# Positive interactions between lactic acid bacteria promoted by nitrogen-based nutritional dependencies

Fanny Canon, Marie-Bernadette Maillard, Gwénaële Henry, Anne Thierry, Valérie Gagnaire

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Fanny Canon, Marie-Bernadette Maillard, Gwénaële Henry, Anne Thierry, Valérie Gagnaire. Positive interactions between lactic acid bacteria promoted by nitrogen-based nutritional dependencies. Applied and Environmental Microbiology, 2021, 10.1128/AEM.01055-21. hal-03330070

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1 **Positive interactions between lactic acid bacteria promoted by nitrogen-**
2 **based nutritional dependencies**

3 Fanny Canon¹, Marie-Bernadette Maillard¹, Gwénaële Henry¹, Anne Thierry¹, Valérie

4 Gagnaire¹*

5 ¹UMR STLO, INRAE, Institut Agro, F35000, Rennes, France

6 *valerie.gagnaire@inrae.fr

7 Abstract

8 Nutritional dependencies, especially those regarding nitrogen sources, govern numerous
9 microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary,
10 organoleptic, and health properties of most fermented products, such positive interactions
11 have previously been studied between yogurt bacteria. However, they have never been
12 exploited to create artificial co-cultures of LAB that would not necessarily coexist naturally,
13 i.e from different origins. The objective of this study was to promote LAB positive
14 interactions, based on nitrogen dependencies in co-cultures, and to investigate how these
15 interactions affect some functional outputs, e.g. acidification rates, carbohydrate consumption,
16 and volatile compound production. The strategy was to exploit both proteolytic activities and
17 amino acid auxotrophies of LAB. A chemically defined medium was thus developed to
18 specifically allow the growth of six strains used, three proteolytic and three non-proteolytic.
19 Each of the proteolytic strains, *Enterococcus faecalis* CIRM-BIA2412, *Lactococcus lactis*
20 NCDO2125, and CIRM-BIA244, was co-cultured with each one of the non-proteolytic LAB
21 strains: *L. lactis* NCDO2111, *Lactiplantibacillus plantarum* CIRM-BIA465 and CIRM-
22 BIA1524. Bacterial growth was monitored using compartmented chambers to compare
23 growth in mono- and co-cultures. Acidification, carbohydrate consumption and volatile
compound production was evaluated in direct co-cultures. Each proteolytic strain induced different types of interactions: either strongly positive, weakly positive, or no interactions, with *E. faecalis* CIRM-BIA2412, *L. lactis* NCDO2125 and *L. lactis* CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations in tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to faster acidification rates, lower pH, higher raffinose utilization and concentrations in five volatile compounds.

### 30 **Importance:**

Lactic acid bacteria (LAB) interactions are often studied in association with yeasts or propionibacteria in various fermented food products and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigates the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and non-proteolytic strains. The results suggest that proteolytic LAB do not equally stimulate non-proteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guaranty their positive interactions.

**Keywords:** lactic acid bacteria, positive interactions, commensalism, nutritional dependency, nitrogen nutrition, functional outputs
45 **1 Introduction**

46 Lactic acid bacteria (LAB) are the most prevalent bacterial actors in fermented foods
47 consumed in Western countries (1). LAB can produce a variety of compounds including weak
48 organic acids, e.g. lactic and acetic acids, aroma compounds such as diacetyl, amino acids,
49 peptides, exopolysaccharides, vitamins, as well as hydrolytic enzymes, hydrogen peroxide
50 and bacteriocins, during fermentations (2). These compounds provide fermented foods with
51 varied desirable functional outputs such as organoleptic, sanitary, nutritional, probiotic, and
52 health properties (3–6). However, their production in LAB-fermented foods is both species-
53 and strain-dependant and, consequently, the “super strain” that would produce all the
54 expected metabolites does not exist (7). To increase the functional outputs, we need to find
55 efficient ways to associate strains with complementary properties in an artificial co-culture,
56 defined as an association of microorganisms that may not necessarily be found in nature (8).
57 Co-culture can also increase substrate conversion, yields and microbial fitness, in particular
58 when microorganisms interact positively with each other, either through commensalism,
59 cooperation, mutualism, or syntrophy (8).

60 The exploitation of nutritional dependencies seems to be one the most promising way for
61 LAB to interact in co-culture. The exchange of nitrogen compounds is of particular interest
62 either as public goods, defined as the pool of molecules available in the medium (9), or
63 through cross-feeding, *i.e* the phenomenon by which one microorganism takes in a primary
64 substrate and converts it into a product excreted as a public good (10). For example,
65 dependencies based on nitrogen nutrients have been observed between the yeast
66 *Saccharomyces cerevisiae* and the LAB *Lactococcus lactis* (11), as well as between the two
67 LAB species, used as a prime example, *Streptococcus salivarius* subsp. *thermophilus* and
68 *Lactobacillus delbrueckii* subsp. *bulgaricus*, associated in yogurt (12). In milk fermentation,
69 the sharing of extracellular protease activity, especially by LAB species such as *L. lactis*, is
paramount to ensure microbial interactions in cheese and fermented milks (13). Protease activity is unevenly distributed among the strains, rendering the growth of the strains that lack this activity dependent on the composition of the medium in peptides and free amino acids.

LAB co-cultures were recently shown to efficiently ferment mixes of milk and plant-based substrates (14, 15) to meet the growing need of new substrate use in the context of food transition. Up to now, most LAB strains were mainly selected from dairy applications, but we need to find more suitable candidates to ferment plant-sourced substrates as well. Efficient co-cultures of LAB strains adapted to both dairy and plant-based resources has been achieved by our team in a previous work in which functional complementarity of selected LAB strains were exploited to design co-cultures able to ferment mixes of milk and lupin (15). More specifically, we associated non-proteolytic strains able to degrade specific carbohydrates with proteolytic strains, the latter being expected to provide the former with nitrogen nutrients. As the culture medium was initially rich in nitrogen compounds, and four to six strains were associated in co-culture, this complicated the study of the nitrogen-related interactions between the LAB.

The objective of this study was to promote LAB positive interactions based on nitrogen dependencies in artificial co-cultures and to investigate the possible functional outputs of such interactions. For this, LAB strains were deliberately associated in a model medium mimicking a mix of milk and plant-based substrate. We selected two types of strains from various dairy and non-dairy/vegetable origins. The first ones, referred as donor strains, were selected for their proteolytic activity and their contribution to the development of flavor. *Lactococcus lactis* and *Enterococcus faecalis* were found to be adequate candidates. Both species possess a very efficient proteolytic system and can modify sensorial profiles in cheeses and other fermented dairy products (16, 17). *E. faecalis* proteolytic system was even shown to be
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responsible for a decrease of allergenicity of bovine milk proteins (18). The second type of strains, referred to as receiver strains, were selected, in contrast, for their lack of proteolytic activity and their ability to consume raffinose-family oligosaccharides, which are responsible for intestinal discomfort. Strains of *L. lactis* and *Lactiplantibacillus plantarum* that were previously shown to exhibit this capacity (15) were selected for the present study (Figure 1).

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In the present study, we first developed a chemically defined medium that contained all necessary vitamins, minerals, nucleic acids, as well as the proteins and carbohydrates of bovine milk and lupin. Second, we associated a donor strain with a receiver one. We used either compartmented chambers or direct co-cultures to study LAB interactions and compared them to monocultures. The present study demonstrated that it is possible to promote positive interactions based on nutritional dependencies between LAB strains by exploiting both their proteolytic activities and their amino acid auxotrophies. Regarding the functional outputs that result from LAB interactions, we investigated in particular the acidification rates to ensure safety, RFO consumption to reduce intestinal discomfort, and production of aroma compounds to improve sensory properties. The results indicate that different proteolytic LAB do not equally stimulate non-proteolytic LAB. They further suggest that the stronger the interactions between LAB are, the more functional outputs we can expect.

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## 2 Results

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### 2.1 Selection of the proteolytic (donor) and non-proteolytic (receiver) strains and validation of a chemically defined medium to study LAB interactions

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The donor strains were selected according to their proteolytic profiles, i.e the NH₂-containing compounds they provided, when grown in the chemically defined medium that contained casein and/or lupin proteins as sole nitrogen source (CDM PROT). The concentrations of free amino acids (FAA) and peptides in the medium after fermentation by each of the donor
118 strains are illustrated in **Figure 2**. FAA and peptides resulted from the hydrolysis of casein
119 and/or lupin proteins present in the CDM PROT. *E. faecalis* CIRM-BIA2412 (Efa2412),
120 appeared as the most proteolytic strain, with proteolytic indices (PI) of 10.8 % ± 0.1 versus
121 5.5 % ± 0.1 and 3.6 % ± 0.3 for *L. lactis* NCDO2125 (Lla2125) and *L. lactis* CIRM-BIA244
122 (Lla244), respectively.

