Lactobacillus Reuteri grows in the Microalga Isochrysis Galbana Generating a Fermented Compound that reduces the AIEC Bacteria Harmfulness

Microalgae are currently considered as alternative sustainable resources for high-value bioproducts such as omega3 polyunsaturated fatty acids (PUFAs). Probiotics are assumed to benet human health by their direct actions on the composition and function of gut microbiota. Aims of the study are: 1) to set up the anaerobic growth of the probiotic Lactobacillus reuteri (L. reuteri) in the omega3-rich microalga Isochrysis galbana (I. galbana); 2) to assess the potential role of the obtained fermented compound (FC) to control the harmfulness of adherent invasive Escherichia coli (AIEC) to intestinal epithelial cells. I. galbana powder solubilized in PBS was used for the anaerobic growth of L. reuteri. The lipidic content of I. galbana and FC was analyzed by GC-MS. Colorectal adenocarcinoma cells CACO2 and the AIEC strain LF82 were used for in vitro experiments. I. galbana is shown to be an excellent culture medium for growing L. reuteri. The obtained FC signicantly reduces the AIEC adhesiveness and invasiveness to intestinal epithelial cells. We show for the rst time that microalgae may represent an innovative culture medium to grow probiotics in anaerobiosis. The obtained FC shows benecial properties for human health by controlling the harmfulness of AIEC bacteria.


Introduction
Among functional foods, microalgae are focusing the attention of the whole world thanks to their high content of elements of high value for the well-being of consumers, such as carotenoids, vitamins, essential amino acids, polyphenols and oils rich in omega [1][2][3][4][5][6][7]. Several studies have found that many of these components have an important impact on human health as they exhibit anti-mutagenic, anticancer, anti-oxidant and anti-in ammatory effects [8,9]. Microalgae rich in polyunsaturated fatty acids (PUFA), in particular omega3-PUFA, which include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have aroused particular interest. Indeed, these bioactive compounds appear to be strategic for their effects on the cardiovascular system and for the prevention of cognitive disorders [10][11][12][13][14].
Microalgae species that produces high PUFA concentrations are Nanochloropsis gaditana, Isochrysis galbana, Phaeodactylum tricornutum, and Crypthecodinium cohnii [15]. In particular, Isochrysis galbana (I. galbana) can synthesize EPA and DHA and contains vitamins, polysaccharides, sterols and carotenoids which make it a valid source for human and animal nutrition [16][17][18]. Interestingly, several studies have also shown that I. galbana has bene cial effects on human health and could be thought as a nutraceutical as well as therapeutic agent for the management of in ammatory and oxidative damagederived disorders [19][20][21].
Therapeutic microbiology is expanding and bene cial bacteria are being implemented as treatment and prevention strategies for immune disorders and infectious diseases. Currently, probiotics have become highly recognized as supplements for humans and animals due to their bene cial outcome on health improvement and well-being maintenance [22][23][24][25]. Lactobacillus reuteri (L. reuteri), a commensal-derived anaerobic probiotic, that resides in the human gastrointestinal tract, shows many bene cial effects, such as prevention and/or amelioration of diverse disorders [26][27][28][29]. Recent evidence highlights the role of L. reuteri in controlling the growth and survival of pathobionts correlated with infectious or chronic gastrointestinal diseases, such as the adherent-invasive Escherichia coli (AIEC) [30,31].
Here, we present an innovative and low-cost method for the growth of L. reuteri in raw seaweed extracts from I. galbana instead of the conventional medium, under conditions of oxygen deprivation (anaerobiosis). We demonstrate for the rst time that I. galbana is an excellent culture medium for the growth of L. reuteri. Moreover, we show that the omega-3 present in the seaweed are still available after the fermentation process and the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis and thus consisting of the probiotic in a medium with available microalgaereleased PUFA-omega3, slows the growth of AIEC bacteria and signi cantly limits their adhesiveness and invasiveness to intestinal epithelial cells.
Anaerobic growth of L. reuteri For anaerobic growth, 2x10 6 CFU/ml of L. reuteri were inoculated in MRS or in 5 ml of I. galbana solubilized in PBS (36 mg/ml). Each vial was capped and incubated anaerobically without agitation at 37 o C for 120 hours (5 days).
The bacterial growth was evaluated at different times (24,48,72, 96, 120 hours) by plating serially diluted samples in PBS on MRS agar plates (1,2% agarose) and incubated at 37°C for 24 hours. Resulting colonies were counted and the viability (CFU/mL) value was calculated based on the plated dilution. After 5 days, the fermentation compound (FC) contained an average concentration of 3.5x10 7 CFU/ml.

