In this review we presented a detailed overview of the different methodologies employed for the discovery of new probiotic strains. The diversity of screening assays is considerable and their efficacy variable. Some assays are more applicable for screening high numbers of strains while others are more appropriate for validating the probiotic properties of a handful of strains. There is no bulletproof procedure or workflow for selecting probiotics than perhaps the actual testing of candidate strains on the target population (Rijkers et al., 2010). However, considering the limitations of human trials, traditional *in vitro* and *in vivo* assays along with novel omics approaches will remain important. To take full advantage of probiotics for the health of humans a methodological evolution is needed. For example, new "humanized" animal models may be necessary to study host–microbe interactions. Such developments need to go hand in hand with improvements in legislation, ethics, if we want to meet the scientific and technological challenges of probiotic research.

Today, in most parts of the Western world, the acceptance of health claim dossiers is very difficult. In Europe the use of the term "probiotic" has been banned in communications toward the consumer. In the food area, except for a health claim on lactose tolerance for yogurt, none of the 300 bundled health claim dossiers have been approved. It can be hoped that a more profound study of the mechanisms of action and a better understanding of the microbiome functioning and its dynamics with the host, will provide the health claim evaluation panels with the necessary evidence to consider a wider legal acceptance of health claims. While some of the technological evolutions mentioned in this review have the potential to contribute significantly to this, the willingness to consider new types of probiotics, such as *F. prausnitzii* or *A. muciniphila*, currently not yet available on the market, will be crucial.

Not treated in this review is the importance of the production process. With these new, highly anaerobic organisms such as *F. prausnitzii* or *A. muciniphila* in mind, it will be important for probiotic producers to come up with new production processes and modified preservation and administration strategies to guarantee the delivery of active strains to the consumer or patient. As several papers have shown (van Baarlen et al., 2009; Lebeer et al., 2011, 2012; Bron et al., 2012; van Bokhorst-van de Veen et al., 2012) the (industrial) processing of a probiotic preparation has a fundamental impact on the functionality in the host. Viability, the presence or absence of pili, the cell wall condition, the matrix or the growth stage of the probiotic, they all seem to have an important influence on its performance and its interaction with the host. Defining the mechanism of action of a probiotic might therefore also include some critical parameters of the production process.

Without any doubt, the continued development of omics technologies will assist in alleviating the shortages currently faced with the traditional *in vitro* and *in vivo* models. Although it may take a while before we can predict probiotic functionality directly

from genomic and metagenomic information, the use of omics approaches to follow up on interesting *in vitro* or *in vivo* observations is very likely going to speed up research progress in the field of probiotics in the near future.

#### **ACKNOWLEDGMENTS**

The present work was cofinanced by the European Social Fund and the National resources EPEAEK and YPEPTH through the Thales project. In addition, this work was cofinanced by the European Regional Development Fund (ERDF) of the EU and by the Greek National Resources under the Operational Program Competitiveness and Entrepreneurship (OPCE II), Action "COOP-ERATION 2011", Project "ProbioDairyMeat". BP is indebted to the "*Fondation pour la Recherche Médicale*" (FRM), the "*Institut Pasteur de Lille"* (IPL), and the "*Centre National de la Recherche Scientifique*" (CNRS) for research funding.

#### **REFERENCES**

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linoleic acid and show anti-obesity effects in diet-induced obese mice. *Biochim. Biophys. Acta* 1761, 736–744. doi: 10.1016/j.bbalip.2006.05.007

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 07 December 2014; paper pending published: 30 December 2014; accepted: 17 January 2015; published online: 17 February 2015.*

*Citation: Papadimitriou K, Zoumpopoulou G, Foligné B, Alexandraki V, Kazou M, Pot B and Tsakalidou E (2015) Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches. Front. Microbiol. 6:58. doi: 10.3389/fmicb.2015.00058 This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2015 Papadimitriou, Zoumpopoulou, Foligné, Alexandraki, Kazou, Pot and Tsakalidou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Edited by:*

Fabio Minervini, Università degli Studi di Bari Aldo Moro, Italy

#### *Reviewed by:*

Marcela Carina Audisio, Instituto de Investigaciones para la Industria Química-CONICET, Argentina Amit Kumar Tyagi, The University of Texas MD Anderson Cancer Center, USA

#### *\*Correspondence:*

Simone Guglielmetti, Division of Food Microbiology and Bioprocessing, Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Via Celoria 2, Milano 20133, Italy<EMAIL_ADDRESS>
> *†Present Address:* Maria C. Remagni, Sacco S.r.l., Cadorago, Italy

#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 30 April 2015 *Accepted:* 28 August 2015 *Published:* 15 September 2015

#### *Citation:*

Balzaretti S, Taverniti V, Rondini G, Marcolegio G, Minuzzo M, Remagni MC, Fiore W, Arioli S and Guglielmetti S (2015) The vaginal isolate Lactobacillus paracasei LPC-S01 (DSM 26760) is suitable for oral administration. Front. Microbiol. 6:952. doi: 10.3389/fmicb.2015.00952

# The vaginal isolate *Lactobacillus paracasei* LPC-S01 (DSM 26760) is suitable for oral administration

Silvia Balzaretti 1 , Valentina Taverniti 1 , Greta Rondini 1 , Giorgio Marcolegio1 , Mario Minuzzo2 , Maria C. Remagni 1 †, Walter Fiore3 , Stefania Arioli 1 and Simone Guglielmetti 1 \*

1 Division of Food Microbiology and Bioprocessing, Department of Food Environmental and Nutritional Sciences, Università degli Studi di Milano, Milano, Italy, 2 Department of Biosciences, Università degli Studi di Milano, Milano, Italy, 3 Sofar S.p.A., Trezzano Rosa, Italy

Bacterial vaginosis is one of the most common urogenital diseases affecting women in reproductive age. The administration of probiotics as vaginal suppository has been proposed as a strategy to cure this condition and reduce its recurrence. Nonetheless, also oral consumption of probiotics, which is a more practical route of administration, proved to be an efficient strategy. In this perspective, we studied Lactobacillus paracasei LPC-S01 (DSM 26760), a human vaginal isolate included in commercial probiotic preparations for topical use, in order to assess if this bacterium can also perform as gastrointestinal probiotic. Comparative genomics revealed the presence of several accessory genes suggesting that LPC-S01 is a niche-generalist member of its species. According to a procedure conventionally used to predict the probiotic potential, we demonstrated that the probiotic properties of strain LPC-S01, with respect to those of the well-known probiotic references L. paracasei Shirota and DG, are equal for the bile tolerance and the reduction of NF-κB activation in Caco-2 cells, or superior for the tolerance to gastric juice and the adhesion to Caco-2 epithelial cells. We then demonstrated that LPC-S01 is susceptible to antibiotics indicated by EFSA and does not produce biogenic amines. Finally, a double-blind cross-over pilot intervention trial on healthy human volunteers showed that, after a 7-days oral consumption of capsules containing about 24 billion live cells, the fecal cell concentrations of strains LPC-S01 and DG (evaluated by qPCR) were not dissimilar. Specifically, both probiotics' cell concentrations were above the detection limit for an average of 5 days from the end of the treatment, corresponding to a mean number of evacuations of 7 ± 2. Taken together, these data demonstrate that the vaginal isolate L. paracasei LPC-S01 possesses safety and functional properties that may support its use as probiotic to be administered per os for potential intestinal as well as vaginal applications.

Keywords: lactobacilli, probiotic, Caco-2 adhesion, NF-κB, *in vivo* trial, gastrointestinal persistence

## Introduction

The genus Lactobacillus is a taxonomically broad and heterogeneous group of Gram positive bacteria, which has important industrial applications as fermented food starter and probiotic adjuncts. Particularly, lactobacilli are the bacteria most frequently employed as probiotics, i.e., "live microorganisms which when administered in adequate amounts confer a health benefit on the host" (FAO/WHO, 2002). Inside the Lactobacillus genus, the L. casei group of species (i.e., L. casei, L. paracasei, and L. rhamnosus) includes some of the most conventional and well-characterized probiotic strains, such as L. paracasei Shirota (considered the first probiotic ever; Nanno et al., 2011), and L. rhamnosus GG (Szajewska and Chmielewska, 2013).

Strains of the L. paracasei species have been isolated from several diverse ecological niches such as raw milk, plants, fermented artisanal products (fermented milk, cheese, sourdough bread starter, and fermented vegetables), and the intestinal tracts and reproductive systems of humans and animals (Cai et al., 2007). The remarkable ecological adaptability of L. paracasei to diverse habitats can be plausibly explained by very frequent accumulation of indels and genome rearrangements, which originated a high level of genotypic and phenotypic diversity in the species (Cai et al., 2007; Smokvina et al., 2013). Given that most effects of probiotics are strain-specific (Azaïs-Braesco et al., 2010), the intrinsic high heterogeneity existing among L. paracasei strains makes this species an optimal source for the selection of novel candidate probiotic strains possessing unique technological and health-promoting traits.

So far, lactobacilli have been predominantly investigated and explicitly proposed as probiotics for their potential beneficial roles on the gastrointestinal tract and its microbiota (Guglielmetti et al., 2011; Ferrario et al., 2014). Nevertheless, the interest for the application of lactobacilli beyond the gut is constantly increasing, leading to the development of new categories of probiotic products that target oral cavity (Guglielmetti et al., 2010a,b; Taverniti et al., 2012; Wescombe et al., 2012), skin (Krutmann, 2009), stomach (Johnson-Henry et al., 2004), urinary tract (Borchert et al., 2008), and vaginal mucosa (Borges et al., 2014).

Interestingly, it was demonstrated that orally administered lactobacilli can be re-isolated from the vagina, presumably as a consequence of the migration from the rectum via perineum (Vásquez et al., 2005). Consistently, bacteria colonizing the vaginal mucosa (both commensals and vaginosis-associated microbes) have been isolated from the rectum and the mouth, suggesting that gut and oral cavity act as extravaginal reservoirs of vaginal microbiota bacteria (van de Wijgert et al., 2014). Therefore, it appears plausible that the oral administration of probiotic bacteria may potentially influence the vaginal microbiota through two possible mechanisms: (i) modification of the intestinal microbiota (e.g., by reducing potentially harmful bacteria and increasing endogenous lactobacilli); (ii) direct migration to the vaginal mucosa via the gastrointestinal route. Furthermore, the possibility to benefit vaginal health by administering vaginal probiotics per os has the additional advantage of favoring the accomplishment of long-term treatments. However, in order to make this strategy effective, selected bacteria must possess properties supporting their survival and activities both in the gastro-intestinal tract and vaginal mucosa. In this perspective, we assessed if L. paracasei LPC-S01 (DSM 26760) (a strain originally isolated from the vaginal mucosa of a healthy woman and included in probiotic preparations for vaginal use marketed in Europe) can also perform as a conventional gastrointestinal probiotic. To this aim, we generated a draft genome of strain LPC-S01 and performed a comparative genomic analysis against other L. paracasei reference strains. In addition, we included LPC-S01 in several in vitro tests conventionally employed to establish the intestinal probiotic potential of microbial strains. Finally, we carried out a double-blind cross-over pilot intervention trial in comparison with the well-known intestinal probiotic strain L. paracasei DG (Ferrario et al., 2014) in order to assess the ability of LPC-S01 to transiently persist in the gastrointestinal tract of healthy adults.

## Materials and Methods

### Bacterial Strains and Culture Conditions

Unless differently specified, lactobacilli were grown at 37◦C in De Man-Rogosa-Sharpe (MRS) broth (Difco Laboratories Inc., Detroit, MI) for 24 h. L. paracasei LPC-S01 and DG were isolated from the commercial products PreGyn R and Enterolactis R Plus (Sofar S.p.A.), respectively; L. paracasei strain Shirota was isolated from Yakult fermented milk; strain FMBr3 was isolated from Raschera artisanal Italian cheese.

### Genome Sequencing, Sequence Annotation, and Comparative Analysis

The draft genome sequences of L. paracasei LPC-S01, DG, and FMBr3 were obtained through Ion Torrent PGM (Life Technologies, Germany) according to a previously described protocol (Milani et al., 2013) by GenProbio Ltd. The raw sequence data were assembled using MIRA v.3.9 (http://www. chevreux.org/projects\_mira.html), applying default parameters recommended for Ion Torrent data processing. Initial automated annotation of the L. paracasei genomes was performed using RAST, combined with BLASTX. Results of the gene-finder program were combined manually with data from BLASTP analysis against a non-redundant protein database provided by the National Center for Biotechnology Information (NCBI). LPC-S01 draft genome sequence was compared with other L. paracasei genome sequences by means of BLAST Ring Image Generator (BRIG; Alikhan et al., 2011). The DNA sequences presented in this study have been deposited in the EMBL database under the accession numbers LN846896 (putative phospho-βgalactosidase operon), LN846897 (putative taurine ABC-type transport and metabolization operon), LN846898 (putative ABCtype Fe3+ transport system), LN846899 (putative nucleotide transport and metabolism operon), LN846900 (putative EPS2 region), and LN846901 (multi-transport region for the uptake of sugars and other small molecules).

## Bacterial Resistance to Simulated Gastric Juices and Bile

One hundred microliters of L. paracasei culture containing 108– 109 CFU/ml were transferred to 6 ml of simulated gastric juice at pH 2 (1 g/l DIFCO peptone, 100 mM KCl, 500 U/ml pepsin) or simulated gastric juice at pH 3 (125 mM NaCl, 45 mM NaHCO3, 7 mM KCl, 500 U/ml pepsin). Cells in 0.1 M phosphate buffer pH 7 were used as control. After 90 min of incubation at 37◦C, viability was monitored by plating on MRS agar. Afterwards, cell suspension was neutralized by adding 1 M phosphate buffer pH 8 and centrifuged at 10,000 × g for 12 min. Bacterial cell pellet was then suspended in 5 ml bile solution (1 g/l peptone, 0.3% Difco Oxgall in 0.1 M phosphate buffer at pH 6.5). Cells in 0.1 M phosphate buffer pH 6.5 in the presence of 1 g/l peptone were used as control. The number of viable cells was determined after 3 h of incubation at 37◦C by plating on MRS agar. Plates were incubated under anaerobic conditions and colonies were counted after 48 h. The ability to grow in presence of bile was also investigated by inoculating bacteria in MRS broth supplemented with increasing concentrations of Oxgall (from 0.0625 to 9.6%). Growth was assessed by measuring the optical density at 600 nm.