123 The concentration of total FAA in CDM PROT increased from 4.8 mg/L initially to 250, 261,
124 and 155 mg/L after fermentation by Efa2412, Lla2125 and Lla244, respectively. The
125 concentration of peptides released by Efa2412 (48.8 mg NH₂/L) was significantly higher,
126 compared to that released by the two other donor strains Lla244 (5.8 mg NH₂/L) and Lla2125
127 (4.0 mg NH₂/L). Among all the free NH₂ groups available in medium after the fermentation
128 by Efa2412, Lla2125 and Lla244, peptides represented 59.4%, 9.6% and 20.7%, for each
129 donor strain respectively.

130 **Figure 3** shows that the three donor strains, Efa2412, Lla2125 and Lla244 in monocultures
131 (dotted lines, lower panel) grew well in the CDM PROT medium, reaching a population of
132 9.1 ± 0.2, 9.0 ± 0.1 and 9.3 ± 0.1 log(CFU/mL), respectively, after 24 h of culture. In contrast,
133 the three strains *L. lactis* NCDO2111 (Lla450), *L. plantarum* CIRM-BIA465 (Lpl465) and *L.
134 plantarum* CIRM-BIA1524 (Lpl1524) did not grow in CDM PROT in monocultures (dotted
135 lines, upper panel). They maintained their cultivable population at the initial level of
136 inoculation after 24 h of culture, and the pH of the medium remained steady.

137 To validate that the factor limiting the growth of the three non-growing strains was the
138 nitrogen source, we added 0.5 g/L of casein tryptone, which supplies casein peptides and free
139 amino acids, in the CDM PROT medium, and referred as CDM TRYP. Growth of the strains
140 Lla450, Lpl465 and Lpl1524 was then restored after 24 h as their cultivable population
reached 9.1 ± 0.1, 8.8 ± 0.2, and 8.9 ± 0.1 log(CFU/mL), respectively. These three strains were then considered as non-proteolytic and referred to as receiver strains.

The CDM TRYP, in which all receiver strains grew, contained 108.9 mg/L of FAA and a peptide concentration estimated at 7.7 mg NH₂/L (Figure 2). It was therefore considered as a positive control for supporting receiver strain growth. The total nitrogen content of CDM TRYP was lower compared to that of the three donor strains. Thus, at first glance, all three donor strains would be capable of providing the receiver strains enough nitrogen compounds for their growth.

## 2.2 Bacterial growth in co-cultures

The maximal counts of the three donor strains Efa2412, Lla2125 and Lla244 were not impacted by the mode of culture, neither mono nor co-culture (Figure 3). They reached in 14 h a plateau of 9.0 ± 0.2 log(CFU/mL) in 8 h, 9.0 ± 0.2 log(CFU/mL) in 12 h, and 9.2 ± 0.1 log(CFU/mL), respectively. However, the population of Lla2125 decreased significantly after reaching the plateau in co-culture with Lla450 and Lpl465.

In co-culture with Efa2412, the counts of Lla450, Lpl465 and Lpl1524 reached a maximum of 9.0 ± 0.1 log(CFU/mL) within 10 h, 8.9 ± 0.3 log(CFU/mL) in 22 h and 9.0 ± 0.1 log(CFU/mL) in 24 h culture, respectively (Figure 3). In co-culture with Lla2125, the counts of Lla450, Lpl465 and Lpl1524 reached a maximum of 8.8 ± 0.2 log(CFU/mL) in 20 h, 8.5 ± 0.6 log(CFU/mL) in 22 h and 8.4 ± 0.2 log(CFU/mL) in 24 h, respectively. The three receivers grew at a higher rate in co-culture with the donor strain Efa2412 compared to the donor strain Lla2125. In co-culture with Lla244, the counts of Lpl1524 remained at the inoculation level of 7.0 ± 0.4 log(CFU/mL), whereas the counts of Lla450 and Lpl465 decreased to 6.5 ± 0.1 log(CFU/mL) (Figure 3).
To summarize, three types of interactions were observed between the receiver strains and the donor strains, according to the donor strain: strong interactions with Efa2412, weak interactions with Lla2125, and no interaction with Lla244.

We checked that bacterial growth was equivalent in co-cultures performed in compartmented chambers and in direct co-cultures. We chose the specific incubation time of 14 h for this verification because it corresponded to the end of exponential growth phase of the donor strains and the middle of the exponential growth phase of the *L. plantarum* receiver strains. This receiver species was chosen to facilitate count in direct culture on both different plate media for donor and receiver. No significant differences were observed between the bacterial counts obtained in direct co-cultures and in compartmented chambers. Therefore, we chose to use direct co-cultures to study the impact of co-cultivation on acidification, carbohydrates hydrolysis, and volatile profiles, for the rest of the study.

## 2.3 Acidification parameters were influenced by the type of the interactions and the intrinsic capacities of the receiver strains

**Table 1.** Maximal acidification rates (dpH/h expressed as absolute values), and pH of CDM PROT after 24 h culture of LAB strains.

<table><thead><tr><th>Receiver strain</th><th colspan="4">Donor strain</th></tr><tr><th></th><th>No</th><th>Efa2412</th><th>Lla2125</th><th>Lla244</th></tr></thead><tbody><tr><td colspan="5">Maximal acidification rates (dpH/h)</td></tr><tr><td>No</td><td>-</td><td>0.60<sup>cd</sup></td><td>1.14<sup>a</sup></td><td>1.05<sup>ab</sup></td></tr><tr><td>Lla450</td><td>0.53<sup>d</sup></td><td>0.99<sup>b</sup></td><td>0.97<sup>b</sup></td><td>1.04<sup>ab</sup></td></tr><tr><td>Lpl465</td><td>0.35<sup>e</sup></td><td>0.69<sup>cd</sup></td><td>1.11<sup>ab</sup></td><td>1.01<sup>ab</sup></td></tr><tr><td>Lpl1524</td><td>0.21<sup>e</sup></td><td>0.69<sup>c</sup></td><td>1.09<sup>ab</sup></td><td>1.02<sup>ab</sup></td></tr><tr><td colspan="5">Final pH after 24 h</td></tr><tr><td>No</td><td>-</td><td>4.20<sup>gh</sup></td><td>4.12<sup>f</sup></td><td>3.94<sup>ab</sup></td></tr><tr><td>Lla450</td><td>4.16<sup>g</sup></td><td>4.07<sup>de</sup></td><td>4.10<sup>ef</sup></td><td>3.94<sup>ab</sup></td></tr><tr><td>Lpl465</td><td>4.22<sup>h</sup></td><td>3.96<sup>b</sup></td><td>4.04<sup>cd</sup></td><td>3.93<sup>ab</sup></td></tr></tbody></table>
<table><tr><td>Lpl1524</td><td>4.55<sup>i</sup></td><td>4.01<sup>c</sup></td><td>4.08<sup>def</sup></td><td>3.91<sup>a</sup></td></tr></table>

181 Donor strains Lla244, Lla2125 and Efa2412 were incubated as monocultures and in co-cultures with Lla450, Lpl465 or Lpl1524. Results for the LAB receiver strains Lla450, Lpl465, Lpl1524 in monoculture were obtained in the CDM TRYP, which corresponds to CDM PROT supplemented with 0.5 g/L of casein tryptone. For strain codes see Table 4. Values followed by different letters indicate significant differences between cultures (p < 0.05).

186 The acidification rates of the different LAB strains in mono- and co-cultures are shown in Table 1 as well as the final pH observed after 24 h of fermentation. In monoculture in CDM PROT, the donor strains Lla2125 and Lla244 showed the fastest acidification, compared to Efa2412. Among the receiver strains, Lla450 showed a faster acidification than Lpl465 and Lpl1524.

191 The acidification rates of the co-cultures depended on the receiver strains associated with the donor strains. The two *L. plantarum* strains did not impact the acidification rates of the donor strains with which they were co-cultured. On the contrary, different acidification rates were observed for Lla450 depending on the associated donor strains. Lla450 induced a +65% increase of the maximal acidification rate in co-culture with Efa2412, while a 15% decrease was observed with Lla2125 and no change of the acidification rate in co-culture with Lla244.
197 The final pH in monocultures ranged from 3.94 to 4.55 for Lla244 and Lpl1524, respectively (Table 1). In the three co-cultures with the donor strain Efa2412, the final pH was significantly lower compared to that of the donor strain in monoculture, in particular with the two *L. plantarum* strains. Considering the donor strain Lla2125, only its co-culture with Lpl465 significantly decreased the final pH, when compared to the monoculture of the donor strain. Concerning the donor strain Lla244, the final pH did not differ in mono- and co-cultures.

