Crypt-like patterned electrospun nanofibrous membrane and probiotics promote intestinal epithelium models close to tissues.

In vitro intestinal epithelium models have drawn great attention to investigating intestinal biology in recent years. However, the difficulty to maintain the normal physiological status of primary intestinal epithelium in vitro limits the applications. Here, we designed patterned electrospun polylactic acid (PLA) nanofibrous membranes with crypt-like topography and mimic ECM fibrous network to support crypt culture and construct in vitro intestinal epithelium models. The patterned electrospun PLA nanofibrous membranes modified with Matrigels at 0 °C showed high biocompatibility and promoted cell growth and proliferation. The constructed duodenum epithelium models and colon epithelium models on the patterned electrospun PLA nanofibrous membranes expressed the typical differentiation markers of intestinal epithelia and the gene expression levels were close to the original tissues, especially with the help of probiotics. The constructed intestinal epithelium models could be used to assess probiotic adhesion and colonization, which were verified to show significant differences with the Caco-2 cell models due to the different cell types. These findings provide new insights and a better understanding of the roles of biophysical, biochemical, and biological signals in the construction of in vitro intestinal epithelium models as well as the potential applications of these models in the study of host-gut microbes interactions. KEY POINTS: • Patterned electrospun scaffold has crypt-like topography and ECM nanofibrous network. • Matrigels at 0°C modify scaffolds more effectively than at 37°C. • Synergy of biomimic scaffold and probiotics makes in vitro model close to tissue.

Joint protection strategies for Saccharomyces boulardii: exogenous encapsulation and endogenous biofilm structure.

Biofilms are heterogeneous structures composed of microorganisms and the surrounding extracellular polymeric substances (EPS) that protect the microbial cells from harsh environments. Saccharomyces boulardii is the first yeast classified as a probiotic strain with unique properties. However, tolerance of S. boulardii biofilms to harsh environments especially during production and in the gastrointestine remains unknown. In this study, S. boulardii cells were encapsulated in alginate microcapsules and subsequently cultured to form biofilms, and their survival and tolerance were evaluated. Microencapsulation provided S. boulardii a confined space that enhanced biofilm formation. The thick alginate shell and the mature biofilm improved the ability of S. boulardii to survive under harsh conditions. The exogenous encapsulation and the endogenous biofilm structure together enhanced the gastrointestinal tolerance and thermotolerance of S. boulardii. Besides, as the alginate shell became thinner with an increase in the subsequent culture duration, the EPS of S. boulardii biofilms exerted an important protective effect in resisting high temperatures. The encapsulated biofilm of S. boulardii after 24-h culture exhibited 60 × higher thermotolerance at 60 °C (10 min), while those after 6-h and 24-h culture showed 1000 × to 550,000 × higher thermotolerance at 120 °C (1 min) compared with the planktonic cells without encapsulation. The present study's findings suggest that a combination of encapsulation and biofilm mode efficiently enhanced gastrointestinal tolerance and thermotolerance of S. boulardii. KEY POINTS: • Encapsulated S. boulardii in biofilm mode showed enhanced tolerance. • Exogenous shell and endogenous biofilm provided dual protection to S. boulardii.

Lactobacillus reuteri biofilms formed on porous zein/cellulose scaffolds: Synbiotics to regulate intestinal microbiota.

Supplementing probiotics or indigestible carbohydrates is a usual strategy to prevent or revert unhealthy states of the gut by reshaping gut microbiota. One criterion that probiotics are efficacious is the capacity to survive in the gastrointestinal tract. Biofilm is the common growth mode of microorganisms with high tolerances toward harsh environments. Suitable scaffolds are crucial for successful biofilm culture and large-scale production of biofilm-phenotype probiotics. However, the role of scaffolds containing indigestible carbohydrates in biofilm formation has not been studied. In this study, porous zein/cellulose composite scaffolds provided nitrogen sources and carbon sources simultaneously at the solid/liquid interfaces, being beneficial to the biofilm formation of Lactobacillus reuteri. The biofilms showed 2.1-17.4 times higher tolerances in different gastrointestinal conditions. In human fecal fermentation, the biofilms combined with the zein/cellulose composite scaffolds act as the "synbiotics" positively modulating the gut microbiota and the short-chain fatty acids (SCFAs), where biofilms provide probiotics and scaffolds provide prebiotics. The "synbiotics" show a more positive regulation ability than planktonic L. reuteri, presenting potential applications in gut health interventions. These results provide an understanding of the synergistic effects of biofilm-phenotype probiotics and indigestible carbohydrates contained in the "synbiotics" in gut microbiota modulation.

