Multifunctional effects of Lactobacillus sakei HEM 224 on the gastrointestinal tract and airway inflammation

Mucosal tissues serve as the first defense line and their commensal microbiota play a role in sustaining of host health. This study aimed to isolate and evaluate a putative probiotic strain on various mucosal regions. Lactobacillus sakei HEM 224 was isolated from traditional Korean kimchi and identified. In the safety assessment L. sakei HEM 224 showed negative results for hemolysis, biogenic amine production and transferable antibiotic resistance. The probiotic potential of strain HEM 224 in diverse mucosal areas was shown in two different models, viz. a murine model with colitis induced by dextran sulfate sodium (DSS) and an allergic airway inflammation model induced by ovalbumin (OVA). In the colitis model, oral administration of L. sakei HEM 224 improved colitis physiology with immunomodulation, enhancing barrier components and gut microbiota alteration. In the allergic airway inflammation model, the intranasal administration of the strain decreased type 2 inflammation and enhanced epithelial barrier integrity from the airways. These results demonstrate that L. sakei HEM 224 can ameliorate inflammatory conditions in both the gastrointestinal and respiratory tracts through the reinforcement of the epithelial barrier and immunomodulation.

Our body is composed of mucosal organs and specialized goblet cells and mucous cells are aligned in epithelia integrated by intercellular epithelial junctions.Their apical surface is covered by the glycocalyx matrix basically separating our body from the environment and trapping the influx of noxious stimuli 1 .Innate immune cells, antibodies and antimicrobial molecules are contained in the mucosal layer, and they counteract against invaders initiating adaptive immunity 2 .Moreover, each mucosal organ harbors a distinct microbiota and they actively interact with the barrier components to maturate host systems 3 .Gut and lungs are representative mucosal organs sharing structural and functional similarities.A proper mucus production is essential to sustain a steady-state of epithelial integrity and microbial homeostasis for both organs 4 .Therefore, the association between gut and lung (gut-lung axis) has been interesting and studied extensively in recent times.
Concerning pathogenesis of inflammatory diseases in the gut and lungs, both display a disrupted homeostasis resulting in mucosal inflammation, loss of barrier integrity, and dysbiosis 5,6 .Especially, the loss of epithelial integrity can trigger the exacerbation of diseases due to the influx of allergens, excess inflammatory cells and mediators, leading to a mucosal barrier damage.Thereby, increased intestinal permeability is one of representative features in inflammatory bowel diseases (IBD) 7 .In case of airway inflammation, inhaled harmful factors can disrupt barrier integrity connecting to the allergen sensitization.The loss and dysfunction of epithelial tight junctions as has been observed in asthmatic patients 8 .
Based on that, the use of probiotics has been proposed as one of the novel future strategies.Probiotics are defined as "living microorganisms, which when administered in adequate amounts, confer a health benefit on the host" 9 .Most probiotic strains belong to the lactic acid bacteria (LAB), especially to the genera Lactobacillus and Bifidobacterium, and they are often isolated from fermented foods 10 .Strains of Lactobacillus sakei have been used in the food industry as natural preservatives and starter cultures 11 .Recently, the aspects of immunomodulators Bacteria L. sakei HEM 224 and L. rhamnosus GG were grown on MRS agar and in broth (BD) at 37 °C for 16 h.As pathogenic controls for hemolysis and the biogenic amine production assay, Escherichia coli ATCC 25922 and Bacillus cereus ATCC 27348 were used.Both strains were cultured in Brain Heart Infusion (BHI) (BD) media at 37 °C for 18 h with agitation.

Whole genome sequencing
The complete genome sequence of L. sakei HEM 224 was generated using the PacBio RS platform with single molecule real-time (SMRT) sequencing at Theragenetex Inc. (Seoul, Republic of Korea) as commercial service.Briefly, L. sakei HEM 224 was cultured on MRS agar, as previously described.Bacterial DNA was extracted from a fresh pellet using the Wizard ® Genomic DNA Purification Kit (Promega, Thermo Fischer Scientific).Subsequently, libraries were prepared for 151 bp paired-end sequencing using the TruSeq Nano DNA Sample Prep kit (Illumina, CA, USA), and sequencing was conducted as paired-end (2 × 151 bp) with the Illumina NovaSeq platform.Following SMRT library preparation and sequencing, a draft genome was assembled using CANU v.1.7.Annotations were performed by merging the results obtained from the Rapid Annotations using Subsystems Technology (RAST) server 14 .