## 2.4 The type of interactions modulates the carbohydrate consumption

205 The consumption of milk carbohydrates, i.e. lactose, and lupin carbohydrates, i.e. raffinose and sucrose are shown in Figure 4. Lactose was hydrolysed by all strains except Lla450, in
agreement with the results observed using API gallery (see material and method section). The
consumption of lactose in the co-cultures with the donor strain Efa2412 significantly varied
depending on the associated receiver strain. 72 % of lactose was consumed with Efa2412
alone, against 30%, 62 %, and 87%, in its co-culture with Lla450, Lpl465 and Lpl1524,
respectively. Lactose consumption was less impacted by the co-cultures involving the donor
strain Lla2125. There was significantly less lactose consumed when Lla2125 was co-cultured
with Lla450 (81 % of lactose consumed in co-culture against 90% with Lla2125 in
monoculture). No significant differences were observed in the co-cultures involving Lla2125
and the two *L. plantarum* strains. The co-cultures involving the donor strain Lla244 did not
change the percentage of lactose consumed (65%).

Sucrose was only consumed by the strains Lla450 (82 %), Lpl465 (72 %) and Lla244 (30 %) in monocultures. Considering the results of the API gallery (see material and method section), a diminution of sucrose was also expected with Efa2412 but was not observed in monoculture. However, its association with Lla450, Lpl465 and Lpl1524 significantly modified sucrose consumption as 47 %, 34 % and 13 % were consumed in the respective co-cultures, showing the individual carbohydrate consumption by the strains. Likewise, the strain Lla2125 did not consume sucrose from the medium, but the association with Lla450, significantly increased sucrose consumption to 12 %. The co-cultures involving the donor strain Lla244 did not change the percentage of sucrose consumed.

Concerning raffinose, even if the three receiver strains were capable to hydrolyse this
carbohydrate, as shown by the API gallery results, less than 8 % was consumed by these
strains in monocultures. The only association that significantly increased raffinose
consumption to 20 % was the co-culture of Efa2412 with Lpl1524.
230 Table 2. Volatile compounds identified in CDM after 24 h of fermentation at 30°C with the six LAB strains in mono and co-cultures and in

231 unfermented CDM used as controls (CDM TRYP and CDM PROT).

<table><thead><tr><th>Compound (trivial name)</th><th>m/z</th><th>Ab.</th><th>Identification</th><th>Associated odor/flavor<sup>a</sup></th><th>CAS n°</th><th>LRI</th><th>Max fold-change<sup>b</sup></th><th>Culture associated to the maximal ratio culture:control</th><th>Origin</th></tr></thead><tbody><tr><td>3-Methylbutanal</td><td>58</td><td>MBT</td><td>S, LRI, DB</td><td>Fruity</td><td>590-86-3</td><td>918</td><td>905 ± 15</td><td>Lla244 x Lla450</td><td>cata Leu</td></tr><tr><td>2,3-Butanedione (diacetyl)</td><td>42</td><td>D</td><td>LRI, DB</td><td>Buttery</td><td>431-03-8</td><td>975</td><td>39 ± 3</td><td>Lla244 x Lla450</td><td>metab C</td></tr><tr><td>2,3-Pentanedione</td><td>100</td><td>PD</td><td>LRI, DB</td><td>Buttery</td><td>600-14-6</td><td>1062</td><td>150 ± 23.4</td><td>Efa2412_Lla450</td><td>metab C</td></tr><tr><td>Hexanal</td><td>44</td><td>H</td><td>LRI, DB</td><td>Grassy</td><td>66-25-1</td><td>1080</td><td>0.06 ± 0.02</td><td>Lla2125</td><td>CDM</td></tr><tr><td>1-Propanol, 2-methyl</td><td>74</td><td>MPP</td><td>LRI, DB</td><td>Fusel</td><td>78-83-1</td><td>1114</td><td>3890 ± 240</td><td>Lla244 x Lpl465</td><td>cata Val</td></tr><tr><td>Heptanal</td><td>44</td><td>HP</td><td>LRI, DB</td><td>Green</td><td>111-71-7</td><td>1183</td><td>0.12 ± 0.06</td><td>Lla2125 x Lla450</td><td>CDM</td></tr><tr><td>2-Hydroxy-3-butanone (acetoin)</td><td>88</td><td>AC</td><td>LRI, DB</td><td>Buttery</td><td>513-86-0</td><td>1277</td><td>22211 ± 3014</td><td>Lla2125 x Lpl1524</td><td>metab C</td></tr><tr><td>2-Nonanone</td><td>58</td><td>NN</td><td>S, LRI, DB</td><td>Cheesy</td><td>821-55-6</td><td>1383</td><td>6.8 ± 0.4</td><td>Lla244_Lla450</td><td>cata FFA</td></tr><tr><td>Acetic acid</td><td>60</td><td>A</td><td>S, LRI, DB</td><td>Sour</td><td>64-19-7</td><td>1476</td><td>558 ± 82</td><td>Efa2412 x Lla450</td><td>metab C</td></tr><tr><td>Benzaldehyde</td><td>106</td><td>BZH</td><td>S, LRI, DB</td><td>Nutty</td><td>100-52-7</td><td>1535</td><td>3.3 ± 0.1</td><td>Efa2412_Lla450</td><td>cata Phe</td></tr><tr><td>2-Methylthiolan-3-one</td><td>60</td><td>MTL</td><td>LRI, DB</td><td>Sulfurous fruity berry</td><td>13679-85-1</td><td>1539</td><td>20.6 ± 2.6</td><td>Lla244</td><td>cata Met</td></tr><tr><td>Benzeneacetaldehyde</td><td>91</td><td>BZAH</td><td>LRI, DB</td><td>Honey</td><td>122-78-1</td><td>1635</td><td>223 ± 7</td><td>Lla244</td><td>cata Phe</td></tr></tbody></table>

232 Receiver strains Lla450, Lpl465 and Lpl1524 were grown on CDM TRYP, which corresponds to CDM PROT supplemented with 0.5 g/L of casein tryptone. The selection

233 criteria for the volatile compounds was a ratio culture/control >5 or < 0.1. Compound name according to IUPAC (International Union of Pure and Applied Chemistry)

234 nomenclature, CAS=Chemical Abstract Service registry number, S = Retention time and mass spectrum from standard, LRI = Linear retention index, DB = mass spectral data

235 library NIST, Ab = abbreviation, cata = catabolism, metab = metabolism, C = carbohydrate, FFA = free fatty acid. For strain codes see Table 4.

236 <sup>a</sup> flavor description according to the goodsentscompany.com

237 <sup>b</sup> ratio culture:control
238 **2.5 Volatile compound profiles in direct co-cultures**

239 Among volatile compounds, twelve compounds exhibiting significant changes in
240 concentration in mono- or co-cultures compared to the unfermented control samples (fold-
241 change >5 or < 0.1) were identified (Table 2). They represented five chemical classes: five
242 aldehydes, one sulphur-containing compound, four ketones, one acid and one alcohol and
243 they derived from different pathways: five from amino acids catabolism, four from
244 carbohydrate metabolism and one from free fatty acid catabolism (Table 2). Two linear
245 aliphatic aldehydes, heptanal and hexanal, were 10-fold more concentrated in the control
246 medium than in LAB cultures. Co-cultures did not contribute to a further decrease in the
247 concentrations of hexanal and heptanal, compared to the monocultures of the donor strains.

248 The ten other volatile compounds were produced by the LAB strains both in mono- and co-
249 cultures. The highest fold-changes between cultures and controls varied from ~3 for
250 benzaldehyde to ~20,000 for acetoin. The greatest fold-changes were observed with the donor
251 strain Lla244 and/or the co-cultures involving the receiver strain Lla450 for most (10 out of
252 12) volatiles (Table 2). Although the volatile profiles differed according to the donor and
253 receiver strains, most of the compounds were produced in various amounts by all strains,
254 except 2,3-pentanedione, only produced by two strains (Lla450 and Lla244).

255 The global profiles of volatile compounds produced mainly depended on the donor strains, as
256 shown on the radar plots in Figure 5A, which illustrates the fold-change in log of the most
257 impacted volatile compounds in mono- and co-cultures. The donor strains presented
258 differences in the volatiles they produced in monocultures. Globally, Lla2125 produced the
259 lowest level of volatiles and Lla244 the highest, especially benzeneacetaldehyde (BZAH), 2-
260 methyl-1-propanol (MPP) and 2,3-pentanedione (PD). Efa2412 produced more benzaldehyde
261 (BZH) and acetic acid (A) compared to Lla2125.
262 The association of Efa2412 with each receiver strain led to higher levels of five volatiles (PD,
263 NN, BZH, BZAH (not shown) and MTL) compared to the monoculture of Efa2412 **(Figure
264 5B)**. PD, NN, BZH, BZAH and MTL were associated with buttery, cheesy, honey, nutty and
265 sulfurous fruity berry flavors, respectively. Concerning the donor strain Lla2125, its co-
266 culture with the receiver strains significantly increased the production of four volatiles (D,
267 NN, AC and MTL). D and AC were associated with buttery flavor. Concerning Lla244 and its
268 co-cultures, there was no significant differences in the volatile compound produced **(Figure
269 5)**.

