Effects of Selenium- and Zinc-Enriched Lactobacillus plantarum SeZi on Antioxidant Capacities and Gut Microbiome in an ICR Mouse Model

Selenium and zinc are essential trace minerals for humans with various biological functions. In this study, selenium- and zinc-tolerant lactic acid bacteria (LAB) isolates were screened out from human fecal samples. Amongst three hundred LAB isolates, the Lactobacillus plantarum SeZi strain displayed the tolerance against selenium and zinc with the greatest biomass production and bioaccumulation of selenium and zinc. To further assess the characteristics of this strain, the lyophilized L. plantarum SeZi were prepared and administered to Institute of Cancer Research (ICR) mice. The mice were divided into four groups, provided with normal chow (Con), or normal chow supplemented with Na2SeO3 and ZnSO4∙7H2O (SZ), L. plantarum SeZi (Lp), or selenium- and zinc-enriched L. plantarum SeZi (SZ + Lp), respectively. After 4 weeks of oral administration, the concentrations of selenium and zinc in blood were significantly increased in the SZ + Lp group when compared to the control or SZ group (p < 0.05). The increased selenium level led to an enhanced glutathione peroxidase activity and decreased blood malondialdehyde level in the SZ + Lp group (p < 0.05). Meanwhile, the results of bacterial community and microbial metabolic pathway analysis via 16S rRNA gene amplicon sequencing showed that L. plantarum SeZi significantly promoted the utilization of selenocysteine, seleno-cystathionine and seleno-methionine in the selenocompounds metabolism. Here, the in vivo antioxidant capacities of the selenium- and zinc-enriched lactobacillus strain showed us the utilization of a unique probiotic as a Se/Zn supplement with high availability, low toxicity, and additional probiotic advantages.


Introduction
Micronutrient deficiencies, known as "hidden hunger", have affected more than 50% of the world's population [1]. Selenium (Se) is a vital trace element, contributing to modulation of growth, regulation of antiviral capacity, and prevention of disease, especially cancer and cardiovascular disease [2][3][4]. The antioxidant activity of selenium is exhibited as a form of selenoenzymes, including glutathione peroxidase (GSH-Px), selenoprotein P, thioredoxin reductase, and methionine sulfoxide reductase [5]. Selenium deficiency can trigger serious health issues such as poor growth, muscle pain, decreased immune responses, and hypofunction of glandula thyreoidea [6,7]. Besides, the deficit of selenium is associated with a cardiomyopathy named as Keshan disease (KD) and an osteoarthropathy named as Kashin-Beck disease (KBD) [8]. Although KD and KBD are just local problems primarily in China and east Serbia due to environmentally low selenium [9], the hypothyreose or insuline secretion impairments associated with the lack of selenium have a global impact. Zinc (Zn) is another essential micronutrient for humans. It is a key component of many metalloenzymes (i.e., superoxide dismutase (SOD), carbonic anhydrase, alcohol dehydrogenase) associated with human growth, immunity, fertility, and reproduction [10]. Additionally, zinc is significant for the correct secretion of hormone isoline by pancreas [11]. The chronic deficiency of zinc can lead to glucose intolerance and pre-diabetic syndromes [12,13]. On the other hand, zinc chronic overdose, which can be caused by some nutraceuticals, might be responsible for some neurodegenerations such as nervus opticus inflammation [14,15].
Inorganic selenium, such as selenate (SeO 4 2− ) and selenite (SeO 3 2− ), are toxic and poorly bioavailable [16]. The reduction of selenium oxyanions largely depends on biotic processes by microorganisms [17]. The utilization of microorganisms as the natural adsorbent for metal ions (i.e., selenium and zinc) is eco-friendly and cost-effective [18]. The bioabsorption capacities are attributed to their intrinsic biochemical and structural properties of the cellular membrane [19]. Lactic acid bacteria (LAB), as important food-grade bacteria with probiotic advantages, have been extensively studied in this field. The selenium concentration in the medium is highly linked to bacterial selenium level, but the growth of most bacterial isolates from the human gut can be inhibited by the addition of inorganic selenium into the medium [20]. However, some LAB strains have been reported to be capable of resisting selenium oxyanions at high concentrations during cultivation [21][22][23]. Especially, Lactobacillus plantarum has been suggested as Se-enriched lactobacilli for food applications [24]. Few studies about Zn-enriched LAB have been conducted, but it has been found that the bacterial growth and probiotic effect of L. plantarum can be enhanced by zinc in the gut [25].
Although the resistances of LAB to selenium and zinc have been reported, the in vivo antioxidant capacities of the SeZn-enriched probiotic products have not been reported. In this study, SeZn-tolerant LAB strains isolated from human feces were screened out to further investigate the effects on selenium and zinc bioaccumulation and related metabolism, antioxidant activities, and compositional changes of intestinal microbiota in vivo in an Institute of Cancer Research (ICR) mouse model.

