Hydrophilic Food-Borne Nanoparticles from Beef Broth as Novel

Article Cite This: J. Agric. Food Chem. 2019, 67, 6995−7004

pubs.acs.org/JAFC

Hydrophilic Food-Borne Nanoparticles from Beef Broth as Novel Nanocarriers for Zinc Jiaxin Geng,†,‡ Xunyu Song,†,‡ Xuedi Zhang,†,‡ Shanshan Tie,†,‡ Lin Cao,†,‡ and Mingqian Tan*,†,‡ †

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Qinggongyuan 1, Ganjingzi District, Dalian, Liaoning 116034, People’s Republic of China ‡ Engineering Research Center of Seafood of Ministry of Education of China, Dalian, Liaoning 116034, People’s Republic of China

Downloaded via BUFFALO STATE on July 24, 2019 at 11:48:54 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Food-borne nanoparticles (FNs) may be used as nanocarriers for metal ion chelation in micronutrient supplements. In this paper, the preparation and characterization of hydrophilic FNs were reported from beef broth cooked with a pressure cooker at 117 °C for different periods (30, 50, and 70 min) and their potential application as nanocarriers for zinc was investigated. The broth FNs are quasi-spherical with good water solubility and ultrasmall size, which can emit a strong sapphire color under 365 nm ultraviolet irradiation. X-ray photoelectron spectroscopy (XPS) analysis showed that there are carboxyl, amino, and hydroxyl groups on the FNs, which are useful for Zn(II) chelation. The vibration band of CO at 1688 cm−1 in the infrared spectrum of FNs shifted to 1718 cm−1 after binding with Zn(II) ions, suggesting the participation of the carbonyl group in Zn(II) ion chelation. The appearance of Zn2p XPS peaks, at 1021.6 and 1045 eV for Zn(II)−FNs, clearly demonstrated the formation of Zn−O between the FNs and zinc ions. Biodistribution of FNs and the Zn(II)−FN complex in normal rat kidney cells demonstrated that they could easily enter normal rat kidney cells. A downfield was found for the signals of Zn(II)−FNs in 1H nuclear magnetic resonance spectroscopy and strongly suggested the binding of Zn(II) ions to FNs through carboxylic acid, hydroxyl, and amine groups. In addition, no obvious cytotoxicity was found for Zn(II)−FNs compared to zinc (ZnSO4) and commercial zinc gluconate. The results revealed that the FNs from beef broth may have a potential as nanocarriers for zinc chelation. KEYWORDS: beef broth, nanoparticles, zinc(II) carriers, food-borne, micronutrient supplements

■

INTRODUCTION Zinc is an essential micronutrient in certain key biological processes in human growth, which is widely used as a nutritional supplement. As a catalytic composition of numerous enzymes, zinc has important structural and biological functions in regulating growth factors and cytokines.1 Subclinical zinc deficiency is prevalent in certain populations of humans, especially among the elderly, infants, children, and pregnant women.2 Severe zinc deficiency is associated with alopecia, diarrhea, delayed sexual maturation, and eczematous skin rash.3,4 Zinc deficiency has been a more prevalent problem in the regions where people eat a high-cereal and low-meat diet or are poorly nourished.5 Zinc supplementation is an effective method for combating zinc deficiency as a result of its good adaptability and availability. Inorganic metal salts are not good micronutrient supplements as a result of the negative effects associated with them, such as the problem of easy crystallization and precipitation and unpleasing taste.6 As an alternative, some chelating peptides with binding groups have been used as carriers to enhance zinc absorption in the digestive tract. For example, ́ Garcia-Nebot et al.7 reported the caseinophosphopeptide effects from casein fractions on zinc retention, transport, and uptake in heterogeneous human epithelial colorectal adenocarcinoma cells. Chen et al.8 described a 10 amino acid peptide from Alaska pollock skin for zinc binding and evaluated its stability during in vitro gastrointestinal enzymatic digestion. Udechukwu et al.9 examined the prospective of food peptides © 2019 American Chemical Society