270 **2.6 Insight on the NH₂-containing compounds available for the receiver strains**

271 Each donor strain showed a particular amino acid profile which could influence the growth of
272 the receiver strains. Regarding the FAA released, we focused on the 13 considered as
273 essential for the growth of *L. lactis* (19) and/or *L. plantarum* (20) and compared their content
274 in the medium after culture of each of the donor strain and in the medium used to grow the
275 receiver strains (CDM TRYP) (**Table 3**). Four FFA were exhausted in the medium after some
276 cultures. Hence, there was no Met and Arg left after the culture of the three donor strains,
277 while both these FAA were present in CDM TRYP. In addition, Efa2412 did not leave Tyr,
278 while the two other donor strains did not leave Trp and Lla244 did not leave Ile. The
279 concentrations of the FAA present also varied according to the donor strain: Efa2412
280 monoculture contained significantly more Val, His, Ile, Leu, Phe, and Trp compared to the
281 two other donor strain monocultures and CDM TRYP; Lla2125 monoculture was
282 characterised by higher concentrations of Asp, Thr, Ser, Glu, and Tyr.

283 In four out of the six co-cultures with the donor strains Efa2412 or Lla2125, the overall
284 amount of FAA was significantly lower compared to the one in the donor strain
285 monocultures: -65 %, -9 % -11 %, and -8 % with Efa2412xLla450, Efa2412xLpl465,
286 Lla2125xLla450, and Lla2125xLpl465, respectively. Arg, Trp, and Ile were no longer
detectable after the culture of Lla450 with Efa2412 could thus have limited the growth of both
287
donor and receiver strains. The remaining essential FAA were detectable (> 1 mg/L) in all
288
nine co-cultures after fermentation, suggesting they were not limiting (data not shown).
289

290 The concentrations in peptides were significantly reduced in the co-cultures of the donor
291 strain Efa2412 with each receiver (-54 %, -16.8 %, and -19.3 %, with Lla450, Lpl465, and
292 Lpl1524, respectively) (data not shown). The ratio FAA:total NH₂ compounds tended to
293 decrease in the cultures involving Lla450 and, in contrast, to increase in the cultures involving
294 Lpl465 and Lpl1524, indicating that the receiver *L. lactis* strain tested preferentially
295 consumed amino acids, whereas the *L. plantarum* strains preferentially consumed peptides.
**Table 3.** Concentrations (mg/L) of the 13 essential amino acids after 24 h fermentation of the CDM PROT by the donor strains Efa2412, Lla2125 and Lla244; and present in the CDM TRYP prior to the culture with the receiver strains. Percentages represent the quantity of each amino acid released from milk and/or lupin proteins of the CDM. For strain codes, see Table 4. The significantly highest concentrations for each amino acid are shown in bold.

<table><thead><tr><th></th><th>Efa2412</th><th>Lla2125</th><th>Lla244</th><th>CMD TRYP</th></tr></thead><tbody><tr><td>Asparagine (Asn)</td><td>6.9<br>(1.7%)</td><td><strong>11.0</strong><br>(2.7%)</td><td>4.7<br>(1.3%)</td><td>1.9</td></tr><tr><td>Threonine (Thr)</td><td>4.8<br>(2.7%)</td><td><strong>7.3</strong><br>(4.2%)</td><td>2.1<br>(1.2%)</td><td>2.4</td></tr><tr><td>Serine (Ser)</td><td>2.3<br>(0.9%)</td><td><strong>6.6</strong><br>(2.7%)</td><td>1.4<br>(0.6%)</td><td>2.4</td></tr><tr><td>Glutamic acid (Glu)</td><td>64.7<br>(6.2%)</td><td><strong>111.9</strong><br>(10.7%)</td><td>61.9<br>(5.9%)</td><td>1.0</td></tr><tr><td>Valine (Val)</td><td><strong>13.1</strong><br>(5.8%)</td><td>5.9<br>(2.6%)</td><td>1.7<br>(0.7%)</td><td>5.1</td></tr><tr><td>Methionine (Met)</td><td>0</td><td>0</td><td>0</td><td><strong>3.9</strong></td></tr><tr><td>Histidine (His)</td><td><strong>7.4</strong><br>(7.2%)</td><td>5.4<br>(5.2%)</td><td>5.3<br>(5.1%)</td><td>1.5</td></tr><tr><td>Isoleucine (Ile)</td><td><strong>5.7</strong><br>(2.8%)</td><td>3.2<br>(1.6%)</td><td>0</td><td>3.4</td></tr><tr><td>Leucine (Leu)</td><td><strong>33.5</strong><br>(9.0%)</td><td>10.0<br>(2.7%)</td><td>2.7<br>(0.7%)</td><td>22.0</td></tr><tr><td>Tyrosine (Tyr)</td><td>0</td><td><strong>13.9</strong><br>(5.5%)</td><td>10.8<br>(4.3%)</td><td>2.7</td></tr><tr><td>Phenylalanine (Phe)</td><td><strong>24.5</strong><br>(11.7%)</td><td>8.8<br>(4.2%)</td><td>8.7<br>(4.2%)</td><td>12.0</td></tr><tr><td>Arginine (Arg)</td><td>0</td><td>0</td><td>0</td><td><strong>15.3</strong></td></tr><tr><td>Tryptophan (Trp)</td><td><strong>10.1</strong><br>(ND)</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

**2.7** The absence of interaction is not due to growth inhibitor production nor isoleucine deficiency

Regarding the lack of interaction between the donor strain Lla244 and the receiver strains, two hypotheses were made: either the production of a growth inhibitor, or a deficiency in available Ile. **Figure 6** shows that all receiver strains reached $10^9$ CFU/mL within 24 h of culture in the CDM TRYP. The receiver strains grew in the culture supernatant of Lla244 supplemented with casein tryptone: bacterial counts exceeded $5.10^8$ CFU/mL and were
similar to the bacterial counts observed in the CDM TRYP. This result suggests that no growth inhibitor was produced by the donor strain Lla244. The receiver strains grew but did not reach $10^8$ CFU/mL when cultured in the culture supernatant of Lla244 supplemented with 5 mg/L of Ile. This result suggests that isoleucine was not the only limiting growth factor explaining why no interactions were observed between the donor strain Lla244 and the receiver strains.

## 2.8 Expression of the functional outputs by the different co-cultures summarised by PCA on the whole dataset

A total of 33 variables of four categories were selected to characterize the functional outputs of the three monocultures of donor strains and the nine co-cultures: two acidification parameters, consumption of three carbohydrates, volatile compounds (n=12), and the nitrogen composition of the medium after 24 h (including the content in each of the 13 amino acids known to be essential to LAB, global content in FAA, peptides, and the percentage of FAA).

The first and second dimensions accounted for 43.6 % and 32.6 % of total variance, respectively (Figure 7). The three replicates of cultures appeared all co-localised, which underlines the good reproducibility of the experiments. In most cases, the donor strain was grouped with its three co-cultures, excepted in one case (Efa2412 associated with Lla450).

Three groups were distinguished by hierarchical clustering. Group 1 gathered Lla244 and its co-cultures and the co-culture Efa2412 with Lla450 and was characterized ($p<10^{-5}$) by high concentrations in most of the volatile compounds (BZH, HP, MTL, BZAH, MBT, H, MPP, and D), low FAA concentrations and more specifically Ile and Val and a higher sucrose consumption. Group 2 gathered Lla2125 and its co-cultures and was characterized ($p<10^{-5}$) by high concentrations in some amino acids (Glu, Thr, Asn, Ser) and AC, and low concentrations in some volatile compounds (A, BZH). Group 3 gathered Efa2412 and its two co-cultures with *L. plantarum* strains, and was characterized ($p<10^{-5}$) by high concentrations in branched-
333 chain (Leu, Val, Ile), aromatic (Phe, Trp) amino acids and His, and globally in free NH₂-
334 containing compounds, more specifically peptides, low acidification rates, and low Tyr
335 concentration.

