Enhanced Viability and Anti-rotavirus Effect of Bifidobacterium longum and Lactobacillus plantarum in Combination With Chlorella sorokiniana in a Dairy Product

Microalgae and probiotics such as Bifidobacterium and Lactobacillus genera are associated with human beneficial effects. The aim of this study was to evaluate the activity of Chlorella sorokiniana on Bifidobacterium longum and Lactobacillus plantarum viability in a dairy product (flan) and its microbial effect against rotavirus, which is one of the major diarrhea-causing pathogens worldwide. Microalge were isolated from a Mexican river and characterized by molecular tools. Their prebiotic activity was evaluated by determining Bifidobacterium longum and Lactobacillus plantarum shelf-life after incorporation in the food matrix. In addition, HT-29 cells were infected with rotavirus Wa and treated with 1 × 109 CFU/mL L. plantarum and B. longum metabolites alone or in combination with 1 × 109 cells/mL Chlorella sorokiniana; viral titers in probiotics- and/or microalgae-treated cells were evaluated for antiviral activity. Results indicated that C. sorokiniana not only significantly (p < 0.05) improved L. plantarum and B. longum viability in flan, but also increased their antiviral activity; potent anti-rotavirus effect of C. sorokiniana alone was observed. Although more studies are needed, results suggest that incorporation of this microalga into a dairy product confers enhanced viability and antiviral effects, which indicates that C. sorokiniana might be used as an ingredient to design products with additional health benefits.


INTRODUCTION
The microalga Chlorella spp. is an important source of bioactive compounds such as proteins, fatty acids, carotenoids, sulfated polysaccharides, vitamins, and other natural bioactive molecules. This microalga is an important ingredient in functional foods because of its nutritional and pharmaceutical properties (de Morais et al., 2015;Silva et al., 2019). In addition, Chlorella spp. extracts have shown potential to modulate human immune response, accelerate dioxin elimination, prevent stress-induced ulcers, and decrease high-fat-diet-induced dyslipidemia (Ciliberti et al., 2019). Furthermore, microalga extracts incorporation into food triggers an array of metabolic and immunological processes in human and monogastric animal intestines stressed by enteric bacterial pathogens (Hulst et al., 2019). Notably, microalga sulfated polysaccharides have been associated with blocking viral entry to cells, thus avoiding infection (Guedes et al., 2011).
Probiotics are often used to prevent enteric diseases (Sales-Campos et al., 2019) and decrease diarrhea severity, days with symptoms, and reinfections (Huang et al., 2002). Particularly, Bifidobacterium and Lactobacillus genera are the most commonly associated probiotics with beneficial effects for humans, mainly against enteric infections, such as the one caused by rotavirus (Gonzalez-Ochoa et al., 2017;Park et al., 2017). These probiotics and their metabolites modulate the cytokine milieu, stimulated by the gut-associated lymphoid tissue and improves intestinal barrier function against pathogens by promoting an efficient immune response (Vlasova et al., 2016;Tang and Lu, 2019).
Although probiotics have shown beneficial effects on human health, they are strain-, dose-, and viability-dependent. For instance, in probiotic fermented milks production, the viability of probiotics is lost during the fermentation process and storage at cold (refrigerator) conditions (Terpou et al., 2019). However, probiotics must be viable and abundant (10 6 CFU/gr) at the time of food product consumption to obtain the health benefits associated with these microorganisms (Terpou et al., 2019). Probiotics effectiveness would be improved by incorporating prebiotics into the food matrix; they produce compounds that stimulate probiotics growth and function (Beheshtipour et al., 2013). The aim of the present study was to evaluate the effect of C. sorokiniana to improve the viability of Bifidobacterium longum and Lactobacillus plantarum in a stored dairy food product and determine its antimicrobial effect against rotavirus, which is one of the main causing-diarrhea pathogens worldwide.

Cells
Human colon cancer (HT-29; ATCC R HTB-38 TM ) and African green monkey kidney (MA104; ATCC R CRL-2378 TM ) cell lines were used in this study. They were grown in RPMI medium (Gibco/Life Technologies, Grand Island, NY, United States) supplemented with 5% of fetal bovine serum (FBS; Mediatech, Herndon, VA, United States), 2 mM L-glutamine, and 1% antibiotic and antimycotic solution (Caisson Laboratories, North Logan, UT, United States), and incubated at 37 • C in 5% CO 2 , until confluence. Cells were then harvested using PBS and 0.25% trypsin (Mediatech, Herndon, VA, United States) at 37 • C, until detachment from the culture flask, and transferred to 6-well plates for probiotics, microalgae, and rotavirus assays or 96well plates for rotavirus microtitration. Probiotics and microalga cytotoxicity to HT-29 and MA014 cells was determined by the MTT reduction assay, as previously described (Mosmann, 1983).

