# Spotlight on the viral diversity of fermented vegetables using viral metagenomics

Julia Gendre, Claire Le Marrec, Stéphane Chaillou, Sophie Landaud, Eric Dugat-Bony

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Julia Gendre, Claire Le Marrec, Stéphane Chaillou, Sophie Landaud, Eric Dugat-Bony. Spotlight on the viral diversity of fermented vegetables using viral metagenomics. PHAGES in LYON 2023, Nov 2023, Lyon, France. hal-04444032

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Distributed under a Creative Commons Attribution 4.0 International License
# Spotlight on the viral diversity of fermented vegetables using viral metagenomics

Julia Gendre¹ (julia.gendre@inrae.fr), Claire Le Marrec², Stéphane Chaillou³, Sophie Landaud¹, Eric Dugat-Bony¹

Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France; ² UMR 1366 Œnologie, INRAE, Univ. Bordeaux, Bordeaux INP, France; ³ Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France

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## Introduction

Fermented vegetables are usually produced using the natural microorganisms present in the raw material. The process is characterized by a first heterofermentative step, generally carried out by *Leuconostoc mesenteroides*, which acidifies the medium and creates anaerobic conditions. The second homofermentative step is mainly carried out by *Lactiplantibacillus plantarum*. The composition of bacterial and fungal communities present in fermented vegetables (cabbage, carrot, turnip) have been widely characterized, whereas only few studies described the viral communities of these ecosystems.

Several phages infecting different bacterial species were previously isolated from sauerkraut during the fermentation process, which revealed for the first time the complex ecology of phages in this ecosystem¹. Later, viral metagenomic analysis performed on different fermented foods including sauerkraut and kimchi highlighted again that complexity and showed that the vast majority of the sequences originated from uncharacterized phages².

The objective of the present study was to describe the viral composition of commercial fermented vegetables (sauerkraut, fermented carrots and fermented turnips) as a first stone to build future research on the phage ecology in this peculiar ecosystem.

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## Material

<figure><img src="image_5.png" /><figcaption>Fermented carrots (3 batches)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fermented turnips (3 batches)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Method</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Epifluorescence microscopy</figcaption></figure>

<table><thead><tr><th>Epifluorescence microscopy -<br>viral load estimation</th><th>Viral load estimation<br>(phages/mL)</th></tr></thead><tbody><tr><td>Cabbage batch 1 (pH 3,39)</td><td>2.14E+08</td></tr><tr><td>Cabbage batch 2 (pH 3,36)</td><td>1.02E+08</td></tr><tr><td>Cabbage batch (pH 3,53)</td><td>4.47E+07</td></tr><tr><td>Carrot batch 1 (pH 3,34)</td><td>6.16E+07</td></tr><tr><td>Carrot batch 2 (pH 3,84)</td><td>1.43E+08</td></tr><tr><td>Carrot batch 3 (pH 3,47)</td><td>2.70E+07</td></tr><tr><td>Turnip batch 1 (pH 3,67)</td><td>1.27E+08</td></tr><tr><td>Turnip batch 2 (pH 3,55)</td><td>6.93E+07</td></tr><tr><td>Turnip batch 3 (pH 3,30)</td><td>NA</td></tr><tr><td>Control buffer</td><td>4.01E+04</td></tr></tbody></table>

## Viral particles extraction method

<figure><img src="image_9.png" /><figcaption>Extraction and concentration of viral fraction from 10 g of fermented vegetables</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Viral metagenomic data</figcaption></figure>

<table><thead><tr><th>Samples</th><th>No. Contigs assembled >2kb</th><th>Longest contig</th></tr></thead><tbody><tr><td>C1_1D</td><td>308</td><td>31 kb</td></tr><tr><td>C2_1D</td><td>35</td><td>27 kb</td></tr><tr><td>C3_1D</td><td>295</td><td>46 kb</td></tr><tr><td>Eau_CD</td><td>307</td><td>5 kb</td></tr><tr><td>Tampon_CD</td><td>219</td><td>8 kb</td></tr><tr><td>N1D</td><td>611</td><td>33 kb</td></tr><tr><td>N2D</td><td>194</td><td>18 kb</td></tr><tr><td>N3D</td><td>0</td><td>-</td></tr><tr><td>Ct1D</td><td>21</td><td>3 kb</td></tr><tr><td>Ct2D</td><td>169</td><td>49 kb</td></tr><tr><td>Ct3D</td><td>40</td><td>10 kb</td></tr><tr><td>WD (water control)</td><td>11</td><td>2 kb</td></tr><tr><td>BD (buffer control)</td><td>179</td><td>6 kb</td></tr></tbody></table>

<table><thead><tr><th colspan="3">Final dataset</th></tr><tr><th>No. Contigs > 2kb</th><th>No. Clustered contigs</th><th>No. Predicted viral contigs</th></tr></thead><tbody><tr><td>2832</td><td>1293</td><td>541</td></tr></tbody></table>

N3D and Ct1D samples: low sequencing quality and poor assembly statistics compared to other samples.