336 Globally, co-cultures of the donor strains Lla2125 and Efa2412 showed lower scores on the
337 first dimension than the corresponding monoculture of the donor strain, indicating that they
338 contained less sucrose, more lactose, had a lower pH and contained less NH₂-containing
339 compounds (FAA and peptides).
340 **3 Discussion**

341 This study aimed at promoting positive interactions based on nitrogen nutritional dependencies between LAB strains in co-culture. This was done in a context of designing starters of new (mixed) food fermentation, and seeking consequences of such positive interactions on the functional outputs of the fermented media.

345 McCully et al. (21) previously determined that a nitrogen starvation response is important for a stable coexistence between *Escherichia coli* and *Rhodopseudomonas palustris*. Thus, we first conceived a chemically-defined medium, called CDM PROT, which contained proteins as the sole nitrogen source, so as to control the nutritional interactions between strains and allow only proteolytic strains to grow. A similar strategy was used by Ponomarova et al. (11) to study the interactions between yeast and LAB strains. Selecting a medium supporting the growth of microorganisms in co-cultures, but not in monocultures allowed evidencing nutrient cross-feeding. While *S. cerevisiae* growth varied very little between monoculture and co-culture with LAB in the CDM used, both *L. lactis* and *L. plantarum* could grow only when co-cultured with the yeast, suggesting metabolic dependencies. In our study, we used a mix of milk and lupin proteins as sole nitrogen source, and a mix of lactose, sucrose, and raffinose as carbon sources, to mimic the content of a mix of milk and lupin. This medium thus both facilitated the study of LAB interactions, and provided some insight on the outputs that could be obtained by fermenting a more complex and mixed food matrix. As expected, only the three proteolytic strains grew in CDM PROT, whereas none of the three non-proteolytic strains tested did. The growth of the latter was restored by supplementing the medium with a casein hydrolysate, which demonstrates that the lack of available nitrogen nutrients was the only factor that prevented their growth in CDM PROT. In co-cultures of pairwise proteolytic and non-proteolytic strains, the former, qualified as donors, were then expected to provide the latter, qualified as receivers, with nitrogen nutrients.
We chose two complementary approaches to explore the interactions between donor and receiver strains. First, co-cultures in compartmented chambers facilitated the enumeration of each strain without needing to develop selective enumeration media, in particular in the case of the *L. lactis*/*L. lactis* co-cultures. These chambers allowed to quickly establish whether both strains grew, when they started to grow, and whether the interactions required or not a physical contact between the strains. In their review on co-culture systems, Goers et al. (22) suggested that such devices were excellent tools to explore cell-cell interactions. The kinetics of diffusion of metabolites from one compartment to the other, e.g. nitrogen nutrients and lactic acid, which are expected to enhance or inhibit the growth of the receiver strains (23), did not seem to alter the donor/receiver interactions, since bacterial counts were identical after 14 h in compartmented chambers and in direct co-cultures. Secondly, direct co-cultures were chosen to investigate the functional outputs that resulted from LAB metabolism, because they are closer to food fermentation than co-cultures in compartmented chambers. Three main outputs were targeted: the acidification rate, which should be quick enough for economic and sanitary reasons, the consumption of lactose and raffinose, because these carbohydrates can induce intestinal discomfort, and the production of aroma compounds susceptible to desirably influence food flavor.

Three cases of interactions were observed, which depended only on the donor strains and not on receiver strains tested. Firstly, co-cultured with the donor strain *E. faecalis* CIRM-BIA2412 (Efa2412), the receiver strains quickly started to grow and reached high counts (above $10^9$ CFU/mL), showing strong interactions. Secondly, co-cultured with the donor strain *L. lactis* NCDO2125 (Lla2125), the receivers grew but stayed below $10^9$ CFU/mL, suggesting weaker interactions. Thirdly, co-cultured with the donor strain *L. lactis* CIRM-BIA244 (Lla244), none of the receivers grew, suggesting the absence of positive interactions. Concerning donor strains, their maximal growth was similar in mono- and co-culture (Figure
390 3), implying that the positive interactions observed were commensalistic (8), i.e. that the
391 fitness of receivers increased with no apparent cost or benefit for the donors. These
392 differences of interactions could be explained by different factors. The proteolytic activity of
393 the donors probably had likely the main impact in terms of quantity and nature of peptides
394 and free amino acids released (Figure 2). This also could be balanced by the respective
395 nitrogen nutritional requirements of donors and receivers, as well as their kinetics of growth
396 that could also influence the interactions observed (Figure 3), as developed below.

397 Regarding the proteolytic activity, the donor strain Efa2412 could be qualified as a “model”
398 donor strain compared to the two other donors, provided the receivers with high
399 concentrations both in several free amino acids (Trp, Leu, Val, Phe, Ile, Arg) and in peptides
400 (Figures 2 and 7). Among the three donors, Lla244 liberated the lowest amounts of nitrogen
401 nutrients, but the concentration in FAA exceeded that of the control medium that contained
402 0.5 g/L casein hydrolysate and the concentration of peptides exceeded that produced by
403 Lla2125. It was thus unexpected that receiver strains, which grew in the control medium and
404 in the co-culture with Lla2125, did not grow in co-culture with Lla244. We first hypothesised
405 that Lla244 would produce a growth inhibitor such as a bacteriocin, but this was invalidated
406 by the results observed in sequential cultures, which showed that receiver strains grew in the
407 culture supernatant of Lla244 supplemented with 0.5 g/L of casein hydrolysate (Figure 6).
408 We also hypothesised that Ile concentration limited receiver growth since Ile was the only
409 essential amino acid lacking in Lla244 monoculture (Table 3). This second hypothesis was
410 also ruled out since Ile addition did not restore the growth of the two L. plantarum strains in
411 direct co-culture with Lla244 (Figure 6). These results suggest that the nutritional
412 dependencies based on nitrogen sources rely on both the nature and concentration of nitrogen
413 nutrients.
414 The nutritional requirements and preferences for peptides or FAA of both donor and receiver
415 strains can also modulate their interactions. In our study, the comparison between FAA and
416 peptide uptakes by the receiver strains suggests that the *L. lactis* receiver had no significant
417 preference, whereas the two *L. plantarum* strains preferred peptides to FFA (data not shown).

418 Such a preference for peptides by a *L. plantarum* strain was demonstrated by Saguir *et al.* (24)
419 who observed that dipeptides were more effective than FAA in sustaining its growth under
420 nutritional stress conditions. This is also in agreement with a higher number of amino acids
421 required for the growth of the species *L. plantarum* compared to *L. lactis* and *E. faecalis* (20,
422 25, 26).

423 Finally, the growth kinetics of donor and receiver strains could also have influenced the
424 interactions observed in the present study. The two *L. plantarum* receivers grew slower than
425 the three donors (Figure 3) and thus could have been inhibited in co-culture by the lactic acid
426 early produced by the donor strain. However, they can support lower pH (27) and thus keep
427 growing even after the donor strain stopped growing. The amensal interactions observed
428 between Lla244 and the receiving strains may also be due to the fastest acidification and
429 lowest final pH obtained with this donor strain compared to the two others (Table 1). To
430 counteract such inhibitions, the inoculation ratios of the donors/receivers associated could be
431 adjusted by increasing the initial counts of the receiver, and/or the donor strains could be
432 chosen among strains that do not exhibit too high acidification rate nor reduce pH to too low
433 values, as it is the case for the donor Efa2412 compared to the donor Lla2125.

434 The stronger the interactions, the more the outputs observed in co-cultures differed from the
435 ones observed in monoculture of the donor strains. In co-cultures with the donor strain
436 Efa2412, which induced the strongest interactions, all the functional outputs characterised in
437 this study were impacted: the rate and degree of acidification, the consumption of three
carbohydrates and the production of some aroma compounds. When compared to the monoculture Efa2412, a lower final pH was observed in the co-cultures with the two *L. plantarum* receivers, a higher acidification rate with Lla450 receiver, and lower final concentrations in lactose, sucrose, and/or raffinose, depending on the receivers. The maximum consumption of raffinose was only 20% in MCD media, although the three receiver strains were capable of using it, as shown with the API 50CHL gallery results (Table 4). This apparent discrepancy most likely results from preferences between the three carbohydrates contained in CDM, i.e. lactose, sucrose and raffinose. In agreement with our results, lactose and/or sucrose were preferentially used, compared to raffinose, by nearly 300 LAB strains of 25 different species grown in soy juice (28). Concerning sucrose and lactose preference, Efa2412 and Lpl1524 preferred sucrose over lactose, whereas Lpl465 preferred lactose over sucrose. The volatile profile was also markedly impacted in the co-cultures with the donor Efa2412, with five out of the 12 aroma compounds significantly more produced, associated with varied potentially desirable aroma: acetoin (milky), 2-nonanone (fruity-cheesy), 2,3-pentanedione (sweet-caramel-buttery), benzaldehyde (almond) and 2-methylthiolan-3-one (fruity). In co-cultures with the second donor strain, Lla2125, which induced only weak interactions, smaller impacts on the functional outputs were observed. When compared to the monoculture of Lla2125, a lower final pH was observed in the co-cultures of the receivers Lpl465, and four out of the 12 aroma compounds were produced in higher concentrations: 2-nonanone (fruity-cheesy), 2,3-pentanedione (sweet-caramel-buttery), 2-methylthiolan-3-one (fruity) and diacetyl (buttery). Finally, in co-cultures with the donor strain Lla244, in which there was no interaction, no further functional outputs were observed compared to the monoculture of this strain.