## Bacterial Adhesion to Caco-2 Cell Line

The adhesion of L. paracasei strains to Caco-2 (ATCC HTB-37) cell layer was assessed as previously described (Guglielmetti et al., 2008). In brief, Caco-2 cells were grown in Dulbecco's Modified Eagle's Medium (MEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.1 mM non-essential amino acids, 2 mM Lglutamine and incubated at 37◦C in an atmosphere of 95% air and 5% carbon dioxide. For adhesion experiments, fully differentiated Caco-2 were used (i.e., 15 days after confluence). Approximately 2×108 cells for each bacterial strain (determined microscopically with Neubauer Improved counting chamber; Marienfeld GmbH, Lauda-Königshofen, Germany) were incubated with a monolayer of Caco-2 cells for 1h at 37◦C. Monolayers were washed three times with phosphate-buffered saline pH 7.3 (PBS) to release unbound bacteria and incubated with 3 ml of methanol for 8 min at room temperature to fix cells. Afterwards, cells were stained with 3 mL of Giemsa stain solution (1:20) (Carlo Erba, Milano, Italy) and left 30 min at room temperature in the dark. Finally, monolayers were washed three times with PBS, dried in an incubator for 1 h, and examined microscopically (magnification, 400×) under oil immersion. All experiments were performed in duplicate.

## Inhibition of Pathogens

We investigated the ability of L. paracasei strains to secret inhibitor molecules against pathogens by disc diffusion assay. Lactobacillus plantarum strain WHE 92 was used as a positive control for bacteriocin production (Ennahar et al., 1996). We used seven indicator microorganisms: Salmonella enterica MIMms, Listeria monocytogenes FMB4b, Escherichia coli VE7108, Streptococcus pyogenes emm TYPE 77, Pseudomonas aeruginosa FMBpa18, Staphylococcus aureus FMBDR, and Candida albicans ATCC MYA2876. Indicator bacteria were cultivated in Brain Heart Infusion (BHI) agar medium (Oxoid S.p.A., Milan, Italy), supplemented with 0.3% yeast extract and 1% glucose (ygBHI), whereas C. albicans was cultivated on Sabouraud dextrose agar (Oxoid S.p.A., Basingstoke, UK). Three different fractions for each Lactobacillus strain (tester bacteria) were used: (i) broth culture at stationary growth phase, (ii) cell-free broth (obtained by centrifugation at 10,000 × g for 10 min and sterilization with a 0.22µm syringe filter), and (iii) cell-free broth neutralized with NaOH to pH 7.4. Sterile MRS was used as negative control. The assay was carried out as follows: 0.1 ml from an over-night culture of the indicator strains were plated on ygBHI (or Sabouraud) agar in order to obtain a confluent growth after incubation. Sterile paper discs (Whatman™ Grade AA 9 mm disc, Maidstone, UK) were dipped into three different preparations from tester bacteria and subsequently positioned on the Petri dishes inoculated with the indicator strain. Plates were kept at 4◦C for 2 h and then incubated at 37◦C. The presence of an inhibition halo was checked after 24 or 48 h.

### Fermentation Profile

Carbon source fermentation was determined in a 96-well microtiter plate in a final volume of 200µl with a basal CHL medium at pH 6.3 (Bio-Merieux, Montelieu-Vercieu, France) containing bromocresol purple as pH indicator, and the desired filter-sterilized carbohydrate at a final concentration of 0.5% (w/v). The 42 different substrates tested (Supplementary Table 1) were from Sigma-Aldrich (St. Louis, MO, USA), with the exception of fructooligosaccharides (FOS) which were from Actilight R (Giulio Gross S.p.A., Trezzano sul Naviglio, Italy). Cells from an over-night culture of L. paracasei were collected by centrifugation, washed with PBS, and used to inoculate the liquid medium in microtiter wells (1/100 inoculation). Plates were examined for color change (from purple to yellow) after 24 and 48 h incubation at 37◦C.

## NF-κB Activation Assay

The activation nuclear factor κB (NF-κB) was studied by means of a recombinant Caco-2 cell line stably transfected with vector pNiFty2-Luc (InvivoGen, Labogen, Rho, Italy) as in Taverniti et al. (2013). In brief, recombinant Caco-2 monolayers (approximately 3 × 105 cells/well), cultivated in the presence of 50µg/ml zeocin, were washed with 0.1 M Tris-HCl buffer (pH 8.0) and then incubated with 3.5 × 108 cells of L. paracasei suspended in fresh DMEM containing 100 mM HEPES (pH 7.4), resulting in a MOI of approximately 1000. In a different set of experiments, Caco-2 monolayers were incubated with 0.1 ml of cell-free broth obtained by centrifugation and filter-sterilization from a stationary-phase culture of L. paracasei. Stimulation was conducted by adding 2 ng/ml of IL-1β. After incubation at 37◦C for 4 h, the samples were treated and the bioluminescence was measured as described by Stuknyte et al. (2011). Two independent experiments were conducted in triplicate for each condition.

## Susceptibility to Antibiotics and Biogenic Amines Production

The minimal inhibitory concentrations (MICs) of L. paracasei have been determined using a micro-dilution method in LSM broth (ISO-Sensitest broth, Oxoid supplemented with 10% v/v MRS Difco) as described in the ISO10932 IDF 223 document and recommended by EFSA (2012). The data are reported as average of two independent assays. The ability to produce cadaverine, tyramine, histamine and putrescine respectively from L-lysine, tyrosine disodium salt, L-histidine monohydrochloride and Lornithine monohydrochloride was investigated by a qualitative test, according to the method proposed by Bover-Cid and Holzapfel (1999). All amino acids were purchased from Sigma. Strains were inoculated (1%) in MRS broth containing 0.1% of each amino acid precursor and incubated at 37◦C for 18 h. The qualitative test to evaluate decarboxylase activity was performed in an agar medium specially formulated (0.5% tryptone, 0.5% yeast extract, 0.5% meat extract, 0.25% NaCl, 0.05% glucose, 0.1% Tween 80, 0.02% MgSO4, 0.005% MnSO4, 0.004% FeSO4, 0.2% ammonium citrate, 0.001% thiamine, 0.2% K2PO4, 0.01% CaCO3, 0.005% pyridoxal phosphate, 1% amino acid precursor, 0.006% bromocresol purple, 2% agar; pH 5.3). 10µL of each culture were placed on the medium containing the same amino acid precursor. The plates were incubated at 37◦C for 4 days. Positive test is indicated by the color change and by aminoacid precipitation around the corresponding spot (for tyramine only).

## Human Intervention Trial

The study protocol was approved by the Research Ethics Committee of the Università degli Studi di Milano (opinion no. 37/12, December 2012). Written informed consent was obtained from all subjects before recruitment. The intervention study consisted of a randomized, double blind, cross-over pilot trial with two parallel groups (Supplementary Figure 1). Each volunteer was asked to participate to 6 visits: before run-in period (visit V0); before the first treatment (V1); after the first treatment (V2); before the second treatment (V3); after the second treatment (V4), and at the end of the trial (V5) (Supplementary Figure 1). After a 4-week pre-recruitment phase, volunteers have been randomized to receive 1 capsule daily of L. paracasei LPC-S01 (product A, 5 subjects) or L. paracasei DG (product B, 6 subjects) every day for 1 week, in addition to their habitual diet. After a 2 week wash-out period, the volunteers received a daily capsule of the other product for 1 week. Volunteers received directions to keep the products at room temperature and to avoid exposure to heat sources. Volunteers received also oral and written instructions to consume the capsule during the morning, while drinking natural water, at least 15 min before breakfast; alternatively, volunteers were allowed to consume capsule in the evening at least 3 h after the last meal of the day. The two probiotic preparations (provided by Sofar S.p.A., Trezzano Rosa, Italy) consisted of a gelatin capsule containing about 24 billion viable cells of the bacterial strain L. paracasei DG (CNCM I-1572) and LPC-S01 (DSM 26760), respectively; silicon dioxide and magnesium stearate were also added inside capsules as antiagglomerants. Capsules were delivered to participants in metal boxes sealed with a plastic cap containing desiccant salts. From the last day of consumption of the probiotic capsules, for the following 7 days, the volunteers provided one stool sample per day. Furthermore, participants provided a fecal sample at visits V1, V3, and V5 (Supplementary Figure 1). The sample was collected in a sterile plastic pot no more than 24 h before visit. Volunteers were asked to preserve the fecal sample at room temperature until delivery to the laboratory, according to the recommendations on storage conditions of intestinal microbiota matter in metagenomic analysis provided by Cardona et al. (2012). During the trial period, the participants compiled a weekly diary (including a Bristol stool chart) of their bowel habits.

## Fecal DNA Extraction and qPCR

Stools were stored at −80◦C until DNA extraction, performed by means of QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). Before the extraction, the sample was homogenized in a Bead Beater Precellys 24 (Bertin Technologies, Montigny le Bretonneux, France) after addition of 0.45 g 0.5 mm glass beads. Subsequently, the extraction proceeded consistently with manufacturer's recommendations. Real-Time quantitative (qPCR) protocols were adopted for the quantification of L. paracasei DG in fecal metagenomic DNA (targeting the glycosyltransferase gene welF, with primers rtWELFf, 5 ′ -TACTAAAGAAATTAGCTTTTGT-3′ and rtWELFr, 5′ - AGTAATGTCTGCATCCTCCA-3′ ; Ferrario et al., 2014) and LPC-S01(targeting an hypothetical protein coding sequence with primers qS01a-F, 5′ -TGGAAGAGACCCTGCGAA-3′ and qS01a-R, 5′ -GAGGTTGATTCACAAACCGTGC-3′ ; this study). These two genes were selected because they resulted unique to the respective strain as revealed by a search in the GenBank nucleotide database. A gradient PCR was initially performed to standardize the qPCR conditions. qPCR amplifications were carried out in a final volume of 15µl containing 7.5µl of EvaGreen R Supermix and 0.5µM of each primer. We used 100 ng of fecal DNA template in each reaction. Samples were amplified with the following programs: for rtWELF primers, initial hold at 95◦C for 3 min, and 44 cycles at 95◦C for 30 s, 58◦C for 30 s and 72◦C for 30 s; for qS01a primers, initial hold at 95◦C for 3 min, and 40 cycles at 95◦C for 30 s, 62◦C for 30 s and 72◦C for 30 s. Melting curves were analyzed to confirm the specificity of the amplification products. To generate the standard curves with spiked feces for DG and LPC-S01 detection, a 200 mg matrix of fecal sample was added with 1 × 109 bacterial cells and DNA was extracted as reported above. Six-fold dilution series of isolated DNA were prepared and used in qPCR reactions. The equation of the derived standard curves was used to calculate the correlation between Ct values and the concentration of bacterial cells per g of feces.

## Results

## Comparative Genomics Revealed the Indels of Several Chromosomal Regions in *L. paracasei* LPC-S01

We generated a draft genome sequence of L. paracasei LPC-S01 consisting of 17 contigs, which in total contained 3.02 Mbp. Comparative genomic analysis was performed on strain LPC-S01 and the following six L. paracasei strains: the type strain of the species (L. paracasei JCM 8130T ), the probiotic strains DG and Shirota, a strain isolated from Raschera Italian artisanal cheese (FMBr3), and two other L. paracasei strains whose complete genome is available in GenBank (strains 8700:2 and N1115) (**Figure 1**). When we used L. paracasei LPC-S01 as reference genome in the comparative analysis, we identified 14 genomic islands with putatively known biological functions, which have not been found in the genomes of type strain JCM 8130T or other L. paracasei strains. These chromosomal islands, which discriminate LPC-S01 genome from the others, include 7 mobile genetic elements (e.g., prophages, phage remnants, and integrated plasmids; **Figure 1**), a transposon containing a phospho-β-galactosidase operon (region "a" in **Figure 1**; **Figure 2A**), a putative taurine transport and metabolization operon (b; **Figure 2B**), a large sugars transport/metabolization island (c; **Figure 2C**), an ABC-type Fe3+ transport system (d; **Figure 2D**), and a putative nucleotide transport and metabolism operon (e; **Figure 2E**). We also found in the genome of strain LPC-S01 two regions putatively involved in exopolysaccharide (EPS) synthesis. Specifically, one of these EPS regions (namely, EPS2; **Figure 1**) was found exclusively in the genome of L. paracasei LPC-S01 and is constituted of 14 putative genes, including six genes potentially coding for glycosyl transferases. Notably, BLASTN search revealed inside EPS2 a region of about 8 kb encompassing eight putative genes, which did not find any significant match in the GenBank database (genes from A to H in **Figure 3**). In subsequent comparative analyses, we used the genome of the other L. paracasei strains as reference to identify missing chromosomal islands in the genome of strain LPC-S01. Largely most of the genomic regions that are absent in LPC-S01 genome refer to mobile genetic elements, DNA restrictionmodification systems, and EPS biosynthesis regions (data not shown).