Hemolysis
The bacterial cultures grown at 37 °C for 16 h were streaked onto blood agar plates containing 5% of sheep blood (Hanil Komed, Republic of Korea), followed by incubation at 37 °C for 24 h to 48 h.The hemolysin production was determined by observation of the clear zone formation.

Biogenic amine formation
L. sakei HEM 224 was cultured in a special medium with some modifications at 37 °C for 48 h 15 .Briefly, 1% of each precursor amino acids [l-tyrosine (Samchun Chemicals, Republic of Korea), l-histidine (Daejung Chemicals, Republic of Korea), l-ornithine (Sigma-Aldrich), and l-lysine (Samchun Chemicals)] was included in each decarboxylase medium to determine the production of tyramine, histamine, putrescine and cadaverine.The production ability was identified by color change of the pH indicator bromocresol purple in the medium.

Antibiotic susceptibility
The agar diffusion method was used to determine the antibiotic susceptibility of the strains suggested by the Clinical and Laboratory Standard Institute (CLSI; http:// www.clsi.org) and/or EFSA.The method previously described was introduced with some modifications 16 .Two-fold serial dilutions of antibiotic solutions were prepared and suspended in MRS and Iso-Sensitest mixture agar (Oxoid, UK).The bacterial suspension was adjusted to desired concentration (1 × 10 7 CFU/mL) and 10 μL (1 × 10 5 CFU/mL) was inoculated onto the plates using a multipin-inoculator.The plates were incubated at 37 °C for 24 h under anaerobic conditions.Antibiotic resistance was determined by a higher concentration of a specific antimicrobial than the cut-off value according to

In vivo DSS-induced colitis model
Seven-week-old female C57BL/6J mice were purchased from Central Laboratory of Animal Inc. (Seoul, Republic of Korea).Mice were adapted for 1 week and housed under controlled environmental condition (23-25 °C, 40-60% of humidity and 12 h light/dark cycle) with chow diet and water ad libitum.To induce colitis in mice, 4% DSS salt (MP Biomedicals, Santa Ana, USA) in drinking water was administered for a period of 6 days, followed by a washout phase using sterile water for 2 days.L. sakei HEM 224 (1 × 10 9 CFU/200 μL/mouse/day) was orally administered from 2 weeks before DSS treatment until the day before sacrifice.Mixture of ketamine hydrochloride (Yuhan, Republic of Korea) and xylazine (Rompun) (Korea branch of Elanco Co., Republic of Korea) was used for anesthetizing and sacrificing by intraperitoneal route.In detail, the composition of ketamine/ xylazine is following: 3.2 mL of ketamine hydrochloride, 1.4 mL of xylazine and 5.4 mL of PBS.The control group received PBS only.All experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) of Handong Global University (approval number HGUIACUC 20191008-021) and all procedure were carried out in compliance with the ARRIVE guideline and the Guidelines for Animal Care and Use at Handong Global University.

In vivo OVA-induced allergic airway inflammation model
Six-week-old female BALB/c mice were housed under specific pathogen-free conditions.The allergic airway inflammation was induced to mice based on the previous protocol with some modifications 22 .Mice were sensitized with 50 μg of OVA (Sigma-Aldrich, USA) with 2 mg of aluminum hydroxide (alum) (Sigma-Aldrich) on days 0 and 7 via the intraperitoneal route.After the last sensitization, the mice were intranasally challenged with 10 μg of OVA on days 14, 17, 21, 24, 28 and 31.On day 32, mice were sacrificed and serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected.L. sakei HEM 224 (1 × 10 7 CFU/mouse) was administered via the intranasal route after the first allergen challenge.The administration was performed after anesthesia for five consecutive days per week for 3 weeks before sacrifice.Mixture of ketamine hydrochloride (Yuhan) and xylazine (Rompun) (Korea branch of Elanco Co.) was used for anesthetizing and sacrificing.All experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) of Handong Global University (approval number HGUIACUC 20190328-013) and all procedure were carried out in compliance with the ARRIVE guideline and the Guidelines for Animal Care and Use at Handong Global University.