## Bioinformatic pipeline

<figure><img src="image_11.png" /><figcaption>Virome beta-diversity</figcaption></figure>

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* The first axis helps discriminating samples according to the vegetable

* Samples N3D and Ct1D cluster together (low quality samples)

Viral composition depending on the plant matrix

<figure><img src="image_13.png" /><figcaption>Viral composition</figcaption></figure>

### 3 contig groups

1. Contigs unique to two carrots samples (cluster 3)

2. Contigs common to most samples

3. Contigs mostly abundant in cabbage samples (cluster 1)

<table><caption>Most abundant viral contigs</caption><thead><tr><th>Contigs identity</th><th>Infection cycle</th><th>Contig quality</th><th>Host order</th><th>Host family</th><th>Host genius</th><th>score_iphop</th></tr></thead><tbody><tr><td>Ct1D_125k_NODE_1_length_2064</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lactiplantibacillus</td><td>94.0</td></tr><tr><td>Ct2R_125k_NODE_1_length_41237</td><td>-</td><td>-</td><td>Enterobacterales</td><td>Enterobacteriaceae</td><td>Rahnella</td><td>91.4</td></tr><tr><td>Ct1_1D_25k_NODE_2_length_23337</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lentilactobacillus</td><td>91.4</td></tr><tr><td>Ct2D_250k_NODE_59_length_2554</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C2_1D_250k_NODE_1_length_27078</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lacticaseibacillus</td><td>99.4</td></tr><tr><td>C3_1D_25k_NODE_2_length_16738</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lacticaseibacillus</td><td>99.4</td></tr><tr><td>N2D_125k_NODE_42_length_2495</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C3_1D_25k_NODE_3_length_5026</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lentilactobacillus</td><td>91.1</td></tr><tr><td>C2_1D_250k_NODE_2_length_13954</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lacticaseibacillus</td><td>96.6</td></tr><tr><td>C1_1D_25k_NODE_1_length_31726</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lentilactobacillus</td><td>91.6</td></tr><tr><td>C3_1D_125k_NODE_58_length_3209</td><td>-</td><td>-</td><td>Lactobacillales</td><td>Lactobacillaceae</td><td>Lentilactobacillus</td><td>91.6</td></tr></tbody></table>

The most abundant contigs are different between samples

The hosts for these contigs, when predicted, correspond to bacteria usually found during the late stage of the fermentation process. Phages infecting *Leuconostoc* were also detected but at a lower abundance (not present in the top 10)

## Conclusion and perspectives

This study provides an overview of the composition of viral communities in different commercial fermented vegetables, with a particular focus on extracellular particles (virion forms). We identify phages in high abundance that infect bacteria belonging to genera found in the late stage of the fermentation process (e.g. *Lactiplantibacillus*, *Lentilactobacillus*,...), and at a lower abundance phages infecting bacteria of the early stage (e.g. *Leuconostoc*) or spoilage bacteria (e.g. *Enterobacter*). Our results suggest that several phages infect most of the dominant lactic acid bacteria involved in the fermentation process and, therefore, may impact the bacterial successions occurring in this system. Since phages are likely to modify fermentation kinetics and consequently the quality of end-products, it is now necessary to better understand their individual ecological roles and ultimately set-up technological strategies to better control their activity and secure fermentation.

1. Lu et al., *Bacteriophage Ecology in Commercial Sauerkraut Fermentations*, 2003, DOI: 10.1128/AEM.69.6.3192-3202.2003

2. Park et al., *Metagenomic Analysis of the Viral Communities in Fermented Foods*, 2011, DOI:10.1128/AEM.01859-10

Funding: This work was funded by the Agence Nationale de la Recherche (ANR) through the MetaSimFood project (ANR-21-CE21-0003).

INRAE

AgroParisTech

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université PARIS-SACLAY

SayFood

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metasimfood anr® agence nationale de la recherche

Unité de recherche œnologie EA4577 - USC 1366 INRA

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Ecole Doctorale ABIES

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