Safety and efficacy of a feed additive consisting of Pediococcus pentosaceusIMI 507024 for all animal species (ALL-TECHNOLOGY (IRELAND) LIMITED [Alltech Ireland]).

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 and efficacy of Pediococcus pentosaceus IMI 507024 as a technological additive for all animal species. The additive is intended to improve the production of silage at a proposed application rate of 1 × 109 colony forming units (CFU)/kg fresh material. The bacterial species P. pentosaceus is considered by EFSA to be suitable for the qualified presumption of safety approach. As the identity of the strain has been established and no antimicrobial resistance determinants of concern were detected, the use of the strain as a silage additive is considered safe for livestock species, for consumers and for the environment. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be a skin/eye irritant or a skin sensitiser. Given the proteinaceous nature of the active agent, the additive should be considered a respiratory sensitiser. The additive at the proposed application rate of 1 × 109 CFU/kg fresh material has the potential to improve the fermentation of the silages from easy to moderately difficult to ensile forages.

Introduction

Background and Terms of Reference as provided by the requestor

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 ALL‐TECHNOLOGY (IRELAND) LIMITED [Alltech Ireland]2 for the authorisation of the product Pediococcus pentosaceus IMI 507024, 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 particulars and documents in support of the application were considered valid by EFSA as of 7 January 2021.

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 product Pediococcus pentosaceus IMI 507024, when used under the proposed conditions of use (see Section 3.1.4).

Additional information

The additive is a preparation containing viable cells of Pediococcus pentosaceus IMI 507024. It has not been previously authorised as a feed additive in the European Union.

Data and methodologies

Data

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 Pediococcus pentosaceus IMI 507024 as a feed additive.

EFSA has verified the European Union Reference Laboratory (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 Annex A.4

Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of Pediococcus pentosaceus IMI 507024 is in line with the principles laid down in Regulation (EC) No 429/20085 and the relevant guidance documents: Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017a), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019) and Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018b).

Assessment

The product under assessment is a preparation of viable cells of Pediococcus pentosaceus IMI 507024 intended for use as a technological additive (functional group: silage additives) in easy and moderately difficult to ensile forages for all animal species.

Characterisation

Characterisation of the active agent

The active agent was originally isolated from fermented sausage. It is deposited in the Centre for Agriculture and Bioscience International culture collection, formerly International Mycological Institute, CABI‐IMI Culture collection, with the accession number IMI 507024.6 It has not been genetically modified.

The taxonomical identification was confirmed with alignment‐free genome distance estimation with Mash using MinHash and OrthoANI to calculate in silico average nucleotide identity (ANI) based on the whole genome sequence. Results showed that P. pentosaceus CGMCC 7049 was the closest matching NCBI RefSeq genome with a Mash distance of 0.00671909 and a calculated OrthoANI value of 98.88% with P. pentosaceus ATCC 25745, respectively.7

The bacterial strain was tested for antibiotic susceptibility using a broth microdilution method.8 The antibiotics used included those recommended by EFSA (EFSA FEEDAP Panel, 2018a). All the minimum inhibitory concentration (MIC) values were equal or fell below the corresponding cut‐off values for Pediococcus spp., except for kanamycin (MIC: 128 mg/L vs cut‐off value: 64 mg/L), streptomycin (MIC: 128 mg/L vs cut‐off value: 64 mg/L), chloramphenicol (MIC: 8 mg/L vs cut‐off value: 4 mg/L) and tetracycline (MIC: 64 mg/L vs cut‐off value: 8 mg/L). 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 susceptible to all the relevant antibiotics except for tetracycline.

The whole genome sequence of the strain was searched for antibiotic resistance genes using the ABRicate tool with thresholds of 70% for both identity and coverage, at nucleotide and protein level.7 The databases used were ARG‐ANNOT, MEGARes, NCBI Bacterial Antimicrobial Resistance Reference Gene Database and ResFinder. No hits of concern were identified.