## *In vitro* Assays Showed That *L. paracasei* LPC-S01possesses Marked Acid Tolerance and Noticeable Adhesion Ability

The probiotic potential of strain L. paracasei LPC-S01 was assessed through in vitro experiments including L. paracasei DG and L. paracasei Shirota as reference probiotic strains. The potential ability to survive the gastro-intestinal transit was assessed in simulated gastric juices at pH 3 or 2 (90 min incubation), followed by 3 h incubation in bile (Oxgall) solution. All strains showed high tolerance to pH 3; cell viability, in fact, was not significantly reduced after 4.5 h (Supplementary Figure 2). Conversely, pH 2 determined a drastic reduction of the viability of all tested strains (**Figure 4**). However, L. paracasei LPC-S01 displayed the highest tolerance, which was significantly higher than that of the reference strain L. paracasei Shirota (**Figure 4**).

Growing concentrations of Oxgall were added to the MRS broth to assess the ability to grow in presence of bile. For all tested

rectangles refers to panels of Figure 2.

#### FIGURE 2 | Continued

rectangles in Figure 1. (A) Putative phospho-β-galactosidase operon. In black are indicated transposase-associated putative genes. In white are indicated putative genes that are also present in the genome of the other L. paracasei strains considered for comparative genomic analysis. (B) Putative taurine ABC-type transport and metabolization operon. (C) Multi-transport region for the uptake of sugars and other small molecules. The membrane transport mechanism is indicated above the picture, whereas the transported molecule is indicated below. ABC, ATP binding cassette transport system, PTS, phosphotransferase transport system. Gal-NAc, N-acetyl galactosamine; Glu-NAc, N-acetyl glucosamine; Co+, cobalt ions. (D) Putative ABC-type Fe3+ transport system. Gene "a," putative acetyltransferase (COG0456) coding gene; gene "b," putative gene coding for an uncharacterized protein (DegV family, COG1307). (E) Putative nucleotide transport and metabolism operon. MFS, major facilitator superfamily. In white are indicated putative genes that are also present in the genome of all the other L. paracasei strains considered.

strains, we observed a dose-dependent inhibition of the bacterial growth, which was completely arrested at Oxgall concentrations higher than 1%. In specific, Shirota displayed the best tolerance to bile, even though not statistically significant (Supplementary Figure 3).

Subsequently, the ability of bacterial strains to adhere to the Caco-2 epithelial cell layer was tested. Only LPC-S01 displayed a marked adhesive phenotype, corresponding to an adhesion index (i.e., bacterial cells per 100 Caco-2 cells) of more than 2000 (**Figure 5A**). Also strain DG displayed adhesion ability, whereas strain Shirota was unable to adhere on Caco-2 cells, as observed in previous studies (Botes et al., 2008; Guglielmetti et al., 2008).

Caco-2 cell layer was also used to explore the immunomodulatory properties of L. paracasei strains by testing the effect of the microorganisms on NF-κB activation in presence of the pro-inflammatory cytokine IL-1β. To this aim, we employed a reporter cell line obtained by transfecting Caco-2 cells with a luciferase reporter vector induced by active NF-κB (Guglielmetti et al., 2010a). For all three L. paracasei strains, both bacterial cells and cell-free neutralized broths were able to significantly decrease the NF-κB-dependent production of bioluminescence. Particularly, we found that the highest ability to reduce NF-κB activation was exerted by DG and LPC-S01 cells, and by broth from Shirota culture (**Figure 5B**).

We also tested the ability of the bacteria under investigation to produce inhibitor molecules against pathogens by disc diffusion assay carried out by using cell-free MRS broths cultures. With the only exception of L. plantarum WHE 92 against L. monocytogenes, none of the neutralized broths was able to prevent pathogens' growth, suggesting that, plausibly, the only inhibitory molecules produced by tested L. paracasei strains were organic acids (Data not shown).

Afterwards, the in vitro probiotic properties of LPC-S01 and reference strains were assessed by testing their ability to produce acid from 44 different carbohydrate sources. The fermentation patterns of L. paracasei strains were very similar. In detail, the three tested strains fermented cellobiose, FOS, fructose, galactose, glucose, inulin, maltulose, mannitol, mannose, ribose, salicine, sorbose, sucrose, trehalose, and turanose (Supplementary Table 1).

In conclusion, the in vitro experiments carried out in this study showed that strain L. paracasei LPC-S01 possesses potential probiotic capabilities to be oral administered comparable to those of the probiotic commercial strains L. paracasei DG and Shirota. Notably, the survival rate of LPC-S01 to simulated gastrointestinal transit at pH 2 was significantly higher than that of the reference probiotic strain Shirota; in addition, strain LPC-S01 displayed marked adhesion ability, corresponding to an adhesion index higher than 2000.

We also studied the antibiotic resistance of L. paracasei LPC-S01by the microdilution assay recommended by International Organization for Standardization (ISO, 2010) with reference to the EFSA breakpoints for L. casei/paracasei (EFSA, 2012). The MICs for strain LPC-S01 was below the breakpoints for all tested antibiotics (**Table 1**). Finally, the safety of L. paracasei strains was also estimated by assessing the bacterial decarboxylation of amino acids, which generates biogenic amines in food (Deepika Priyadarshani and Rakshit, 2014). According to our experiments, none of the tested L. paracasei strains produced histamine, tyramine, putrescine and cadaverine (Data not shown), which are among the most common biogenic amines found in food products (Naila et al., 2010).

## *L. paracasei* LPC-S01 Persists in the Gut of Healthy Adults

The ability of L. paracasei LPC-S01 to colonize the human gastrointestinal tract was tested with a pilot intervention trial based on a double-blind, randomized, cross-over design, in comparison with the probiotic strain L. paracasei DG. We enrolled 11 healthy adult volunteers, who were asked to consume for 1 week a capsule per day containing LPC-S01 or DG cells. Out of 11 enrolled volunteers, 8 concluded the study. Three volunteers dropped out of the study due to non-conformity (n = 1) and protocol violation (missed probiotic consumption; n = 2) (Supplementary Figure 1). Capsules, which contained about 24 billion CFU of L. paracasei, were well tolerated by all participants and no adverse events were reported. In order to quantify L. paracasei cells in the fecal samples all over the trial, we used qPCR with primers targeting gene welF (specific for strain DG; Ferrario et al., 2014) and a gene coding for a hypothetical protein (specific for strain LPC-S01; primers designed in this study). According to repeated measure ANOVA, we did not find significant difference in the persistence of the two L. paracasei strains (**Figure 6**). Specifically, the concentrations of LPC-S01 and DG cells were above the qPCR detection limits for an average of 5 days from the end of treatment; in addition, L. paracasei cells were not detectable in the fecal samples after 7 ± 2 evacuations from the last capsule consumption (**Figure 6A**).

## Discussion

Bacterial vaginosis (BV) is one of the most common urogenital diseases affecting women of reproductive age (Allsworth and Peipert, 2007). BV is typically associated to the disruption of the healthy vaginal microbiota, which is normally dominated by lactobacilli (Ma et al., 2012). Due to the dominant role of dysbiosis in the onset of BV, probiotics, which are regularly used to prevent and treat dysbiosis in the intestine, have been proposed as a strategy to cure this urogenital condition and reduce its recurrence (Bodean et al., 2013). Several studies documented the beneficial effects on BV of viable Lactobacillus cells directly introduced in the vagina as suppository (Patel et al., 2008; Donders et al., 2010). Nonetheless, also the oral administration of probiotics has been demonstrated to be an efficient strategy to alleviate BV symptoms and prevent recurrence (Reid, 2006; Bodean et al., 2013). In fact, it was proposed that ingested lactobacilli, similarly to colonic pathogenic bacteria, once excreted from rectum, may ascend to the vagina through perineum (Reid et al., 2001; Bastani et al., 2012). Then, once in the vagina, lactobacilli create a protecting barrier against the translocation from the gut of possible detrimental bacteria such as Prevotella spp. or E. coli, and, at the same time, limit the possible migration of uropathogens from vagina to the bladder (Reid et al., 2001).

The oral administration of probiotics is a more practical route than their direct introduction in the vagina, and can also provide consumers with the gastrointestinal health benefits typically associated with the consumption per os. In this context, this study aimed to the identification of a probiotic strain

FIGURE 5 | Experiments on human epithelial colorectal Caco-2 cell layer. (A) Adhesion of Lactobacillus paracasei strains to the Caco-2 epithelial cell layer as observed with Giemsa staining under a light microscope. Bars, 25 µm. One Caco-2 nucleus for each layer is indicated with the letter N. LPC-S01 adhesion was specific to Caco-2 cells: no adhesion was detected on the cover glass underlying Caco-2 cells (G; top part of the panel on the left). (B) Effect of L. paracasei strains on Caco-2 cells stably transfected with an NF-κB/luciferase reporter vector, in the presence of 5 ng/ml of IL-1β. Data in the histograms are the means (± standard deviations) from two independent experiments conducted in triplicate. RLU, relative luminescence units. Asterisks indicate statistically significant differences according to two-tailed unpaired Student's t-test: \*\*\*P < 0.001; \*\*P < 0.01; \*P < 0.05.

TABLE 1 | Antibiotic sensitivity of *Lactobacillus paracasei* LPC-S01 determined according to the microdilution assay recommended by EFSA (2012).

Data are reported as the lower concentration (mg/l) inhibiting bacterial growth (minimal inhibitory concentration, MIC).

possessing a wide range of properties, which may support its (i) industrial production, (ii) gastrointestinal administration, and (iii) vaginal colonization. To this aim, we focused our attention on Lactobacillus paracasei, a species characterized by marked ecological flexibility and extensive genetic diversity, which is widely used in industry (Cai et al., 2007). Particularly, we studied strain L. paracasei LPC-S01, which was originally isolated from the vagina of a healthy adult and has had a several-years history of commercial use as a vaginal probiotic for topical application in Europe. Specifically, the experiments carried out in this study were designed to evaluate if L. paracasei LPC-S01 has probiotic properties that can support its administration per os.

At first, the draft genome of LPC-S01 was analyzed by comparative genomics. We found the presence of numerous accessory genes (i.e., unique to this particular strain) that suggests the niche-generalists nature of LPC-S01; such potential ability to adapt to a wide range of environmental conditions is evidenced, for instance, by the presence of large chromosomal regions including genes for the utilization of numerous sugars (**Figure 2C**). Similar regions are also present in the genome of the probiotic strains DG and Shirota, which are of human intestinal origin. On the contrary, it was proposed that the adaptation of L. paracasei to the nutrient-rich milk environment had been gone along with extensive decay of genes involved in carbohydrate utilization (Broadbent et al., 2012). Accordingly, we found the lack of genes for sugar transport and metabolism in the chromosome of the dairy strains L. paracasei FMBr3 and N1115 compared to LPC-S01, DG, and Shirota strains (**Figure 1**).

The in vitro characterization of L. paracasei LPC-S01 was conducted in accordance with a procedure conventionally used to predict the probiotic potential of a microbial strain; such process consists in the assessment of sensitivity to acidity and bile, adhesion ability, pathogen inhibition, sugar fermentation profile, and immunomodulatory properties. In order to better predict the probiotic capabilities of LPC-S01, the reference strains Shirota and DG were also included in the study. L. paracasei Shirota is one of the most intensively studied probiotics (van den Nieuwboer et al., 2015; Wang et al., 2015) that has been used in the production of the fermented milk Yakult since 1935. L. paracasei DG is a bacterial strain with demonstrated probiotic properties (D'Incà et al., 2011; Tursi et al., 2013; Ferrario et al., 2014), which is included in Enterolactis R , one the most popular probiotic supplements in Italy. We observed that the in vitro performances of LPC-S01 were basically very similar to those of the reference strains. One exception was the significantly higher ability of LPC-S01 to survive in simulated gastric juice at pH 2 compared to L. paracasei Shirota, which is an acid-tolerant bacterial strain with demonstrated survivability to human gastrointestinal transit (Tuohy et al., 2007; Wang et al., 2015). In addition, LPC-S01 showed a much higher ability to adhere to Caco-2 epithelial cell layer than DG and Shirota. Bacterial adhesion properties mainly depend on the molecules exposed at the external cell surface (Guglielmetti et al., 2008; Polak-Berecka et al., 2014). Genome analysis revealed the presence in LPC-S01 of putative exopolysaccharide coding region (EPS2) containing a cluster of five genes which are not present in the other known L. paracasei genomes. Therefore, EPS2 could potentially support the synthesis of a peculiar exopolysaccharide molecule, which could plausibly contribute to the adhesion ability of the LPC-S01. Exopolysaccharides have been proposed to promote Lactobacillus adhesion (Ren et al., 2014), although, more frequently, the presence of a polysaccharide capsule has been shown to reduce adhesion by masking cell surface Lactobacillus adhesins determinants (Lebeer et al., 2012; Horn et al., 2013; Dertli et al., 2015), which are most commonly proteins (Lebeer et al., 2012; Polak-Berecka et al., 2014), (lipo)teichoic acids (Granato et al., 1999), and/or fatty acids (Polak-Berecka et al., 2014).

The acid tolerance and adhesion properties of LPC-S01 suggest that this bacterium might tolerate and adapt to the gastrointestinal environment. To test this hypothesis, we performed an intervention trial, in which we compared the gut persistence of L. paracasei LPC-S01 with that of strain DG. Following a daily consumption of 24 billion viable cells for 1 week, we estimated that strain LPC-S01 reached a concentration of about 108 cells per g of feces, persisting above the detection limit (105 cells per g of feces) for about 5 days, which is not significantly dissimilar to the persistence of the reference L. paracasei DG, a strain demonstrated to colonize the human gut of healthy adults and induce specific modifications in the microbiota composition (De Vecchi et al., 2008; Ferrario et al., 2014). Our data on L. paracasei persistence are in agreement with other studies, which observed that the persistence of L. paracasei Shirota in the gastrointestinal tract of healthy adults is lower than 1 week after cessation of the probiotic ingestion (Tuohy et al., 2007; Wang et al., 2015). The persistence of a certain probiotic strain in the gut plausibly depends on the evacuation frequency. In our study, we found persistence of strains LPC-S01 and DG as average for 7 evacuations. No data correlating the day of persistence with the number of evacuations were found in literature.

probiotic-intake period. Vertical bars denote 0.95 confidence intervals. Current effect according to repeated measures ANOVA performed to determine the statistical

## Conclusions

The results of this study can be summarized as follows:

## References

performances not dissimilar to those of the probiotic commercial strain L. paracasei DG.