Histology
The distal colon (~ 2 cm) and left superior lobes of the mouse lung were fixed in 10% formaldehyde for histological analysis including hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining at KP&T Inc. (Cheongju, Republic of Korea) as commercial service.In brief, the fixed tissues were embedded in paraffin, and 3-μm-thick slices were prepared.After deparaffinization and dehydration, H&E or PAS staining was carried out.

ELISA
The protein levels of IL-4 and IL-5 in BALF were measured by using ELISA MAX™ Deluxe Set Mouse IL-4 and IL-5 (BioLegend, USA) according to the manufacturer's protocol.For serum IgE analysis, the obtained serum was stored at − 80 °C and measured using ELISA MAX™ Deluxe Set Mouse IgE (BioLegend) according to the manufacturer's instructions.

Short chain fatty acid (SCFA) analysis
SCFA analysis was performed using Headspace sampler-gas chromatography-Flame ionization detector (HSS-GC-FID) analysis.To provide a brief overview, all SCFAs were extracted from 0.1 g of mice fecal samples in 1 mL of distilled H 2 O.These samples were vortexed and then centrifuged at 13,000 rpm for 3 min at room temperature.The resulting supernatant was transferred to a vial containing a GC buffer solution, and 2-ethylbutyric acid was added as an internal standard 23 .The samples were analyzed using an Agilent 7890B GC system equipped with a 7697A headspace sampler and FID (Agilent Technologies, USA).Data acquisition and processing were performed using ChemStation software from Agilent Technologies.

DNA extraction and next generation sequencing (NGS) analysis
DNA extraction and NGS analysis were conducted by HEM Pharma Inc.Briefly, bacterial DNA in feces was extracted and the fecal sample was homogenized by bead-beating.After isolating, cleaning, and eluting procedures based on the manufacturer's protocols, the variable V3-V4 regions of the 16S ribosomal RNA gene was sequenced by Illumina Miseq System (Illumina).Sample DNAs were cleaned and the Index PCR was carried out.The concentration of libraries was verified and sequenced using Illumina Miseq system.Reads were sorted using the unique barcodes for each PCR product.The barcode, linker and primer sequences were then eliminated from the original sequencing reads for the next analysis step.

Bioinformatic analysis
Single end FASTQ files were imported into the Quantitative Insights into Microbial Ecology 2 (QIIME2, ver.2021.2) via the FASTQ manifest protocol 24,25 .Demultiplexed files were denoised with DADA2 26 .All amplicon sequence variants were aligned with MAFFT plugin and a phylogeny analysis was performed with Fasttree2 based on the Greengenes database 27,28 .For all analyses, all sampling depths reached to 19,400 reads.Alpha-diversity metrics (Shannon index 29 , Simpson index 30 , Chao1 index 31 , Evenness 32 and Faith's Phylogenetic Diversity 33 ), beta diversity metrics (weighted UniFrac 34 , unweighted UniFrac 35 ), and principal coordinate analysis (PCoA) were performed to evaluate the microbial distances between groups.

Statistical analysis
Data were analysed by GraphPad Prism software 6.0 and 9.3.1 (GraphPad Software, USA) and expressed as the mean ± SD.Statistical significance was calculated by one-way ANOVA with Fisher's LSD test.

Isolation and identification of L. sakei HEM 224
Lactic acid bacterial strains were isolated from a vegetable-based kimchi ingredient and predominantly identified as L. sakei by comparative 16S rRNA sequencing.Among the identified isolates, a strain with negative catalase reaction, L. sakei HEM 224, was selected for further investigation (data not shown).To obtain entire genome information of L. sakei strain HEM 224, whole genome sequencing was performed.The genome of the strain was composed of a circular DNA chromosome with a total size of 2,079,011 bp with a G + C content of 41.2 mol%.Three contigs were found with the varying in range from 11,771 to 1,966,385 bp (Fig. 1).