Intestinal models based on biomimetic scaffolds with an ECM micro-architecture and intestinal macro-elasticity: close to intestinal tissue and immune response analysis.

The synergetic biological effect of scaffolds with biomimetic properties including the ECM micro-architecture and intestinal macro-mechanical properties on intestinal models in vitro remains unclear. Here, we investigate the profitable role of biomimetic scaffolds on 3D intestinal epithelium models. Gelatin/bacterial cellulose nanofiber composite scaffolds crosslinked by the Maillard reaction are tuned to mimic the chemical component, nanofibrous network, and crypt architecture of intestinal ECM collagen and the stability and mechanical properties of intestinal tissue. In particular, scaffolds with comparable elasticity and viscoelasticity of intestinal tissue possess the highest biocompatibility and best cell proliferation and differentiation ability, which makes the intestinal epithelium models closest to their counterpart intestinal tissues. The constructed duodenal epithelium models and colon epithelium models are utilized to assess the immunobiotics-host interactions, and both of them can sensitively respond to foreign microorganisms, but the secretion levels of cytokines are intestinal cell specific. The results demonstrate that probiotics alleviate the inflammation and cell apoptosis induced by Escherichia coli, indicating that probiotics can protect the intestinal epithelium from damage by inhibiting the adhesion and invasion of E. coli to intestinal cells. The designed biomimetic scaffolds can serve as powerful tools to construct in vitro intestinal epithelium models, providing a convenient platform to screen intestinal anti-inflammatory components and even to assess other physiological functions of the intestine.

Lactobacillus reuteri Biofilms Inhibit Pathogens and Regulate Microbiota in In Vitro Fecal Fermentation.

Bacteria colonizing the gastrointestinal tract generally grow well in biofilms. In recent years, probiotic biofilms have been considered the most promising fourth-generation probiotics. However, the research into the functions of probiotic biofilms is just starting. In this study, Lactobacillus reuteri DSM 17938 biofilms formed on electrospun cellulose acetate nanofibrous scaffolds were contrasted with planktonic cells. Pathogen inhibition analysis of Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes suggested a significant distinction between the planktonic and biofilm groups. In human fecal fermentation, L. reuteri remodeled the microbiota by decreasing the relative abundances of Proteobacteria, Escherichia-Shigella, and Desulfovibrio and increasing the relative abundances of Phascolarctobacterium, Bacteroides, and Lactobacillus. Moreover, L. reuteri biofilms played more positive roles in microbiota modulation and short-chain fatty acid production than planktonic L. reuteri. These findings provide an understanding of the beneficial effects of probiotic biofilms, laying a foundation for the application of probiotic biofilms as a health promoter.

Enhanced antibacterial efficacy and mechanism of octyl gallate/beta-cyclodextrins against Pseudomonas fluorescens and Vibrio parahaemolyticus and incorporated electrospun nanofibers for Chinese giant salamander fillets preservation.

A series of alkyl gallates were evaluated for the antibacterial activity against two common Gram-negative foodborne bacteria (Pseudomonas fluorescens and Vibrio parahaemolyticus) associated with seafood. The length of the alkyl chain plays a pivotal role in eliciting their antibacterial activities and octyl gallate (OG) exerted an excellent inhibitory efficacy. To extend the aqueous solubility, stability, and bactericidal properties of octyl gallate (OG), an inclusion complex between OG and ß-cyclodextrin (ßCD), OG/ßCD, was prepared and identified with various methods including X-ray diffraction (XRD), differential scanning calorimeter (DSC) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the enhanced inhibitory effect and potential antibacterial mechanism of OG/ßCD against two Gram-negative and Gram-positive foodborne bacteria were comprehensively investigated. The results show that OG/ßCD could function against bacteria through effectively damaging the membrane, permeating into cells, and then disturbing the activity of the respiratory electron transport chain to cause the production of high-level intracellular hydroxyl radicals. Moreover, the reinforced OG/ßCD-incorporated polylactic acid (PLA) nanofibers were fabricated using the electrospinning technique as food packaging to extend the Chinese giant salamander fillet's shelf life at 4 °C. This research highlights the antibacterial effectiveness of OG/ßCD in aqueous media, which can be used as a safe multi-functionalized food additive combined with the benefits of electrospun nanofibers to extend the Chinese giant salamander fillets shelf life by 15 d at 4 °C.