In conclusion, this study demonstrates that the vaginal isolate L. paracasei LPC-S01 possesses safety and functional properties that may support its use as probiotic to be administered per os for potential intestinal as well as vaginal applications. In this perspective, it would be of interest to carry out a clinical trial consisting of the oral administration to healthy adult women of L. paracasei LPC-S01 cells, in order to verify the potential ability of this probiotic bacterium to (i) modulate the intestinal and vaginal microbiota, and (ii) colonize the human vagina via gastrointestinal route.

## Acknowledgments

This work was financially supported by Sofar S.p.A. (grant cod.16693). We thank Valentina Balzaretti for her assistance during manuscript revision.

## Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.00952

**Conflict of Interest Statement:** Research was financially supported by Sofar S.p.A., which is the company that produces and sells the probiotics products employed in the intervention study. WF is an employee of Sofar S.p.A. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Balzaretti, Taverniti, Rondini, Marcolegio, Minuzzo, Remagni, Fiore, Arioli and Guglielmetti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Fortification of table olive packing with the potential probiotic bacteria Lactobacillus pentosus TOMC-LAB2

## *F. Rodríguez-Gómez†, V. Romero-Gil †, P. García-García, A. Garrido-Fernández and Francisco N. Arroyo-López\**

Department of Food Biotechnology, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Seville, Spain

#### *Edited by:*

Fabio Minervini, Università degli Studi di Bari Aldo Moro, Italy

#### *Reviewed by:*

Chrysoula C. Tassou, National Agricultural Research Foundation, Greece Aldo Corsetti, University of Teramo, Italy

#### *\*Correspondence:*

Francisco N. Arroyo-López, Department of Food Biotechnology, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Avda. Padre García Tejero 4, 41012 Seville, Spain e-mail<EMAIL_ADDRESS>
†F. Rodríguez-Gómez and V. Romero-Gil have contributed equally to this work.

Dairy products are currently the main carriers of probiotic microorganisms to the human body. However, the development of new matrices for probiotic delivery is convenient for intolerant to milk (or its derivatives) and those requiring low-cholesterol diet consumers.The present work focused on the fortification of previously fermented green Spanish style olives with the autochthonous putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2. The fortification was carried out by inoculating the bacteria into the packing brines using Manzanilla fruits from three different processes: (i) spontaneously fermented (F1), (ii) fermented using L. pentosusTOMC-LAB2 as starter (F2), and (iii) spontaneously fermented and then thermally treated (F3). Data showed that all inoculated treatments had higher population levels (5.49, 4.41, and 6.77 log10 cfu/cm2) than their respective controls (1.66, 4.33, and 0.0 log10 cfu/cm2, for F1, F2, and F3 treatments, respectively). The presence of L. pentosus TOMC-LAB2 on olive surface was confirmed by rep-PCR, with a recovery frequency at the end of the shelf life (200 days) of 52.6, 57.9, and 100.0% for F1, F2, and F3 treatments, respectively. Thus, results obtained in this work show the ability of this microorganism to survive under packing conditions for long period of times as well as to colonize the olive surface which is the food finally ingested by consumers. This opens the possibility for the development of a new and simply probiotic fortified olive product.

**Keywords: fortification,** *Lactobacillus pentosus***, olive packing, probiotic vegetables, table olives**

#### **INTRODUCTION**

Table olive is one of the most important fermented vegetable of the food industry in the western countries, especially in the Mediterranean basin, with Spain, Turkey, Egypt, Italy, and Greece as the main producers. The International Olive Oil Council (IOC) estimated that the last consolidate balance of table olive production (2011/2012 season) was above 2.4 million tons (International Olive Council [IOC], 2014). Approximately 60% of this amount was processed according to green Spanish-style table olives, which implies a lye treatment of fruits (1.8–2.5% NaOH, w/v), followed by washing and brining (10–11% NaCl, w/v) where fruits undergo a typical lactic acid fermentation (Garrido-Fernández et al., 1997).

Lactic acid bacteria (LAB) exhibit diverse characteristics with potential benefits on human health. Among others, the following favorable effects have been documented: improvement of food digestibility and lactose assimilation, modulation of the immune response, reduction of hypercholesterolemia, prevention of intestinal infections, cancer, food allergies, and constipation (Champagne and Gardner, 2005). To these favorable aspects, we have to add that table olives might also be considered as a functional food itself because of their high content in antioxidant compounds, vitamins, dietary fiber, and anticancer compounds (Garrido Fernández et al., 2001; Reyes-Zurita et al., 2009).

Fermented vegetables are nowadays being considered as a splendid source and vehicle of probiotic microorganisms (Peres et al., 2012). Probiotic vegetables have the potential to attract more consumers who demand functional products, since vegetables provide to those who are intolerant to milk and its derivatives, or require low-cholesterol diets, access to new probiotic formulations (Lavermicocca et al., 2010; Peres et al., 2012). Packed green Spanish-style table olives can be thermally treated for preservation, but they can also be stabilized simply by physicochemical characteristics, being in this case considered as ready-to-eat product directly consumed without any prior cooking. This makes them very convenient carriers of beneficial microorganisms to the human body.

Ranadhera et al. (2010) considered that the type of food carrier plays an essential role in buffering the probiotic during its passage throughout the gastrointestinal tract, regulating their colonization or interacting with other microorganisms to alter functionality. Lavermicocca et al. (2005) used table olives as a vehicle to incorporate exogenous probiotic bacteria species into the human body. Particularly, one strain of *Lactobacillus rhamnosus* was remained invariant until the end of the experiment and showed a good recovery after fermentation. *L. paracasei* IMPC2.1 successfully colonized the olive surface and dominate the natural LAB population until the end of the fermentation (De Bellis et al., 2010), making the product a suitable carrier for its delivery to humans. Recently, Argyri et al. (2013a) and Bautista-Gallego et al. (2013) have isolated diverse LAB strains from table olive processing with promising characteristics for their use as probiotic agent.

Usually, the development of probiotic table olives has been focused on the application of these microorganisms as starter cultures at the onset of fermentation. A recent study showed that the inoculation with multifunctional lactobacilli starters led to a decrease in the *Enterobacteriaceae* populations and to higher LAB and yeast populations as well as to a faster acidification of the brines (Rodríguez-Gómez et al., 2013). Argyri et al. (2014) and Blana et al. (2014) also evaluated with promising results diverse potential probiotic LAB strains, originally isolated from olive fermentation, as starter cultures in table olive fermentation.

On the contrary, the present study is completely novel because is focused on the application of autochthonous putative probiotic microorganisms isolated from olive processing not during fermentation, but at the moment of packing closing with the aim of: (i) the development of a new and easy method for the fortification of table olives with potential probiotic, (ii) studying the survival of bacteria under the packing conditions to determine shelf life of the functional product.

## **MATERIALS AND METHODS**

#### **OLIVE PACKING CONDITIONS**

The fruits used in the present study were of the Manzanilla variety (*Olea europaea pomiformis*), previously fermented according to the green Spanish-style (Garrido-Fernández et al., 1997). To mimic industrial packing conditions, after 6 months of fermentation (from October 2010 to March 2011), 175 g of olives were introduced into A314 jars (314 mL volume, 75 mm diameter × 103.5 mm high) and covered with 145 mL of new fresh brine. This brine had the adequate concentrations of salt and lactic acid to reach 5.5% NaCl and 0.6% titratable acidity, expressed as lactic acid, in the equilibrium. The final pH was, approximately, 3.8 ± 0.1.

#### **FORTIFICATION OF OLIVES**

Packed olives were fortified with overnight cultures of strain *L. pentosus* TOMC-LAB2 (henceforth LAB2), previously selected because of their potential probiotic characteristics (Bautista-Gallego et al.,2013), ability of adhesion to olive epidermis (Arroyo-López et al., 2012), good performance as starter in previous fermentation trials performed at laboratory scale (Rodríguez-Gómez et al., 2013), and high survival to simulated *in vitro* human digestions (0.74%). Inoculum was grown until early stationary phase and then an aliquot of the suspension was added to the packing brines to reach an initial inoculum level of approximately 6.5 log10 cfu mL−1. The addition was essayed using three different types of fruits:

further addition of LAB, and fruits fortified with LAB2 strain after cooling of brines (F3-F).

Each treatment was carried out in duplicate, making a total of 12 packing containers which were kept during the entire process at the Instituto de la Grasa pilot plant (CSIC, Seville, Spain) at room temperature (variable from 18◦C in March/2011 to 31◦C September/2011). The experiment was monitored for 200 days.

#### **MICROBIOLOGICAL ANALYSES OF FRUITS**

Olive samples, or their decimal dilutions, were plated using a Spiral Plating System model dwScientific (Don Whitley Scientific Ltd., Shipley, UK) on the media described below at 0, 38, 80, and 200 days of packing. Plates were counted using a CounterMat v.3.10 (IUL, Barcelona, Spain) image analysis system. *Enterobacteriaceae* were counted on VRBD (Crystalviolet Neutral-Red bile glucose)-agar (Merck, Darmstadt, Germany), LAB on MRS (de Man, Rogosa and Sharpe)-agar (Oxoid) supplemented with 0.02% (w/v) sodium azide (Sigma, St. Louis, MO, USA), and yeasts on YM (yeast-malt-peptoneglucose) agar (DifcoTM, Becton and Dickinson Company, Sparks, MD, USA) supplemented with oxytetracycline and gentamicin sulfate as selective agents for yeasts. Plates were incubated at 37◦C for 24 h (*Enterobacteriaceae*) or 30◦C for 48 h (yeasts and LAB).

To determine the number of microorganisms adhered to the olive epidermis, the protocol developed by Böckelmann et al. (2003) was slightly adapted to the specific characteristics of table olives. Briefly, two fruits from each packing container were randomly taken at different sampling times and washed for 1 h with 250 mL of a sterile PBS buffer solution (8.0 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2HPO4, 0.24 g L−1 KH2PO4, pH finally adjusted to 4.7 with HCl 1 M) to remove non-adhering cells. Then, olives were transferred to 50 mL of a PBS solution added of the following enzymes: 14.8 mg L−1 lipase (L3126), 12.8 mg L−1 βgalactosidase (G-5160) and 21 μL L−1 α-glucosidase (G-0660) (Sigma–Aldrich, St. Louis, MO, USA). To achieve biofilm disintegration and removal of the adhered cells, the fruits were incubated at 30◦C in this enzyme cocktail with slight shaking (150 rpm). After 12 h, the olives were removed and the resulting suspension was centrifuged at 9,000 × *g* for 10 min at 4◦C. Finally, the pellet was re-suspended in 2 mL of PBS and spread onto the different culture media described above. Olive microbial counts were expressed as log10 cfu/cm2, using the formula of a prolate spheroid for the approximate calculus of olive surface from the longitudinal and transverse axes of fruits (Weisstein, 2013). For the Manzanilla fruits used in the present study, the average area was 10.99 ± 1.01 cm2 and the weight 4.08 ± 0.46 g.

#### **CHARACTERIZATION OF THE LACTIC ACID BACTERIA POPULATION**

For characterization of the lactobacilli population, repetitive bacterial DNA element fingerprinting analysis (rep-PCR) with primer GTG5 was followed using the protocol described in Gevers et al. (2001). The PCR reaction in a final volume of 25 μL contained: 5 μL of 5x MyTaq reaction buffer (5 mM dNTPs and 15 mM MgCl2), 0.1 μL of My Taq DNA polymerase (Bioline reactives, UK), 1 μL GTG5 primer (25 μm), 13.9 μL desionized H20, and

5 μL DNA (∼20 ng/μL). PCR amplification was carried out in a thermal cycler (Master Cycler Pro, Eppendorf) with the following program: 95◦C for 5 min as initial step, plus 30 cycles at: (1) 95◦C for 30 s, (2) annealing at 40◦C for 30 s, and (3) 65◦C for 8 min, with a final step of 65◦C for 16 min to conclude the amplification. This methodology was used to determine the recovery frequency of the inoculated strain at the end of packing. Firstly, the repeatability of the technique was determined using the LAB2 strain as internal control, obtaining a 86.9% ± 3.4% of similarity for this bacteria in ten different gels (**Figure 1**). Then, diverse isolates were randomly picked from each treatment at the end of experiment (200 days), making a total of 88 lactobacilli to analyze. They were named with the name of treatment (F1, F2, or F3) and with a "F" in the case of fortified/reinforced olives. Their pattern profiles of bands (from 100 up to 3,000 bp) were compared with the strain used to fortify the treatments (LAB2). For this purpose, PCR products were electrophoresed in a 2% agarose gel and visualized under ultraviolet light by staining with ethidium bromide. The resulting fingerprints were digitally captured and analyzed with the BioNumerics 6.6 software package (Applied Maths, Kortrijk, Belgium). The similarity among digitalized profiles was calculated using the Pearson product-moment correlation coefficient. Dendrograms were obtained by means of the Unweighted Pair Group Method using Arithmetic Average (UPGMA) clustering algorithm and the automatic calibration tool for the determination of the optimization and curve smoothing parameters.

#### **STATISTICAL ANALYSIS**

Analysis of variance was performed by means of the one-way ANOVA module of Statistica 7.1 software to check for significant differences among treatments. For this purpose, a *post hoc* comparison was applied by means of the Scheffé test.

### **RESULTS AND DISCUSSION**

Afterfermentation (6 months), three different types of fruits where placed in new fresh brine and inoculated with a putative probiotic bacteria (LAB2) just before closing the containers. Initial physicochemical conditions (pH, salt, free, and combined acidity) were stable during the whole period of packing (data not shown). Below, the main results obtained for the changes in the microbial populations and the presence of the inoculated strain after 200 days of shelf life are shown.

