Cytoprotection of Probiotic Lactobacillus acidophilus with Artificial Nanoshells of Nature-Derived Eggshell Membrane Hydrolysates and Coffee Melanoidins in Single-Cell Nanoencapsulation

One-step fabrication method for thin films and shells is developed with nature-derived eggshell membrane hydrolysates (ESMHs) and coffee melanoidins (CMs) that have been discarded as food waste. The nature-derived polymeric materials, ESMHs and CMs, prove highly biocompatible with living cells, and the one-step method enables cytocompatible construction of cell-in-shell nanobiohybrid structures. Nanometric ESMH-CM shells are formed on individual probiotic Lactobacillus acidophilus, without any noticeable decrease in viability, and the ESMH-CM shells effectively protected L. acidophilus in the simulated gastric fluid (SGF). The cytoprotection power is further enhanced by Fe3+-mediated shell augmentation. For example, after 2 h of incubation in SGF, the viability of native L. acidophilus is 30%, whereas nanoencapsulated L. acidophilus, armed with the Fe3+-fortified ESMH-CM shells, show 79% in viability. The simple, time-efficient, and easy-to-process method developed in this work would contribute to many technological developments, including microbial biotherapeutics, as well as waste upcycling.

The one-step approach is especially beneficial in the field of single-cell nanoencapsulation (SCNE) [15][16][17][18][19][20], where cytoprotective nanometric-shells are constructed on individual living cells, leading to the creation of cell-in-shell nanobiohybrids. Its procedural simplicity also would be seamlessly coupled with the existing industrial processes, for instance, for fabrication of probiotic capsules, as well as enhancing biocompatibility with living cells, probiotics in this study. As a related work, tea polyphenols and poly(N-vinylpyrrolidone) (PVPON) were added simultaneously to an aqueous cell suspension for cytocompatible formation of cell-in-shell structures with a polymeric complex of tea polyphenols and simplicity also would be seamlessly coupled with the existing industrial processes, for instance, for fabrication of probiotic capsules, as well as enhancing biocompatibility with living cells, probiotics in this study. As a related work, tea polyphenols and poly(N-vinylpyrrolidone) (PVPON) were added simultaneously to an aqueous cell suspension for cytocompatible formation of cell-in-shell structures with a polymeric complex of tea polyphenols and PVPON [21], as an alternative to the LbL-based SCNE with TA and PVPON [22]. It is envisaged that the identification of other nature-derived materials, structurally different from polyphenolics, would further upscale the usability and applicability of onestep film construction in the practical applications as well as in the research domain of single-cell surface engineering including SCNE [23]. In this work, we constructed a cellin-shell structure of probiotic Lactobacillus acidophilus by one-step, cytocompatible formation of the cytoprotective shells comprising nature-derived eggshell membrane hydrolysates (ESMHs) and coffee melanoidins (CMs) (Figure 1). ESMHs, hydrolyzed ESMs, have recently been suggested as nature-derived biomaterials in the LbL-based formation of thin films [24]. In consideration of high content of proteins (80-85%) in ESMs, including collagens (types I, V, and X) and glycosaminoglycans, ESMHs could serve as extracellular matrix-mimetic materials in the manipulation of living cells. The LbL-SCNE methods with ESMH-TA [25] and ESMH-CM pairs [26] have previously been demonstrated to show the great cytocompatibility of ESMHs. Utilization of ESMHs as biomaterials also would contribute to the waste upcycling in the aspect of circular economy; eggshells, comprising about 11% of egg weight, have been discarded as food waste [27,28]. In the aspect of waste upcycling, spent coffee grounds also have been explored as a sustainable solution in various fields, such as photothermal materials in sterilization, fertilizers and soil conditioners in the agricultural industry, raw materials for biodiesel and biochar production, and sorbents in water management [29][30][31][32]. CMs, a product of the Maillard reaction during coffee-roasting process, exhibit antioxidant activity as well as containing biologically active molecules [33]. Aside from potential use as nutraceuticals [34], CMs, extracted from spent coffee ground, form thin films with Fe 3+ , which has been utilized in SCNE [35]. The combined use of ESMHs and CMs as next-generation biomaterials would widen the material scope in biomedical engineering and related fields, assisted by the development of simple, one-step methods for forming ultrathin films and shells.