Safety evaluation of L. sakei HEM 224
To determine the safety of the isolated strain, hemolytic activity, biogenic amine production and antibiotic susceptibility were evaluated.B. cereus ATCC 27348 served as a positive control in the hemolysis test and L. sakei HEM 224 showed no hemolysis reaction.In the biogenic amine production assay, strain HEM 224 was confirmed be negative in presence of the precursor amino acids compared to a positive control, E. coli ATCC 25922 (Table S2).The strain L. sakei HEM 224 was also examined for antibiotic susceptibility and all the minimum inhibitory concentration (MIC) values of the target strain were below or similar to the compared EFSA cut-off values, indicating its susceptibility to all tested antibiotics (Table S3).Moreover, the presence of acquired antibiotic resistance genes was assessed through the ResFinder program and L. sakei HEM 224 was confirmed to negative for any resistance genes of various classes of antibiotics (Table S4).These data suggested L. sakei HEM 224 to be safe for human consumption and thereby it qualified for further evaluation as a probiotic candidate.

Adaptability of L. sakei HEM 224 to GIT
In vitro SSDP assay was performed to determine the potential viability of L. sakei HEM 224 under conditions of the upper GIT.The strain was exposed to a simulated stomach-and duodenum-like environment in sequence and counted at each timepoint.L. sakei HEM 224 showed a higher survival rate after the stomach-and duodenum-simulated environment pass than that of LGG (Table S5).Additionally, the adhesion ability of strain HEM 224 was also investigated using Caco-2 cells.After incubation, its adhesion rate of was similar to that of LGG (Table S6).These data suggest that L. sakei HEM 224 has a comparable or superior adaptability to upper GIT conditions as the probiotic control.

L. sakei HEM 224 confers protective effect against DSS-induced colitis
To evaluate the functionality of L. sakei HEM 224 in an in vivo system, its alleviative potential was examined against a DSS-induced colitis model.The oral consumption of L. sakei HEM 224 ameliorated body weight loss and recovered colon length significantly (Fig. 2A-C).Histological specimens showed that L. sakei HEM 224 treatment improved a deteriorated structure of epithelial barrier (Fig. 2D).In the colon, L. sakei HEM 224 downregulated the mRNA expression of major pro-inflammatory cytokines (Fig. 2E).

L. sakei HEM 224 improves gut epithelial barrier by upregulation of TJPs in the gut
Moreover, the TJP expression levels in the colon were measured and the strain significantly upregulated main TJP markers (Fig. 3).In case of mucin, the gene expression of MUC4 showed an increasing tendency only.Collectively, this strain may reinforce the epithelial barrier integrity with the upregulation of TJPs, thereby resulting in a protective effect against colitis.

L. sakei HEM 224 modulates gut microbial metabolites and gut microbiota
In the SCFA profiles of the three experimental groups, butyrate production level was significantly increased in the L. sakei HEM 224-treated group suggesting the possible modulation of the gut microbiota (Table S7).Subsequently, L. sakei HEM 224 ingestion resulted in increased values in every alpha diversity index (Fig. 4A).The microbial community structure was analyzed based on beta diversity including unweighted and weighted UniFrac distances and represented by principal coordinates analysis (PCoA) plot.Each group presented a distinct clustering and especially the L. sakei HEM 224 treatment group showed a clear difference compared to the DSS + PBS group (Fig. 4B).In the taxonomical analysis the L. sakei HEM 224 group augmented the abundance of Lactobacillus and Clostridium species groups (data not shown).The microbial data suggest that the L. sakei HEM 224 induced the modulation of the gut microbiota.

