plant material. The additive is intended for use with 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. coryniformis 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 the animal species, for consumers of products from animals fed the treated silage and for the environment. Regarding user safety, the additive containing Loigolactobacillus coryniformis DSM 34345 should be considered as a potential skin and respiratory sensitiser, and any exposure through skin and respiratory tract is considered a risk. One preparation 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 Loigolactobacillus coryniformis DSM 34345 has the potential to improve the production of silages prepared from all fresh plant materials at a minimum concentration of 1 × 108 CFU/kg fresh material.

Tables

TABLE 1. Physico‐chemical properties of PEG 4000.

Property	Value	Unit
Molecular weight a	4000	g/mol
Estimated K oc	10	L/kg
Water solubility a	500	g/L
Vapour pressure a	1	Pa

TABLE 2. PEC values for PEG 4000 in Phase I assessment.

	Dairy cows	Cattle for fattening
PECsoil	0.79 μg/kg	1.02 μg/kg
PECgw	0.59 μg/L	0.76 μg/L

TABLE 3. Characteristics of the fresh material samples used in the three ensiling experiments.

Study	Test material	Dry matter content (%)	Water‐soluble carbohydrate content (% fresh matter)
1 30	Grass	34.7	3.80
2 31	Grass	25.8	2.36
3 32	Grass	22.4	1.26

TABLE 4. Effects of L. coryniformis DSM 34345 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	9.8	4.48	1.57	0.76	6.44
	1 × 108	7.4*	4.04*	2.75*	0.12*	2.58*
2	0	10.9	4.54	0.68	0.67	9.89
	1 × 108	10.6	4.18*	1.30*	0.56*	7.06*
3	0	20.3	5.03	0.51	0.90	13.90
	1 × 108	11.8*	4.18*	1.72*	0.43*	6.95*

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 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 Loigolactobacillus coryniformis DSM 34345, 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 16 May 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00362. The particulars and documents in support of the application were considered valid by EFSA as of 09 October 2023.

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 Loigolactobacillus coryniformis DSM 34345, when used under the proposed conditions of use (see Section 3.1).

Additional information

1.2

The additive is a preparation containing viable cells of Loigolactobacillus coryniformis DSM 34345. 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 Loigolactobacillus coryniformis DSM 34345 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.

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 25 March to 15 April 2024 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 09 October 2023 to 09 January 2024; the comments received 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 active substance in animal feed.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of the additive is in line with the principles laid down in Regulation (EC) No 429/20087 and the relevant guidance documents: Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018a), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018b), 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, 2021), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023).

ASSESSMENT

3

The additive under assessment is a preparation of viable cells of L. coryniformis DSM 34345 to be added to fresh plant material to promote ensiling (category: technological additives, functional group: silage additives) with the eventual use of the silage for all animal species.

Characterisation

3.1

Characterisation of the active agent

3.1.1

The active agent was originally isolated from maize silage and is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) with the accession number DSM 34345.8 The applicant claimed that it has not been genetically modified.9

The taxonomic identification of the strain was done by digital DNA–DNA hybridisation (dDDH) based on the whole genome sequence (WGS) data.10 ■■■■■

The antimicrobial susceptibility of the strain DSM 34345 was tested against the battery of antibiotics recommended by the FEEDAP Panel (EFSA FEEDAP Panel, 2018b) for facultative heterofermentative lactobacilli.11 All the minimum inhibitory concentration values for the strain fell below the corresponding cut‐off values. Therefore, the strain is considered to be susceptible to all relevant antibiotics.

The WGS data of the strain DSM 34345 were interrogated for the presence of antimicrobial resistance (AMR) genes by a search against the ResFinder and NCBI databases.12 No hits of concern were identified exceeding the thresholds recommended by EFSA (EFSA, 2021).

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. This mixture is freeze‐dried and ground to powder. The final feed additive consists of ~ 35%–50% active agent and 50%–65% cryoprotectants/carriers. All the proposed cryoprotectants and carriers are feed materials or authorised feed additives,13 with the exception of ■■■■■ which safety is assessed in the corresponding section (see Section 3.2). The applicant states that no antimicrobial substances are used during the manufacturing process.14

The additive is specified to contain a minimum concentration of active agent of 2 × 10^11^ colony forming unit (CFU)/g of additive. Analysis of five batches of the additive (containing whey powder as carrier) confirmed compliance with specifications of the additive with a mean value of 3.7 × 10^11^ CFU/g (range 2.7–4.3 × 10^11^ CFU/g).15

