Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Probiotics Biofilm-Integrated Electrospun Nanofiber Membranes: A New Starter Culture for Fermented Milk Production Meng-Xin Hu,* Ji-Nian Li, Qian Guo, Ya-Qian Zhu, and Hong-Mei Niu School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, P. R. China
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ABSTRACT: 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 biofilmintegrated 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. KEYWORDS: electrospun nanofiber membrane, scaffold, biofilm, probiotics, fermented milk have been evaluated for biofilm formation.21 It has been realized that material surface structures, including chemical structures and physical structures, play an important role in biofilm formation. Surface charge, wettability, roughness, topographic patterns, and functional chemical modifications are known to affect biofilm formation. Generally speaking, material surfaces with positive charge, low surface-energy, and micro- and nanosized roughness facilitate biofilm formation.22,23 However, these strategies are not applicable to all bacteria due to the enormous differences between bacteria.24 Thus, pursuing new materials for biofilm formation is imperative. Electrospun nanofiber membranes have loosely connected 3D porous structure with high porosity and high surface area. The unique structure can mimic natural ECM structure, which supports cells and presents an instructive background to guide their behavior.25−27 Hence, an electrospun nanofiber membrane is an excellent candidate material for cell adhesion, proliferation, and expression of matrix components.28 Cells are inherently sensitive to local mesoscale, microscale, and nanoscale patterns of chemistry and topography.28 On the basis of many studies, nanofiber membranes with the bionic structure of ECM are the best choice for cell adhesion. However, most of the studies focused on animal cells with
1. INTRODUCTION Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix (ECM).1 Biofilm colonies will in many cases form a smart material capable of responding to external threats dependent on their size and internal state.2 Ordered structures and differentiated functions provide protection and resistance mechanisms to bacteria in biofilms.3,4 Material surfaces are indispensable to formation and development of biofilms. Regulating the biofilm formation process on material surfaces to meet practical demands is of great importance. However, most of the biofilm research was focused on the pathogenic mechanisms of pathogenic bacterium3,5−8 and the approaches to inhibit biofilms formation and destroy and detach biofilms.9−12 The important beneficial functions of biofilms in biology are overlooked.13 In fact, biofilms of nonpathogenic bacterium and genetically engineered bacterium used as biocatalysts possess great potential for industry.14−16 Biofilms have inherent characteristics of self-immobilization, high resistance to reactants, high biomass density, high activity and stability, and potential for long-term fermentation and bioconversion.17 Accordingly biofilms immobilized on materials are regarded as potential functional biomaterials, which have great potential as industrial workhorses for the sustainable production of chemicals and target substances.14 In recent years, biofilms-integrated materials have been increasingly employed in the treatment of wastewater, biological fermentation, and microbial fuel cells.18−20 Wood chips, porous bricks, cotton cloth, glass and ceramics, and foams © XXXX American Chemical Society
Received: September 14, 2018 Revised: December 21, 2018 Accepted: February 11, 2019
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DOI: 10.1021/acs.jafc.8b05024 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