that cound improve zinc bioavailability and concluded that the chelating capability of peptide and zinc played an important role during the development of human nutritional supplement. However, the solubility of certain peptides with hydrophobic amino acids, such as valine, leucine, methionine, phenylalanine, and tryptophan, is limited, which significantly influences solubility and chelating ability. Moreover, the preparation and separation of the peptides are time-consuming and costly. Therefore, the exploration of zinc chelators with good chelating ability derived from food components using a costeffective strategy is more acceptable in terms of general health considerations and dietary preferences. The artificial nanospheres have shown positive effects on improving food quality, and their potential beneficial impacts have drawn considerable attention.10,11 Recently, a new type of nanoparticle, namely, food-borne nanoparticles (FNs), was discovered in our daily foods. For the first time, Sk et al.12 examined the physicochemical properties of the FNs in bread. Jiang et al.13 found FNs in commercial Nescafé instant coffee that is consumed in everyday life. Liao et al.14 directly extracted the FNs from several commercial beverages, such as Kvass, Pilsner beer, Pony Malta, Vivant Storm, and Profit. Li et al.15 demonstrated the properties of nitrogen-containing FNs Received: Revised: Accepted: Published: 6995

February 28, 2019 May 30, 2019 June 3, 2019 June 3, 2019 DOI: 10.1021/acs.jafc.9b01372 J. Agric. Food Chem. 2019, 67, 6995−7004

Article

Journal of Agricultural and Food Chemistry

Figure 1. TEM images of FNs obtained from beef broth cooked at (a) 30 min, (b) 50 min, and (c) 70 min. High-resolution TEM images are displayed as insets. Histograms of size distribution of FNs extracted from beef broth cooked in (d) 30 min, (f) 50 min, and (g) 70 min. in Shanghai, China. Potassium bromide was acquired from Beijing Biocity Technology Co., Ltd. The NRK cell line was provided by Dr. Li Yu’s lab in Tsinghua University, China. The 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for the cell viability assay was bought from Aladdin Reagent Co., Ltd. in Shanghai, China. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Biological Industries Israel Beit Haemek, Ltd. Commercial zinc gluconate was bought from Harbin Pharmaceutical Group Co., Ltd. (Harbin, China), which contains 3.5 mg of zinc gluconate/mL. Unless otherwise specified, all of the chemicals were of analytical grade. Instrumentation. JEM-2100 transmission electron microscopy (TEM) from JEOL in Tokyo, Japan, was used to characterize the size of FNs and Zn(II)−FNs. Fourier transform infrared spectroscopy (FTIR) spectra were measured by mixing the testing sample into potassium bromide with a PerkinElmer Frontier spectrometer (Norwalk, CT, U.S.A.). Fluorescence spectra of the FNs were recorded by a F-2700 fluorescence spectrometer produced by Hitachi Corporation in Tokyo, Japan. Ultraviolet−visible (UV−vis) absorption spectra of FNs and Zn(II)−FN samples were measured by a PerkinElmer UV−vis spectrophotometer (Lambda 35) manufactured in Cambridge, MA, U.S.A. An ESCALAB 250 X-ray photoelectron spectroscopy (XPS) spectrometer produced from Thermo Scientific Corporation (Waltham, MA, U.S.A.) was used to characterize the surface chemical composition using an Al Kα excitation source (1486.6 eV). 1H nuclear magnetic resonance (NMR) spectra of the FNs and Zn(II)−FN complex were measured by a Bruker AVANCE III 400 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany).A total of 6 mg of FNs or Zn(II)−FN sample was dissolved in 600 μL of deuterium oxide (D2O), separately, and transferred to 5 mm sample tubes for NMR analysis with a total 16 scans. Fluorescence lifetime of the FNs and Zn(II)−FNs was measured by

in grilled hamburger beef that was cooked at different temperatures. Song et al.16 reported endogenous FNs in roasted chicken breasts and described their physicochemical properties. Accordingly, endogenous FNs are universally present in our daily food and are intrinsic components of food that may offer nutritional value. Certain types of FNs produced during food processing have good water solubility, compact structure, and abundant functional groups for zinc chelation. To date, no study has been reported on the potential application of FNs from broth as a zinc carrier for use as a micronutrient supplement. In this study, we reported the preparation of FNs from beef broth and their evaluation as Zn(II) nanocarriers. The generation of FNs at different time periods (30, 50, and 70 min) and the formation of Zn(II)−FN complexes were studied by examining their particle size, fluorescent behavior, fluorescent lifetime, and elemental composition. The functional groups of FNs and their binding with zinc were thoroughly characterized using various analytical techniques. In addition, the biodistribution and toxicity of FNs and Zn(II)− FN complexes were studied in normal rat kidney (NRK) cells. Our result indicated that the FNs derived from beef broth may have great potential as nanocarriers for zinc.