L. sakei HEM 224 ameliorates allergic airway inflammation
In order to assess the immunomodulatory effect of the strain in a local mucosal organ, an allergic airway inflammation model with induction by OVA was applied.Intranasal administration of L. sakei HEM 224 successfully decreased IL-4 and IL-5 in BALF and IgE in the serum (Fig. 5A,B).IL-13 decreased at a transcript level but with no significance.The mRNA expression of chemokines was also altered and especially, C-C motif chemokine ligand (CCL) 24 was significantly reduced (Fig. 5C).In the histological analysis, the infiltration of inflammatory cells was mitigated around the alveoli in the L. sakei HEM 224 treated group (Fig. 5D).

L. sakei HEM 224 improves airway epithelial barrier by upregulation of TJPs and inhibition of mucus hyperproduction in the lungs
Next, the effects of intranasal treatment of L. sakei HEM 224 on airway epithelial barrier health were evaluated about expression of TJP genes and mucus hypersecretion.The mRNA expression levels of occludin and ZO-1 were significantly increased compared with the OVA + PBS group.Moreover, the MUC5AC mRNA expression levels showed a decreasing tendency (p = 0.1719) suggesting the improvement of the epithelial barrier integrity in the airways (Fig. 6).Intranasal treatment of L. sakei HEM 224 still showed the increased amount of mucus along with the alveoli surface (violet), but the intensity was alleviated compared to the OVA + PBS group (Fig. 7).These results demonstrate that the intranasal administration of L. sakei HEM 224 ameliorated