Rotavirus Strains
The rotavirus strain Wa was kindly provided by Dra. Susana López from the Instituto de Biotecnología at Universidad Nacional Autónoma de México. Viral particles were propagated in MA104 cells and activated with 10 µg/mL of trypsin, incubated at 37 • C in 5% CO 2 for 1 h, the inoculum replaced by RPMI medium without FBS, and incubated at 37 • C in 5% CO 2 , until cell lysis. Viral titers were calculated as focus forming units per milliliter (FFU/mL), using an immunochemistry assay protocol (Chasey, 1980). In brief, lysates from virus propagation or cells infected with rotavirus and treated with probiotics and the microalga were used to infect MA104 in 96-well plates; after 14 h of incubation, cells were fixed with a cold acetone-PBS solution and incubated for 45 min at room temperature. Monolayer was then washed twice with PBS, primary anti-rotavirus antibodies (Invitrogen, Carlsbad, CA, United States) were added, and cells incubated 1 h at 37 • C and washed. Next, horseradish peroxidase (HRP)-anti-sheep IgG conjugate (Invitrogen, Carlsbad, CA, United States) was added to the cells and incubated 1 h at 37 • C, followed by the addition of the substrate aminoethylcarbazole (0.64 mg/mL; Sigma-Aldrich, St. Louis, MO, United States), prepared in dimethylformamide (Sigma-Aldrich) with acetate buffer (30 mM sodium acetate and 12 mM acetic acid), pH 5.0, plus 0.36% hydrogen peroxide. Infected cells were counted using optical microscopy and identified by their brown color, indicating presence of viral antigens. FFU/mL were calculated using the following formula: FFU/mL = 20 × (microscope objective) × 5.5 (well diameter) × average number of focus (duplicate, 100-200 focus count/well) × dilution (focus count). Rotavirus multiplicity of infection (MOI) was 0.01 in each assay.

Microalgae Growth Conditions and Characterization
Microalgae were isolated in the river San Juan Cadereyta in Nuevo León, Mexico (Reyna-Martinez et al., 2018). They    , 2010). Microalgae were microscopically classified as the green microalga Chlorella spp. and was genetically classified by molecular tools as follows: 40 mg of biomass in 1 mL of PBS were frozen with liquid nitrogen, homogenized at 8,000 rpm for 1 min, followed by DNA extraction, using the Purelink Genomic DNA kit (Invitrogen, Carlsbad, CA, United States). Purified DNA was used to amplify the 18S ribosomal RNA gen, using the primers P2F 5 -GGCTCATTAAATCAGTTATAG-3 /P2R 5 -C CTTGTTACGA(C/T)TTCTCCTTC-3 (Lee and Hur, 2012). PCR amplicon (1700 bp) was purified with the Wizard SV Gel and PCR clean-up system Kit (Promega, Madison, WI, United States) and sequenced twice by the dideoxynucleotide chain termination method, using an ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, United States). The sequence of the isolated microalgae was submitted to the GenBank database with the access (1 × 10 6 Cells/g), and bold black represent L. planatrum or B. longum (1 × 10 9 CFU/g) + C. sorokiniana (1 × 10 9 Cells/g). Data were analyzed by two-way ANOVA with subsequent Fisher ìs LSD test using GraphPad Prism 7.0.
number MN011866. This was analyzed and compared with other sequences reported in GenBank database; phylogenetic relationships between strains were reconstructed using the maximum likelihood method with 1,000 replicates using MEGA X software (Kumar et al., 2018).

Proximate Composition of Microalgae Biomass
Association of Official Analytical Chemist (AOAC) methods were followed in detail. In brief, moisture was evaluated by drying the microalgae at 105 • C for 24 h (method 925.09B); crude protein was assessed using the micro-Kjeldahl assay (method 960.52); lipids were evaluated using a Soxhlet apparatus and petroleum ether as solvent (method 920.39C); ashes were calculated after incineration at 550 • C (method 923.03), and carbohydrates by difference of the other components (AOAC, 1999). The results were expressed in mg/g on a dry-matter basis. All evaluations were determined in triplicate.