#### **EVOLUTION OF MICROBIAL POPULATIONS THROUGH PACKING**

Diverse works have reported that the survival of microorganisms through table olive packing is considerable. Argyri et al. (2013b) showed that microbial population after 40 days of Halkidiki green table olive packing was above 7 and 5 log10 cfu g−1 for LAB and yeasts, respectively. Bautista-Gallego et al. (2011) reported that after 60 days of Aloreña directly brined packing, the counts for LAB and yeasts were above 5 and 6 log10 cfu mL−1, respectively. For this reason, it can be expected that probiotic bacteria could also survive for long period of time under packing conditions, and thereby, be used for fortification purposes.

**Figure 2** shows the evolution of the LAB and yeasts population on olive surface for fruits spontaneously fermented (F1) and fortified (F1-F) with LAB2 strain at the moment of packing. A clear and statistical significant difference between the changes in both LAB populations in the two types of treatments was noticed (**Figure 2A**). In the case of F1 fruits, a marked decline of population was observed through packing, while in fortified fruits (F1-F), LAB counts were always higher and practically constant during the shelf life. Thereby, at the end of the studied period (200 days), the population for fortified fruits was 5.5 against 1.7 log10 cfu/cm2 for non-fortified fruits. In the case of yeast population (**Figure 2B**), the evolution between both treatments was similar but with slight differences. A marked decline of yeast population was observed through packing in both cases, from the initial 5.8 to 1.3 (F1- F) or 0.0 (F1) log10 cfu/cm2. Then, yeast survival was also slightly (but statistical significant) favored by the addition of the bacteria.

**Figure 3** shows the evolution of the LAB and yeast populations on olive surface for fruits previously fermented using LAB2 as starter culture, packaged just as they were processed (F2) or reinforced with the same strain just before closing of containers (F2-F). The changes in the LAB population was very similar (no statistical differences) between reinforced and non-reinforced fruits, with a progressive reduction from the initial 7.0 to around 4.4 log10 cfu/cm2 at 80 days. Later on, the population remained,

**FIGURE 2 | Changes in the LAB (A) and yeast (B) populations on olive surface during shelf life.** Control (F1) corresponded to just packaged spontaneously fermented fruits, while fortified (F1-F) were those inoculated with L. pentosus TOMC-LAB2 strain.

in practice, constant until the end of experiment. The evolution of the yeast population was very similar as in the previous case, with a clear decline through packing period (from the initial 4.3 to around 2.0 log10 cfu/cm2 at the end of study), although a higher population was observed during most of the shelf life.

**Figure 4** shows the evolution of the LAB and yeast populations on olive surface for fruits spontaneously fermented and thermally treated before packing. As commented, some fruits were packed without LAB addition (F3), while others were fortified with LAB2 strain (F3-F). Apparently, the thermal treatment (85◦C for 5 min) was very efficient for killing the vegetative cells of both LAB and yeasts adhered to the olive surface, and no microbial cells were detected, except in the treatments inoculated with LAB2 strain (**Figure 4A**). In this case, the LAB added to the brine was able to colonize the olive surface in less than 38 days. In fact, the population increase from 0.0 to approximately 6.8 log10 cfu/cm2 was very fast and persisted up to the end of the shelf life.

Finally, *Enterobacteriaceae* were not detected in any of the packing treatments assayed through shelf life. This is in agreement with the low pH obtained in the packing conditions (pH < 4.0), which is a very inhibitory factor for the survival of this group of microorganism (Garrido-Fernández et al., 1997), but also due to the lack of appropriate nutrients because the sugars were previously exhausted during fermentation process (data not shown).

Therefore, data obtained in this work show the ability of the LAB2 strain to move from packing brine onto the fruits and to colonize the olive surface, which was especially evident in the case of F3-F treatment. This is in agreement with results obtained recently by Arroyo-López et al. (2012) and Domínguez-Manzano

et al. (2012) who demonstrated that diverse *L. plantarum* and *L. pentosus* strains were able to establish polymicrobial communities with yeasts on the surface of olives processed according to green Spanish-style. Peres et al. (2012) mention diverse types of technological processes (microencapsulation, vacuum, and immersion impregnation, cell immobilization, etc.) to fortify plant matrices with probiotic microorganism. In this work, the simply preparation of a packing brine with a concentration of ∼ 6.5 log10 cfu mL−1 of LAB2 strain, resulted in a single and easy method for the fortification of olives with the desire bacteria by means of a surface adhesion mechanism.

#### **PRESENCE OF THE INOCULATED STRAIN AT THE END OF SHELF LIFE**

The presence of the inoculated LAB2 strain at the end of packing was corroborated by molecular methods (rep-PCR) using a total of 88 lactobacilli isolates obtained from F1, F2, F3, and their respective fortified/reinforced treatments.

**Figure 5** shows the dendrogram generated using the patterns profile of 29 LAB isolates randomly obtained from epidermis of F1 (10) and F1-F (19) fruits plus the inoculated LAB2 strain. The cluster analysis showed that 10/19 isolates obtained from the fortified F1 treatment shared a 87.4% similarity with the inoculated LAB2 strain, which was above repeatability of the technique (86.8%). Thereby, LAB2 strain showed in F1-F treatment a recovery frequency of 52.6%, while 9/19 isolates (47.4%) where grouped in different clusters together with many of the isolates obtained from the spontaneous and non-fortified F1 treatment.

**Figure 6** shows the dendrogram generated using the patterns profile of 39 lactobacilli isolates randomly obtained from epidermis of F2 (20) and F2-F (19) fruits plus the inoculated LAB2 strain. The cluster analysis showed that 11/19 isolates obtained from the reinforced F2-F treatment shared a 88.8% similarity with the inoculated LAB2 strain, which again was above repeatability of the technique. Thus, LAB2 strain showed in the reinforced treatment a recovery frequency of 57.9% at 200 days of packing, while 8/19 isolates (42.1%) where grouped in different clusters with many of the isolates obtained from the non-reinforced F2 treatment. It is remarkable to verify that, in the case of no inoculation, it was not possible to recover the profile of the LAB2 strain from the F2 treatment even when this microorganism was used as starter culture at the onset of fermentation. Thus, a reinforcement step with this strain at the beginning of packing to assure its presence during shelf life it is always advisable.

Finally, **Figure 7** shows the dendrogram generated using the patterns profile of 20 lactobacilli isolates randomly obtained from olive epidermis of F3-F treatments plus the inoculated LAB2 strain. In this case, because of the thermal treatment, no LAB isolates were obtained from F3 treatment. The cluster analysis showed that 20/20 isolates obtained from the F3-F treatment shared a 86.5% similarity with the inoculated LAB2 strain, which is in the limit of repeatability of the technique. Thereby, LAB2 strain showed in the fortified treatment a recovery frequency of 100.0% at 200 days, which is logical because, in this case, the survival of other microorganism was prevented by the thermal treatment.

Taking into account the average surface values obtained for the Manzanilla fruits used in the present study (11 cm2), a total of ∼ 108 LAB cells could be ingested by consumers who eat only one fortified olive with LAB2 strain after thermal treatment. Data could also be converted to cfu g−1 taking into account the average weight of fruits (4.08 g).  The Spanish Association of Processors and Exporters (ASEMESA, 2014) considers 7 olives (about 25 g of olive flesh) as a satisfactory daily intake. Olives thermally treated and fortified with

LAB2 strain clearly exceed the number of recommended probiotic microorganism. Pasteurization of Spanish green table olives using glass and tin as packing material is usually carried out by industry to enlarge the shelf life of the product using a minimum amount of preservatives (Garrido-Fernández et al., 1997). Thus, this type of product could be a convenient carrier of probiotic to the human body because of the high number of microorganisms that potentially could be ingested by consumers with a larger shelf life compared to dairy product.

#### **CONCLUSION**

Data obtained in this work show that fortification of table olives with *L. pentosus* TOMC-LAB2 is possible because of the considerable survival of this microorganism during shelf life at room temperature without chill chain. Among the different types of fortification assayed, a thermal treatment after fermentation to eliminate microorganism and later inoculation with the bacteria is desired because of the high counts and recovery frequency of the added LAB bacteria obtained at the end of the packing period. However, sensorial studies should be also carried out to determine

**digitalized rep-PCR fingerprints with primer GTG5 of 39 lactobacilli isolates obtained from fruits previously fermented using** *L. pentosus* **TOMC-LAB2 as starter culture.** Part of fruits were just

closing (F2-F), while 1–20 is the number for the isolates obtained within each treatment. Clustering parameters: 0.5% optimization and 5.0% curve smoothing.

the influence of the thermal treatment or bacteria growth on the sensory profile of packaged olives.

#### **ACKNOWLEDGMENTS**

The research leading to these results has received funding from the EU's Seventh Framework Programme [FP7/2007-2013] under grant agreement no 243471 (PROBIOLIVES). We also thank the Spanish Government for financial support (projects AGL2009- 07436/ALI, and AGL2010-15529/ALI partially financed by European regional development funds, ERDF), and the Junta de Andalucía (through financial support to group AGR-125). Thanks to JOLCA and ASEMESA for supplying the fruits and their own expertise for the development of this work. Francisco N. Arroyo-López wishes to express thanks for his Ramón y Cajal postdoctoral research contract (Spanish government).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 June 2014; paper pending published: 11 July 2014; accepted: 18 August 2014; published online: 03 September 2014.*

*Citation: Rodríguez-Gómez F, Romero-Gil V, García-García P, Garrido-Fernández A and Arroyo-López FN (2014) Fortification of table olive packing with the potential probiotic bacteria Lactobacillus pentosus TOMC-LAB2. Front. Microbiol. 5:467. doi: 10.3389/fmicb.2014.00467*

*This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Rodríguez-Gómez, Romero-Gil, García-García, Garrido-Fernández and Arroyo-López. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Survival of pathogenic and lactobacilli species of fermented olives during simulated human digestion

*Francisco N. Arroyo-López1\*, Stéphanie Blanquet-Diot 2 , Sylvain Denis 2 , Jonathan Thévenot 2,3 , Sandrine Chalancon2 , Monique Alric 2 , Francisco Rodríguez-Gómez 1, Verónica Romero-Gil 1, Rufino Jiménez-Díaz1 and Antonio Garrido-Fernández1*

1 Biotecnología de Alimentos, Instituto de la Grasa – Consejo Superior de Investigaciones Científicas, Seville, Spain

2 Centre de Recherche en Nutrition Humaine Auvergne, EA 4678, Conception Ingénierie et Développement de l'Aliment et du Médicament, Clermont Université – Université d'Auvergne, Clermont-Ferrand, France

3 Centre de Recherche en Nutrition Humaine Auvergne, M2iSH, UMR INSERM/Université d'Auvergne U1071 USC-INRA 2018, Clermont Université – Université d'Auvergne, Clermont-Ferrand, France

#### *Edited by:*

Fabio Minervini, Università degli Studi di Bari Aldo Moro, Italy

#### *Reviewed by:*

Chrysoula C. Tassou, Hellenic Agricultural Organization "DEMETER", Greece Zhao Chen, Clemson University, USA

#### *\*Correspondence:*

Francisco N. Arroyo-López, Biotecnología de Alimentos, Instituto de la Grasa – Consejo Superior de Investigaciones Científicas, Avenida Padre García, Tejero n◦4, 41012 Seville, Spain e-mail<EMAIL_ADDRESS>
The present survey uses a dynamic gastric and small intestinal model to assess the survival of one pathogenic (Escherichia coli O157:H7 EDL 933) and three lactobacilli bacteria with probiotic potential (Lactobacillus rhamnosus GG, L. pentosus TOMC-LAB2, and L. pentosusTOMC-LAB4) during their passage through the human gastrointestinal tract using fermented olives as the food matrix. The data showed that the survival of the E. coli strain in the stomach and duodenum was very low, while its transit through the distal parts (jejunum and ileum) resulted in an increase in the pathogen population. The production of Shiga toxins by this enterohemorrhagic microorganism in the ileal effluents of the in vitro system was too low to be detected by ELISA assays. On the contrary, the three lactobacilli species assayed showed a considerable resistance to the gastric digestion, but not to the intestinal one, which affected their survival, and was especially evident in the case of both L. pentosus strains. In spite of this, high population levels for all assayed microorganisms were recovered at the end of the gastrointestinal passage. The results obtained in the present study show the potential use of table olives as a vehicle of beneficial microorganisms to the human body, as well as the need for good hygienic practices on the part of olive manufacturers in order to avoid the possibility of contamination by food-borne pathogens.

**Keywords: food carrier, lactobacilli, pathogen, probiotic, survival, Shiga toxins, table olives,TIM system**

## **INTRODUCTION**

Green Spanish-style table olives are considered ready-to-eat products which are directly consumed without any prior cooking, and often, without any pasteurization or sterilization treatment. This makes olives a splendid vehicle of microorganisms (beneficial or harmful) to the human body. Thereby, the analysis of the microbiota adhered to the fruits acquires a relevant importance, although, incomprehensibly, this point has received scarce attention by researchers. Fermented olives contain biofilm structures formed mainly by lactic acid bacteria (LAB) and yeasts, which can reach population levels up to 8 log10 CFU g−1 in the epidermis of the fruits (Nychas et al., 2002; Arroyo-López et al., 2012; Domínguez-Manzano et al., 2012). Native LAB or yeast strains isolated from olive microbiota which show potential probiotic characteristics could be used as starters either to initiate fermentation or to obtain a functional product after adhesion to the fruits (Rodríguez-Gómez et al., 2013; Blana et al., 2014). To exert their health effect, microorganisms to be evaluated as probiotics should be able to survive the harsh conditions found in the human digestive tract such as acidic pH or high concentrations of digestive enzymes and bile salts.