■

EXPERIMENTAL SECTION

Materials. Beef was bought from a local grocery market in Dalian, China. Zinc(II) sulfate (ZnSO4) was bought from Kemiou Chemical Reagent Co., Ltd. in Tianjin, China. Dialysis bags with molecular weight cutoff at 0.5 kDa were purchased from Aldrich Chemical Co. 6996

DOI: 10.1021/acs.jafc.9b01372 J. Agric. Food Chem. 2019, 67, 6995−7004

Article


Figure 2. (a) FTIR spectra and (b) UV−vis absorption spectra of FNs derived from beef broth at different time periods.

Figure 3. Fluorescence emission spectra of FNs processed at (a) 30 min (b) 50 min, and (c) 70 min. Insets show the photographs of FNs in water solution under daylight and UV light. (d) Fluorescence lifetime decay curves of the FNs (30, 50, and 70 min). off of 0.5 kDa) to remove unbinding free zinc ions. The dialyzed fluid was collected and freeze-dried to yield 90.3 mg of the Zn(II)−FN complex. Isothermal Titration Calorimetry (ITC) Characterization. The binding affinity between FNs and zinc was evaluated by the ITC technique at 25 ± 0.2 °C using an Affinity ITC calorimeter produced by TA Instruments, Ltd. (New Castle, DE, U.S.A.). The FNs and ZnSO4 were dissolved in deionized water and filtrated with a 0.22 μm Millipore membrane to remove the larger particles. After degassing, the zinc solution (1 mM) was titrated into FN solution (7 mM) using the titration of zinc aqueous solution into deionized water as the blank control. The obtained ITC data were then fitted using the Nano Analyze software to calculate the association constants (Ka) between zinc and FNs. Biodistribution of FNs and in Zn(II)−FN NRK Cells. A total of 2 mg/mL FNs and Zn(II)−FN aqueous solution was added to NRK cells, separately, and incubated in the coverglass-bottom confocal dish at 37 °C for 24 h. After the NRK cells were washed with phosphatebuffered saline (PBS) buffer thrice, the cells were imaged with a SP8 inverted laser scanning confocal microscope from Leica (Wetzlar, Germany) under the excitation wavelength of 408 nm.17 Cytotoxicity Assay. The potential cytotoxicity of FNs and Zn(II)−FNs was evaluated by a MTT assay in NRK cells. The NRK cells were trypsinized and cultured at a cell density of 5 × 104 cells per

a FLS980 spectrometer made by Edinburgh Instruments Co., Ltd., Edinburgh, U.K., using a 320 nm laser as the excitation source. Extraction of the Nanocarrier. Beef (266 g) was cut into pieces at a size of 2 × 2 × 2 cm and cooked at 117 °C in deionized water (0.8 L) by a pressure cooker for 30, 50, and 70 min, respectively. The pressure was set at 181 kPa (absolute pressure). After the beef broth was cooled to room temperature, 2000 mL of ethanol was added in the broth. The resulting precipitate was removed by filtration. After removal of solvent by a rotary evaporator, the raw product was extracted with 50 mL of ethyl acetate 5 times to separate the hydrophobic components. Then, UV230II semi-preparative highperformance liquid chromatography produced by Elite Co., Ltd. (Dalian, China) was used to separate the hydrophilic FNs by a SinoChrom octadecyl-silica-bonded phase column (20 × 300 mm, 10 μm) using 10% (v/v) methanol as the mobile phase at a flow rate of 20 mL min−1 for 40 min.5 The fractions with UV absorbance at 254 nm were collected, concentrated by a rotary evaporator, and freezedried, yielding yellow−white powder of 1.326, 1.534, and 1.895 g for broth of 30, 50, and 70 min, respectively. Preparation of the Zn(II)−FN Complexes. For preparation of the Zn(II)−FN complex, 200.4 mg of FNs was mixed with 201.0 mg of ZnSO4 in 40 mL of deionized water. The binding reaction was performed at 60 °C with a water bath for 2 h. Thereafter, the resulting Zn(II)−FN complex was purified by dialysis (molecular weight cut6997