No resistance determinants were found to justify the tetracycline resistance, thus the Panel considers that it is unlikely that this resistance may raise safety concerns.

Characterisation of the additive

The inoculum of the active agent is prepared in ■■■■■ to guarantee a minimum concentration of active agent of 1 × 1010 CFU/g of additive.

Analysis of five batches showed a mean value of 1.5 × 1011 CFU/g (range 1.3–1.8  ×  1011 CFU/g).9

A total of four batches were analysed for microbiological contamination and mycotoxins, heavy metals and arsenic concentrations. Regarding the specifications for the microbiological contaminants, limits are set for total coliforms (1,000 CFU/g), β‐glucuronidase‐positive Escherichia coli (100 CFU/g), coagulase‐positive staphylococci (including Staphylococcus aureus) (10 CFU/g), Salmonella spp. (no detection in 25 g), Listeria monocytogenes (no detection in 25 g), Clostridium perfringens (100 CFU/g), anaerobic sulfite reducers (100 CFU/g), yeasts (1,000 CFU/g) and filamentous fungi (1,000 CFU/g). Analysis of four batches of the additive showed compliance with these limits. The same batches of the additive were tested for aflatoxins (B1, B2, G1, and G2), ochratoxin A, fumonisins B1+B2, HT‐2 toxin, T‐2 toxin, deoxynivalenol, zearalenone, lead, mercury, cadmium and arsenic; results showed levels below the respective limits of detection, except for arsenic (average 0.043 mg/kg, range 0.041–0.047 mg/kg) and lead (average 0.026 mg/kg, range 0.013–0.041 mg/kg).10 , 11 The levels of the detected impurities do not raise concerns.

The additive has an average density of 1,292 kg/m3 (all three batches the same value) and an average bulk density of 411 kg/m3 (all three batches the same value).12 The dusting potential of the additive was measured in three batches (Stauber–Heubach) and showed a mean value of 8.58 g/m3 (range: 6.52–10.39 g/m3).12 The same three batches were tested for particle size distribution by laser diffraction; results showed that approximately 21% of the additive consists of particles with diameters below 100 μm, 15% below 50 μm and 8% below 10 μm.12

Stability

Four batches of the additive were tested for shelf‐life by storing in sealed aluminium foil bags at 4°C for 3 months,13 at 25°C for 2 months14 and at 30°C for 3 months.15 Negligible losses were observed under the above mentioned conditions (< 0.5 log of the initial value).

The stability in water was studied by suspending 1 g of the additive (one batch) in 1 L of water and then storing for 48 h at 4°C and 20°C. Negligible losses were observed for both temperatures tested, with < 0.5 log of the initial value.16

Conditions of use

The additive is intended for use in easy and moderately difficult to ensile forages at a proposed minimum inclusion level of 1 × 109 CFU/kg fresh material for all animal species. It is to be applied as such or as an aqueous suspension.

Safety

Safety for the target species, consumer and the environment

The species P. pentosaceus is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA, 2007; EFSA BIOHAZ Panel, 2020). This approach requires the identity of the strain to be conclusively established and evidence that the strain lacks acquired determinants for resistance to antibiotics of human and veterinary importance. In the view of the FEEDAP Panel, the identity of the strain was established as P. pentosaceus and the antibiotic resistance qualification has been met. Consequently, Pediococcus pentosaceus IMI 507024 is presumed safe for the target species, consumers and the environment.

Safety for user

No studies were submitted regarding the effects of the additive to the respiratory tract, skin or eyes.

The dusting potential reported is high (8.6 g/m3), thus exposure by inhalation is possible. Owing to the proteinaceous nature of the active agent, the additive should be considered a respiratory sensitiser.

Once an active agent has been authorised as a silage additive, different formulations can be placed on the market with reference to that authorisation. For this specific product, the excipients used in the preparation of the final formulation are not expected to introduce additional risks.