The analysis of three batches of the additive (containing whey powder as carrier) confirmed compliance with the specifications set by the applicant for Enterobacteriaceae (< 1000 CFU/g), yeasts and filamentous fungi (< 1000 CFU/g) and Salmonella spp. (no detection in 25 g).16

Results of the analyses of three batches of the additive (containing whey powder as carrier) for detection of arsenic, cadmium, mercury, lead, aflatoxins (B1, B2, G1, and G2), zearalenone and deoxynivalenol showed levels below the respective limits of quantification (LOQ) of the analytical methods with the only exception of cadmium, which ranged 0.031–0.035 mg/kg.17 ^,^ 18

The FEEDAP Panel considers that the microbial contamination and the amounts of the detected impurities do not raise safety concerns.

The analysis of three batches of the additive containing whey powder as carrier showed a bulk density of 644–652 kg/m^3^. The dusting potential of the same three batches tested using the Stauber‐Heubach method showed values of 1780–2690 mg/m^3^. The particle size distribution of the additive in the same three batches measured by laser diffraction, showed that ~ 53% of the particles have diameters < 100 μm, 29% < 50 μm and 6% < 10 μm, respectively.19

Stability and homogeneity

3.1.3

The shelf‐life of the additive containing whey powder as carrier (three batches) was tested when stored in its original packaging (Alu‐PE bag) at 20°C for up to 12 months.20 No losses (< 0.5 log of the initial value) were observed under the above‐mentioned conditions.

The stability of the additive in water was studied in three batches with whey powder as carrier. One gram of the additive was suspended in 19 mL of tap water and half of the sample was stored for 48 h at 20°C, while the other half for 7 days at 4°C.21 No losses (< 0.5 log of the initial value) were observed at the end of the respective storage periods.

Conditions of use

3.1.4

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

Safety for the target species, consumers and users

3.2.1

The species L. coryniformis is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA BIOHAZ Panel, 2023). 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. coryniformis and the antibiotic resistance qualification met. Consequently, the active agent L. coryniformis DSM 34345 is considered safe for the target species and consumers.

All the proposed cryoprotectants and carriers are feed materials or authorised feed additives, with the exception of ■■■■■ ■■■■■23 The safety of ■■■■■ in the formulation of another silage additive was already assessed by the FEEDAP Panel in a previous opinion (EFSA FEEDAP Panel, 2018c). The FEEDAP Panel concluded based on a worst‐case scenario that, at this concentration, PEG 4000 is safe for target animals, consumers and users.

Considering all the above, the Panel concludes that the use of preparations formulated with the cryoprotectants and carriers listed by the applicant are safe for the target animals and consumers.

With regard to user safety, no specific studies investigating the effects of the additive on the respiratory system were submitted. The Panel notes that the highest dusting potential measured was 2690 mg/m^3^, suggesting that exposure by inhalation is likely.

The skin irritation potential of L. coryniformis DSM 34345 was investigated in one batch of the additive containing whey powder as carrier in an in vitro test performed according to OECD TG 439.24 The results of the study showed that the test item is classified as non‐irritant to the skin (UN GHS ‘No Category’).

The eye irritation potential of L. coryniformis DSM 34345 was investigated in one batch of the additive containing whey powder as carrier in an in vivo test according to OECD TG 405.25 Based on the results of the study, the test item is considered non‐irritant to eyes.

Therefore, the additive containing whey powder as carrier is not a skin or eye irritant. However, the Panel further notes that once an active agent has been authorised as a technological additive, different preparations can be placed on the market with reference to that authorisation. Consequently, the Panel cannot assess the irritation potential of other possible preparations.

The additive is a preparation containing a microorganism, and therefore should be considered as a potential skin and respiratory sensitiser. Any exposure through skin and respiratory tract is considered a risk.

Safety for the environment

3.2.2

Since the identity of the strain has been established as L. coryniformis and the antibiotic resistance qualification met, the active agent L. coryniformis DSM 34345 is suitable for QPS and considered safe for the environment (EFSA BIOHAZ Panel, 2023).

All the proposed cryoprotectants and carriers are feed material or authorised feed additives, except for PEG 4000. The additive may contain up to 9.1% of PEG 4000 and the FEEDAP Panel considered that its safety for the environment should be assessed.

The applicant provided information consisting of a Phase I environmental risk assessment of PEG 4000 and information obtained from the scientific literature on its biodegradability.