Article


Figure 4. (a) XPS spectrum and high-resolution (b) C1s, (c) N1s, and (d) O1s spectra of the FNs from beef broth produced at 30 min. (e) XPS spectrum and high-resolution (f) C1s, (g) N1s, and (h) O1s spectra of the FNs from beef broth produced at 50 min. (i) XPS spectrum and highresolution (j) C1s, (k) N1s, and (l) O1s spectra of the FNs from beef broth produced at 70 min. well overnight. Then, the NRK cells were incubated with different concentrations (0, 0.31, 0.63, 1.25, 2.5, 5.0, and 10.0 mg/mL) of FNs, Zn(II)−FNs, zinc (ZnSO4), and commercial zinc gluconate for 24 h, followed by adding 20 μL of MTT (5 mg/mL) solution. After the cells were further incubated for 4 h, and the culture medium was replaced with 100 μL of dimethyl sulfoxide (DMSO). The mixture optical density (OD) at 490 nm was measured using an Infinite 200 multimode microplate reader (Tecan, Hombrechtikon, Switzerland). Cell viability was calculated according to the previous method.15

Table 1. XPS Elemental Analysis of the FNs from Beef Broth Produced at 30, 50, and 70 min

■

element

30 min

50 min

70 min

C1s (%) N1s (%) O1s (%)

61.34 11.40 25.63

66.23 8.30 24.14

66.85 10.41 22.66

(panels d−f of Figure 1). Clearly, the FN size decreased gradually with the extension of the cooking time. In addition, it was found that the FNs for 30 and 50 min cooking time have lattice fringes with an interplanar spacing of 0.145 and 0.096 nm, respectively. However, the FNs of 70 min cooking time have no noticeable lattice fringe. The lattice fringe was also found in the FNs extracted from the grilled hamburger after heating at 220, 260, and 300 °C.15 The appearance of lattice fringes indicated that the degree of structural order increased,

RESULTS AND DISCUSSION The morphology of FNs separated from beef broth at different cooking time periods (30, 50, and 70 min) was characterized by TEM. The FNs were nearly spherical and well-dispersed without obvious aggregation (panels a−c of Figure 1). The mean particle size was 5.4, 4.9, and 2.4 nm, respectively, for the FNs derived from beef broth cooked for 30, 50, and 70 min 6998


Article


Figure 5. (a) Fluorescence spectra of FNs from beef broth produced at 70 min and corresponding Zn(II)−FNs. (b) UV−vis spectra of the FNs from beef broth produced at 70 min and corresponding Zn(II)−FNs. (c) Fluorescence lifetime decay curve of the FNs from beef broth produced at 70 min and corresponding Zn(II)−FNs. (d) FTIR spectra of the FNs from beef broth produced at 70 min and corresponding Zn(II)−FNs.

yield (QY) of FNs derived from beef broth after cooking for 30, 50, and 70 min was 2.0, 2.1, and 2.5%, respectively. The fluorescence lifetime was 6.49, 7.26, and 8.32 ns, respectively. The lifetime is an inherent characteristic that reflects the period for an electron to return to its ground state.22 The results revealed that both the QY and lifetime increased with the extension of the cooking time. The elemental composition of FNs derived from beef broth was analyzed by XPS. The peaks observed at 284.87, 399.61, and 531.11 eV were ascribed to C1s, N1s, and O1s, respectively (panels a, e, and i of Figure 4). In detail, there are four components with characteristic peaks at 284.5, 285.4, 286.3, and 287.8 eV in the high-resolution C1s spectrum of FNs (30 min; Figure 4b), which indicates the presence of C−C or C C, C−N, C−O, and CO chemical bonds, respectively.23,24 The high-resolution N1s spectrum (Figure 4c) contains two major peaks at 399.3 and 400.4 eV, associated with pyridinic− N and N−H, respectively.25 Similar results in the C1s and N1s spectra were obtained for FNs from broth cooked for 50 and 70 min (panels f, g, j, and k of Figure 4). The O1s XPS spectrum of FNs was decomposed into two peaks at 530.1 and 532.3 eV, indicating the presence of O−H and C−O (30 min; Figure 4d). However, the high-resolution O1s spectrum of FNs (50 and 70 min) contained three major peaks at 530.6, 531.7, and 533.2 eV (panels h and l of Figure 4), suggesting the presence of O−H, OC−O, and O−C chemical bonds.17 The elemental contents of the FNs based on the calculation from the integral area of the XPS spectra are summarized in Table 1. The FNs from broth cooked for 30 min show a lower content (61.34%) of carbon than that (66.23−66.85%) of the FNs from broth cooked for 50 or 70 min. These finding indicated that the carbon content of FNs exhibited an increased trend with the extension of processing time. The oxygen content of FNs was in the range of 22.66−25.63%, while the nitrogen content was in the range of 8.30−11.40%. All of the results revealed that the FNs showed a carbon-rich nature, which are similar to the result of carbon dots (CDs)