Lipid extraction
Lipids were extracted from I. galbana (36 mg/ml) solubilized in PBS and FC of a single experiment and the analysis was performed in duplicate.
Samples were freeze-dried for 2 days at -40 o C and 60 mBar pressure by freeze-dryer (Edwards). Each sample (5 mg) was resuspended with 1 ml of dichloromethane (DCM) and 0.5 ml of methanol/sulfuric acid (MeOH/ H 2 SO 4 ) and sonicated for 1 hour at 50 o C, 40 KHz frequency. Hexane (1 ml) was used as extracting solvent and, after agitation, calcium carbonate (16 mg) and H 2 O (1 ml) were added and samples were centrifugated for 5 min at 2000 rpm. The separation of polar from apolar phase was repeated twice and nally the latter was dried with nitrogen ow (4 ml for each sample).
Brie y, infected cells were washed twice in PBS and lysed for 10 minutes with 0.5 ml of 0.1% Triton X-100 in PBS buffer. Adherent bacteria were recovered and plated on LB agar plates. The latter were incubated at 37°C overnight and then the colonies were counted for statistical analysis.
Invasion assay CACO2 cells were infected and incubated as above. For invasion assay, we followed the protocol of A. Darfeuille-Michaud et al [32]. Brie y, after incubation, cells were washed twice in sterile PBS and then incubated in DMEM and McCoy's medium, respectively with 0.1 mg/ml gentamicin for 1 hour to kill the extracellular bacteria. Cells were washed twice in sterile PBS. Lysis, incubation and counts were performed as in the adhesion assay. To ensure maximum reproducibility, accuracy and statistical signi cance, adhesion and invasion assays were carried out simultaneously in triplicates. To obtain an accurate count of adhesive bacteria, the number of invasive colonies was subtracted from the number of the adhesive ones.

Statistics
Data are given as mean ± standard deviation. All experiments were repeated three times. Comparison between groups was performed by a two-tailed Student t-test (signi cance taken as P<0.05).

Results
The unicellular microalga I. galbana is an excellent culture medium for the growth of L. reuteri I. galbana extracts were obtained from dehydrated whole seaweed. L. reuteri was inoculated at a concentration of 2x10 6 CFU/ml in physiological solution containing I. galbana (36 mg/ml) or commercial medium (MRS) and placed at 37°C. The growth was followed for 5 days, that is the time necessary for the bioavailable sugars to guarantee the growth of the lactobacillus in anaerobiosis. Results showed that L. reuteri grows in the microalga medium similarly as in MRS reaching the nal concentration of 3.5x10 7 and 1.9x10 7 , respectively (Fig. 1).
The PUFA-omega3 content of the microalgae I. galbana are still mostly available after the fermentation by L. reuteri The GC-MS lipidomic analysis of I. galbana con rmed that the microalga is rich in omega3, especially DHA. More interestingly, the analysis showed that the availability of DHA and EPA is similar before and after fermentation by L. reuteri. Indeed, the amount of EPA is unchanged, while alpha-linoleic acid (ALA) and DHA undergoes a small variation between 15 and 20% less after fermentation (Fig. 2). Therefore, the FC, in its fullness, contains the probiotic but is also rich in omega3 and, thus, might represent a very innovative and bene cial compound for the human health.
I. galbana favors the growth of the probiotic L. reuteri respect to that of the pathobiont AIEC LF82 The AIEC bacteria, known as potent pro-in ammatory microorganisms, strongly increase in the gut of people with intestinal in ammatory disorders and represent a signi cant challenge for clinicians. Then, we investigated whether I. galbana could also represent an optimal culture medium for the growth of harmful bacteria. Then, L. reuteri and the AIEC prototype, LF82, were grown together in the microalgae.
Interestingly, although LF82 had been inoculated at a concentration of 1 log higher, in order to mimic the selective advantage this bacterial group has in the in amed environment, however, the growth curve of LF82 decreases after the rst 24 hours while that of L. reuteri improves and at the end of fermentation the two species are present at roughly the same concentration (9.9x10 6 and 6X10 6 , respectively), clearly suggesting that the probiotic has an advantage in the microalgae culture medium (Fig. 3).
The FC derived from the 5 days-growth of L. reuteri in I. galbana strongly limits the adhesiveness and invasioness of LF82 to intestinal epithelial cells The human epithelial colorectal adenocarcinoma cells, CACO2, are a recognized in vitro model of intestinal epithelial barrier. Hence, we used con uent CACO2 cells to assess the ability of the FC, administered as such, to control the adhesiveness and invasiveness of AIEC LF82, better that the probiotic alone. Con uent CACO2 were exposed for 3 hours to LF82 alone (3x10 6 CFU) or LF82 + L. reuteri (3,5x10 6 CFU) or LF82 + I. galbana (100µl) or LF82 + FC (100µl).
Results showed that the FC signi cantly reduces the adhesion (P = 0.002) and invasion (P = 0.0002) of LF82 compared to the probiotic alone. Surprisingly, I. galbana shows a very good capacity to decrease AIEC harmfulness even if lower than that of the FC (Fig. 4).