One-Step Formation of ESMH-CM Films and Shells on Abiotic Substrates
The ESMHs and CMs were prepared according to our previous reports [26,35]. The stock solution of ESMHs or CMs was made to the final concentration of 2 mg/mL in a sodium chloride (NaCl) solution (50 mM). Prior to use, gold substrates were cleaned with ethanol and acetone. The cleaned gold substrates were immersed in a 1:1 mixture of the ESMH and CM stock solutions (500 μL each), stirred at 120 rpm for 3 h, washed with deionized (DI) water, and dried under a stream of argon gas. The same protocol was employed for other flat substrates (silver, aluminum, copper, nickel, tin, titanium, silicon, ESMHs, hydrolyzed ESMs, have recently been suggested as nature-derived biomaterials in the LbL-based formation of thin films [24]. In consideration of high content of proteins (80-85%) in ESMs, including collagens (types I, V, and X) and glycosaminoglycans, ESMHs could serve as extracellular matrix-mimetic materials in the manipulation of living cells. The LbL-SCNE methods with ESMH-TA [25] and ESMH-CM pairs [26] have previously been demonstrated to show the great cytocompatibility of ESMHs. Utilization of ESMHs as biomaterials also would contribute to the waste upcycling in the aspect of circular economy; eggshells, comprising about 11% of egg weight, have been discarded as food waste [27,28]. In the aspect of waste upcycling, spent coffee grounds also have been explored as a sustainable solution in various fields, such as photothermal materials in sterilization, fertilizers and soil conditioners in the agricultural industry, raw materials for biodiesel and biochar production, and sorbents in water management [29][30][31][32]. CMs, a product of the Maillard reaction during coffee-roasting process, exhibit antioxidant activity as well as containing biologically active molecules [33]. Aside from potential use as nutraceuticals [34], CMs, extracted from spent coffee ground, form thin films with Fe 3+ , which has been utilized in SCNE [35]. The combined use of ESMHs and CMs as next-generation biomaterials would widen the material scope in biomedical engineering and related fields, assisted by the development of simple, one-step methods for forming ultrathin films and shells.

One-Step Formation of ESMH-CM Films and Shells on Abiotic Substrates
The ESMHs and CMs were prepared according to our previous reports [26,35]. The stock solution of ESMHs or CMs was made to the final concentration of 2 mg/mL in a sodium chloride (NaCl) solution (50 mM). Prior to use, gold substrates were cleaned with ethanol and acetone. The cleaned gold substrates were immersed in a 1:1 mixture of the ESMH and CM stock solutions (500 µL each), stirred at 120 rpm for 3 h, washed with deionized (DI) water, and dried under a stream of argon gas. The same protocol was employed for other flat substrates (silver, aluminum, copper, nickel, tin, titanium, silicon, stainless steel, poly(acrylic acid), polycarbonate, polyethylene, polyurethane, and polytetrafluoroethylene). Calcium carbonate (CaCO 3 ) particles were prepared by mixing 4 mL of an aqueous poly(sodium 4-styrenesulfonate) solution (PSS, MW: ca. 70,000, 2 mg/mL), 48 µL of an aqueous sodium carbonate solution (Na 2 CO 3 , 1 M), and 96 µL of an aqueous calcium chloride solution (CaCl 2 , 1 M) under vigorous stirring for 40 s, incubating for 7 min at room temperature, and calcinating at 450 • C for 2 h. ESMH-CM shells were formed

Single-Cell Nanoencapsulation (SCNE) and Characterizations
A single colony of Saccharomyces cerevisiae, picked from the YPD agar plate, was cultured for 30 h in a YPD broth medium at 33 • C. After washing with DI water, S. cerevisiae were immersed for 3 h in a 1:1 mixture of the ESMH and CM stock solutions (500 µL each) and washed with DI water three times. The same SCNE protocol was employed for L. acidophilus and Levilactobacillus brevis, after culturing for 24 h in an MRS broth medium at 33 • C. For viability assay of S. cerevisiae, 5 µL of the stock solution of fluorescein diacetate (FDA, 10 mg/mL in acetone) and 2 µL of an aqueous solution of propidium iodide (PI, 1 mg/mL) were added to a S. cerevisiae suspension (1 mL), and the mixture was incubated for 15 min at 33 • C. SYTO  was added to 150 mL of the MRS broth medium (final OD 600 = 0.001) and incubated at 33 • C. The 100 µL of the culture mixture was picked at the predetermined time, and the cell density was measured at 600 nm with a microplate reader. Linear fitting of ln(OD 600 ), from −4.0 to +1.0, with incubation time (in hour) gave t OD 600 −2.0 , the time for ln(OD 600 ) of −2.0. For cytoprotection studies, cells were suspended in 1 mL of an aqueous polyethylenimine solution (PEI, 0.5, 1, 10, or 50 mg/mL) for 30 min or 1 mL of an aqueous TA solution (1, 5, 10, 25, or 50 mg/mL) for 1 h. The simulated gastric fluid (SGF) was prepared by dissolving NaCl (0.2% (w/v)) and pepsin (3 mg/mL) in DI water, followed by pH adjustment to 2 with 1 M HCl. The cells were suspended in the SGF solution and incubated for 2 h at 37 • C for cytoprotection studies against the SGF.