Conclusion and discussion
Gut and lungs possess several similarities connecting to the overlapped characteristic regarding pathogenesis, therefore, the connection between two distinct organs has been highlighted.Inflammatory diseases in both sites accompany dysregulation not only of the immune response but also of the epithelial barrier and mucus production.It was reported that patients with IBD suffer from barrier dysfunction with the reduction of goblet cells and reduced thickness of the mucus layer, and of impaired TJPs leading to increased severity 36 .The relationship between epithelial permeability and inflammation is similar to that of the respiratory system.The loss or dysfunction in TJPs is linked to the pathogenesis of various airway disorders.A reduced gene expression of TJPs such as ZO-1 and occludin was observed as inflammatory phenotypes of an airway inflammation in vivo model 37,38 .Therefore, the well-tightened epithelial lining might be considered a key factor for protection in mucosal diseases.
Probiotics have been suggested as alternatives for treatment of various inflammatory diseases in mucosal sites due to their advantages; the modulation of the (gut) microbiota, the maturation of the immune system and reinforcement of the epithelial barrier 39 .Here, we suggested that the novel putative probiotic strain L. sakei HEM 224, assessed for its safety and functionality, to have beneficial potential for alleviating various mucosal diseases.
This study successfully isolated a novel L. sakei strain and acquired its genomic information through whole genome sequencing (Fig. 1).Although most LAB are considered as safe, safety assessment on a strain level is essential to determine any potential risk to consumers 40 .The biogenic amine formation assay aims to evaluate whether the tested strain can produce specific biogenic amines by decarboxylating of precursor amino acids in the test medium.Biogenic amines exist in low concentrations in nature and low consumption does not elicit harmful effects; however, elevated levels in the diet and the human body can be toxic leading to a health hazard, especially for sensitive persons 41 .L. sakei HEM 224, was identified to be negative to the tested biogenic amines (Table S2).
Antibiotic resistance may be a critical issue especially when related to the presence of acquired resistance genes, indicating the problematic potential of transferability 42 .L. sakei HEM 224 was susceptible to all tested antibiotics recommended by EFSA 17 (Table S3).Furthermore, the presence of acquired resistance genes regarding several antibiotic classes was screened for using ResFinder program and no acquired resistance genes were detected (Table S4).
Regarding functionality evaluation, tolerance to the acidic environment of the stomach is a desirable requirement of putative probiotics to reach the intestine and ultimately elicit beneficial effects 43 .The adherence of a strain to the gut epithelium is closely associated with colonization and eventually the expression of beneficial functions 44 .Our strain showed notable survivability after passage of the gastric-and duodenum-like environments and a similar attachment ratio to intestinal epithelial cells, comparable to that of the representative commercial probiotic strain, LGG, in an in vitro study (Tables S5, S6).
The probiotic potential of L. sakei HEM 224 was further investigated in an in vivo mouse model with induced colitis and allergic airway inflammation.The DSS-induced colitis model has been reported to closely resemble human ulcerative colitis and has therefore been extensively used in IBD research 45 .In our experiments, DSStreated mice showed a significant weight loss, colon shortening and a disrupted villi structure in histological analysis, however, L. sakei HEM 224 supported the recovery of physiology markers (Fig. 2A-D).The mRNA expression of pro-inflammatory cytokines was also suppressed and that of IL-10, the well-known anti-inflammatory regulatory cytokine, was increased (Fig. 2E).It was reported that a specific L. sakei strain can modulate the immune system by boosting the number of regulatory T cells (Treg) and IL-10 production 46 .However, our strain could not influence the increase IL-10 production in the colon (data not shown).Therefore, we expected epithelial barrier markers may be responsible for the mechanism(s).TJPs exist in the distal colon and decreased levels of them indicate the disruption of the gut barrier 47 .The loss of mucin production leads to the weakening of the intestinal barrier and to increased permeability, a typical feature of IBD 48 .In this study, L. sakei HEM 224 increased the mRNA expression level of both TJPs and mucin markers (Fig. 3).These data strongly suggest that L. sakei HEM 224 can reinforce the gut barrier integrity by upregulation of TJPs and mucin.
Another possible mechanism for alleviating colitis in mice is through the modulation of gut microbiota.It was reported that the application of probiotic strain could modulate gut microbiota with the increase in diversity and the ratio of Bacteroidetes and Firmicutes, and eventually attenuated the diseased conditions 49 .In this study, the gut microbiota diversity of the L. sakei HEM 224 treated group was increased and showed a clear clustering in beta diversity (Fig. 4).These data confirm that L. sakei HEM 224 oral administration induced the gut microbiota modulation.Taxonomic analysis did not show any significant difference at the phylum level, however, an increased abundance of Lactobacillus and Clostridium species was observed (data not shown), suggesting that our strain may modulate the gut microbiota and contribute to forming a favorable environment for LAB.
Bacterial metabolites have been suggested as possible factors for strengthening the epithelial barrier by upregulating tight junctions.Probiotics can induce the production of metabolites such as SCFA and stimulate mucin secretion and repair of tight junctions 50 .SCFAs are representative and effective metabolites that can directly or indirectly affect gut barrier integrity.Specifically, butyrate can induce the tight junction component gene contributing to the enhancement of the epithelial barrier function.Moreover, butyrate production is closely related to the gut microbiota modulation in the chronic inflammatory state 51 .In our study, the L. sakei HEM 224 treated mice showed an increased butyrate production more specifically than for any other SCFAs (Table S7).We assumed that butyrate may be involved in the upregulation of tight junction genes and gut microbiota modulation as well.
L. sakei has been applied not only to the gut but also other local mucosal organs, but the application to pulmonary system is sparse.The respiratory tract is the second-largest mucosal organ after the GIT and is endlessly challenged by numerous external factors.The innate immune system competes against inhaled antigens and both resident and infiltrated immune components sustain a proper immune tone to avoid unwanted airway inflammation 52 .However, the immune balance can be disrupted for various reasons including the genetic background and external influences and can lead to respiratory inflammatory conditions.In the inflamed condition, infiltrated immune cells worsen the response, impacting the epithelial barrier structurally and functionally 53 .Similar to IBD studies involving probiotics, beneficial microbes have been proposed as potential alternatives for treatment of airway inflammation.The administration of several Lactobacillus strains showed alleviative effects including immunomodulation and gut microbiota modulation against allergic airway inflammation model 54 .
Research on probiotics and airway inflammation predominantly focused on the oral administration of bacterial strains, while direct targeting of respiratory sites has become a recent focus when beneficial effects of direct contact have been observed 55 .In this study, L. sakei strain HEM 224 was administered intranasally to an OVAinduced allergic airway inflammation model.The results showed successful suppression of Th2-related allergic reaction markers and improvement in physiology was observed in the histological analysis (Fig. 5).Interestingly, when the strain was applied by the oral route, the strain could not modulate biased-Th2 reaction (data not shown).These results indicate that the treatment with L. sakei HEM 224 can impact the target site when it is in direct contact with the organ.
Another key point of this study is that L. sakei HEM 224 upregulated TJP genes expression in the lungs, similar to the colitis results.Chronic airway inflammation can cause airway epithelial damage together with the downregulation of specific TJPs 37 .L. sakei HEM 224 augmented the mRNA expression of claudin-18, occludin, and ZO-1 in the lungs (Fig. 6), suggesting the recovery effect of the strain on the impaired epithelial integrity.Unlike the colitis model, mucin hypersecretion is problematic in the allergic airway inflammation model.MUC5AC/muc5ac, the representative secretory mucin in the airway, has been reported as a marker for goblet cells on the airway surface and patients with asthma showed abnormal production 56 .In our airway inflammation model, intranasal treatment with L. sakei HEM 224 downregulated the mRNA expression of MUC5AC (Fig. 6) and confirmed the suppressed production by histological analysis (Fig. 7).It is meaningful that L. sakei HEM 224 can also affect epithelial barrier health in airways, still confirmation on the protein level for each protein should be provided and bacterial factor(s) are responsible for the reaction need to be clarified.
In this research, we have identified a unique and novel strain that demonstrates beneficial effects on two distinct mucosal sites.What's particularly intriguing is that the observed beneficial effects in each disease model were dependent on the route of administration.In the case of the colitis model, the oral administration of L. sakei HEM 224 successfully attenuated colitis by modulating the gut microbiota.However, as mentioned earlier, the oral application of the strain did not ameliorate allergic airway inflammation.Based on these findings, we hypothesize that L. sakei HEM 224 may exert its modulatory effects on colitis through a secondary route by stimulating changes in the gut microbiota.Conversely, in the context of airway conditions with allergic inflammation, direct contact between the strain and host components may lead to different regulatory mechanisms.Nevertheless, a comprehensive mechanistic study of L. sakei HEM 224 regarding these intriguing responses is still warranted for further applications.
Overall, this study confirmed the safety of L. sakei HEM 224 for use as a putative probiotic.Its beneficial functions were evaluated in in vitro and in vivo models.Two inflammatory models targeting the gut and lung were used and L. sakei HEM 224 successfully attenuated each inflammatory state with the improvement of epithelial barrier components, including TJPs and mucins.Therefore, L. sakei HEM 224 has probiotic potential for use against various mucosal inflammatory disorders. https://doi.org/10.1038/s41598-023-45043-0