Electrospun Nanofibrous Membranes Accelerate Biofilm Formation and Probiotic Enrichment: Enhanced Tolerances to pH and Antibiotics.

Biofilms are the oldest, most successful, and most widely distributed form of microorganism life on earth, existing even in extreme environments. Presently, probiotics in biofilm phenotype are thought as the most advanced fourth-generation probiotics. However, high-efficiency and large-scale biofilm enrichment in an artificial way is difficult. Here, fibrous membranes as probiotic biofilm-enriching materials are studied. Electrospun cellulose acetate nanofibrous membranes with nano-sized fibers show outstanding superiority over fibrous membranes with micron-sized fibers in Lactobacillus paracasei biofilm enrichment. The special 3D structure of electrospun nanofibrous membranes makes other facilitating biofilm formation factors insignificant. With a suitable scaffold/culture medium ratio, nearly 100% of L. paracasei cells exist as biofilm phenotype on the membrane from the very beginning, not planktonic state. L. paracasei biofilms possess a potential for long-term survival and high tolerances toward strong acidic and alkali conditions and antibiotics. RNA sequencing results explain why L. paracasei biofilms possess high tolerances toward harsh environments as compared to planktonic L. paracasei. Electrospun nanofibrous membranes can serve as powerful biofilm-enriching scaffolds for probiotics and other valuable microbes.

Probiotics Biofilm-Integrated Electrospun Nanofiber Membranes: A New Starter Culture for Fermented Milk Production.

Electrospun nanofiber membranes are widely investigated in the past few decades as candidates for tissue engineering, which can mimic natural extracellular matrix (ECM) and improve cell adhesion, proliferation, and expression on nanofiber membranes. However, the formation of bacterial biofilms on nanofiber membranes and application of the biofilm-integrated nanofiber membranes remain largely unknown. Here, electrospun cellulose acetate nanofiber membranes are first utilized as scaffold materials for Lactobacillus plantarum ( L. plantarum) biofilm formation. Nanofiber membranes proved to be an excellent scaffold for bacteria biofilm with high stability, where biofilms were interlocked with nanofibers forming a cohesive structure. In comparison with planktonic bacteria, L. plantarum biofilms on nanofiber membranes show excellent gastrointestinal resistance. Instead of decreasing, the number of viable cells increased after 3 h digestion in vitro. The L. plantarum biofilm-integrated nanofiber membranes were used as reusable starter cultures for fermented milk production showing excellent fermentative ability and higher survival of L. plantarum during shelf life. The viable cells in fermented milk remained at 11 log CFU/g throughout the reusable batches, which is far above the required value of 7 log CFU/g in commercial products. In addition, the produced fermented milk possesses shorter fermentation time and higher survival of probiotics during shelf life. The results suggest electrospun nanofiber membranes are ideal scaffold materials for bacteria biofilms immobilization in biotechnology and fermentation engineering, which broaden the potential use of electrospun nanofiber membranes in microbiology and strengthen the application of biofilms in fermentation engineering.

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation.

Hydrophilic modification of PVDF microfiltration membranes by adsorption of facial amphiphile cholic acid.

Amphiphilic molecules have been widely used in surface modification of polymeric materials. Bile acids are natural biological compounds and possess special facial amphiphilic structure with a unusual distribution of hydrophobic and hydrophilic regions. Based on the facial amphiphilicity, cholic acid (CA), one of the bile acids, was utilized for the hydrophilic modification of poly(vinylidene fluoride) (PVDF) microfiltration membranes via the hydrophobic interactions between the hydrophobic face of CA and the membrane surfaces. Ethanol, methanol, and water were respectively used as solvent during CA adsorption procedure. Their polarity affects the CA adsorption amount, as similar to CA concentration and adsorption time. There are no changes on the membrane surface morphology after CA adsorption. The hydrophilicity of PVDF membranes is greatly enhanced and the water drops permeates into the CA modified membranes quickly after modification. All these factors benefit to the permeation flux of membrane for water. When CA concentration is higher than 0.088 M, the water permeation flux is doubled as compared with the nascent PVDF membrane and shows a good stability during filtration procedure. These results reveal the promising potential of facial amphiphilic bile acids for the surface modification of polymeric materials.