Probiotics and Microalgae Combination in a Dairy Product
Combinations were made with probiotics at 1 × 10 9 CFU/mL and Chlorella spp. at 1 × 10 6 cells/mL or 1 × 10 9 cells/mL in 2 g of a dairy product such as flan and stored at 4 • C. Probiotics viability was monitored every 72 or 96 h of storage as follows: 1 g of flan containing the probiotic and/or microalgae was disrupted in PBS and diluted to calculate CFU/g. Microalgae were cultured independently from bacteria, thus, live microalgae were combined with probiotics. Assays were designed to take up to 34 days; nevertheless, results provided in figures indicated different treatment times, because experiments were ended once probiotic counts were lower than 10 6 UFC/gr.

Statistical Analysis
Data were reported as mean ± SD of triplicate determinations from three independent experiments. Statistical analysis was determined by two-way ANOVA and Fisher's LSD multiple comparisons or Kruskal-Wallis and Dunn ìs multiple comparisons, using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, United States).

Incorporation of Chlorella sorokiniana Into a Dairy Product Increases Probiotics Viability
The probiotics Lactobacillus plantarum and/or Bifidobacterium longum in combination with Chlorella sorokiniana were added to flan and stored at 4 • C and probiotics viability evaluated every 72 or 96 h. L. plantarum and B. longum showed viability up to 1 × 10 6 CFU/g at 10 and 7 days, respectively, whereas addition of C. sorokiniana (1 × 10 9 cells/mL) into the dairy product extended L. plantarum viability up to 18 days (p < 0.05) (Figure 2a); in contrast, it did not prolong that of B. longum (Figure 2b). On the other hand, L. plantarum, B. longum, and C. sorokiniana combination in flan showed longer viability (>34 days) than that of probiotic combinations without microalgae (p < 0.05) (Figure 2c).
L. plantarum metabolites alone or in combination, respectively (Figure 4). Each probiotic and combination significantly reduced rotavirus infectivity (p < 0.05), with no significant difference between the use of B. longum or L. plantarum. After adding C. sorokiniana metabolites to each treatment, infectivity was significant reduced to 30, 8, and 5%, for B. longum, L. plantarum, or both probiotics, respectively (Figure 4). Notably, treatment with C. sorokiniana metabolites alone caused significant (p < 0.05) 4% reduction of rotavirus infectivity (Figure 4).