Figure 1 .
Figure 1.The circular genome map of L. sakei HEM 224.The first circle (starting with the inner circle) represents the genome size.The second circle indicates the GC content.The third circle depicts the GC skew (G + C/G − C).The fourth circle shows the number and location of contigs.

Figure 3 .
Figure 3. Effects of L. sakei HEM 224 on tight junction protein and mucin expression in mice with DSSinduced colitis.The relative expression of genes regarding tight junction proteins in murine colon tissue was measured by qRT-PCR.CTRL vehicle-treated control, DSS + PBS DSS + vehicle-treated group, DSS + LS224 DSS + L. sakei HEM 224 treated group.Data show the mean ± SD (n = 6-8 mice per group) and the difference compared to the DSS + PBS group was analyzed by one-way ANOVA with Fisher's LSD test.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6 .
Figure 6.Effects of intranasal administration of L. sakei HEM 224 on the tight junction proteins and mucin expression in an OVA-indued allergic airway inflammation murine model.The relative mRNA expression of cytokines in murine lung tissue was measured by qRT-PCR.CTRL vehicle-treated control group, OVA + PBS OVA + vehicle-treated group, OVA + LS224 OVA + L. sakei HEM 224 treated group.Data show the mean ± SD (n = 3-6 mice per group) and the difference compared to the OVA + PBS group was analyzed by one-way ANOVA with Fisher's LSD test.*p < 0.05, **p < 0.01, ***p < 0.001.