Glycosylation of the polypropylene membrane surface via thiol-yne click chemistry for lectin adsorption.

Glycosylated membrane, as one of the most important affinity membranes, permits affinity separation/purification of proteins based on carbohydrate-protein interactions. It is an important scientific issue to screen facile method for fabricating the glycosylated membrane surface with high glycosyl density. Such a surface can be fabricated by the direct covalent immobilization of carbohydrate ligands on the surfaces of microporous polypropylene membrane (MPPM). First, alkyne-functionalized membrane surface was fabricated by plasma pretreatment combined with UV-induced graft polymerization of 3-(trimethylsilyl) propargyl methacrylate. Then, the glycosylated membrane surface was directly fabricated with the thiol-yne click reaction to ensure rapid process, improved efficiency, and high glycosyl density. Chemical and physical properties of the membrane surface were characterized by ATR/FT-IR, XPS, FESEM and water contact angle measurement. Static lectin adsorption indicates that the glycosylated membrane can specifically adsorb lectin concanavalin A (Con A) other than peanut agglutinin (PNA). Break through curves from dynamic Con A adsorption show the membrane has unique properties such as strong specificity, high adsorption capacity, and reversible binding capability. We suggest that the prepared glycosylated membrane is of great potentials in affinity membrane chromatography for rapid and high-resolution separation/purification of lectins.

Carbohydrate decoration of microporous polypropylene membranes for lectin affinity adsorption: comparison of mono- and disaccharides.

Carbohydrates (saccharides) are ubiquitous on the extracellular surface of living cells and mediate a myriad of biological recognition and signaling processes. Carbohydrate decoration of polymer surfaces with covalent attachment of saccharides offers a new realm of opportunities to mimic cellular events such as protein recognition and binding. We describe the carbohydrate decoration (surface glycosylation) of poly(2-hydroxyethyl methacrylate)-grafted microporous polypropylene membranes (poly(HEMA)-g-MPPMs) with mono- and disaccharides. Galactose, lactose, glucose, and maltose were covalently attached on the surfaces of poly(HEMA)-g-MPPMs and were compared in detail. The process was verified by solid state (13)C NMR spectra. Membranes with high binding degree (BD) of saccharide ligands on the surfaces were facilely prepared from poly(HEMA)-g-MPPMs with high grafting degree (GD) of poly(HEMA). For poly(HEMA)-g-MPPM with the same GD of poly(HEMA), the BD of disaccharides is lower than that of monosaccharides and the disaccharide-decorated MPPMs are more hydrophilic than the monosaccharide-decorated ones. The carbohydrate-decorated MPPMs prepared from galactose, lactose, glucose, and maltose (denoted as MPPM-Gal, MPPM-Lac, MPPM-Glc and MPPM-Mal, respectively) recognize and adsorb specifically one of the two lectins, concanavalin A (Con A) and peanut agglutinin (PNA). As the BD of saccharide increases, the "glycoside cluster effect" plays a primary role in lectin adsorption. MPPM-Lac has enhanced affinity to PNA as compared with MPPM-Gal having similar BD of saccharide., on the other hand, MPPM-Mal shows no enhanced affinity to Con A in comparison with MPPM-Glc as the BD of saccharide is above 0.9 µmol/cm(2), where the "glycoside cluster effect" occurs.

Construction of a comb-like glycosylated membrane surface by a combination of UV-induced graft polymerization and surface-initiated ATRP.

Carbohydrate residues are found on the extracellular side of the cell membrane. They form a protective coating on the outer surface of the cell and are involved in intercellular recognition. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this work, the surface of a polypropylene microporous membrane (PPMM) was modified with comb-like glycopolymer brushes by a combination of UV-induced graft polymerization and surface-initiated atom-transfer radical polymerization (ATRP). 2-Hydroxyethyl methacrylate (HEMA) was first grafted to the PPMM surface under UV irradiation in the presence of benzophenone and ferric chloride. ATRP initiator was then coupled to the hydroxyl groups of poly(HEMA) brushes. Surface-initiated ATRP of a glycomonomer, D-gluconamidoethyl methacrylate, was followed at ambient temperature in aqueous solvent. Water had a significant acceleration effect on the ATRP process; however, loss of control over the polymerization process was also observed. The addition of CuBr2 to the ATRP system largely increased the controllability at the cost of the polymerization rate. The grafting of HEMA, the coupling of ATRP initiator to the hydroxyl groups, and the surface-initiated ATRP were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy.