while the disappearance of lattice fringes demonstrated that the FNs changed unordered with the extension of the heating time. A rational conjecture was that lipids, polysaccharides, and proteins in beef were broken down from the bulk size muscle and polycondensed into small size FNs. The universal presence of the foodborne FNs provided good candidates for metal ions chelating in the development of micronutrient supplements. The functional groups of the FNs produced at different cooking time periods (30, 50, and 70 min) were determined by FTIR. The broad absorption at 3200−3600 cm−1 can be ascribed to N−H and O−H stretching vibrations (Figure 2a). The peak at 1688 cm−1 is due to amide CO bending, and the peak at 1598 is ascribed to CC stretching vibrations. The various peaks from 1522 to 1233 cm−1 may be caused by the C−O vibration. Additionally, the peaks appearing near 1120 cm−1 are generated by the vibration of C−N. All of these data indicated the existence of numerous groups on the FN surfaces, which are useful for chelation of metal ions.18 The UV−vis spectra were further measured to study the optical properties of FNs from beef broth in different cooking time periods. The FNs displayed a broad absorption band in the region of 220−260 nm, which is possibly due to the π−π* or n−π* electronic transitions of functional groups on the FNs, indicating that the FNs may have unique fluorescence emission ability.15 Figure 3 shows that the FNs emit fluorescence in the region of 370−500 nm with an excitation-dependent emission property, when the excitation wavelength varied from 280 to 400 nm. When the emission spectra shifted to a longer wavelength, the fluorescence intensity of the FNs gradually declined. The bathochromic emission phenomenon was similar to that of the widely reported carbon dots19 as a result of the complexity of the surface state that affected the band gap of the FNs.20−22 The aqueous solution of FNs is colorless and transparent under daylight, as displayed in insets of panels a−c of Figure 3. Under the UV light (365 nm), strong sapphire fluorescence of the aqueous FNs was observed. The quantum 6999


Article


Figure 6. (a) TEM image of Zn(II)−FNs (scale bars = 200 nm). (b) XPS spectrum of the Zn(II)−FNs and high-resolution (c) C1s, (d) N1s, (e) O1s, and (f) Zn2p XPS spectra. (g) Schematic illustration of Zn(II) ions attached to the FNs through the coordination of Zn−O and Zn−N bonds.

obtained from instant coffee.13 Noteworthy, the XPS results and FTIR data fit well, suggesting that the FNs contain carbon, nitrogen, and oxygen, which are probably derived from the protein, polysaccharide, and fat of the beef broth. The abundant functional groups present on the FNs are very useful for the conjugation of zinc ions. The FNs derived from beef broth cooked for 70 min possess the smallest size, the highest QY, and the longest lifetime; therefore, they were selected as candidate nanocarriers for loading Zn(II). The steady-state fluorescent spectra (Figure 5a) of the Zn(II)−FNs demonstrated that the maximum emission is not changed and the fluorescence intensity enhanced significantly. The QY for Zn(II)−FNs increased from 2.5 to 5.3%. The reason is likely due to the passivation effect of zinc, thus enhancing the radiative recombination of

the energy-trapping sites on the FNs after forming Zn(II)−FN complexes.25 The electronic structure of Zn(II) is unique, which is critical for chelation and, thus, facilitating the radiative recombination of electrons on the energy-trapping sites. The UV−vis spectra of the FNs and Zn(II)−FNs are depicted in Figure 5b. A new absorption peak appeared at approximately 246 nm in the spectrum of Zn(II)−FNs, which indicated the structural change of FNs by the chromospheres (−CO and −COOH) and auxochromes (−OH and −NH2) after binding the zinc ion.26,27 The new absorbance peak appeared indicating the formation of the complex of FNs and Zn(II).28 The interaction between FNs and Zn(II) was necessary for the formation stable Zn(II)−FN complexes when using the beef broth FNs as a nanocarrier. Also, the fluorescence lifetime of 7000


Article


Figure 7. 1H NMR spectra of the FNs and Zn(II)−FN complex in D2O.