Phase I assessment of PEG 4000

3.2.2.1

Cattle for fattening, followed by dairy cows, are considered the worst‐case scenario species/categories for this assessment. The predicted environmental concentrations (PECs) in soil (PEC_soil_) and groundwater (PEC_gw_) were calculated according to the criteria described in EFSA guidance on environmental risk assessment of feed additives (EFSA FEEDAP Panel, 2019). The calculations conducted by the applicant were based on the concentration of the additive in fresh silages (concentration of the additive in dry matter (DM) silage was not calculated) and under the assumption that the diet of the animals would consist of 100% silage.26 Those calculations were reviewed by the FEEDAP Panel and updated based on the concentration of the additive on DM silage and considering that usually silage constitutes 50%–60% of the animals' diet (the categories mentioned above) on a DM basis.

The physico‐chemical properties of PEG 4000 for Phase I calculation were obtained from the safety data sheet of the substance27 and from the PubChem database and are summarised in Table 1.

Considering that the additive contains the minimum granted concentration of the active agent of 2 × 10^11^ CFU/g of additive, that PEG 4000 represents 9.1% of the additive, and that the additive is used at the proposed minimum concentration of 1 × 10^8^ CFU/kg fresh material, the mg of PEG 4000/kg DM complete feed estimated for cattle for fattening and for dairy cows were 0.060 and 0.066, respectively.28 These concentrations were used to calculate the PEC_soil_ and PEC_gw_ reported in Table 2.

The PEC_soil_ are below the trigger value of 10 μg/kg soil dry weight; therefore, no risk is expected for terrestrial organisms. Considering groundwater, the calculated PEC_gw_ values exceed the trigger value of 0.1 μg/L.

The applicant provided a scientific report by Menzies et al. (2023) in which the ready biodegradability and the inherent biodegradability of PEG 4000 had been tested following OECD 301B and 302B technical guidance, respectively. The results of the study showed more than 80% biodegradation with negligible dissolved organic carbon remaining at study completion. Consequently, PEG 4000 is considered readily biodegradable.

Therefore, considering that PEG 4000 will represent 9.1% of the additive, and the fact that it is readily biodegradable, no safety concerns are expected for both groundwater and aquatic species.

Conclusions on safety for the environment

3.2.2.2

The active agent is considered safe for the environment. No safety concerns are expected for the environment for the ingredients present in the preparation described by the applicant (PEG 4000 used at concentrations up to 9.1% of the additive). Therefore, the silage additive is considered safe for the environment at the proposed conditions of use.

Conclusions on the safety

3.2.3

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, the additive is a preparation containing a microorganism, and therefore should be considered as a potential skin and respiratory sensitiser. Any exposure through skin and respiratory tract is considered a risk. One preparation was shown not to be irritant to skin or eyes. However, the Panel cannot assess the irritation potential of other possible preparations.

Efficacy

3.3

Three laboratory experiments were made with grass samples representing materials easy to ensile (studies 1), moderately difficult to ensile (study 2) and difficult to ensile (study 3) as specified by Regulation (EC) No 429/2008 (Table 3).29 All the studies included a control and a group inoculated with L. coryniformis DSM 34345. The additive was dissolved in water and sprayed on the forage at an intended inclusion rate of 1 × 10^8^ CFU/kg fresh matter (not confirmed by analysis). The fresh material for the control silos was sprayed with an equal volume of water but without the additive. Samples of fresh material were ensiled for 90 days in 6.5 L mini‐silos (study 1) or 1.5 L mini‐silos (study 2 and 3) (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 dry matter (DM), pH, lactic and acetic 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 one‐sided 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 4.

In the three studies, the addition of L. coryniformis DSM 34345 to the fresh grass resulted in lower pH, ammonia‐N production and acetic acid concentration and higher lactic acid concentration in the silage compared to the control. In studies 1 and 3, lower DM loss was observed in the silage supplemented with the additive compared to the control.

Conclusions on efficacy

3.3.1

The use of L. coryniformis DSM 34345 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 Loigolactobacillus coryniformis DSM 34345 and the formulated additive are safe for all animal species, consumers and the environment.

Regarding user safety, the additive should be considered as a potential skin and respiratory sensitiser, and any exposure through skin and respiratory tract is considered a risk. One preparation was shown not to be irritant to skin or eyes. However, the Panel cannot assess the irritation potential of other possible preparations.

The addition of L. coryniformis DSM 34345 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 resistanceCFUcolony forming unitDMdry matterEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedLODlimit of detectionLOQlimit of quantificationMICminimum inhibitory concentrationQPSqualified presumption of safetyWGSwhole genome sequence

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00362

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Maryline Kouba, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Roberto Edoardo Villa, and Ruud Woutersen.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.