static conditions. The inoculated broth in the glass tube was incubated statically for 16−20 h at 37 °C. Porcine trypsin and pepsin were purchased (Aladdin, China). All the ingredients used in the cultivation step were of pharmaceutical quality and were purchased as standard pharmaceutical ingredients for food supplements production or drug production. 2.2. Biofilm Culture. Cellulose acetate nanofiber membranes were cut into round samples with a diameter of 25 mm and sterilized by ultraviolet radiation for 1 h in the laminar flow cabinet. The sterile cellulose acetate nanofiber membranes were dipped into 5 mL MRS broth and then 1% L. plantarum inoculated broth was added under static conditions at 37 °C. The MRS broth was changed every 24 h until the culture process was finished. 2.3. Quantitation of Bacteria in Biofilm. Biofilms formed on nanofiber membranes were rinsed with 0.85% saline three times. Different from quantitation of planktonic bacteria, quantitation of biofilm rely on detachment followed by conventional plate counting. The used detachment methods include ultrasonic detachment protocol, enzymatic detachment protocol, and enzymatic and ultrasonic combined protocol. The difference between three detachment protocol was studied. The detached L. plantarum supernatant was serially diluted 10-fold and 100 μL aliquots were plated onto MRS agar plates. Colony-forming units (CFU) were counted after 24 h of colony growth at 37 °C. 2.4. Scanning Electron Microscope. Morphology of biofilms on cellulose acetate nanofiber membranes was characterized by scanning electron microscope (SEM, Phenom Pro). First, biofilm samples were fixed by 2.5% glutaraldehyde for 6−8 h, then dehydrated in a graded series of ethanol aqueous solutions, and finally air-dried at room temperature. After coating a layer of gold, the biofilm samples were observed under SEM. 2.5. In Vitro Gastrointestinal Tolerance Assay. L. plantarum in the planktonic state and in the biofilm state were all freeze-dried with 11% nonfat dry milk, 8% ascorbic acid, and 8% sorbitol as the freezedrying protective additives. L. plantarum in the planktonic state was used as controls. After lyophilization, the biofilms integrated with cellulose acetate nanofiber membranes were crisp, which turn into powders with gentle pressure. The lyophilized powders were used to evaluate the gastrointestinal tolerance in the simulated gastrointestinal medium. In vitro gastrointestinal tolerance of L. plantarum assays were carried out following a protocol described previously with some modifications.43 Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were freshly prepared before each experiment. For SGF, HCl was diluted with deionized water to gain solutions with pH 2.5, 3.5, and 4.5, separately. Then pepsin was added in these solutions at a fixed concentration 10 g/L. For SIF, 3.4 g KH2PO4 was dissolved in 250 mL of deionized water and 4 g/L NaOH solution was added to adjust pH to 6.8. Then, trypsin was mixed in the solution with a concentration of 10 g/L. Finally, deionized water was added to make the total volume to 500 mL. All the SGF and SIF were filtrated with the membrane (pore size 0.20 μm) before digestion experiments. To evaluate the gastrointestinal tolerance of L. plantarum, 0.1 g lyophilized powders were mixed into SGF and SIF, separately. L. plantarum lyophilized powders were then digested on a shaker at a rate of 100 r/min at 37 °C. At predetermined time points (0, 1, 2, and 3 h), the viable bacteria count of solutions was determined by conventional plate counting method with an MRS agar plate. All the experiments were performed in triplicate. Survival of bacterial in SGF/ SIF was expressed in terms of log (N/N0) and % Survival (i.e., N/N0 × 100%), where N0 and N represent the viable CFUs of samples before and after digestion. 2.6. Fermented Milk Production. Fermented milk was manufactured using the procedures stated by Aryana.44 Whole milk powders (112 g) and white granulated sugar (100 g) were mixed in Milli-Q water and homogenized at 60−65 °C for 20 min. After 30 min standing, the solution was pasteurized at 90−95 °C for 5 min and rapidly cooled to 40 °C. Lyophilized powders of planktonic L. plantarum and L. plantarum biofilms containing about 107 CFU bacteria were used as the starter cultures for 100 mL milk, separately.