Isolation of Probiotic Strains from Human Feces
According to the protocol approved by the Institutional Review Board of Seoul National University (IRB No. 1702/002-013), fresh fecal samples were obtained from five children (1-6 years old) in Korea and stored at 4 • C during transportation. Each fecal sample (1 g) was serially diluted with a sterilized phosphate buffered saline solution (pH 7.4). The suspension was plated on Lactobacillus Selection (LBS) agar (Difco, Sparks, MD, USA) to isolate Lactobacillus spp. The plates were incubated anaerobically at 37 • C for 48 h [26]. Three hundreds of morphologically different microbial colonies were collected and cultured for further tests. The isolated LAB strains were then cultured in De Man, Rogosa and Sharpe (MRS) medium (Becton Dickinson, Cockeysville, MD, USA) containing 0.05% L-cysteine hydrochloride anaerobically at 37 • C. The bacterial stocks were stored at −80 • C with 17% glycerol utilized as a cryoprotectant.

Screening of SeZn-Tolerant LAB Isolates from Human Feces
To identify selenium-tolerant strains, the isolates were plated on the MRS agar in the presence of 60 mM Na 2 SeO 3 (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C for 24 h under anaerobic condition. When the concentration of selenium in the medium is high, strains convert inorganic selenium into element of selenium (red color) in the medium [27]. Thus, the strains with selenium resistance were selected based on the results of bacteria growth and color changes.
Thereafter, the screened strains were further tested for zinc-tolerant abilities by culturing in the MRS agar with 100 mM ZnSO 4 ·7H 2 O (Sigma-Aldrich) at 37 • C for 24 h under anaerobic condition. Strains with strong zinc tolerance were selected by observing the bacterial growth. The final screened SeZn-tolerant bacteria were identified by phylogenetic analysis of 16S rRNA gene sequence.