Novel photoinduced grafting-chemical reaction sequence for the construction of a glycosylation surface.

Carbohydrates play a major role in many recognition events, such as blood coagulation, immune response, fertilization, cell growth, embryogenesis, and cellular signal transfer, which are essential for the survival of living entities. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this work, we present a facile strategy for the formation of glycopolymer tethered on polypropylene microporous membrane surface. Acrylamide was grafted onto the polypropylene microporous membrane surface by photoinduced graft polymerization in the presence of benzophenone. The amide groups of grafted poly(acrylamide) were then transformed to primary amine groups by the Hofmann rearrangement reaction. Quantificational evaluation of the rearrangement reaction was carried out by ninhydrin method and mass weighting. Sugar moieties were coupled with the grafted functional layer to form glycopolymer by the reaction between primary amine groups and carbohydrate lactones. The grafting of acrylamide, the conversion of amide groups to amine groups, and the coupling of sugar moieties were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy combined with surface morphology observation by scanning electron microscopy.

Fabrication of glycosylated surface on polymer membrane by UV-induced graft polymerization for lectin recognition.

Increasingly, carbohydrate-protein interactions are viewed as important mechanisms for many biological processes such as blood coagulation, immune response, viral infection, inflammation, embryogenesis, and cellular signal transfer. However, the weak affinity of the interactions and the structural complexity of carbohydrates have hindered efforts to develop a comprehensive understanding of carbohydrate functions. Fortunately, synthetic polyvalent glycoligands give us a chance to reveal the nature of these biological processes. In this work a sugar-containing monomer (alpha-D-allyl glucoside (AG)) was grafted onto polypropylene microporous membrane (PPMM) by UV-induced graft polymerization to generate a glycosylated porous surface for the first time. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy were employed to confirm the glycosylation. Water contact angle measurement was used to evaluate the hydrophilicity change of the surfaces before and after the graft polymerization of AG. It was found that the grafting density increased reasonably with the increase of AG monomer concentration, and then this increase slowed when the AG concentration exceeded 80 g/L. At the same time a 20-25 min UV irradiation was enough for the grafting polymerization. The photoinitiator concentration also influenced the grafting density obviously, and there was an optimal concentration of the photoinitiator for the grafting process. The water contact angle of the polyAG-tethered membrane surface decreased from 149 degrees to 80 degrees with the increase of grafting density from 0 to 187.76 microg/cm2, which indicated a hydrophilic variation of the membrane surface by the grafting of AG. Results also indicated that the surface-grafted polyAG chains showed weak interaction with Con A when the grafting density was low. However, when the sugar density exceeded 90 microg/cm2, the binding affinity increased dramatically which was the due to the "glycoside cluster effect".

Novel sequence for generating glycopolymer tethered on a membrane surface.

Cell surface carbohydrates, usually binding with other biomacromolecules (such as lipids and proteins), are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation, and inflammation. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this study, a novel two-step sequence for the generation of a glycopolymer layer tethered on a polypropylene microporous membrane is described. First, a UV-induced graft polymerization of 2-aminoethyl methacrylate hydrochloride (AEMA) was carried out on the membrane to generate an amino-functionalized surface, and the effects of polymerization factors (monomer/initiator concentration and UV irradiation time) on the grafting density were studied. Second, sugar moieties were bound with the grafted functional layer to form glycopolymer by the reaction between the amino groups on the membrane surface and carbohydrate lactones. Chemical analysis by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy combined with surface morphology observation by scanning electron microscopy confirmed the graft polymerization of AEMA and the formation of glycopolymer. The decreases of water contact angle and protein adsorption on the membrane revealed the enhancement of hydrophilicity and protein resistance due to the typical characteristics of the glycopolymer tethered on the surface. These results indicated that the novel sequence reported in this work is a facile process to form glycopolymer-modified surfaces.