Zn(II)−FNs (Figure 5c) was 7.921 ns, which was comparable to that of FNs. The FTIR spectra of FNs and the Zn(II)−FN complexes are displayed in Figure 5d. Shifting in the FTIR peaks of the FNs revealed the interaction of Zn(II) with FNs. The band strength at 3393 cm−1 in the spectrum of FNs is ascribed to −OH decreasing, while the peak at 3266 cm−1 is due to the vibration of N−H for Zn(II)−FNs, indicating that the nitrogen atoms formed coordination bonds with Zn(II) ions by offering their electron pairs and the N−Zn(II) bond replaced the N−H hydrogen bonds. The vibration band of CO at 1679 cm−1 in the spectrum of the FNs shifted to 1718 cm−1 after binding with Zn(II) ions. This result suggested the participation of the carbonyl (CO) group in the interaction between the FNs and Zn(II) ions. In addition, the peak at 1400 cm−1 of the C− O group changed to 1426 cm−1 for Zn(II)−FN complexes, suggesting that the C−O bond was important in binding with zinc. The bands near 1120 cm−1 in the FN spectrum are usually attributed to the C−O and C−N stretching vibrations.29 A significant enhancement of the intensity of these characteristic bands was found in the spectrum of the Zn(II)−FN complex, which means that C−O and C−N groups are probably involved in Zn coordination.30 Together, these results indicated that the Zn(II) ions bound to the FNs primarily through interactions with amino nitrogen and carboxyl oxygen atoms, similar to other metal-binding protein hydrolysates or peptides.18,31,32

TEM analysis of the morphological feature of the Zn(II)− FN complexes is shown in Figure 6a. The Zn(II)−FNs exhibited aggregate morphology, with the size increasing significantly, and are quite different from the FNs. Similar morphological changes have also been found in the coordination chemistry of Zn(II) with the amyloid β (Aβ) peptide.33 The reason is likely due to the interaction between the FNs and Zn(II). Scanning electron microscopy (SEM) characterization (Figure S1 of the Supporting Information) shows that the FNs are too small to be detected (Figure S1a of the Supporting Information), while the Zn(II)−FNs display broccoli-like morphology (Figure S1b of the Supporting Information). The elemental composition was determined on the basis of the XPS spectrum analysis of Zn(II)−FNs, which shows four predominant peaks at 284.12, 400.16, 531.58, and 1021.76 eV (Figure 6b), namely, elements C, N, O, and Zn(II), with a relative content of ca. 59.44, 5.0, 28.6, and 3.7%, respectively. The energy-dispersive X-ray (EDX) mapping element analysis shows that the FNs from beef broth are mainly composed of C, O, and N (Figure S1c of the Supporting Information), while the Zn(II)−FNs contain C, O, N, and Zn, which is in line with that of XPS analysis (Figure 6b). The EDX result revealed that a significant amount of zinc was loaded into the complex of Zn(II)−FNs (Figure S1f of the Supporting Information) compared to FNs (Figure S1e of the Supporting Information). Noteworthy, besides pyridinic N and N−H (Figure 6d), a new peak appeared at 397.7 eV, which 7001


Article


Figure 8. Confocal microscopy images of the FNs and Zn(II)−FNs in the NRK cells excited by a 405 nm laser and overlay images. (a) Bright field, (b) fluorescence, and (c) overlay image of panels a and b for NRK cells incubated with FNs extracted from beef broth after cooking for 70 min. (d) Bright field, (e) fluorescence, and (f) overlay image of panels d and e for NRK cells incubated with Zn(II)−FNs. Cells without FNs and Zn(II)− FNs were used as a control in panels g−i.

Figure 9. Cytotoxicity of (a) FNs and (b) Zn(II)−FNs, zinc (ZnSO4), and commercial zinc gluconate with increasing concentrations from 0 to 10 mg/mL in NRK cells. 1

indicates the presence of N−Zn in the high-resolution N1s spectrum.34−36 This is strong evidence that the Zn(II)−FN complexes have formed. The Zn2p XPS spectrum (Figure 6f) of Zn(II)−FNs can be decomposed into two peaks, at 1021.6 and 1045 eV, respectively, which is typical of the bimodal structure of zinc ions, indicating the presence of Zn−O.37 These results suggested that the Zn(II) ions that bind to the FNs may primarily interact with oxygen from the carboxyl or hydroxyl group and nitrogen from the amino group. These results agree well with the FTIR findings described above. The schematic diagram in Figure 6g further explains the binding between the FNs and the Zn(II) ions. The zinc ions attach to the FNs through the coordination of Zn−O and Zn−N bonds.