Assessing Bioaccumulation of Selenium and Zinc in LAB Strains During the Cultivation
Considering the application for food and feed additive, the initial concentrations of Na 2 SeO 3 and ZnSO 4 ·7H 2 O were set at 0.01 mM and 3.5 mM, respectively. After 24 h anaerobic culture in MRS broth, the LAB strains were centrifuged (15,600× g, 5 min) to gain pellets. The bacterial pellets were washed three times with phosphate buffered saline (PBS) and frozen at −80 • C for lyophilization. One liter of PBS buffer (pH 7.4) was prepared by dissolving NaCl (8 g), KCl (200 mg), Na 2 HPO 4 (1.44 g) and KH 2 PO 4 (245 mg) in the distilled water and autoclaved at 121 • C for 15 min.
The concentrations of zinc and selenium in the bacterial biomass were measured using an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Optima-4300 DV, Perkin Elmer, Waltham, MA, USA). The lyophilized sample (400 mg) was digested with HNO 3 (5 mL) and HClO 4 (0.5 mL) by heating in a Multiwave 3000 microwave. After the cool-down to room temperature, the solution was diluted with deionized water to reach a final volume of 20 mL, and mineral levels were assessed by the Inductively coupled plasma atomic emission (ICP-AES). The bioconversion rates of Se and Zn were calculated by dividing the Se or Zn content in dry cell mass by the total Se or Zn content added in the broth. The strain with the highest levels of selenium and zinc bioaccumulation was selected as the experimental strain for in vivo study. The genomic DNA of pure cultured L. plantarum SeZi isolate was extracted by using MG™ Cell Genomic DNA Extraction SV kit (MGmed, Seoul, Korea), following the manufacturer's instructions. Whole genome sequencing was carried out by using a Nextera XT Library Preparation kit (Illumina, San Diego, CA, USA) and sequenced at a read length of 300 bp with paired-end library via an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). The Illumina sequencing raw data in the FASTQ format were assembled with SPAdes 3.9.0. Gene-finding and functional annotation pipeline of whole genome assemblies used in EzBioCloud genome database (http://www.ezbiocloud.net, ChunLab Co., Ltd., Seoul, Korea) [28]. The tRNA genes were investigated via tRNAscan-SE 1.3.1 [29]. The rRNA and other non-coding RNAs were explored by using a Rfam covariance model version 12.0 [30]. Protein-coding sequences (CDSs) were predicted via Prodigal 2.6.2 [31], and classified into different functional groups (EggNOG 4.5; http://eggnogdb.embl.de) [32]. In order to obtain more functional annotation, the UBLAST program [33] was utilized to search and compare the predicted CDSs in the protein databases, including Swissprot [34], Kyoto Encyclopedia of Genes and Genomes (KEGG) [35] and subsystems-based annotations (SEED) [36]. The comparative genomics analysis was conducted by using the genome sequences of closely related Lactobacillus plantarum strains from the EzBioCloud database and analyzed via ChunLab's comparative genomics tool (http://www.ezbiocloud.net/contents/cg). The L. plantarum SeZi strain was anaerobically grown at 37 • C for 24 h in MRS medium with the addition of 0.01 mM Na 2 SeO 3 and 3.5 mM ZnSO 4 ·7H 2 O. For harvesting probiotic powder, the bacterial pellets were collected by centrifugation (15,600× g, 5 min) after SeZn enrichment, thoroughly washed with the PBS buffer, and frozen at −80 • C for lyophilization.

Animals and Diets
Seven-week old male ICR mice were purchased from Central Lab Animal (Seoul, Korea). The animal breeding environment was adjusted to a dark cycle of 12 h light/12 h dark at a temperature of 23 ± 1 • C and a humidity of 40-60%. The mice were acclimatized in the laboratory room for one week and then randomly divided into four groups (n = 8/group). The control group was provided with a normal chow diet AIN-93G purchased from Doo Yeol Biotech (Seoul, Korea). The treatment groups (SZ, Lp, SZ + Lp) were fed the same normal chow diet mixed with Na 2 SeO 3 (1.2 µg/g Se 4+ ) and ZnSO 4 ·7H 2 O (5 µg/g Zn 2+ ), 10 12 CFU/mouse Lactobacillus plantarum SeZi, and 10 12 CFU/mouse SeZn-enriched L. plantarum SeZi, respectively. The daily administration was conducted for 4 weeks. The protocols and facilities utilized in this animal experiment were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-180403-2-2).

Blood Analysis
To assess Se and Zn concentrations and oxidative stress-related parameters in mouse blood, blood samples were collected into a 1.5 mL heparinized tube from the mouse heart by cardiac puncture. Approximately 0.9 mL of the blood samples were centrifuged (2500 rpm, 10 min) to separate serum. The serum and whole blood samples were stored at −80 • C. The concentrations of selenium and zinc in the whole blood were measured using ICP-AES. GSH-Px activity, SOD activity, and malondialdehyde (MDA) level in serum were assessed via antioxidant enzyme detection kits purchased from Jiancheng Bioengineering Institute, Nanjing, China.

Bacterial Community Analysis by 16S rRNA Gene Amplicon Sequencing
Fecal DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Manchester, UK). The V3-V4 hypervariable regions of the 16S rRNA genes in the stool DNA samples were targeted and amplified using interest-specific primers. A pooled library was constituted by attaching specific barcode sequences to the 16S rRNA amplicons. The denatured and diluted pooled library and PhiX control (Phix control v3, 30%, v/v) library were mixed and loaded onto a MiSeq v2 (500 cycle) reagent cartridge (Illumina, San Diego, CA, USA). The primers and methods were as described in our previous study [37]. After the metagenomic sequencing, paired-end FASTQ files were collected and imported into Quantitative Insights Into Microbial Ecology 2 (QIIME2) (ver. 2020.6, https://qiime2.org) for analysis. Operational taxonomic unit (OTU) taxonomy and related analysis were performed using QIIME2 as described in our previous study [37]. KEGG associated with selenocompounds metabolism pathways were assessed by conducting phylogenetic investigation of the community by reconstruction of unobserved states (PICRUSt) with the entire picrust2 pipeline command [38].