One-Step Formation of ESMH-CM Films and Shells on Abiotic Substrates
Prior to SCNE, we investigated the feasibility of one-step film formation with ESMH-CM complex and optimized the reaction conditions, with a gold substrate as a model. The concentrations of ESMHs and CMs were set to be 1 mg/mL each. The ellipsometric thickness measurement, after 3 h of reaction, indicated that a film was formed with 6.2 nm of thickness. It was also found that the addition of NaCl to the coating mixture increased film thickness, and 50 mM of NaCl was chosen as an optimized concentration in this study. The film thickness significantly increased to 10.3 nm with 50 mM of NaCl as an additive after 3 h of reaction (166% increase). Detailed thickness analysis showed that the film growth stopped after about 3 h without NaCl (thickness: 7.1 nm), but in stark contrast, the ESMH-CM film grew continuously at least up to 24 h (thickness: 14.0 nm) ( Figure 2a). As a control, we used only a single component (ESMHs or CMs) in the film formation. The thickness analysis showed that 1.6-nm-thick and 0.8-nm-thick films were formed with ESMHs and CMs, respectively, under the same conditions, confirming the significance of pre-association of ESMHs and CMs in solution ( Figure S1). On the other hand, it was observed that the pre-mixed ESMH-CM pair also could be utilized in the LbL-type film formation: film thickness increased in a linear fashion, with 2.8 nm per 10 min of incubation, making 28 nm thick films after 10 deposition cycles ( Figure S2).
pre-association of ESMHs and CMs in solution ( Figure S1). On the other hand, it was observed that the pre-mixed ESMH-CM pair also could be utilized in the LbL-type film formation: film thickness increased in a linear fashion, with 2.8 nm per 10 min of incubation, making 28 nm thick films after 10 deposition cycles ( Figure S2).  Figure S3b,c) [35,36]. The FE-SEM and AFM analysis showed that the ESMH-CM films were composed of nanoparticulates, clearly distinct from the bare gold surface (Figure S3d,e). The water-contact angle measurements also indicated the successful formation of ESMH-CM films: the contact angle was changed to 28.2 • from 76.6 • , after film formation.
We also examined whether our one-step method for ESMH-CM-film formation was universal, applicable to interface engineering of various different substrates, including silver, aluminum, copper, nickel, tin, titanium, silicon, stainless steel, poly(acrylic acid), polycarbonate, polyethylene, polyurethane, and polytetrafluoroethylene (PTFE), in addition to gold. The water-contact angle measurements clearly showed that the one-step method was material-independent ( Figure 2c). The water-contact angles of all the substrates tested were changed to be below 60 • after film formation, regardless of their intact angles: for example, the contact angle of PTFE was changed to 52.8 • from 115.7 • . In addition to the flat substrates, our one-step method was employed for the construction of core-shell structures in particle engineering, exemplified with CaCO 3 and amine-terminated silica (SiO 2 ) particles. Changes in the zeta (ζ) potential indicated the formation of ESMH-CM shells on the particles: −15.7 eV from +4.1 eV for CaCO 3 particles (diameter: 2-4 µm) and −35.5 eV from +44.3 eV for SiO 2 particles (diameter: 3.92 µm) ( Figure S4a). It is of note that the shell formation occurred regardless of the surface charge of particles. The formation of ESMH-CM shells was visualized with a rhodamine-linked ESMH (ESMH_TAMRA, λ emission : 575 nm) [25,26] by confocal laser-scanning microscopy (CLSM) ( Figure S4b). The ability to form shells on individual particles under biocompatible synthetic conditions suggested the potential of our system in the SCNE of living cells.

