the Function of Mixed Postbiotic Pe0401 in improving intestinal health via elevating Anti-inflammation, Anti-oxidation, epithelial tight Junction Gene expression and Promoting Beneficial Bacteria Growth

Dysbiosis of intestinal microbiota may cause irregular digestive function, and intestinal wall inflammation. Over the past few years, probiotics generate bioactive metabolites, named postbiotics, have been discovered its crucial roles in modulation of intestinal microbiota. single-strain postbiotics have positive effect on health of host, but the functions of multi-strain postbiotics remain unclear. this study proposed a useful application of multi-strain postbiotics and thereby establish the developing foundation of multi-strain postbiotics. initially, various probiotics and postbiotics were screened for anti-inflammatory activity through inducing the transforming growth factor beta (tGF-β)and interleukin-10 (il-10) in peripheral blood mononuclear cells (PBMCs). then, we detailed the synergistic effects of 4Mix postbiotics (named as Probiotic extracts of 4 strains- number 1, Pe0401) consisted of metabolites generated from Lactobacillus salivarius AP-32, Lactobacillus acidophilus tYCA06, Lactobacillus plantarum lPl28, Bifidobacterium longum subsp. infantis Bli-02 on anti-inflammatory activity, antioxidative capacity, regulation of tight junction proteins. the results displayed that anti-inflammatory activity of 4Mix postbiotics Pe0401 was stronger than other mixed postbiotic combinations. the antioxidative capacity, which correlated to anti-inflammation, also significantly increased as shown in DPPh and FRAP assays. the epithelial tight junction proteins expressed in mRNA levels (ZO-1, ZO-2, Occludin, JAM-A, and Claudin) were highly potent modulated by Pe0401. in addition, Pe0401 selectively promoted the growth of intestinal bacterial strains including Lactobacillus, Bifidobacterium strains and other beneficial bacteria. therefore, this study provides a fascinating insight into the strategy to the treatment of the intestinal disorders. Pe0401 may deliver as health functional food ingredient.


iNtRODuCtiON
Chronic inflammatory bowel disease (IBD) were usually caused by the repetition of symptoms of intestinal wall inflammation. Patients with Crohn's disease or ulcerative colitis are prone to present complications such as diarrhea, abdominal pain, weight loss, malnutrition, growth retardation in children, osteoporosis, higher risk of developing cancer. 1 Tight junction proteins on intestinal wall including Occludins, Junctional adhesion molecule A protein (JAM-A), Claudin and Zonula occludens (ZO) contribute to hold the integrity of gut barriers and maintain healthy intestinal function. Chronic inflammation of Crohn's disease would lead to damage and erosion of tight junctions on intestinal tract. 2 The elevated anti-inflammatory cytokines such as transforming growth factor beta (TGF-β) and Interleukin 10 (IL-10) may benefit to maintain intestinal barrier function and repair epithelial wound. 3 The postbiotic is defined as the metabolites secreted by probiotic strains, 4 which has been reported functions of anti-inflammation, anti-oxidation, growth promotion, antidiarrheal activity and digestion. 4,5 Probiotic strains secrete amino acids, vitamins, bacteriocins, peptidoglycanderived muropeptide, short chain fatty acids (SCFAs) and promote anti-inflammatory and anti-oxidant effect in human gut. 6,7 Additionally, researchers discovered that Bacillus strains generating metabolic enzymes including amylase, protease, catalase and superoxide dismutase facilitate the growth of Lactobacillus. 8 However, previous studies haven't tested the functions of multi-strain postbiotic mixture.
Therefore, in this study, we aimed to (i) screen potential multi-strain probiotics presented anti-inflammatory activities, (ii) test the synergistic anti-inflammatory effect of multi-strain postbiotic mixture, which mixed from several individual probiotic metabolites (Lactobacillus salivarius AP-32, Lactobacillus acidophilus TYCA06, Lactobacillus plantarum LPL28, Bifidobacterium longum subsp. infantis BLI-02), (iii) measure the anti-oxidative effect of multi-strain postbiotic mixture, (iv) verify whether postbiotic mixture promote intestinal health via regulating tight junction protein genes in epithelial cells, and (v) observe whether postbiotic mixture facilitate the growth of other intestinal beneficial bacterial strains.
Following the in vitro results, the four strains mixed postbiotics (named Probiotic Extracts of 4 strains-number 1, PE0401) showed the synergistic effect on promoting growth of beneficial bacterial strains and anti-inflammatory activity for alleviating chronic inflammatory bowel disease (IBD) and other inflammatory disorders.
The strains were identified using 16s rRNA sequencing and the API 50CHL kit (BioMerieux, France). The four strains mixed postbiotics (named as Probiotic Extracts of 4 strains-number 1, PE-0401) was developed as followed: four probiotic strains including Lactobacillus salivarius AP-32, Lactobacillus acidophilus TYCA06, Lactobacillus plantarum LPL28, Bifidobacterium longum subsp. infantis BLI-02 were cultured with 1:1:1:1 ratio and growing in culture medium under anaerobic conditions at 37°C for 16 hrs. Then, centrifuging the fermented broth at 18000 rpm for 20 min, and collecting supernatant part of fermentation solution as the PE0401. The abbreviation of the four strains mixed postbiotics, PE0401, will be used for the following sections.