Many LAB properties are strain-dependent (29), and consequently so are the changes in the functional outputs observed. This gives great opportunity to choose receivers able to modulate
463 the functional outputs targeted. In our study, for example, the growth of the receiver Lla450
464 increased the concentration of some desirable aroma compounds such as 2,3-pentanedione
465 and 2-methylthiolan-3-one in co-culture. The two *L. plantarum* receivers led to a lower final
466 pH, while Lpl1524 receiver was associated with a decrease in raffinose content. Although
467 residual raffinose remained in the medium, the raffinose-hydrolysis activity of Lpl1524 (α-
468 galactosidase) may also remain active in the intestine, as observed for lactase activity (β-
469 galactosidase) after yogurt ingestion (30). In conclusion, our attempt to enforce nitrogen-
470 based nutritional dependencies between LAB strains did not necessarily ensure positive
471 interactions. The resulting functional outputs of fermented media depend on the strength of
472 the interactions binding the LAB strains in co-culture. We showed that the amount and the
473 nature of the FFA and peptides released by donor strains impacted the growth of receivers.

474 Further investigation of the peptides produced and consumed would be required to better
475 understand the interactions observed in this study. Omic studies such as transcriptomics,
476 proteomics, peptidomics, and/or metabolomics could also be of great interest to investigate
477 the mechanisms of the interactions observed (31, 32). Genetic insight could also be useful to
478 manipulate the genomes to confirm the mechanisms observed. In regard to better understand
479 complex microbial communities, future studies are required, including an increased genetic
480 diversity, by adding multiple strains of different genera and species.

481 **4 Materials and Methods**

482 **4.1 Bacterial collection**

483 Six mesophilic LAB strains were tested in the following experiments: two *Lactiplantibacillus plantarum*, one *Lactococcus lactis* and one *Enterococcus faecalis* strains belonging to the
484 collection of CIRM-BIA: International Centre for Microbial Resources dedicated to bacteria
485 of food interest, (INRAE Rennes, France, https://www6.reennes.inrae.fr/stlo_eng/ ), and two *L. lactis* strains from NCDO: National Collection of Dairy Organisms; now NC of Food
486
487
488 Bacteria, (Berkshire, UK). The strains were selected on their proteolytic capacities in order to
489 either obtain donor strains able to furnish peptides from the medium, and receiver strains
490 unable to grow without an external source of peptides or free amino acids.

491 **Table 4.** Origin and characteristics of the strains used for proteolytic activity and

<table><thead><tr><th>Genus</th><th>Species</th><th>Strain number</th><th>Origin</th><th>Strain code</th><th>PI (%)</th><th>LAC</th><th>SUC</th><th>RAF</th></tr></thead><tbody><tr><td>Enterococcus</td><td>faecalis</td><td>CIRM-BIA2412</td><td>NA</td><td>Efa2412</td><td>10.8</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Lactococcus</td><td>lactis</td><td>NCDO2125</td><td>Termite Gut</td><td>Lla2125</td><td>5.5</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Lactococcus</td><td>lactis</td><td>CIRM-BIA244</td><td>Raw milk</td><td>Lla244</td><td>3.6</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Lactococcus</td><td>lactis</td><td>NCDO2111</td><td>Pea</td><td>Lla450</td><td>0</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Lactiplantibacillus</td><td>plantarum</td><td>CIRM-BIA465</td><td>Sauerkraut</td><td>Lpl465</td><td>0</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Lactiplantibacillus</td><td>plantarum</td><td>CIRM-BIA1524</td><td>Silage</td><td>Lpl1524</td><td>0</td><td>+</td><td>+/-</td><td>+</td></tr></tbody></table>

492 carbohydrate consumption using API 50CHL™ galleries.

493 LAC = lactose, SUC = sucrose, RAF = Raffinose. PI = proteolytic indices, determined after 24 h of culture in a chemically defined medium containing a mix of caseins and lupin proteins, expressed as a percentage of free NH₂-containing compounds liberated relative to the calculated maximal amino groups that can be released. NA = non-available data.

497 **4.2 Composition of the chemically defined medium**

498 The chemically defined medium (CDM) was developed in order to fulfil all the nutritional requirements of lactic acid bacteria (mainly lactococci and lactiplantibacilli) (33) in terms of vitamins, mineral salts and nucleic acids. The nitrogen source was solely in the form of protein thus limiting access to amino acids to proteolytic strains. The protein and carbon resources were chosen to mimic the composition of both resources: lactose and caseins for milk part, sucrose, raffinose and lupin proteins for the legume part.

504 The final composition of the medium used was the following:

505 **Buffer**: K₂HPO₄/KH₂PO₄: 50 mM, pH 6.9 ± 0.1 ; **Sugars**: Lactose⁽²⁾: 5 g/L, sucrose⁽²⁾: 5 g/L, raffinose⁽²⁾: 5 g/L ; **Nitrogen source**: sodium caseinate (Eurial, Nantes, France): 2,5 g/L, lupin protein isolate (homemade LPI, obtained from Protilup 450 flour (Lup'ingrédient, Martigne-Ferchaud, France) by precipitating the proteins at pH 4,6 (15) 2,5 g/L ; **Mineral salts**: CaCl₂⁽¹⁾: 25 mg/L, Cl₂Co⁽²⁾: 1 mg/L, CuCl₂*2H₂O⁽¹⁾: 4 mg/L, MgCl₂*6H₂O⁽²⁾: 25 mg/L,
510 (NH₄)₆Mo₇O₂₄·4H₂O(1): 1 mg/L, ZnSO₄·7H₂O(2): 1 mg/L, MnSO₄(1): 100 mg/L, FeSO₄(1): 5 mg/L; **Vitamins**: Riboflavin(2): 3 mg/L, Nicotinic acid(2): 3 mg/L, Ca-pantothenate(2): 3 mg/L, Pyridoxine(2): 1 mg/L, Biotin(2): 0,5 mg/L, Folic acid(2): 1 mg/L, Thiamine, HCl(2): 0,5 mg/L, p-aminobenzoic acid(2): 1 mg/L, Pyridoxal, HCl(2): 1 mg/L; Nucleic acids: Adenosine(2): 10 mg/L, Guanine(2): 10 mg/L, Uracil(2): 10 mg/L, Inosine(2): 10 mg/L, Orotic acid(2): 10 mg/L, Thymidine(2): 10 mg/L; **Fat**: Tween 80(2): 0,5 g/L, provided by (1) Merck, Darmstadt, Germany and (2) Sigma - Aldrich, Munich, Germany.

517 The protein fraction was prepared separately from the other constituents. A twofold concentrated solution of lupin proteins and caseins was prepared in osmosed water and sterilized by autoclaving at 115°C during 20 min. In parallel, a twofold concentrated solution of vitamins, mineral salts, sugars, Tween 80 and buffers were sterilized by filtration through a 0.2 µm PES membrane filter (Thermo Scientific, Waltham, Massachusetts, USA). Then these two solutions were mixed together to constitute the CDM PROT, which was stored at 4 °C for less than 1 month and isolated from the light with aluminium foil.

524 A supplementary CDM, referred to as CDM TRYP was also prepared to enable the growth of the non-proteolytic strains. It consists in the same CDM preparation, supplemented with 0.5 g/L of casein tryptone (Biokar, Allonne, France). The receiver strains were always grown in the CDM TRYP when tested in monocultures.

528 Two media were used to verify the absence of growth inhibitor in *Lactococcus lactis* CIRM-BIA244 cultures and to verify the deficiency in isoleucine in these cultures. The two media first consisted in the culture supernatant of *L. lactis* CIRM-BIA244 in CDM PROT incubated for 24 h at 30 °C, harvested by centrifugation at 5000 g x 10 min and adjusted at pH 7 with NaOH 5 N. The supernatant was sterilized using a 0.22 µm filter. For the medium used for the verification of the absence of growth inhibitor, a sterile 10 % tryptone solution was added to
reach a final concentration of 0.5 g/L. For the medium used for the verification of the
deficiency of isoleucine, a sterile solution of isoleucine 10 % was added to reach a final
concentration of 5 mg/L

537 **4.3 Culture conditions**

538 **4.3.1 Pre-cultures**

539 LAB strains were conserved in cryotubes at -80 °C. One cryotube was used for each replicate
culture. Bacteria were cultured once in a rich medium: M17 for lactococci (34) and de Man
540
541 Rogosa and Sharpe broth (MRS) for lactobacilli (35). Two pre-cultures were made in the
542 CDM PROT in order to adapt the proteolytic strains to the medium. The receiver strains that
543 are not proteolytic were cultured on CDM TRYP to enable their growth.