One-Step SCNE of S. cerevisiae
After confirming the one-step formation of films and cells on abiotic substrates with ESMHs and CMs, the protocol was applied to the SCNE with S. cerevisiae as a model. S. cerevisiae was chosen for investigation of cytocompatibility of our method and cytoprotectability of the formed ESMH-CM shell, because of the availability of numerous reference reports on the SCNE of S. cerevisiae [37,38].
S. cerevisiae was incubated for 3 h in a 50 mM NaCl solution of ESMHs (1 mg/mL) and CMs (1 mg/mL), leading to the construction of S.cerevisiae@ESMH-CM. The cell viability, after SCNE, was analyzed with FDA (λ emission : 521 nm; for live cells) and PI (λ emission : 636 nm; for dead cells). FDA is a membrane-permeable, fluorogenic viabilityprobe that measures both enzymatic activity and membrane integrity, and PI is a membraneimpermeable, nucleic-acid-intercalating agent that is commonly used to detect dead cells. The CLSM images showed that most S.cerevisiae@ESMH-CM cells were viable (Figure 3a), and the quantitative analysis showed 98.7% of %viability (calculated by dividing the viability of S.cerevisiae@ESMH-CM (97.3 ± 0.9%) by the viability of intact, bare S. cerevisiae as a reference (98.6 ± 1.0%)), indicating no noticeable harm to the cells. That is, the viability assay confirmed that the one-step shell formation with ESMHs and CMs was extremely cytocompatible. The ESMH-CM shells on S. cerevisiae were visualized with use of ESMH_TAMRA by CLSM, which showed green/red core/shell structures of FDA-stained S. cerevisiae (Figure 3b).
Cytoprotectability of the ESMH-CM shells on S. cerevisiae was tested and demonstrated with PEI (branched, MW: 25,000). The viability of bare S. cerevisiae decreased significantly with the PEI concentration, after 30 min of incubation in a PEI solution (pH 7, in DI water) (Figure 3c): for instance, the viability was calculated to be 18.1 ± 2.1% in the case of 1 mg/mL, and no bare S. cerevisiae survived the PEI concentration of 10 mg/mL. In striking contrast, the viability of S.cerevisiae@ESMH-CM was 75.2 ± 5.1% (ca. 5-fold increase in viability) for 1 mg/mL of PEI, and 27.6 ± 8.8% of S.cerevisiae@ESMH-CM was viable in the case of 10 mg/mL of PEI. In addition, the enhanced viability against various concentrations of TA was also observed for S.cerevisiae@ESMH-CM (Figure 3d). The SCNE results with S. cerevisiae arguably confirmed that our one-step ESMH-CM method created cytoprotective shells in the cytocompatible manner, which is the primary requirement of the first-generation cell-in-shell nanobiohybrids or artificial spores [19,20]. It was also noticeable that the ESMH-CM shell of only ca. 10 nm in thickness had such cytoprotective power. The cytoprotectability and durability of the shells could be enhanced further by forming ESMH-CM shells for a longer time than 3 h and/or repeating the shell-forming process. For instance, the thickness of ESMH-CM films on gold increased to 67.5 nm after 10 cycles of the film deposition ([ESMH] = [CM] = 1 mg/mL; 3 h of reaction) ( Figure S5).

One-Step SCNE of Probiotic L. acidophilus and L. brevis
The ESMH-CM shells were formed on L. acidophilus, producing L.acidophilus@ESMH-CM. L. acidophilus is a gram-positive, microaerophilic probiotic bacterium, naturally present in the gastrointestinal (GI) tract, vagina, and others. It is one of the major probiotic species in commercialized products, such as yogurt and probiotic capsules, along with L. bulgaricus, Streptococcus thermophilus, and Bifidobacterium bifidum.
Innumerable encapsulation methods for L. acidophilus (and also other probiotics) have been attempted and reported to enhance the survival during food/nutraceutical processing as well as against harsh conditions in the stomach and GI tract [39][40][41]. In addition to the long-pursued microencapsulation approach [42,43], recent research efforts have intensively been devoted to the formation of nanometric shells on probiotics in SCNE [44][45][46]. Notable examples include the recent utilization of TA-Fe 3+ nanoshells [47][48][49] for cytoprotective SCNE of anaerobic Bacteroides thetaiotaomicron [50] and L. casei [51] for potential development of microbial biotherapeutics, in addition to the autonomous nanoencapsulations of L. rhamnosus with polydopamine [52] and engineered S. thermophilus with hyaluronic acid [53]. Nanoshells of metal-organic frameworks and silica nanoparticles also have been used for potential cytoprotection of L. acidophilus, B. infantis, and B. breve [54,55].