Preparation of Peripheral Blood Mononuclear Cells
The human peripheral blood was gained from blood samples of healthy donors (Tainan Blood Center, Taiwan Blood Services Foundation). The peripheral blood mononuclear cells (PBMCs) were isolated from low-density fraction (around 42.5%-50% interface) by centrifugation (3000rpm, 10 min) through a Ficoll-Hypaque gradient (Pharmacia, Sweden). The RPMI-1640 medium containing 10% fetal bovine serum (FBS) was used

Anti-inflammatory Cytokine Assay
Loading 100 μl isolated PBMCs into 96 well plate (No. 167008, MicroWell™ 96-Well, Thermo Fisher, USA) at 4x10 5 cells/100μl/well. Then co-culturing PBMCs with 20 μl of the postbiotics or probiotics (4x10 6 CFU) at ratio of 1:10 (postbiotics: PBMCs) in the 5% carbon dioxide (CO 2 ) at 37°C for 48hrs. The control group was treated with 20 μl of PHA (Phytohemagglutinin, 0.2 μg/ml), and the medium control group was treated with 20 μl of cell culture medium. After 48 hrs of incubation, collecting the supernatant of coculture medium by centrifuging the 96 well plate at 3000 rpm for 10 min at 4°C. Then, using ELISA assay (eBioscience) to measure IL-10 and TGF-β of the collected supernatant. Finally, cytokines levels were measured by μQuant microplate spectrophotometer (BioTek, USA) at absorbance value of 450 nm. The experimental protocol was according to previous research. 6

Cells Culturing
The Caco-2 cells (HTB-37™) are epithelial cells isolated from colorectal adenocarcinoma and was obtained from American Type Culture Collection (USA). The Eagle's Minimum Essential Medium (ATCC No. 30-2003) with final concentration of 20% fetal bovine serum (ThermoFisher, USA) was used to culture Caco-2 cells. The incubation condition was under 5% carbon dioxide (CO 2 ) at 37°C. The following tests of anti-oxidative assay and tight junction proteins in mRNA levels were based on Caco-2 culturing platform.

Free Radical scavenging Assay
DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) is a stable nitrogen-centered radical (Sigma-Aldrich, USA). When the antioxidant compounds bind to DPPH, it causes a decreased level in OD517, which determined the free radical scavenging activity. The DPPH assay is performed as following description: 1:1 mixed 0.2mM DPPH in methanol solution with 0.5% postbiotics or 10 ug/ ml of vitamin C (positive control) or medium (blank group), respectively. The mixture was incubated for 30 min at room temperature in the dark before centrifuging at 12000rpm for 2 min at 4°C. The sample was measured at absorbance of 517nm with μQuant microplate spectrophotometer (BioTek, USA). The calculation formula is as follows: Free radical scavenging ability = OD blank -OD sample ) /OD blank X 100 Where OD Blank is the absorbance value of the cell medium, and OD sample is the absorbance value of the tested samples.