Statistic Analysis
Differential abundance analyses were performed by non-parametric one-way analysis of variance (ANOVA) using the Kruskal-Wallis test, or non-parametric t-test with Mann-Whitney test. Other analyses were conducted by one-way ANOVA with Tukey's multiple comparisons test or paired t-test analysis. All statistical analyses were carried out via Graph-Pad Prism 8. Statistically significant difference was accepted at p < 0.05.

Screening and Selection of SeZn-Tolerant LAB Strains
Amongst the three hundreds of isolated strains, only four LAB species grew in the presence of 60 mM selenite, including L. plantarum, L. pentosus, L. fermentum, and L. rhamnosus. All of these four strains are able to resist 100 mM ZnSO 4 ·7H 2 O. Among these four SeZn-tolerant strains, L. plantarum SeZi yielded the greatest dry cell mass with the best selenium and zinc bioaccumulation capability as shown in Table 1. Based on the results of whole genome sequencing analysis in Tables 2 and 3, the genes coding DedA and CysA proteins related to Na 2 SeO 3 uptake and detoxification, and zinc uptake regulation protein ZUR (zinc uptake regulation) and zinc resistance protein MerR (Mercury resistance) were found in L. plantarum SeZi genome. Thus, L. plantarum SeZi was selected for in vivo mouse study. The abundances of these microbial selenium/zinc metabolism-related genes in genomes from other LAB strains were investigated using publicly deposited genome databases (NCBI genome datasets, https://www.ncbi.nlm.nih.gov/genome/). Among the 1912 genome assemblies available, only a few Lactobacillus genomes contain genes encoding DedA, CysA, ZUR, or MerR proteins (Table 4).

Increased Concentrations of Selenium and Zinc in Blood after L. plantarum SeZi Administration
The selenium and zinc contents in mouse blood are presented in Figure 1. The blood selenium and zinc levels in the SZ + Lp group were significantly higher than that of the control group and the SZ group, respectively (p < 0.05). The differences between the control, SZ and Lp groups were not significant (p > 0.05).

Increased Antioxidant Activities in Mice after L. plantarum SeZi Administration
GSH-Px and SOD are imperative antioxidant defenses against oxidative stress [39,40], and MDA is the most commonly utilized biomarker of oxidative stress [41]. As shown in Figure 2, the GSH-Px activity was highest in the SZ + Lp group, followed by the SZ group when compared with other groups (p < 0.05). The activity of SOD was significantly increased in the SZ + Lp group compared to the Lp group (p < 0.05). Meanwhile, significant decreases in the MDA level were observed in the SZ group and SZ + Lp group compared to other two groups (p < 0.05).

Changes in the Gut Microbiota after L. plantarum SeZi Administration
Gut microbiota alpha diversities were assessed by richness (Faith-pd) and Pielou's evenness analyses, which represent the number of species and the degree of species homogeneity, respectively. No significant difference was observed between the groups in the alpha diversity of richness ( Figure 3A), while the evenness in the SZ group was significantly larger than Lp and SZ + Lp groups ( Figure 3B). The results of beta diversity (Bray-Curtis dissimilarity) indicated that the clustering in microbial communities in the Lp and SZ + Lp group was distinct from that in the control and SZ group ( Figure 3C).
The average relative abundances of the final day fecal samples at the phylum level ( Figure 3D) and the genus level ( Figure 3E) suggested the different microbial compositions amongst the groups after the oral administration of SZ and SZ + Lp. To further evaluate the effects of treatments on microbial compositional changes, the three significantly different genera between the groups were identified and are displayed in Figure 4A-C. The relative abundance of Lactobacillus in the SZ + Lp group was significantly higher than that of the control and SZ groups, and the Lactobacillus level in the Lp group was also significantly higher than the SZ group. Adlercreutzia was significantly abundant in the SZ group compared to the SZ and SZ + Lp groups. Interestingly, Lactococcus was highly enriched only in the SZ group with the relative abundance at 4.15%. In addition, the relative abundance of Allobaculum in the SZ group was much larger than the SZ + Lp group, although the difference was not statistically significant ( Figure 4D, p > 0.05).