Conclusions on safety for user

The additive should be considered a respiratory sensitiser. No conclusions can be drawn on the potential of the additive to cause skin and eye irritation or skin sensitisation.

Efficacy

Five laboratory studies were conducted with different forages representing materials easy to ensile (study 1, 2 and 3)17 and moderately difficult to ensile (study 417 and 518), as specified by Regulation (EC) No 429/2008 (Table 1). All the studies included a control group and a group in which Pediococcus pentosaceus IMI 507024 was applied to the forage at a concentration of 1 × 109 CFU/kg of fresh forage. Analytical confirmation of the counts of the two batches of the additive used for the studies was provided. An aqueous suspension of the mixture was prepared and then sprayed onto the forage prior to ensiling. In the control silos, the same volume of water without the additive was added. In studies 1–3 and 5, forage was ensiled for 90 days in mini‐silos (five replicates per treatment) with a capacity of 1.75 L with the potential to vent gas. In study 4, forage was ensiled for 100 days in mini‐silos (four replicates per treatment) with a capacity of 20 L and a device to vent gas. All experiments were conducted at 20 ± 1°C.

Table 1

Characteristics of the forage samples used in the five ensiling experiments

Study	Test material	Dry matter content (%)	Water‐soluble carbohydrate content (% fresh matter)
1	Grass‐clover (2nd cut)a	40.1	5.3
2	Grass‐clover (2nd cut)a	39.8	3.4
3	Grass‐clover (2nd cut)a	27.1	3.6
4	Meadow grassb	26.5	2.2
5	Grass‐clover (2nd cut)a	24.0	2.1

Grass‐clover consisting of timothy (Phleum pratense), perennial ryegrass (Lolium perenne), meadow fescue (Festuca pratensis), red clover (Trifolium pratense) and white clover (Trifolium repens).

Meadow grass consisting of Italian ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), white clover (Trifolium repens), common dandelion (Taraxacum officinale), ribwort plantain (Plantago lanceolate) and common vetch (Vicia sativa).

After 90 days (or 100 days for study 4), the silos were opened and the contents were analysed for dry matter (DM), pH, lactic, acetic and propionic acids and ethanol concentrations, and ammonia. Aerobic stability was assessed at the end of each experiment by taking samples from each silo and exposing to air with continuous monitoring of temperature. A rise of 3°C above room temperature was considered as an indicator of silage deterioration, and the time at which that rise was observed was taken as a measure of the aerobic stability of treated and control silages. A minimum increase of stability of the treated silage of two days compared to that shown by the untreated control is considered as evidence of aerobic stability.

Data were analysed using the non‐parametric Wilcoxon signed‐rank test (studies 1–3 and 5) or Mann–Whitney (study 4)19 and significance for all studies was declared at p < 0.05. Results are shown in Table 2.

Table 2

Summary of the analysis of ensiled material recovered at the end of the ensiling period with Pediococcus pentosaceus IMI 507024

Study	Application rate (CFU/kg forage)	Dry matter (DM) loss (%)	pH	Lactic acid (%)a	Acetic acid (%)a	Ammonia‐N (% of total N)	Aerobic stability (h)
1	0	0.9	5.10	2.1	0.3	4.5	171
	1 × 109	0.6*	4.16*	3.6	0.2	3.4	131
2	0	0.9	4.81	2.6	0.6	6.6	230
	1 × 109	0.5*	4.17*	3.7*	0.2*	4.2*	144
3	0	1.1	4.43	2.8	0.4	10.1	207
	1 × 109	0.4*	3.97*	3.5*	0.1	5.7*	67*
4	0	2.8	3.89	7.3	1.3	6.4	57
	1 × 109	2.6	3.88	7.3	1.3	6.2	107*
5	0	0.8	4.25	2.9	0.5	9.4	218
	1 × 109	0.4*	3.96*	3.4	0.2*	6.4*	126*

CFU: colony forming unit; DM: dry matter.

Expressed as percentage in silage juice for studies 1, 2, 3 and 5 and in percentage of dry matter for study 4.