Measurement of Antioxidative Activity by FRAP Assay
Ferric-reducing ability of power assay (FRAP assay; Sigma-Aldrich, St. Louis, MO, USA) is a common method used to test the reducing power of antioxidants. In an acidic environment (under pH 3.6), the trivalent iron (Fe 3+ ) in the FRAP reagent will be reduced to divalent iron (Fe 2+ ) by binding with antioxidants, and caused the change of color. The 0.5% postbiotics, 10 ug/ml vitamin C (positive control group), and medium control (blank group) were added to Fe 3+ -TPTZ solution, and detected the OD593 value, respectively. When the Fe 3+ -TPTZ complex is reduced to Fe 2+ -TPTZ, the solution color will turn from yellow to blue. The deeper blue color indicated the stronger antioxidant capacity. The measured value is compared with the standard calibration curve.

Real-time PCR technique Detected tight Junction Proteins in mRNA levels
Seeding and culturing 3 x 10 5 Caco-2 cells on a 6-well cell culture plate for 6 days (change the medium every 3 days). Co-culturing postbiotics treated Caco-2 cells for 5 hrs. Washing cell culture medium with PBS twice then extracting mRNA by adding 500 μl Trizol (Invitrogen™, Waltham, Massachusetts, USA) to each well. Using High-Capacity RNA-to-cDNA™ Kit (Thermo, Waltham, Massachusetts, USA) to invert extracted mRNA into cDNA. The PowerTrack™ SYBR Green Master Mix (Thermo, Waltham, Massachusetts, USA) and specific primers were used to perform probe-based qPCR assay on the QuantStudio 6 Pro Real-Time PCR platform (Thermo, Waltham, Massachusetts, USA). The experimental protocol and specific primer sequences were according to the Figure 1 of previous study. 9

Detecting Growth Rate of Probiotic strains
The 4Mix postbiotic PE0401 and seven prebiotics were prepared in 4.5 ml of culture medium with a content of 0.25 wt% and 0.5 wt%, respectively. Seven prebiotics were listed as below: human milk oligosaccharides (HMO; DuPont, USA), Maltodextrin (UNION FOOD, Taiwan), FOS (fructooligosaccharides; GreenGo Biomedical international, Taiwan) and GOS (galactooligosaccharides; GreenGo Biomedical international, Taiwan), sorbitol (SHENG YUANG FOOD INDUSTRIAL CO., LTD, Taiwan), erythritol (Buildmore Enterprise Co., Ltd, Taiwan), and inulin (BIOMED HERBAL RESEARCH BIOMEDICAL GROUP, Taiwan). Next, preparing 0.5 ml bacterial liquid of Lactobacillus rhamnosus MP108 (1×10 9 CFU/ml), Bifidobacterium animalis subsp. lactis CP-9 (1×10 9 CFU/ml) and other probiotic strains listed in Table were added in each test medium, respectively. All the test samples were incubated at 37°C for 6 hrs. After that, 0.02ml of the co-culture medium (prebiotics or 4Mix postbiotics obtained in each probiotic medium) was measured with a spectrophotometer (MQX200, BioTek) at 600 nm. The relative growth fold of probiotic strain was presented by following formula: Formula: A = (B -C) / (C -D) The radical scavenging activity of single probiotic or 4Mix postbiotics (a and b) was assessed by DPPH assay, and the ferric-reducing ability was evaluated by FRAP assay (c and d). The positive control was treated with vitamin C (0.5%) and the blank group was treated with medium only.

symbol Definition
A indicated relative growth rate; B indicated OD 600 absorbance value of each group; C indicated OD 600 absorbance value of medium control; D indicated OD 600 absorbance value of blank (ddH 2 O).

statistics
The statistical differences between the experimental groups and the control group was analyzed by Student's t test (SPSS 12, IBM, USA). p < 0.05 (*), p< 0.01 (**), and p<0.001 (***) indicated statistical significance. All experiments were performed in triplicate and presented as mean±SD.