One-Step SCNE of Probiotic L. acidophilus and L. brevis
The ESMH-CM shells were formed on L. acidophilus, producing L. acidophilus@ESMH-CM. L. acidophilus is a gram-positive, microaerophilic probiotic bacterium, naturally present in the gastrointestinal (GI) tract, vagina, and others. It is one of the major probiotic species in commercialized products, such as yogurt and probiotic capsules, along with L. bulgaricus, Streptococcus thermophilus, and Bifidobacterium bifidum.
Considering the importance of sustained survival after passage through the stomach in the development and formulation of probiotic capsules, viabilities of bare L. acidophilus, L. acidophilus@ESMH-CM, and L. acidophilus@ESMH-CM[Fe 3+ ] were measured and compared after incubation in SGF (pH 2) (Figure 4c). After 1 h of incubation, the viability of bare L. acidophilus was calculated to be 69.5 ± 1.7%, in comparison with 78.3 ± 1.5% for L. acidophilus@ESMH-CM and 92.1 ± 1.3% for L. acidophilus@ESMH-CM[Fe 3+ ], signifying the Fe 3+ -fortified L. acidophilus@ESMH-CM would be protected effectively during the passage though the stomach. The cytoprotection of L. acidophilus@ESMH-CM[Fe 3+ ] was much more discernable for 2 h of incubation. The viability decreased significantly to 29.6 ± 2.8% and 30.0 ± 5.8% for bare L. acidophilus and L. acidophilus@ESMH-CM, respectively (not significant between the two values, based on Student's t-test). In stark contrast, 78.5 ± 1.3% of %viability was observed for L. acidophilus@ESMH-CM[Fe 3+ ]. The results clearly confirmed the cytoprotection capability of ESMH-CM and ESMH-CM[Fe 3+ ] shells, suggesting great potential in the construction of probiotic capsules and/or microbial biotherapeutics [65]. It is yet to mention that the cytoprotection degree of ESMH-CM-based shells was species-dependent in the absolute sense. For example, L. brevis, a species in vaginal microbiota [66], was observed to be more labile than L. acidophilus in SGF. Neither bare L. brevis nor L. brevis@ESMH-CM survived after 1 h of incubation, whereas 42.7 ± 6.0% of L. brevis@ESMH-CM[Fe 3+ ] were viable even after 2 h of incubation in SGF (Figure 4d). Although the value (ca. 43 %) was less than the %viability for L. acidophilus (ca. 79%), the ESMH-CM[Fe 3+ ] showed reasonable protection of L. brevis against the attack of SGF.
It could be thought that the observed enhancement in cell viability against SGF was attributed to the stability of ESMH-CM-based shells under acidic conditions, and a model study was carried out. The ESMH-CM and ESMH-CM[Fe 3+ ] films on gold were incubated for 2 h at various pH values (1, 2, 3, 4, 5, 6, and 7) as well as in SGF, and % decrease in film thickness (%∆Th) was calculated after ellipsometric-thickness measurements (Figure 4e). The ESMH-CM film was observed to be stable at pH 3 and 4, but not at pH 2 and below. Accordingly, about 24% of the ESMH-CM film remained after 2 h of SGF treatment. In contrast, the Fe 3+ fortification made the film much more durable at all the pHs tested, as well as in SGF. For example, 84% of the ESMH-CM[Fe 3+ ] film was maintained after 2 h of SGF treatment, in a good agreement with the viability studies above. Of interest, the ESMH-CM[Fe 3+ ] film decreased to about 74% in thickness at pH 7, implying that the nanoencapsulated probiotics could inhabit the gut epithelium after protected passage of the stomach.

Conclusions
In summary, we developed a simple but scalable method for constructing ultrathin films and shells, based on nature-derived biomaterials-eggshell membrane hydrolysates (ESMHs) and coffee melanoidins (CMs). The process developed proved extremely biocompatible with living microbial cells, applied seamlessly to probiotic bacteria, Lactobacillus acidophilus (in gut microbiota) and Levilactobacillus brevis (in vaginal microbiota). The

Conclusions
In summary, we developed a simple but scalable method for constructing ultrathin films and shells, based on nature-derived biomaterials-eggshell membrane hydrolysates (ESMHs) and coffee melanoidins (CMs). The process developed proved extremely biocompatible with living microbial cells, applied seamlessly to probiotic bacteria, Lactobacillus acidophilus (in gut microbiota) and Levilactobacillus brevis (in vaginal microbiota). The ESMH-CM shells protected the probiotic bacteria in the simulated gastric fluid, suggesting potential in the probiotics nanoencapsulation. The cytoprotectability was further augmented by the Fe 3+ -mediated cross-linking of ESMH-CM shells. Considering that multivalent metal cations are present in body fluids, such as Fe 3+ , Ca 2+ , Mg 2+ , Zn 2+ , and Cu 2+ , the shell augmentation also could occur autonomously in the body, which is our next research thrust. In addition, our formulation might be combined with mineral supplements for shell augmentation during oral administration. Furthermore, in the aspect of biomaterials, ESMHs and CMs would add to the set of nature-derived biocompatible materials for fabrication of nanobiohybrid structures, with a characteristic of waste upcycling.

Conflicts of Interest:
The authors declare no conflict of interest.