nanofiber membranes in tissue engineering. Adhesion of microorganisms and biofilm formation on nanofiber membranes are still in the start-up stage. It has been reported that electrospun fiber nonwovens with small fiber diameter (600− 900 nm) favor the formation of thick and continuous biofilm, which makes the microbial fuel cells have the highest current density.29 However, Abrigo et al. have found that circular polystyrene fiber diameter affected the ability of bacteria to proliferate within the fibrous membranes.30 Membranes with an average fiber diameter close to bacterial size were found to offer the best support for bacterial adhesion and spreading, constituting a scaffold that bacteria use as a framework for forming colonies. Fiber diameters smaller than the bacterial length resulted in deformation and death of bacteria. The data suggest that simply tuning the morphological properties of electrospun fibers may be one strategy used to inhibit biofilm formation within wound dressings. Besides, there are only a few pioneering studies examining single bacterial attachment mechanisms on material surfaces, which are regulated and influenced by the morphology, roughness, diameter, spacing, and curvature of fiber.31−33 Study on the promotion of probiotics biofilm formation on electrospun fiber membranes and the relative application is a rarity. In the present work, electrospun cellulose acetate nanofiber membranes mimicking natural ECM structure were chosen as a model system for Lactobacillus plantarum biofilm formation. The L. plantarum biofilms-integrated nanofiber membranes were used as reusable starter cultures for fermented milk production to study the fermentation properties. Fermented milk contains viable, active, and abundant probiotics in the product to the date of minimum durability.34 An accumulating body of epidemiologic and clinical evidence suggests that fermented milk consumption may act beneficially on obesity, metabolic risk factors, and cardiovascular risk.35−37 The unique properties of fermented milk have captured the interest of the scientific community.37,38 So far, the mechanisms linking fermented milk to gut health are largely based on the assumption that all fermented milk contains probiotic bacteria and that commercial fermented milk contains sufficient probiotics to exert a physiologic effect.39 Several public organizations, including the International Dairy Federation (IDF), state that a food product must contain a minimum of 107 colony-forming units per gram of food (CFU/g) to ensure sufficient bioavailable bacteria to exert a functional effect within the body.40 However, insufficient viability and survival of these bacteria remain a problem in commercial food products.41,42 In this work, the influence of culture conditions on the live cell density of biofilms formed on nanofiber membranes was studied in detail. Gastrointestinal resistance and fermentation properties of biofilm-integrated nanofiber membranes in fermented milk production were investigated to demonstrate the superiority of biofilm starter culture in the viability of probiotics during manufacture, storage, and digestion and the reusability of biofilm on nanofiber membranes.
2. EXPERIMENTAL SECTION 2.1. Materials. Electrospun cellulose acetate nanofiber membranes were kindly donated by Professor Zhi-Kang Xu at Zhejiang University. L. plantarum (No. 23941) obtained from the China Center of Industrial Culture Collection (CICC) was used in this work. L. plantarum was grown in deMan-Rogosa-Sharpe (MRS) broth. Inoculated plates were incubated for 1−3 days at 37 °C under B
DOI: 10.1021/acs.jafc.8b05024 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. (a) SEM images of the L. plantarum biofilms on cellulose acetate nanofiber membranes with different culture time; (b) effects of culture time (24 h) on the viable cell on nanofiber membranes; (d) dry weight of biofilm on cellulose acetate nanofiber membranes (P < 0.05). The inoculation concentration of bacteria was 1% (v/v), and the bacteria were detached from biofilm by ultrasonic method. Milk batches were inoculated with starter cultures. Lyophilized powders of L. plantarum biofilms were placed in a filter bag. The mixes were sealed and then incubated at 37 °C. When fermented milk reached the appropriate pH (typically about 4.5), the fermented milk was transferred to refrigerated storage (4 °C) for 3 weeks to monitor the viable cells in shelf life. When fermented milk production was finished, the filter bag containing lyophilized powders of L. plantarum biofilms was taken out, rinsed three times with sterile saline solution, and added into fresh milk solutions to produce fermented milk again. The number of viable cells in fermented milk was determined by CFU method. 2.7. Statistical Analysis. All the statistical approaches, the statistics program EXCEL was utilized. Differences between means were considered significant at p < 0.05.
by ECM. When the culture time was prolonged to 24 h, nanofibers were no longer visible in the biofilms. Channels and pores existed in the biofilms, which belongs to one of the biofilm characteristic structures. Quantitation of biofilms on materials was simultaneously studied by plate counting and dry weight protocols. As shown in Figure 1(b), culture time (