Microbial Function Analysis Related to Selenocompounds Metabolism
To investigate the functional changes in selenocompounds metabolism of the gut microbiome, KEGG analysis was performed by phylogenetic investigation of the community by reconstruction of unobserved states (PICRUSt).
To detoxify selenite and selenate in the selenocompounds metabolism pathway, selenite is converted to selenate directly or via an intermediate, and selenate is further metabolized into hydrogen selenide. As shown in Figure 6, the relative abundances of enzymes related to oxidation of selenite to selenate (EC: 2.7.7.4, EC: 1.97.1.9) were significantly reduced in the Lp and SZ + Lp groups. Arrows indicate the related genes that are involved in the corresponding pathway. Error bars represent means ± SD. Significance was accepted at * p < 0.05, ** p < 0.01 (n = 8).

Discussion
In this study, L. plantarum SeZi isolate was screened out from human fecal bacteria by the seleniumand zinc-tolerant abilities. The selenium-and zinc-enriched L. plantarum SeZi strain increased the levels of selenium and zinc and presented antioxidative properties in an ICR mouse model. A thorough search of the literature reporting the bioavailability and functionality of Se-and Zn-enriched microorganisms using a mouse model yielded only one related article [42]. To determine appropriate concentrations of selenium and zinc for mice, we referenced this article and 300 µg of selenium and 1.5 mg of zinc (/kg body weight/day) were used in our study. Yan et al. reported the antioxidant and antitumor activities were significantly increased by supplementation with Se/Zn-enriched mushrooms. However, the in vivo antioxidant activity of Se/Zn-enriched LAB has not been reported yet.
Selenium is an essential element that must be exogenously provided to reach the requirement of human and animal health [43]. The toxicity order of selenium species from high to low is selenate, selenite, nano-selenium, and lactomicro-selenium [44]. The accumulation of selenium in bacteria is processed by extracellular binding via active groups in the cell-membrane conjunction or intracellular binding via ion transportation on the membrane [45]. Many Lactobacillus strains are well-known to accumulate and biotransform toxic selenite into non-toxic seleno-amino acids (i.e., selenocysteine and selenomethionine) and selenoprotein [46,47] The utilization of Se-enriched Lactobacillus possesses unique advantages, including low toxicity, low cost production and additional probiotic effects.
In this study, we focused on the changes in the gut microbial composition as well as functional metabolism associated with Se/Zn uptake and utilization. Interestingly, Se/Zn supplementation greatly induced the enrichment of specific genus, Lactococcus (belonging to LAB), which confirmed the previous reports regarding in vitro tolerance of LAB to selenium [46,47]. However, this indigenously enriched LAB by inorganic Se/Zn supplement showed significantly different patterns in the microbial selenocompounds metabolism compared with Se/Zn-bioaccumulated L. plantarum SeZi strain.
To further investigate strain-specific functions, whole genome sequencing of L. plantarum SeZi strain was conducted and analyzed based on public databases. The genes coding cysA and dedA were observed in the whole genome sequencing of L. plantarum SeZi. Both of the cysA and dedA genes are associated with selenite uptake and detoxification. These genes were abundantly present in L. plantarum SeZi strain, but not observed in Lactococcus or other Lactobacillus spp. According to previous studies, selenite may enter the cells of E. coli through the sulfate permease CysA [48]. DedA can uptake selenite into cells as a direct transporter or a cofactor. Additionally, the dedA gene-contained mutant E. coli displayed selenite resistance [17]. Based on the results of targeted metagenome sequencing, the L. plantarum SeZi strain promoted the utilization of selenocysteine, seleno-cystathionine and seleno-methionine in selenocompounds metabolism pathway of gut microbiome. Besides, the transformation between the toxic inorganic selenium was reduced by L. plantarum SeZi in the detoxification process. These are consistent with the selenium-related function detected in the genome sequencing of L. plantarum SeZi.