DISCUSSION
In this study, previously isolated microalgae were molecularly characterized by sequence homology and phylogeny of its 18S ribosomal RNA gene (Reyna-Martinez et al., 2018). The sequence was clustered with other sequences previously reported as Chlorella sorokiniana (Figure 1c). This microalga species has been used for wastewater treatment, biodiesel production, and as a traditional dietary supplement due to its antitumoral, antibacterial, and antiviral activities (Reyna-Martinez et al., 2018;Ciliberti et al., 2019;Hulst et al., 2019). The biochemical components of C. sorokiniana biomass grown under photoautotrophic conditions in our laboratory (Table 1), showed lower lipid, but higher protein, carbohydrate, and mineral levels than other microalgae such as Chlorella vulgaris cultured under photoautotrophic or mixotrophic conditions (Abreu et al., 2012;Dineshkumar et al., 2017).
In addition to C. sorokiniana characterization, its effect on probiotic viability was evaluated with B. longum and L. plantarum in flan (Figure 2). Among treatments, L. plantarum showed up and down numbers over time when supplemented with C. sorokiniana, both at 1 × 10 9 cells/mL rate (Figure 2a). This observation may be related to population secondary successions. In this regard, nutrition source availability should be responsible, since other physicochemical factors did not change during the experiment. The results indicated that this microalga improved L. plantarum viability alone or in combination with B. longum (p < 0.05) (Figure 2). In this regard, previous studies with C. sorokiniana and C. vulgaris also showed prebiotic activity by enhancing Lactobacillus rhamnosus growth; in addition, the prebiotic effect of C. sorokiniana was higher than that of C. vulgaris (Lai et al., 2019). Other genera of Chlorella have shown potential for improving L. acidophilus and B. lactis viability in yogurt (Beheshtipour et al., 2012(Beheshtipour et al., , 2013. Although in the present study the mechanisms underlying the improved viability of probiotics after the addition of microalgae were not addressed, these could involve microalgae production of substances responsible for the stimulatory properties from a nutritional factor, such as adenine, hypoxanthine, and free amino acids (Varga et al., 2002;Gyenis et al., 2005;Beheshtipour et al., 2013). In this study, improved probiotics growth by C. sorokiniana was observed with metabolically active microalgae. Our results demonstrated that C. sorokiniana might be used as an ingredient in dairy food products in order to improve the probiotics such as B. longum and L. plantarum shelf-life. This study supports other reports describing the potential use of microalgae and blue-algae biomasses in milk-based functional fermented feeds (Varga et al., 2002;Gyenis et al., 2005;Beheshtipour et al., 2013). Although Chlorella is not yet formerly defined as prebiotic, it was recently considered as a functional food. Indeed, Chlorella used as feeding supplement in pigs have shown gastrointestinal effect in the manage of mild digestive disorders and in the fecal microbiota (increasing Lactobacillus concentration and reducing E. coli) (Yan et al., 2012;Furbeyre et al., 2017). Following in vitro digestion and fermentation, Chlorella was observed to increase propionate-producing microorganisms population in the intestines, which was associated with postbiotic gut health (Jin et al., 2020). Chlorella sorokiniana postbiotic effects represent one of our further research interests.
In addition to the increased viability of probiotics by Chlorella sorokiniana, other studies with this microalgae genus demonstrated its antibacterial activity against intestinal pathogens (Beheshtipour et al., 2012;Lai et al., 2019). In this study, B. longum and/or L. plantarum plus C. sorokiniana antiviral activity was evaluated in rotavirus-infected HT-29 cells (Figures 3, 4). Probiotics/microalga and cell metabolites showed potential to reduce rotavirus cytopathic effect, however, assays were performed using only metabolites due to interferences of probiotics/microalga cells with the immunochemistry stain of rotavirus-infected cells in viral titration. Results described in Figure 4 indicated that rotavirus titers decreased in cell cultures treated with B. longum or L. plantarum metabolites or after treatment with both probiotics' metabolites. Furthermore, the anti-rotavirus effect was improved after C. sorokiniana metabolites addition. Nevertheless, such improved anti-rotavirus effect cannot be fully attributed to the prebiotic properties of C. sorokiniana, since microalga metabolites showed a very potent antiviral effect per se, reducing rotavirus infectivity.
This study was designed to test the effect of C. sorokiniana on viability and antiviral activity of probiotics. Results showed an improved antiviral effect of probiotics, but unexpectedly, microalgae alone showed significant antiviral effect, which represents the first report on this matter to date. Recent reports showed C. sorokiniana effect against bacterial pathogens (Dineshkumar et al., 2017;Lai et al., 2019), whereas other microalgae species showed antiviral effects against murine cytomegalovirus (M-CMV) and herpes simplex virus type 1 (HSV-1) (Ibusuki and Minamishima, 1990;Shalaby, 2011;Santoyo et al., 2012). A recent study with C. sorokiniana demonstrated that the use of this microalga, before and after rotavirus infection in in vitro assays, increased type-I interferon expression levels, improving antiviral response and reducing the cytopathic rotavirus effect on HT-29 cells (Romero-Argüelles, 2020). Although Bifidobacterium spp. (Kim et al., 2014;Li et al., 2016;Olaya-Galan et al., 2016;Gonzalez-Ochoa et al., 2017) and Lactobacillus spp. (Ang et al., 2016;Olaya-Galan et al., 2016;Sunmola et al., 2019) antiviral effect, as well as the antimicrobial activity of Chlorella spp. have been documented, the present study showed that C. sorokiniana might be used as an ingredient in dairy food products to improve probiotics' viability and their effect against pathogens. As far as we know this is the first C. sorokiniana report demonstrating antiviral effect; although, more studies are needed to confirm C. sorokiniana specific effect against rotavirus.

DATA AVAILABILITY STATEMENT
The sequence of microalga Chlorella sorokiniana (18S ribosomal RNA gen partial gene) analyzed in this study can be found in the GenBank database with the access number MN011866 (https: //www.ncbi.nlm.nih.gov/nuccore/MN011866).

AUTHOR CONTRIBUTIONS
GG-O and PT-G conceived the study, designed, and supervised the experiments. SC-B and MD-G worked with the probiotics and microalga assays. IM-P, EA-U, and RG-F contributed with viral propagation and microtitration assays. NO contributed to growth experiments and biomass analysis. LF-M contributed in the sequence analysis and statistics. All the authors contributed to writing the manuscript, read, and approved the manuscript.