Discussion
To date, probiotic production has almost exclusively been carried out using conventional batch fermentation and suspended cultures, but there is an emerging interest from the scienti c community and increasing demand from the business world to explore and set up innovative fermentation technologies.
Here, we present a new method to grow the probiotic L. reuteri in the microlaga I. galbana, under anaerobiosis condition. The advantages of this protocol are several. First, the cost is low. Besides, the probiotic can be administered without being previously puri ed from its culture medium, which, on the contrary, being made up of an omega3-rich microalga, could show bene cial properties for the host organism. Finally, since probiotics must colonize an oxygen-deprived gut environment, the fermentation of the microalga in anaerobiosis can be considered a form of pre-adaptation of probiotics that could improve their survival in the bowel.
Interestingly, our results show for the rst time that the microalgae I. galbana is an excellent medium to grow the probiotic L.reuteri, which in fact lives and proliferates in the microalgae as in the conventional medium.
Recent data demonstrate that adding the microalgae Chorella vulgaris to the Lactobacillus spp. growth medium accelerates the growth and metabolic activity of bacteria, suggesting that the combination allows for the creation of innovative, functional products which confer favorable properties to the nal product [33].
In addition to proposing the microalgae as a growth medium for Lactic acid bacteria, we also support the idea that the microalgae-probiotic combination shows great potential for generating a novel functional product. Actually, we show that, after fermentation, the PUFA-omega3, of which Isochrysis is rich, are not consumed by L. reuteri, who instead prefers the consumption of sugars, and remain almost all available in the FC, giving the latter bene cial properties.
Accordingly, we aimed to investigate whether the FC had the ability to impair AIEC-induced in ammation with greater effectiveness than the probiotic alone. AIEC bacteria represent a particular pathotype of Escherichia coli abnormally colonizing the intestinal mucosa of patients with chronic intestinal in ammation, such as those affected by Crohn's disease [34]. Recent evidence suggests that perturbation of the microbial community (dysbiosis) favors the emergence of opportunistic pathogens, in particular AIEC, that can increase the incidence and severity of gut in ammation, since they strongly adhere to and invade intestinal epithelial cells, inducing in ammatory cytokine secretion [35]. Using an in vitro model of gut epithelial barrier, we rst con rmed previous evidence that L. reuteri is able to reduce the pathogenicity of enteroinvasive Escherichia coli [36,37], including AIEC [31]. More interestingly, we showed that the treatment with FC prevented the AIEC adhesiveness and invasiveness to epithelial cells more effectively than the probiotic alone. We speculate that this improved effect is due to the omega3 present in the compound that act in combination with the probiotic.
Furthermore, we showed for the rst time that I. galbana itself signi cantly reduces the adhesiveness and invasiveness of AIEC LF82 to epithelial cells. Although previous authors demonstrated that several microalgae, including I. galbana, are prospective candidates to inhibit the growth of gram-positive bacteria [38], however, to our knowledge, their potential in controlling pathobionts has never been reported yet. It is worth noting that in our experimental set up, I. galbana had already proven to be a deterrent for the AIEC group, hampering their growth while favoring that of L. reuteri.

Conclusions
Current evidence indicates that microalgae have the potential to become a novel source of bioactive molecules, especially for those who might wish to enhance the nutritional and functional quality of foods. Even further, we show that microalgae may represent an innovative culture medium to grow probiotics in anaerobiosis and that the obtained FC should be administered as such without separating the probiotic from the culture medium which, indeed, by selecting a proper microalga, could be a useful resource of bene cial bioactive molecules such as omega3. Really, we demonstrate that the FC has bene cial properties by decreasing the harmfulness of AIEC bacteria to gut epithelial cells.
These ndings are important in the development of novel tools for the production of probiotics, whose market is growing dynamically, as well as for the supply of novel fermented products that should represent in the future safe therapeutic agents to be utilized for the management of oxidative damagederived or in ammatory disorders.
Finally, the L. reuteri grown in I. galbana should be considered a true novel vegetarian probiotic since free from all animal-derived ingredients differently from probiotics grown in the traditional culture medium.

Declarations Data Availability
The data that support the ndings of this study are available from the corresponding author [L. S.], upon reasonable request.