Zinc plays an essential role in catalytic, structural, and regulatory functions in enzymes and protein domains [49]. Similar to the other trace elements (i.e., selenium), zinc in the organic forms have more bioavailability its inorganic forms [49]. Pharmacological zinc supplements often have low bioavailability and are easily overdosed [50]. Although internalizing zinc by Lactobacillus has not been studied in depth, the utilization of certain Lactobacillus species can be a promising alternative to deliver zinc in a highly organic form [51].
The selenium and zinc levels in the blood can be affected by dietary supplementation and related metabolism. In this study, the concentrations of selenium and zinc in the Se/Zn-enriched Lactobacillus group were significantly higher than the control or SZ group. This is consistent with a recently published study which indicates that feeding a diet supplemented with Se/Zn-enriched probiotics, 0.3 mg/L selenium, and 100 mg/L zinc significantly enhanced the blood selenium and zinc concentrations in Wistar rats [52]. It is also the only published study referred to Se/Zn-enriched probiotics in a murine model to date. However, the assessment of antioxidant capability and its potential mechanisms was not reported yet.
In this study, the enhancement of antioxidant ability in the SZ + Lp group was probably triggered by the increased selenium level. When selenium is incorporated into the selenoenzymes (i.e., GSH-Px), it enhances the antioxidant activities by suppressing the nuclear factor-kappa B (NF-κB) signal pathway [53]. Most selenoproteins take part in the defense against oxidative stress, protecting tissues and cells from oxidative damages [47]. Although the SZ group was administered with inorganic forms of Se/Zn, the SZ + Lp group was administered with bio-accumulated Se/Zn in bacteria. To understand the mechanisms of bioavailability and functionality related to inorganic/organic forms of trace nutrients, it is necessary to investigate the changes in selenium and zinc metabolites (e.g., selenoproteins, zincproteins, etc.) from blood and fecal samples.
Alteration of microbial communities was evaluated by 16S rRNA community analysis in this study. Up to now, the 16S metagenomic technique is still rarely used in the selenium or zinc-related animal studies. Obviously, the significant surge of Lactobacillus levels in the Lp and SZ + Lp groups was caused by the oral administration of L. plantarum SeZi. The relative abundance of Adlercreutzia was significantly higher in the SZ + Lp group compared to the control and SZ groups. According to the previous studies, a decreased level of Adlercreutzia was observed in multiple sclerosis patients and Alzheimer's disease patients [54,55]. Amongst Adlercreutzia species, A. equolifaciens is an equol-producing bacteria, promoting intestinal health [56]. Besides, a high relative abundance of Lactococcus (approximately 4.15%) was found in the SZ group, while this bacterium was almost undetectable in the other groups. It might be caused by utilization of selenium and zinc by Lactococcus spp. According to the previous studies, Lactococcus lactis is capable of selenium biotransformation and zinc uptake [57,58]. The relative abundance of Allobaculum spp., which was markedly reduced in the SZ + Lp group compared with other groups, was reported to be adversely associated with mRNA expression levels of tight junction protein genes (zo-1 and occludin) and anti-inflammatory genes (foxp3 and Il-10) in the colon of rats [59]. In some inflammatory bowel disease (IBD) patients, Allobaculum spp. was one of the uniquely observed species [60].
To the best of our knowledge, this study is the first attempt to evaluate the effects of Se/Zn-enriched LAB on in vivo gut microbiome changes. The microbiome analysis suggests a microbial aspect of selenocompound metabolism, however, lacks information on host's Se/Zn metabolic process.
Future studies on evaluating functionality of L. plantarum SeZi in a disease-induced mouse model should include host's metabolites analysis as well.

Conclusions
In conclusion, the selected strain L. plantarum SeZi is able to resist and biotransform inorganic selenium into organic selenium. In the in vivo study, the selenium and zinc-enriched L. plantarum SeZi increased blood selenium level, antioxidant capability and the utilization of seleno-amino acids. Therefore, the L. plantarum SeZi strain is a potential selenium and zinc-enriched probiotic for application as functional food ingredients in the future.