Regarding safety issues, it is necessary to provide more knowledge about the behavior of pathogens in ready-to-eat fermented olives for future risk assessments. Enterohemorrhagic *Escherichia coli* is a major food-borne pathogen that causes hemorrhagic colitis and a life-threatening sequelae, the hemolytic uremic syndrome (Pennington, 2010). The behavior of the enterohemorrhagic *E. coli* O157:H7 during olive processing (fermentation and packing) has been reported by several authors (Spyropoulou et al., 2001; Skandamis and Nychas, 2003; Argyri et al., 2013; Grounda et al., 2013) but there is hitherto no available information about the survival of this pathogen in the case of a hypothetical intake of contaminated olives. To cause human illness, *E. coli* O157:H7 should not only survive its passage through the gastrointestinal tract but also coordinate the expression of virulence genes especially that of encoding Shiga toxins (Foster, 2013).

Most of the available data on probiotic or pathogen survival in the human digestive tract has been obtained in static *in vitro* systems that are not representative of the continuously changing variable during the gastrointestinal transit. The TNO gastrointestinal tract model (TIM system, Zeist, Netherlands) is an alternative dynamic multi-compartmental *in vitro* system which currently allows the closest simulation of *in vivo* physiological processes occurring within the stomach and small intestine of humans (Minekus et al., 1995; Guerra et al., 2012).

In this work, we used the TIM system to assess the survival and toxin production of an enterohemorrhagic *E. coli* strain inoculated in fermented olives during its transit through the upper human gastrointestinal tract. The survival of two potential probiotic strains isolatedfrom table olives and belonging to *Lactobacillus pentosus* species (never studied before in dynamic digestive assays) was also evaluated and compared to that of a well-recognized probiotic microorganism (*L. rhamnosus* GG).

## **MATERIALS AND METHODS**

#### **OLIVES AND MICROORGANISMS**

Fermented olives of the *Manzanilla* variety (*Olea europaea pomiformis*) were used in the present study. The fruits were previously fermented for two months according to the Spanish-style (Garrido-Fernández et al., 1997) and then pasteurized at 80◦C for 15 min to avoid any microbial presence on the olive surface. Twenty-five grams of pasteurized olives were then homogenized with 250 mL of sterile water and independently inoculated with aerobic cultures of the reference strain *E. coli* O157:H7 EDL 933 (LB, 24 h, 37◦C), the probiotic *L. rhamnosus* GG, and the olive isolates *L. pentosus* TOMC-LAB2 and *L. pentosus* TOMC-LAB4 (MRS broth, 24 h, 37◦C) to reach a final population in the mix of 8.2, 9.1, 9.7, and 9.4 log10 CFU, respectively. Except for the pathogen strain, these inoculation levels are similar for the lactic acid population usually present in 25 g of fermented olives. The two *L. pentosus* strains, originally isolated from Spanish-style table olive fermentations and belonging to the table olive microorganisms collection (TOMC) from Food Biotechnology Department of Instituto de la Grasa (CSIC-Seville), were selected because of their previously described probiotic characteristics (Bautista-Gallego et al., 2013) and good performance as starter in previous fermentation trials carried out at laboratory scale (Rodríguez-Gómez et al., 2013). **Table 1** shows the main physicochemical conditions of the fermented olives at the moment of introduction into the TIM system.

#### **SIMULATED HUMAN DIGESTIVE CONDITIONS**

The TIM system, which simulates the physiological processes occurring in the stomach, duodenum, jejunum, and ileum, was programmed to reproduce the digestion of a solid food matrix in a healthy human adult using the protocol described by Etienne-Mesmin et al. (2011). The total duration of the digestions was 300 min with *n* = *2* digestions for each different microorganism. The parameters used for *in vitro* digestion are described in **Table 2**. Samples were taken in the test meal (initial intake) after inoculation and before its introduction into the artificial stomach, and regularly collected during digestion in the different compartments of the system (stomach, duodenum, jejunum, and ileum) as well as in the cumulative ileal effluents (CIE). Microbial counting was performed on LB agar (for *E. coli*) or on MRS agar (for the lactobacilli strains; Oxoid LTD, Basingstoke, Hampshire, England). Because food and microorganisms are in continuous transit from one compartment to the next, in order to assess microbial survival rates in the TIM system, control digestions (*n* = *2*) were carried out with the same protocol conditions used

for microorganisms, but with water containing only 0.8% (w/v) of blue dextran (Minekus et al., 1995). This compound is a non absorbable transit marker, which will represent a 100% survival percentage for bacteria. Thereby, curves below the transit marker will represent the mortality of the microorganism, while curves above the transit marker will be indicative of bacteria growth renewal.

#### **DETERMINATION OF THE PRODUCTION OF SHIGA TOXINS BY THE** *E. coli* **STRAIN**

Shiga toxins produced by *E. coli* O157:H7 EDL 933 in the ileal effluents of the TIM system were dosed by enzyme linked immunosorbent assay (ELISA) using the Ridascreen®Verotoxin kit (R-Biopharm, Darmstadt, Germany). Purified toxin Stx2 (Toxin Technology®, Sarasota, FL, USA) was used to establish standard calibration curves. Samples were diluted to half with the provided diluent and the dosage was made according to the manufacturer's instructions. Each digestive sample was analyzed in duplicate and serial dilutions of purified toxin were analyzed in triplicate.

#### **STATISTICAL ANALYSIS**

Significant differences among treatments (*p* ≤ 0.05) were tested by analysis of variance (ANOVA) followed by a Fisher-LSD *post hoc* comparison test carried out with STATISTICA 7.0 software package (Statsoft Inc, Tulsa, OK, USA).

### **RESULTS AND DISCUSSION**

In the present study, fermented olives were independently inoculated with a food-borne pathogen and three potential beneficial microorganisms in order to determine their survival during their passage through the upper gastrointestinal tract of humans and the pathogen's ability to produce toxins. So far, there is no information available on the survival of microorganisms through the different compartments of the human gastrointestinal tract using table olives as the food matrix. The only related study was carried out by Lavermicocca et al. (2005), who fed volunteers with olives inoculated with the human origin strain *L. paracasei* IMPC2.1. These authors were able to recover the mentioned microorganism from fecal samples, but they did not obtain information on its resistance to specific gastric and intestinal conditions.

The behavior of the major food borne pathogen *E. coli* O157:H7 (strain EDL 933) during its transit through the different compartments of the TIM system is shown in **Figure 1**. In the stomach and duodenum, its population decreased considerably, probably due to the occurrence of stringent conditions such as gastric acidity, digestive enzymes, or bile salts (Smith, 2003). The highest cell mortality was observed in the artificial stomach from 90 min onward, when the pH fell below 3 units (see **Table 2**). At this time, the population decreased by more than 2 log10 CFU compared to the transit marker used as control, and no living cells were recovered from this compartment at 150 min. In the duodenum, at 180 min, around 3 log10 CFU were lost compared to the marker.

On the contrary, in the distal parts of the artificial gastrointestinal tract (jejunum and ileum), *E. coli* O157:H7 resumption was observed especially at the end of digestion. Thereby, at time 300 min, the counts for bacteria even exceeded that of

**Table 1 | Experimental design and physicochemical conditions of the fermented olives used in the present work as food matrix.**

\*Fruits had an average size of 4.06 ± 0.49 g and area surface of 10.93 ± 1.07 cm2.

**Table 2 | Parameters of gastrointestinal digestion in the TIM system when simulating digestive conditions of a healthy adult after intake of a solid food matrix.**

For each digestive compartment, the table gives the volume (initial volume for gastric compartment, maximal volume for intestinal compartments), the pH (depending on the time of digestion in the stomach), and the flow rates of digestive enzymes and bile salts. HCl and NaHCO3 are added to the gastric and intestinal compartments, respectively, if the measured value of the pH deviates from the set point. The power exponential equation (f = 1–2−(t /t 1/2)β, where f represents the fraction of meal delivered, t the time of delivery, t1/2 half the time of delivery, and β a coefficient describing the shape of the curve) was used for the computer control of gastric and ileal deliveries into the TIM system. Parameters used for t1/2 and β for gastric and ileal deliveries are mentioned in the table.

the transit marker, chiefly in the jejunum (increase of 0.5 log10 CFU compared to the control marker). At 300 min, a high amount of viable cells were recovered from the ileal effluents, decreasing only by 0.2 log10 CFU with respect to the blue dextran. Bacterial growth renewal has been previously observed in the distal compartments of the TIM system for other *E. coli* strains (Ganzle et al., 1999; Etienne-Mesmin et al., 2011; Miszczycha et al., 2014). This growth resumption was probably linked to less stringent environmental conditions, such as a pH closer to neutrality, lower concentrations of bile salts (owing to their passive reabsorption in the TIM system), and/or an increase in the residence time of bacteria. This event led to an increase in the number of bacteria which could potentially enter into the colon and may potentiate the harmfulness of the food-borne pathogen in the hypothetical case of an intake of contaminated olives.

Large variations in survival rates have been obtained for *E. coli* O157:H7 in diverse *in vitro* digestion assays (Arnold and Kaspar, 1995; Takumi et al., 2000; Foster, 2004; Tamplin, 2005; Etienne-Mesmin et al., 2011). This wide range of response may be explained by differences between culture conditions, digestive

systems (static or dynamic) and parameters, food matrices and also bacterial strains. Interestingly, the only studies that evaluated the behavior of *E. coli* O157:H7 in dynamic conditions gave survival rates after gastric digestion close to those obtained in the present work (Takumi et al., 2000; Etienne-Mesmin et al., 2011; Miszczycha et al., 2014). In table olive packing, this microorganism has shown also a considerable survival being present until 19th days of storage on olive fruits at low pH (4.2) and high salt concentration (60 g/l; Argyri et al., 2013).

Hitherto, there is no available data regarding the production (site and amount) of Shiga toxins by enterohemorrhagic *E. coli* strains during their transit through the human gastrointestinal tract. This study is the first to investigate toxin production by this food-borne pathogen in a human simulated digestive environment. Toxin levels in the ileal effluents of the TIM system were too low to be quantified by ELISA assays, which had a detection threshold value of 0.31 ng/ml (data not shown), indicating that under the tested conditions the pathogen was not able to produce Shiga toxins, its main virulence factor, at least at sufficient levels to be detected. It also suggests that toxin production, if it occurs, would rather

take place in the colon, which is already described as the main site of pathogenicity for EHEC strains (Shigeno et al., 2002).

This study also aims to assess the survival of two *L. pentosus* strains isolated from table olives for their use as putative probiotics. LAB species are well known for their ability to resist and live in many different acidified fermented vegetables, among them table olives (Hurtado et al., 2012). The *L. pentosus* strains assayed in this work were originally isolated from table olive fermentations, so, presumably, they should be well adapted to the acidic environment which governs this type of product. **Figures 2** and **3** show the results obtained for the survival in the TIM system of *L. pentosus* TOMC-LAB2 and TOMC-LAB4, respectively. Both bacteria showed a considerable resistance to gastric digestion with a slight decrease at time 150 min of less than 0.1 log10 CFU with respect to the transit marker. However, their survival through the duodenum was considerably lower, especially evident in the case of the *L. pentosus* TOMC-LAB2 strain with a reduction of more than 3 log10 CFU at 240 min. On the contrary, the survival of neither microorganism was affected by the conditions encountered in the distal parts of the gastrointestinal tract (jejunum and ileum). At 300 min, compared to the transit marker, the amount of bacteria recovered from the ileal effluents was reduced by 2.1 and 1.3 log10 CFU for *L. pentosus* TOMC-LAB2 and TOMC-LAB4 strains, respectively.

To be effective and confer health benefits on the host, probiotics must be able to survive passage through the human stomach and small intestine and be present in sufficient number to colonize the colonic environment (Del Piano et al., 2006). The survival of the *L. pentosus* strains isolated from table olives was compared to that of the well-known probiotic *L. rhamnosus* GG (**Figure 4**). This microorganism was slightly affected during its transit through the stomach (only 0.1 log10 CFU reduction at time 150 min). Pitino et al. (2012) also found a high resistance of this species to stomach digestion in a dynamic gastric model. On the contrary, the population decreased in higher proportions at the end of its transit through the duodenum (∼1 log10 CFU at time 240 min) and it seemed to be not affected by the conditions occurring in

the jejunum and ileum (no additional mortality was observed in either compartment). At 300 min, high levels for this LAB species were recovered from the ileal effluents, decreasing by only 0.3 log10 CFU compared to the transit marker.