DisCussiON
Inflammation is an immunovascular response for human body to deal with harmful stimuli including pathogens, irritants, toxins and injuries. When irritants enter and damage cells, inflammatory response functions to release chemicals that enhance vascular permeability for facilitating migration of leukocytes to injured site. Through the inflammatory activities, the necrotic cells will be cleared up and tissue repair will be initiated. 11 However, prolonged inflammation correlated with Crohn's disease, 1 ulcerative colitis, 1 oxidative stress, 12 aging, 13 atherosclerosis, 14 osteoarthritis, 15 chronic pain, 16 and cancer. 17 Cytokines are peptides (5-20 kDa) produced by various cells, which play important roles in communicating between cells, mediating autocrine, paracrine and endocrine signaling, especially involved in inflammatory and antiinflammatory responses. 18,19 TGF-β and IL-10 belong to anti-inflammatory cytokines, which prohibit inflammatory response through regulating pro-inflammatory cytokines. 20 The secretion of IL-10 increases the expression of specific endogenous receptors, and suppresses inflammatory cytokines. An animal model confirmed IL-10 protein regulated neuropathic and inflammatory pain. 21 In addition, TGF-β, a cytokine induces Treg cells, was found to suppress macrophage and Th1 cell activity which produced pro-inflammatory cytokines. 22 At present study, results showed individual probiotic strains of TYCA06, BLI-02, LPL28, and AP-32 effectively increased the TGF-β and IL-10 levels in PBMCs (Figure 1). In vitro test presented that the postbiotics of TYCA06, BLI-02, LPL28, and AP-32 significantly improved the TGF-β and IL-10 levels (Figure 2a and d). The 4Mix postbiotics (PE0401) demonstrated excellent anti-inflammatory effect among other mixed postbiotics (Figure 2b and e), in which the PE0401 revealed better anti-inflammatory function than upper layer and lower layer of re-centrifuged PE0401 (Figure 2c and f). Besides, accumulated free radicals in human body would further lead to various inflammatory-associated diseases. 23 Here, PE0401 presented anti-oxidative effect (Figure 3 a and c), in which the upper layer of re-centrifuged PE0401 may play the major function in free radicals scavenging (Figure 3b and d). It's presumed that four viable probiotic strains secreted anti-oxidative components to the upper layer of re-centrifuged PE0401. However, the major components of the 4Mix postbiotics involved in anti-inflammation and anti-oxidation should be analyzed in the future.
The inflamed colonic mucosa usually altered tight junction protein structure and impaired barrier function. 24 A leaky intestinal barrier caused permeation of endotoxins, bacteria, and luminal antigens into the blood stream, which contributed to chronic intestinal inflammation and diarrhea. 25 Previous study discovered that tight junction proteins of JAM-A, Occludin, and ZO-2( in mRNA levels) would significantly increase by treating probiotic strains of B. animalis subsp. lactis CP-9, L. salivarius subsp. salicinius AP-32, L. johnsonii MH-68, and L. reuteri GL-104. 9 Here, we discovered PE0401 also improved the expressions of Occludin, JAM-A, Claudin, ZO-1 and ZO-2 in mRNA levels (Figure 4).
Prebiotic products such as Human milk oligosaccharide (HMO) have been identified its benefit on promoting the growth in some probiotic strains. 10,26 Postbiotics also functioned in facilitating growth of viable probiotic strains at previous study, 27 and inhibiting the growth rate of pathogenic bacteria via short chain fatty acids (SCFAs). 27,28 The present study discovered that PE0401promoted the growth rate of several beneficial microbial strains of Lactobacillus, Bifidobacterium and Streptococcus thermophilus (Table), notably L. rhamnosus MP108 and B. animalis subsp. lactis CP-9 ( Figure 5). Our results suggested that the probiotic growth promotion rate of PE0401 was superior to other prebiotic products ( Figure 5). PE0401 facilitated the growth of MP108 and CP-9, whereas seven selected prebiotics only promoted the growth of MP108. The functional components of the PE0401 should be measured by LC-MS and HPLC method in the future. 29 In conclusion, PE0401 synergistically promotes growth of beneficial bacterial strains and anti-inflammatory activity, which will be a potential functional food for alleviating chronic inflammatory bowel disease (IBD) and other inflammatory dysfunctions.
performed data analysis and project administration. WYL and CWC visualized the data. HHH performed supervision. WYL and YCH wrote the original draft. YWK, CWC and YCH reviewed the manuscript. YCH edited the manuscript. All authors read and approved the final manuscript for publication.

FuNDiNG
None.

DAtA AvAilABilitY
All datasets generated or analyzed during this study are included in the manuscript.

ethiCs stAteMeNt
Not applicable.