544 **4.3.2 Compartmented chambers set-up**

545 The compartmented chambers (C.P.I.L, Issoire, France), similar to the one used by Paul et al.
546 (36) had a usable volume of 25 mL (30 mL total) and were fixed together with a clamp. O-
547 rings were placed between the two chambers so as to guaranty the sealing of the system. The
548 membrane used to separate the two compartments was a 0.4 µm polycarbonate membrane
549 (Isopore™, Merck Millipore, Darmstadt, Germany). Prior to inoculation, the chambers as
550 well as the filters were sterilised at 121°C for 15 min. O-rings were decontaminated using
551 ethanol and rinsed with sterile deionised water. The whole set-up was installed under sterile
552 conditions.

553 **4.3.3 Co-cultures in compartmented chambers and direct co-cultures**

554 The three proteolytic strains: Lla244, Lla2125, Efa2412, referred to as donors, were
555 associated in direct co-cultures or in compartmented chambers, each with each of the three
556 non-proteolytic strains: Lla450, Lpl465 and Lpl1524, referred to as receivers, thus generating
nine pairs of donor/receiver strains. In all cases, the strains were then inoculated at a total count of 10⁷ colony-forming units (CFU) /mL of CDM PROT or CDM TRYP, either in chambers (25 ml in each chamber) or in Falcon tubes of 15 mL. One falcon per time and several chambers were used so that volumes would not be limiting for the sampling (up to 6 samples of 2 mL were taken per chambers).

In the chambers, the strains were incubated at 30°C for 24 h, with a low orbital shaking of 65 rpm in order to limit the medium aeration, while improving diffusion. The CDM were also incubated as an unfermented control.

Culturable bacterial counts were determined with appropriate diluted suspensions of the samples in 1 g/L tryptone + 8.5 g/L NaCl solution in micro-plates (37). Lactococci and enterococci were incubated for 24 to 48 h under aerobic conditions in M17-glucose, and lactiplantibacilli were cultured for 48 h anaerobically using CO₂ generators (BD Biosciences, San Jose, USA) in MRS, both at 30°C.

Direct co-cultures were incubated at 30°C for 24 h, and samples were then collected for further carbohydrate and volatile analyses. The growth of donor strains was controlled after reaching their maximum population, i.e. after ~ 14 h of culture in both mono and co-cultures using a selective medium constituted by the CDM PROT medium added with 12 g/L agar, incubated for 72 h. Cultures were made in triplicates, independently.

## 4.4 Biochemical analyses

### 4.4.1 Acidification parameters

Acidification kinetics were established in direct co-cultures, using a wireless iCINAC (AMS, Frépillon, France) to estimate the maximal acidification rates by calculating the slope between pH 5.5 and pH 5 in all graphs. The final pH was measured using a pHmeter (WTW, Weilheim, Germany) after 24 h of incubation.
581 **4.4.2 Carbohydrate analysis**

582 100 µL of sulfosalicylic acid 2.3 M were added to 1 mL of sample for deproteinization. The
583 samples were then placed at 4°C for 1 h prior to centrifugation at 10,000 g for 15 min. The
584 Supernatants were then filtered through a 0.22 µm membrane and stored at -20°C prior to
585 analyses. Lactose, sucrose, raffinose, were quantified by anion exchange chromatography
586 ICS-5000+ Dionex (Thermo Electron SA Courtaboeuf France) fitted with a CarboPac PA210-
587 4µm (2 × 150 mm) analytical column (preceded by a corresponding guard column 2 × 30
588 mm). The eluent used was KOH generated with the eluent source Dionex EGC 500 KOH+
589 Eluent Generator Cartridge and ultrapure water from arium® pro system, Sartorius. HPLC-
590 EC was run at 30 °C with a the flow rate of 0.2 ml/min and the gradient was as follows: initial
591 conditions 13 mM of KOH maintained for 32 min then a linear rise to 42 min up to 100 mM
592 KOH maintained from 42 to 52 min followed by reversion to the initial conditions with a
593 linear decrease from 52 to 60 min. Quantification was performed with an external calibrating
594 using carbohydrate standards (Sigma-Aldrich) prepared at 2, 5, 10, 20 and 40 mg/L (linearity
595 range).

596 **4.4.3 Volatile compound analysis**

597 Volatile compounds were extracted using a Turbomatrix HS-40 trap automatic headspace
598 sampler and analysed using a Clarus 680 gas chromatograph coupled to Clarus 600T
599 quadrupole mass spectrometer, operated within a mass range of m/z 29-206 and ionisation
600 impact of 70 eV (PerkinElmer, Courtaboeuf, France) as detailed in (38). In brief, 2.5 g of
601 samples were placed in 20 mL PerkinElmer vial and stored at -80°C prior to analysis.
602 Compounds were eluted on an Elite WAX ETR column (30 m × 0.25 mm × 0.25 µm;
603 PerkinElmer, Waltham, MA, USA), with helium as the mobile phase, in the following
604 conditions: initial temperature 35°C maintained for 10 min, then increase at 5°C/min up to
605 230°C. Volatile compounds were identified using the NIST 2008 Mass Spectral Library data
606 (Scientific Instrument Services, Ringoes, NJ, USA) and by comparing the retention indexes
607 and mass spectral data of standards. Volatiles were semi-quantified from the abundance of
608 one specific mass fragment (m/z), in arbitrary units. MS data were processed using XCMS on
609 the R software (39). The full width at half maximum was set to 5, the maximum number of
610 peaks per ion to 100, the interval of m/z value for peak picking was set to 0.4, the signal to
611 noise ratio threshold to 6, the group band-width to 3 and the minimum to 0.4. The other
612 parameters were those by default. The results are expressed as fold-change, i.e. ratio between
613 their concentration in cultures and in unfermented CDM used as controls.

614 **4.4.4 Amino acids analysis**

615 **Free amino acid content** was determined after deproteinization as described for the
616 carbohydrate analyses (2.4.2 material and methods section). After filtration through a 0.45 µm
617 pore size membrane (Sartorius, Palaiseau, France), the supernatants were diluted three times
618 with 0.2 M lithium citrate buffer (pH 2.2) prior to injection. Amino acids were analysed using
619 cation exchange chromatography on a Biochrom 30 AA analyser (Biochrom Ltd, Cambridge,
620 UK) according to Spackman et al. (40) with lithium citrate buffers as eluents and the
621 ninhydrin as a post-column reaction system.

622 **Total amino acid content** in the LPI and caseinate was determined with a complete
623 hydrolysis of the proteins with concentrated HCl. These samples were hydrolyzed at 110°C
624 for 24 h in presence of 6 N HCl using 1.5 mL acid for an equivalent of 2 mg of total nitrogen.
625 Tubes were then dried and samples were re-suspended in 5 mL of the 0.2 M lithium citrate
626 buffer (pH 2.2) prior to injection on the Biochrom 30 AA analyser. For sulfurous amino acids,
627 samples were initially oxidized overnight at 0°C with 2 mL of performic acid then dried
628 before the acid hydrolysis.
629 **4.4.5 Free amino groups dosage and proteolytic indices calculation**
630 The changes in the amount of nitrogen compounds, i.e. peptides and free amino acids present
631 in the CDM fermented or not after 24 h of incubation were measured in triplicates using the
632 o-phtaldialdehyde (OPA) method of Church et al. (41) adapted to microplate. The proteins
633 were precipitated prior to the assay by half-diluting samples with 2% (w/w) trichloroacetic
634 acid final concentration for allowing the free NH₂ groups present at the N-terminal extremity
635 of the peptides and amino acids to be preferentially detected by the OPA. The results were
636 expressed as mg of free NH₂ / mL. Methionine was used as a standard.

637 The proteolytic indices (PI), represents the number of free amino groups relative to the total
638 amino groups. It is therefore available calculating the ratio of OPA response of fermented
639 samples (OPA<sub>sample</sub>) relative to that of acid hydrolysates OPA<sub>max</sub>, as follows:

$$640 \qquad PI = OPA_{sample}/OPA_{max}, \text{ expressed in \%}$$

641 The quantified FAA and the overall NH₂ group values were used to calculate the content in
642 peptides by difference between the total NH₂ values and the FAA values, converted in NH₂
643 content.