**Table 3** shows the percentage of survival obtained for the four assayed bacteria after their transit through the different compartments of the TIM system as well as in the ileal effluents at the end of digestion. As can be clearly deduced even though *E. coli* O157:H7 is considered a highly acid resistant pathogen (Foster, 2004), this microorganism was the most affected bacteria by gastric digestion, with statistically significant differences (*p* < 0.05) compared to lactobacilli strains. *L. rhamnosus GG* was the most resistant bacteria during its transit through duodenum, although without

significant differences according to a Fisher-LSD *post hoc* comparison test. This is in agreement with previous results obtained by Silva et al. (1987) and Pitino et al. (2012) who cataloged *L. rhamnosus* as an acid and bile-resistant species. Bile salts are described as toxic at high concentrations for bacterial cells by disorganizing the lipid bi-layer structure of the cellular membranes (Thanassi et al., 1997). This is presumably the reason why all assayed microorganisms reduced their population during their transit through the duodenum. On the contrary, the food-borne pathogen was the

most adapted microorganism in the distal parts of the gastrointestinal tract (jejunum and ileum), with significant differences (*p* < 0.05) compared to the lactobacilli species in the ileum. Thereby, after gastric and small intestinal transit, the survival rate was statistically higher for *E. coli* O157:H7 EDL 933 (117.5%) than for the rest of bacteria (from 0.7 to 40.5%), indicating that a higher percentage of the pathogen population could enter into the colon compared to the lactobacilli species in the case of a hypothetical intake of contaminated olives. *L. pentosus* TOMC-LAB2

**Table 3 | Survival (%) of the four tested strains at the end of** *in vitro* **digestions in the different compartments and ileal effluents (CIE) of the TIM system, using fermented** *Manzanilla* **olives as food matrix.**

E150, D240, J300 and I300 stands for instantaneous percentages in the stomach (150 min), duodenum (240 min), jejunum (300 min), and ileum (300 min), respectively. CIE300 and TF325 stands for cumulative percentages from ileal effluents (0−300 min) and cumulative percentages from ileal effluents (0−300 min) plus final gastrointestinal residue, respectively. SD in parentheses. Values followed by different superscript letters, within the same column, are significantly different (p < 0.05) according to a Fisher-LSD post hoc comparison test.

and TOMC-LAB4 have already shown promising probiotic characteristics (Bautista-Gallego et al., 2013) as well as a considerable capacity for adhesion to the olive epidermis (Arroyo-López et al., 2012). Population levels higher than 7.5 log10 CFU were recovered for both strains after their transit through the artificial ileum and before entering into the colon (survival percentages of 0.6 and 3.5% for *L. pentosus* TOMC-LAB2 and TOMC-LAB4, respectively), compared to 8.5 log10 CFU obtained for *L. rhamnosus* GG (survival percentage of 31.3%). This makes them good candidates for use as probiotic agents, which could increase the functional value of table olives.

#### **CONCLUSION**

In summary, these results encourage further research on the development of table olives as a functional food, because, apparently, it is a good vehicle of microorganisms to the human body. Nevertheless, at the same time, they also show the necessity of good hygienic practices by olive manufacturers to avoid any possibility of intake of EHEC-contaminated olives, even though, apparently, the pathogen was not able to produce toxins during its transit through the upper human gastrointestinal tract following olive intake.

### **ACKNOWLEDGMENTS**

The research leading to these results has received funding from the EU's Seventh Framework Program (FP7/2007-2013) under grant agreement n◦ 243471 (PROBIOLIVES). Francisco N. Arroyo-López wish to express his gratitude to the Spanish Government for his Ramón y Cajal postdoctoral research contract, as well as CSIC for financial support of the research mobility program in foreign countries (A12012PM2-PA1003309). Verónica Romero-Gil thanks to the Agrifood Campus of International Excellence (ceiA3) for her predoctoral fellowship (program of formation of Ph.D. in companies).

#### **REFERENCES**

young and elderly people. *J. Appl. Microbiol.* 89, 935–943. doi: 10.1046/j.1365- 2672.2000.01193.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 23 July 2014; paper pending published: 26 August 2014; accepted: 29 September 2014; published online: 14 October 2014.*

*Citation: Arroyo-López FN, Blanquet-Diot S, Denis S, Thévenot J, Chalancon S, Alric M, Rodríguez-Gómez F, Romero-Gil V, Jiménez-Díaz R and Garrido-Fernández A (2014) Survival of pathogenic and lactobacilli species of fermented olives during simulated human digestion. Front. Microbiol. 5:540. doi: 10.3389/fmicb.2014.00540 This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Arroyo-López, Blanquet-Diot, Denis, Thévenot, Chalancon, Alric, Rodríguez-Gómez, Romero-Gil, Jiménez-Díaz and Garrido-Fernández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## *Xinheng Yu1,2,3 , Lei Chen1,2,3 and Weiwen Zhang1,2,3 \**

1 Laboratory of Synthetic Microbiology, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China

2 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, China

3 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, China

#### *Edited by:*

Maria De Angelis, University of Bari Aldo Moro, Italy

*Reviewed by:*

Ebrahim Hadavi, Islamic Azad University, Iran Zhao Chen, Clemson University, USA

#### *\*Correspondence:*

Weiwen Zhang, Laboratory of Synthetic Microbiology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China e-mail<EMAIL_ADDRESS>
Photosynthetic microalgae have attracted significant attention as they can serve as important sources for cosmetic, food and pharmaceutical products, industrial materials and even biofuel biodiesels. However, current productivity of microalga-based processes is still very low, which has restricted their scale-up application. In addition to various efforts in strain improvement and cultivation optimization, it was proposed that the productivity of microalga-based processes can also be increased using various chemicals to trigger or enhance cell growth and accumulation of bioproducts. Herein, we summarized recent progresses in applying chemical triggers or enhancers to improve cell growth and accumulation of bioproducts in algal cultures. Based on their enhancing mechanisms, these chemicals can be classified into four categories:chemicals regulating biosynthetic pathways, chemicals inducing oxidative stress responses, phytohormones and analogs regulating multiple aspects of microalgal metabolism, and chemicals directly as metabolic precursors. Taken together, the early researches demonstrated that the use of chemical stimulants could be a very effective and economical way to improve cell growth and accumulation of high-value bioproducts in large-scale cultivation of microalgae.

**Keywords: chemicals, microalgae, growth, accumulation, bioproducts**

#### **INTRODUCTION**

Microalgae are autotrophic organisms, which utilize light energy, and inorganic nutrients such as CO2, nitrogen and phosphorus, to generate biomass and synthesize valuable metabolites. Some algal species cultivated under stress conditions accumulate specific secondary metabolites (i.e., pigments, vitamins, or lipids), which are high-value bioproducts that can be applied in the cosmetic, food, or pharmaceutical sectors (Skjanes et al., 2013). In contrast to higher plants that contain large amount of cellulose and hemicellulose, larger portion of algal biomass can be directly converted into biofuels or other high-value bioproducts *via* downstream processes (Wijffels et al., 2010; Vanthoor-Koopmans et al., 2013;Yen et al., 2013). One well-known area of such applications is microalgae-based biodiesel that has been proposed as good alternative to non-renewable fossil fuels (Sheehan et al., 1998), and another area of commercial exploitation of microalgae is the production of pharmaceutically and high-value industrial chemicals (Leu and Boussiba, 2014).

Although microalgae are known to synthesize a variety of bioproducts with potential commercial values, only a few of them, such as β-carotene and astaxanthin, have been produced at an industry-scale (Ben-Amotz, 1995; Sheehan et al., 1998; Borowitzka, 2013), which may be due to the low productivity of these products in the native microalgae and the difficulty in isolating them by economically feasible means (Clarens et al., 2010; Norsker et al., 2011; Razon and Tan, 2011; Soratana and Landis, 2011). While significant efforts have been undertaken to select high-yield strains, optimize cultivation and even modify the strains by genetic

engineering in the past decades (Suen et al., 1987; Cerón Garcìa et al., 2005; Kilian et al., 2011), progress has yet to be fully satisfied.

As an alternative method to improve production or accumulation of bioproducts, chemicals as metabolic triggers or enhancers that are able to directly modulate cellular metabolism have been proposed and applied in various commercially viable microalgae. Unlike genetic modification, this approach relies on phenotypic screening and does not require specific knowledge of molecular targets in metabolic and catabolic pathways involved in synthesis of bioproducts. In a recent study, Franz et al. (2013) described a phenotypic screening of 42 chemicals for their roles on lipid metabolism in microalgae, and identified 12 chemicals that are capable of enhancing intracellular lipid levels by *>*100%, with three compounds (i.e., epigallocatechin gallate, CDK2 inhibitor 2 and cycloheximide) increasing intracellular lipids by 200–400% based on Nile Red fluorescence intensity. In addition, the researchers took a further step to verify these chemicals in large-scale cultures and concluded that propyl gallate and butylated hydroxyanisole could be used in large-scale applications considering the low cost of the chemicals and the lipid content increases (Franz et al., 2013), demonstrating that the application of chemical enhancer could be a valuable and practical approach in addressing the low productivity issue with microalgae-based processes. In this article, we review the recent progresses in applying chemicals to improve cell growth and accumulation of high-value bioproducts in microalgae (**Table 1**), with a focus on the molecular mechanisms of their stimulatory roles.

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**Table**

#### **PHYTOHORMONES AND ANALOGS REGULATING MULTIPLE ASPECTS OF METABOLISM**

**TARGETING ON BIOSYNTHETIC PATHWAYS OF HIGH-VALUE PRODUCTS** It has been established that plants have developed a broad spectrum of molecular mechanisms to resist unfavorable environmental perturbations (Ren et al., 2009). Microalgae that share the evolutionary merits with plants also have mechanisms to deal with various environmental stress. One well-studied example is antioxidant pigment astaxanthin that plays a critical role in response to various stress conditions, such as high light, salinity, nutrient stress, and high carbon/nitrogen ratio, in chlorophyceae *Haematococcus pluvialis* (Tripathi et al., 1999; Sarada et al., 2002). The pathway of astaxanthin synthesis in *H. pluvialis* has been deciphered (Grünewald et al., 2000; Vidhyavathi et al., 2008) and several biosynthetic genes related to carotenoid have also been cloned and characterized (Lotan and Hirschberg, 1995; Sun et al., 1998; Linden, 1999; Steinbrenner and Linden, 2003; Huang et al., 2006). To increase the astaxanthin productivity, chemicals as metabolism enhancers were also evaluated recently. In one study, Lu et al. (2010) reported that gibberellic acid (GA3) and methyl jsmonate (MJ) played roles in regulating gene expression of *bkts* that catalyzes β-carotene to canthaxanthin in the astaxanthin biosynthetic pathway (Lu et al., 2010). More recently, Gao et al. (2012a,b, 2013a,b) found that chemicals jasmonic acid (JA), salicylic acid (SA), GA3, and 2, 4-epibrassinolide (EBR) can enhance astaxanthin production to 1.458 mg/L, 2.74 mg/L, 2.39 mg/L, 2.26 mg/L, respectively; and further analysis showed that the enhancing mechanisms of chemicals were concentrationdependent. For example, the results showed that 25 mg/L JA up-regulated the transcriptional expression of *pds*, *crt*R-B, and *lyc* of the astaxanthin biosynthetic pathway (*>*10-fold up-regulation) the most, while 50 mg/L JA impacted the transcriptional expression of *ipi-1*, *ipi-2*, *psy*, *crt*R-B, and *crt*O than on *pds*, *lyc,* and *bkt2* more significantly (Gao et al., 2012b). Based on a correlation analysis between their maximum mRNA transcripts of five carotenoid genes and astaxanthin production, Li et al. (2010) proposed that multiple regulatory mechanisms at transcriptional, translational, and post-translational levels of astaxanthin biosynthetic genes co-existed in controlling the overall carotenogenesis process in *H. pluvialis* (Li et al., 2010). Interestingly, different modes of regulation can be issued by the same chemical in *H. pluvialis*, such as JA that up-regulated *psy*, *pds*, *crt*R-B, *lyc*, *bkt,* and *crt*O genes at the transcriptional level, and up-regulated *ipi-1* and *ipi-2* genes at both transcriptional and post-transcriptional levels, respectively; and SA up-regulated *ipi-1*, *ipi-2*, *psy*, *crt*R-B, *bkt,* and *crt*O gene at the transcriptional level, and *lyc* at the post-transcriptional level and *pds* at both levels, respectively (Gao et al., 2012a,b).

#### **INDUCING OXIDATIVE STRESS RESPONSES**

Photosynthetic algae, like higher plants, generate reactive oxygen species (ROS) through chloroplast photosynthesis and mitochondrial respiration under stress condition, and ROS will then to be used as signal molecules to initiate production and accumulation of many bioproducts (Asada, 1994). The effects of SA and MJ on the antioxidant systems in *H. pluvialis* were investigated, and the results showed that at low concentrations, 100 μM SA

increased astaxanthin content to 6.8-fold under low light (30μmol m−2 s −1), while 10 μM MJ showed marginal increase in astaxanthin. However, at high concentration of 500 μM, both SA and MJ reduced the growth of microalgae and inhibited astaxanthin accumulation. Further mechanism analysis showed that SA at high concentrations increased superoxide dismutase activity to 4.5- and 3.3-fold and ascorbate peroxidase (APX) activity to 15.5- and 7.1-fold under low and high light, respectively, while MJ increased catalase activity (1.4-fold) under high light and APX activity (5.4-fold) under low light, suggesting the low astaxanthin accumulation may be due to the free radicals being scavenged (Raman and Ravi, 2010).

#### **REGULATING OTHER ASPECTS OF CELLULAR METABOLISM**

Phytohormones are signal molecules synthetized by plants, and capable of efficiently regulating cellular metabolism at very low concentrations (Park et al., 2013). The application of phytohormones to improve growth and productivity has been reported, and the results with *Chlorella* species showed that use of natural and synthetic auxins, as well as their precursors, have considerable stimulating effects on algal growth and biomass composition (Czerpak et al., 1994, 1999; Czerpak and Bajguz, 1997; Hunt et al., 2010). In addition, a combination of chemicals from within the auxin family as well as with that of other families, such as 5 ppm 1-naphthaleneacetic acid (NAA) + 10 ppm GA3 + 1 ppm zeatin (ZT), dramatically increased biomass productivity by 170% over the control in *Chlorella sorokiniana* (Hunt et al., 2010). Another study investigated the effects of phytohormones on microalgal growth and oil accumulation for biodiesel production in *Chlamydomonas reinhardtii*. The results indicated that all five of the tested phytohormones (i.e., indole-3-acetic acid, gibberellic acid, kinetin, 1-triacontanol, and abscisic acid) promoted cell growth. In particular, hormone treatment increased biomass production by 54–69% relative to the control growth medium, demonstrating their values in decreasing cost of commercial biodiesel production (Park et al., 2013).

Brassinosteroids (BRs) are hydroxylated derivatives of 5 cholestane and important plant growth regulators in multiple developmental processes, such as cell division and cell elongation (Bajguz and Czerpak, 1996; Bajguz and Tretyn, 2003). A recent study found that BRs cooperated synergistically with auxins in stimulating cell proliferation and endogenous accumulation of proteins, chlorophylls, and monosaccharides in *C. vulgaris* (Bajguz and Piotrowska-Niczyporuk, 2013).