Means in a column within a given trial are significantly different to the control p < 0.05.

The addition of the additive resulted in a significant reduction of dry matter loss and pH in all easy to ensile forages and in one study with moderately difficult to ensile forages, and of ammonia‐N production in two studies (studies 2 and 3) with easy to ensile and in one study with moderately difficult to ensile forages. In addition, in two (2 and 3) out of three studies with easy to ensile forages a significant increase of lactic acid was observed. Regarding the aerobic stability, a positive outcome was observed only in study 4.

Conclusions on efficacy

The use of Pediococcus pentosaceus IMI 507024 at the proposed inclusion rate in the ensiling process has the potential to improve the production of silage with easy and moderately difficult to ensile materials by enhancing the preservation of nutrients.

Conclusions

Based on the QPS approach to safety assessment, Pediococcus pentosaceus IMI 507024 is presumed safe for the target species, consumers and the environment.

The additive should be considered a respiratory sensitiser. No conclusions can be drawn on the eye and skin irritancy, or skin sensitisation potential of the additive.

Pediococcus pentosaceus IMI 507024 at a concentration of 1 × 109 CFU/kg plant material has the potential to improve the preservation of nutrients in silage prepared with easy and moderately difficult to ensile material.

Documentation as provided to EFSA/Chronology

Abbreviations

average nucleotide identity

colony forming unit

coefficient of variation

dry matter

European Union Reference Laboratory

EFSA Panel on Additives and Products or Substances used in Animal Feed

minimum inhibitory concentration

pulsed‐field gel electrophoresis

qualified presumption of safety

In the current applications authorisations are sought under Article 4(1) for Pediococcus pentosaceus IMI 507024 (FAD 2020‐0076) and for Pediococcus pentosaceus IMI 507025 (FAD 2020‐0077) under the category/functional group 1(k) “technological additives”/“silage additives”, according to Annex I of Regulation (EC) No 1831/2003. Specifically, the authorisations are sought for the use of the feed additives in silage for all animal species.

According to the Applicant, the feed additives contain as active substance viable cells of the strains Pediococcus pentosaceus IMI 507024 or Pediococcus pentosaceus IMI 507025. The feed additives are to be marketed as preparations with a minimum content of 1 × 1010 Colony Forming Units (CFU) of Pediococcus pentosaceus IMI 507024 or Pediococcus pentosaceus IMI 507025/g feed additive. Both feed additives are intended to be used at a minimum dose of 1 × 106 CFU/kg fresh silage.21

For the identification of Pediococcus pentosaceus IMI 507024 and Pediococcus pentosaceus IMI 507025, the EURL recommends for official control Pulsed‐Field Gel Electrophoresis (PFGE), a generally recognised methodology for the genetic identification of bacterial strains.

For the enumeration of Pediococcus pentosaceus IMI 507024 and Pediococcus pentosaceus IMI 507025 in the feed additives, the EURL recommends for official control the ring‐trial validated spread plate method EN 15786.

Since the unambiguous determination of the content of Pediococcus pentosaceus IMI 507024 or Pediococcus pentosaceus IMI 507025 initially added to silage is not achievable by analysis, the EURL cannot evaluate nor recommend any method for official control for the determination of Pediococcus pentosaceus IMI 507024 or Pediococcus pentosaceus IMI 507025 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, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Date	Event
14/10/2020	Dossier received by EFSA. Pediococcus pentosaceus IMI 507024. Submitted by ALL‐TECHNOLOGY (IRELAND)
19/10/2020	Reception mandate from the European Commission
07/01/2021	Application validated by EFSA – Start of the scientific assessment
11/02/2021	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: characterisation, efficacy
09/04/2021	Comments received from Member States
16/03/2021	Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives
07/04/2021	Reception of supplementary information from the applicant ‐ Scientific assessment re‐started
23/06/2021	Opinion adopted by the FEEDAP Panel. End of the Scientific assessment