644 **4.5 Statistical analyses**

645 Analyses of variance (ANOVA) were performed to determine whether the acidification and
646 growth parameters, the carbohydrate and volatile contents differed according to the mode of
647 culture used (mono or co-culture), using the function aov of R (R version 3.5.1 (2018-07-02),
648 RStudio, Inc.). Means were compared using the Tukey posthoc test from the package car of R
649 (p-value < 0.05). ANOVA for the acidification parameters and carbohydrates hydrolysis were
650 made on the whole dataset gathering the three monocultures of donor strains, the three
651 monocultures of the receiver strain and the nine co-cultures. For volatiles, ANOVA were
652 made on the ratio of each compounds compared to the control (fold-change) within four data

653 subsets: Efa2412 mono- and co-cultures; Lla2125 monoculture and co-cultures; Lla244

654 mono- and co-cultures; the three receiver strains grown in CDM TRYP.

655 Principal component analyses (PCA) and a hierarchical clustering on principle components

656 (HCPC) were performed using the FactoExtra package of R.

657 **5 Acknowledgment**

658 This study is part of a Ph.D. project funded by the French National Research Institute for

659 Agriculture, Food and Environment (INRAE) and the Brittany region. The authors would like

660 to warmly thank Gwenaël JAN for the thorough proofreading he provided.
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780
781 **7** Figures and captions

782

783 **Figure 1.** Schematic representation of the strategy used to favor positive interactions between two lactic acid bacteria strains.

785 A first strain is selected for its proteolytic activity and its capacity to produce the output A (e.g. aroma compounds). Its protease(s) will degrade the proteins present in the medium into 786 peptides and free amino acids. The first strain is hence referred to as « donor ». A second 787 strain is selected for its capacity to produce the output B (e.g. carbohydrates hydrolysis), its 788 lack of proteolytic capacity and its amino acid auxotrophies. It will then benefit from the pool 789 of peptides and free amino acids available, and is hence referred to as « receiver ». The 790 objective is to favor commensalism or mutualism between the two strains in co-culture. 791

792

793 **Figure 2.** Donor strains were selected for their distinct proteolytic profiles

794 Concentrations of free NH₂ groups (mg/mL) after 24 h fermentation of the CDM PROT by 795 the donor strains Efa2412, Lla2125 and Lla244, and those present in the CDM TRYP and 796 CDM PROT prior to culture. The proteolytic indices (PI) obtained for each donor strain 797 cultured in the CDM PROT was calculated as the ratio of the peptides and free amino acids 798 (FAA) measurable by OPA during fermentation (OPA₃ₐₘₐ) and the OPA₄ₐ. The total 799 amount of free NH₂ has been separated into the part related to free amino acids and the one 800 related to peptides, calculated by difference between total and free amino acid-related NH₂ 801 groups, converted in OPA response using the free amino acids dosage. For strain code, see 802 Table

4.

803 **Figure 3.** Three different donor stains resulted in three different types of interactions

804 Growth curves of the six LAB strains used in the study incubated in compartmented chambers 805 at 30 °C for 24 h.
806 The proteolytic (donor) strains (bottom): *Enterococcus faecalis* CIRM-BIA2412 (Efa2412, ,
807 full purple squares), *Lactococcus lactis* NCDO2125 (Lla2125, , blue circles) and *L. lactis*
808 CIRM-BIA244 (Lla244, , orange triangles),

809 The non-proteolytic (receiver) strains (top): *L. lactis* NCDO2111 (Lla450, orange stars),
810 *Lactiplantibacillus plantarum* CIRM-BIA465 (Lpl465, empty green squares) and *L.
811 plantarum* CIRM-BIA1524 (Lpl1524, red diamonds), in mono and co-cultures. Each one of
812 the proteolytic strains was associated with one non-proteolytic strain. Bacterial counts were
813 made on MRS agar for *L. plantarum* strains and M17 for the rest of the strains.

814

815 **Figure 4. Carbohydrates consumption can vary according to the strains associated**

816 Percentages of lactose, sucrose and raffinose consumed in mono- and co-cultures of LAB
817 donor and receiver strains during 24 h incubation at 30°C. Percentages were calculated as a
818 ratio of the amount in the carbohydrate after fermentation relative to the amount of the non-
819 inoculated media used as a control: CDM PROT was used as the control for the donor strains
820 Efa2412, Lla2125, Lla244 and the co-cultures, and CDM TRYP for the receiver strains
821 Lla450, Lpl465 and Lpl1524 monocultures. Initial concentrations in g/L: [lactose] = 4.78 ±
822 0.50, [sucrose] = 4.03 ± 0.21, [raffinose] = 4.63 ± 0.23.

823 **Figure 5. Co-cultures increased the concentrations of volatile compounds potentially**
824 associated with pleasant flavors

825 A) Radar charts of the abundance ratio (culture:control) of the 12 volatile compounds
826 selected, expressed in log for the LAB donor strains Efa2412 and its co-cultures, Lla2125 and
827 its co-cultures, and Lla244 and its co-cultures, all cultured in CDM PROT. Ø represents the
828 absence of the receiving strain, i.e. the monocultures of the donor strains. The * represents the
829 volatile compounds that are significantly increased in at least one co-culture compared to the
830 donor strain in monoculture. B) Fold changes (culture:control) of 6 volatile compounds whose
831 concentrations were significantly increased with co-cultures. For the strain codes, see Table 4.

832 H = hexanal, HP= heptanal, MBT= 3-methylbutanal, BZH = benzaldehyde, BZAH = benzene
833 acetaldehyde, MTL = 2-methylthiolan-3-one, D = diacetyl, PD = 2,3-pentanedione, AC =
834 acetoin, NN = 2-nonanone, A = acetic acid, MPP = 2-methyl-1-propanol.

835

836 **Figure 6. The absence of interaction is not related to a growth inhibitor factor nor a**
837 **deficiency in isoleucine**

838 Bacterial counts of the three receiver strains cultured in CDM + TRYP: CDM PROT
839 supplemented with 0.5 g/L of casein tryptone; (Lla244) +TRYP: supernatant of the donor
840 strain Lla244 cultured in CDM PROT for 24 h, and supplemented with 0.5 g/L of casein
841 tryptone; (Lla244) + Ile: supernatant of the donor strain Lla244 cultured in CDM PROT for
842 24 h, and supplemented with 5 mg/L of isoleucine.

843

844 **Figure 7. Parameters differentiating the cultures**

845 Biplot of the first two dimensions of principal component analysis performed using 32
846 variables: 12 selected volatile compounds expressed as a culture:control (non-inoculated
847 CDM PROT) ratio, two acidification parameters: maximal acidification rate (Vmax) and final
848 pH, three carbohydrates, expressed as the percentage of carbohydrate consumed (LAC, SUC,
849 RAF), and 15 variables that describe the content in free amino acids and peptides: 13 essential
850 amino acids, free NH₂ groups (NH2), concentration in free amino acids (FAA), concentration
851 in peptides (PEPT) measured in CDM PROT fermented by the monocultures of the strains
852 Efa2412, Lla2125 and Lla244 and their co-cultures with three receiver strains Lla450,
853 Lpl465, Lpl1524. Replicate experiments are represented using the same symbols. For
854 compounds abbreviation see Table 2. The ellipses result from a hierarchical clustering
855 performed on the PCA dataset. For strain codes, see Table 4. H = hexanal, HP= heptanal,

856 MBT= 3-methylbutanal, BZH = benzaldehyde, BZAH = benzene acetaldehyde, MTL = 2-

857 methylthiolan-3-one, D = diacetyl, PD = 2,3-pentanedione, AC = acetoin, NN = 2-nonanone,

858 A = acetic acid, MPP = 2-methyl-1-propanol.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

Proteins

<figure><img src="image_4.png" /></figure>

Proteases

<figure><img src="image_5.png" /></figure>

Peptides and free amino
acids transporters

<figure><img src="image_6.png" /></figure>

Peptidases

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

Peptides and free amino acids
<figure><img src="image_8.png" /><figcaption>Nitrogen content provided by the donor strains and media</figcaption></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

a

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

Receiver
strains

Efa2412
cocultures

Lla2125
cocultures

Lla244
cocultures

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>
<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

D → Buttery

<figure><img src="image_42.png" /></figure>

PD → Buttery

<figure><img src="image_43.png" /></figure>

AC → Buttery

<figure><img src="image_44.png" /></figure>

NN → Cheesy

<figure><img src="image_45.png" /></figure>

BZH → Nutty

<figure><img src="image_46.png" /></figure>

MTL → Sulfurous fruity berry

<figure><img src="image_47.png" /></figure>
<figure><img src="image_48.png" /><figcaption>Receiver strains growth in CDM TRYP and culture supernatant of Lla244</figcaption></figure>
<figure><img src="image_49.png" /></figure>