In terms of the molecular mechanisms, auxins and their analogs have been found to affect photosynthetic efficiency and CO2 fixation in microalgae. For example, a study showed that auxins had incentive effects on reactions of bonding CO2 to 1, 5-biphosphoribulose and photosynthetic phosphorylation. As expected, the increase in intensity of photosynthesis reactions correlated well with higher contents of chlorophylls, pheophytins, and total carotenoids in cells treated with indomethacin that shares structural similarity with natural auxins (Piotrowska et al., 2008). Other studies also indicated that low concentrations of synthetic auxins, such as 2-(2,4-dichlorophenoxy) acetic acid (2,4-D), NAA and 2-phenylacetic acid (PAA), stimulated the photosynthetic rate and chlorophylls as well as carotenoids synthesis in green algae *C. pyrenoidosa*, *Scenedesmus acuminatus,* and *S. qadricauda* (Czerpak et al., 1994, 1999, 2002; Wong, 2000).

Diamines and polyamines are polycation nitrogen compounds presented in almost all prokaryotic and eukaryotic microorganisms and belonged to specific cellular regulators of growth and metabolism (Rayle and Cleland, 1992). The study showed that in *C. vulgaris* treated with diamines and polyamines, the content of monosaccharides, primary products of Calvin cycle were intensively stimulated on 3 days of *C. vulgaris* culture, while chlorophyll content was enhanced on 9 days of *C. vulgaris* culture, indicating that the amines stimulated the dark phase of photosynthesis in the young cells, and the light synthesis phase in aging cells, respectively (Czerpak et al., 2003).

An acid growth theory has been proposed to explain the cell elongation triggered by auxins in plant cells, which refers to the auxin-induced acidification of free space in cell wall. The decrease of pH enhances the plasticity of cell wall thus contributes to the increased elongation rate of the plant tissues, and the phenomenon is presumably related to the activation of membrane-binding proton pumps by auxin (Rayle and Cleland, 1992; Hobbie et al., 1994). A study with algal *C. vulgaris* also showed that BR-stimulated cell growth depended at least partly on acid growth theory (Bajguz and Czerpak, 1996).

Cell phase and mitosis regulated by phytohormones was also reported in microalgae. A recent study showed that NAA (30 ppm) treatment stimulated higher biomass productivity between days 5 and 10 while PAA (5 ppm) treatment effected on the first 5 days in in *C. sorokiniana*, suggesting that NAA might prolong exponential phase and PAA might short initial lag phase before initiation of cell division. The combination of NAA (5 ppm) + PAA (30 ppm) showed 104% increase of biomass and demonstrated that auxins enhanced biomass growth by reducing generation time thus contributing to reducing generation time (Hunt et al., 2010). Another study on the synchronous culture of *C. pyrenoidosa* showed that the time to incipient cell division was reduced by GA and 6 furfurylaminopurine, suggesting these two phytohormones had played roles in eliminating the initial lag phase (Vance, 1987). Similarly, the cell number and dry weight of *C. vulgaris* was also significantly increased in response to optimal dose of IM (10−7 M) on a 5-day cultivation, suggesting that growth elicited by natural and synthetic auxins encompassed the stimulation of mitosis (Piotrowska et al., 2008).

Chlorophyll pigment presents challenges to lipid extraction and biodiesel conversion in downstream processing of algal biomass. Hence, chemicals led to higher biomass and lower pigment production will bring benefits. A study showed that the addition of NAA (30 ppm) and PAA (5 ppm) significantly increased biomass production, meanwhile decreased chlorophyll a synthesis in *C. sorokiniana* (Hunt et al., 2010). In addition, auxins at high concentrations can activate key regulatory enzyme in ethylene biosynthesis (Grossmann, 2000), and large amount of ethylene could then induce the degradation of photosynthetic pigments (Sunohara and Matsumoto, 1997).

As for other regulatory functions, an exposure of *C. vulgaris* cells to exogenous IM, synthetic analog of IAA, has been reported to increase cellular DNA level up to 48% and 20–43% more soluble proteins excreted to the environments (Piotrowska et al., 2008); and cytokinins and allantoin (AT) were found to stimulate carotenoids content by 185–190% and 124% in *C. pyrenoidosa*, possibly due to their inhibition of oxidases and dehydrogenases that are responsible for oxidation process and degradation of chlorophylls and carotenoids (Czerpak and Bajguz, 1997).

### **OTHER CHEMICALS INDUCING OXIDATIVE STRESS RESPONSES**

Apart from phytohormones and analogs, other chemicals capable of inducing oxidative response for enhanceing microalgal growth and accumulation of high-value bioproducts were also investigated. An early study showed that Fe2+, methylene blue (MB) for singlet oxygen (1O2), methyl viologen (MV) for superoxide anion radical (O2 −), H2O2, and 2,2- -azo-*bis*(2-amidinopropane) dihydrochloride (AAPH) for peroxy radical (AO2·), were capable of triggering astaxanthin biosynthesis in *H. pluvialis*, in which Fe2+ possibly served as an HO· generator *via* an ironcatalyzed Fenton reaction (Kobayashi et al., 1993). HO· or other active oxygen species (1O2, O2 −, H2O2, and AO2·) might then enhance carotenoid formation in algal cyst cells by participating directly in the carotenogenic enzyme reactions as an oxidizer or an H acceptor (Beyer and Kleinig, 1989). In a recent study, Ip and Chen (2005) proposed sodium hypochlorite (NaClO) as another oxygen species to enhance astaxanthin production of *C. zofingiensis* in the heterotrophic cultivation medium.

#### **CHEMICALS AS METABOLIC PRECURSORS**

An early study showed that an addition of 100 mM pyruvate into the culture medium of *C. zofingiensis* enhanced the yield of astaxanthin from 8.36 to 10.72 mg/L. In addition, citrate and malic acid also had the similar stimulatory effects on the formation of astaxanthin. Pyruvate might serve as a precursor for isopentenyl pyrophosphate (IPP), the carotenoid precursor in *C. zofingiensis* and *H. pluvialis*, while the stimulatory effects of citrate and malic acid on astaxanthin biosynthesis in *C. zofingiensis* could be due to their conversions to pyruvate (Chen et al., 2009). For docosahexaenoic acid (DHA) accumulation in *Schizochytrium* sp. HX-308, an addition of 4 g/L malic acid to the culture medium at the rapid lipid accumulation stage can increase DHA content of total fatty acids from 35 to 60%. In addition to functioning as a possible carbon precursor, it was speculated that malic acid added at rapid lipid accumulation stage could activate malic enzyme activity and enhance NADPH generating reaction from malic acid to pyruvate (Ren et al., 2009). In addition, ethanol was also found to enhance lipid content by 35% in *Crypthecodinium cohnii*, in which ethanol can be converted to acetyl-CoA directly and in its metabolism might generate additional reducing power NADPH for lipogenesis (Lolke et al., 2005).

To aid in identifying metabolites associated with enhanced production of bioproducts, metabolomics, a measurement, and study of the small-molecule metabolites that constitute cellular metabolic networks, has been recently applied. In one study, Cheng et al. (2012) compared the metabolites between two cyanobacteria *Synechocystis* sp. PCC6803 and *Anabaena* sp. PCC 7120, and one microalga *S. obliquus* by gas chromatography coupled with time-of-flight mass spectrometry to detect important metabolites intricately tied to the lipid content in cyanobacteria and microalgae. The results showed that nine metabolites including ethanolamine were associated with the different lipid accumulation, and further study confirmed that addition of exogenous ethanolamine (2 mmol/L) could increase the lipid content by 22% in *S. obliquus* (Cheng et al., 2012). In another study, Su et al. (2014) investigated mechanism of astaxanthin induction under various stress conditions using a metabolomics and network analysis, and found that several metabolites, such as D-(+) altrose, D-ribose 5-phosphate, *L*-glutamic acid, and α-ketoglutaric acid, were positively associated with the increased astaxanthin accumulation in *H. pluvialis*. Although further confirmation is still needed, it was speculated that the increased abundances of these metabolites might contribute to the enhanced carbon flow into the astaxanthin biosynthesis (Su et al., 2014). Taken together, these early studies demonstrated that metabolomics could be a valuable tool in

identifying potential metabolites for enhancing target production in algae (Zhang et al.,2010). Effective mechanisms of the chemicals were schemed in **Figure 1**.

#### **CONCLUSION**

To produce bioproducts form microalgae in an economically feasible and sustainable way, one major hurdle that needs to be overcome is the low productivity. To address the issues, efforts have been undertaken to identify and apply chemical triggers or enhancers to enhance cell growth and accumulation of bioproducts in microalgae, and the studies have demonstrated that application of chemical triggers or enhancers could be a very practical method in large-scale fermentation of microalgae. In addition, the possible stimulatory mechanisms were also partially deciphered for some of the chemicals. However, to uncover new chemicals and expand the application, it is necessary to determine more accurately the metabolic mechanisms related to cell growth,

#### **FIGURE 1 | Scheme of enhancing mechanisms of chemicals on**

**microalgae.** The major stimulatory mechanisms were indicted inside the cell. **(I)** Chemicals targeting on biosynthetic pathways of high-value product, such as JA, SA, GA, and EBR controlling the overall carotenogenesis process in H. pluvialis; **(II)** Chemicals inducing oxidative stress responses, including direct or indirect addition of active oxygen species and chemical triggers inducing antioxidant production; **(III)** Phytohormones and analogs effecting on photosynthetic efficiency, namely the light phase, including photosynthetic phosphorylation, photosynthetic rate, and chlorophylls synthesis; **(IV)** Phytohormones and analogs impacting CO2 fixation, namely the dark phase of

photosynthesis, such as diamines and polyamines stimulating production of Calvin cycle; **(V)** Phytohormones and analogs encompassed acid growth theory, alternating the plasticity of cell wall thus contributing to cell elongation; **(VI)** Degradation of photosynthetic pigments due to large amount of ethylene caused by high concentration of auxins; **(VII)** Phytohormones and analogs regulating genome and protein expression, such as IM modulating DNA and protein content in C. vulgaris; **(VIII)** Chemicals as metabolic precursors, such as pyruvate serving as a precursor of carotenoid synthesis thus stimulating the formation of astaxanthin and NADPH (led by malic acid) acting as a precursor of fatty acid synthesis increasing DHA content.

production and accumulation of bioproducts, and the modes of action (MOA) of chemicals in microalgae. For this regard, the application of various global-focused technologies, such as genomics, proteomics, and metabolomics, could be valuable tools in the future research.

#### **ACKNOWLEDGMENTS**

The research was supported by grants from the National High-tech R&D Program (No. 2012AA02A707), and the Doctoral Program of Higher Education of China (No.<PHONE_NUMBER>0020 and<PHONE_NUMBER>0022).

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Kobayashi, M., Hirai, N., Kurimura, Y., Ohigashi, H., and Tsuji, Y. (1997). Abscisic acid-dependent algal morphogenesis in the unicellular green alga Haematococcus pluvialis. *Plant Growth Regul.* 22, 79–85. doi: 10.1023/A:1005862809711

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 November 2014; accepted: 16 January 2015; published online: 17 February 2015.*

*Citation: Yu X, Chen L and Zhang W (2015) Chemicals to enhance microalgal growth and accumulation of high-value bioproducts. Front. Microbiol. 6:56. doi: 10.3389/fmicb.2015.00056*

*This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2015 Yu, Chen and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Is lactate an undervalued functional component of fermented food products?

#### *Graciela L. Garrote1, Analía G. Abraham1,2 and Martín Rumbo3\**

*1 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA - CONICET), Universidad Nacional de La Plata, La Plata, Argentina, 2 Área Bioquímica y Control de Alimentos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina, 3 Instituto de Estudios Inmunológicos y Fisopatológicos (IIFP - CONICET), Universidad Nacional de La Plata, La Plata, Argentina*

#### *Edited by:*

*Fabio Minervini, Università degli Studi di Bari Aldo Moro, Italy*

#### *Reviewed by:*

*Giorgio Giraffa, Consiglio per la Ricerca e la Sperimentazione in Agricoltura – Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, Italy Carmen Wacher, Universidad Nacional Autónoma de México, Mexico*

#### *\*Correspondence:*

*Martín Rumbo, Instituto de Estudios Inmunológicos y Fisopatológicos (IIFP - CONICET), Universidad Nacional de La Plata, Calle 47 y 115, 1900 La Plata, Buenos Aires, Argentina<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology*

*Received: 30 April 2015 Accepted: 09 June 2015 Published: 19 June 2015*

#### *Citation:*

*Garrote GL, Abraham AG and Rumbo M (2015) Is lactate an undervalued functional component of fermented food products? Front. Microbiol. 6:629. doi: 10.3389/fmicb.2015.00629* Although it has been traditionally regarded as an intermediate of carbon metabolism and major component of fermented dairy products contributing to organoleptic and antimicrobial properties of food, there is evidence gathered in recent years that lactate has bioactive properties that may be responsible of broader properties of functional foods. Lactate can regulate critical functions of several key players of the immune system such as macrophages and dendritic cells, being able to modulate inflammatory activation of epithelial cells as well. Intraluminal levels of lactate derived from fermentative metabolism of lactobacilli have been shown to modulate inflammatory environment in intestinal mucosa. The molecular mechanisms responsible to these functions, including histone deacetylase dependent-modulation of gene expression and signaling through G-protein coupled receptors have started to be described. Since lactate is a major fermentation product of several bacterial families with probiotic properties, we here propose that it may contribute to some of the properties attributed to these microorganisms and in a larger view, to the properties of food products fermented by lactic acid bacteria.

#### Keywords: fermentation, lactate, functional food, probiotics, bioactive properties

## Lactate is a Major Component of Lactic-Acid Bacteria Fermented Foods