H NMR spectroscopy was also used to investigate the binding of Zn(II) to FNs, and a downfield was found for the signals of Zn(II)−FNs (Figure 7), which was consistent with a similar finding in a previous study.38 The doublet signal for methylene of alkanol appeared at 1.299 and 1.317 ppm, and amine or aliphatic acid in the free FNs shifted to 1.357 and 1.375 ppm, respectively. The protons at 3.011 ppm, which were ascribed to the hydroxyl or methylene group next to the nitrogen atom in the amine backbone, moved to 3.083 ppm after forming Zn(II)−FNs. The signal of −CH2− in the ethyl ester chain at 4.086 ppm showed a significant shift of 0.114 ppm after conjugation with Zn(II). The phenyl or phenol moieties, which appear as a multiplet in the range of 6.069− 6.121 ppm, changed from 5 to 4 peaks after binding with 7002


Article


commercialized zinc-gluconate-treated cells decreased from 80 to 55% with an increasing concentration to 10 mg/mL. This indicated that the cytotoxicity of Zn(II)−FNs was less than that of ZnSO4 and gluconate. Because the FNs are derived beef broth and are the intrinsic nutritional components, the nanoparticles have great potential to be used as carriers for Zn(II). These results showed that the Zn(II)−FN complexes originating from the beef broth had good biocompatibility, without obvious cytotoxicity. In summary, the presence of FNs in beef broth was found for the first time and the beef broth FNs showed an ultrasmall size, a strong fluorescence, and good biocompatibility for application as a zinc nanocarrier. The numerous functional groups of FNs may play crucial roles in the chelation of the Zn(II) ions. The zinc-binding sites of the FNs are primarily considered to be the carboxyl or hydroxyl groups and amino nitrogen atoms. The properties of FNs from beef broth suggest that they have great potential for use as nanocarriers for loading zinc in the development of a zinc supplement.

Zn(II), strongly indicating the interaction between FNs and Zn(II) (Figure 7d). The protons of the aromatic rings of FNs exhibit a minor change from 7.239 to 7.243 ppm (Figure 7e). The signal at 8.324 ppm of FNs is likely due to benzoic acid or naphthalenol, which shifted to 8.348 ppm in the spectrum of Zn(II)−FNs. The proton chemical shift, likely originating from the nitrogen-containing heterocycle, moved from 8.540 to 8.555 ppm. These obvious chemical shift changes strongly suggested the binding of Zn(II) ions to FNs through carboxylic acid, hydroxyl, and amine groups for Zn(II) coordination. The binding affinity of FNs toward zinc was measured by the ITC technique, and the association constants (Ka) of zinc to FNs was determined to be 1.38 × 105, 2.21 × 105, and 2.41 × 106 M−1 for FNs derived from beef broth cooked for 30, 50, and 70 min, respectively. The constant (2.41 × 106 M−1) of Zn−FNs (cooking for 70 min) was much greater than that (1.000 × 103 M−1) of the peptide−Zn complex.39 This indicated that the Zn−FNs are more stable as a result of the interaction of FNs with Zn(II) ions. Moreover, beef broth is an easily obtained food resource, which can be effectively produced on a large scale. The FN nanocarriers derived from beef broth are more cost-effective than the peptides purified from food resources. In addition, unloading of zinc in digestion juices was also investigated using the artificial intestinal and gastric fluids, respectively. For detailed information, please see the Supporting Information. The unloading rate of zinc from Zn(II)−FNs in intestinal and gastric fluids was 74 and 34%, respectively, after subtracting the value obtained in water. Although the unloading of Zn(II) from Zn(II)−FNs occurred in digestion fluids, in vivo assessment is required to study the release behavior of Zn(II) in our next work. The unique fluorescence properties and ultrasmall size of FNs and Zn(II)−FNs are useful properties to investigate their distribution in cells. The NRK cells were used to examine the biodistribution of FNs and Zn(II)−FNs. An in vitro cell-uptake study showed blue fluorescence in the cells treated with FNs and Zn(II)−FNs (panels b and e of Figure 8) under the excitation wavelength of 405 nm. This finding indicated that the fluorescent FNs were internalized by the cells and distributed within the cytoplasm. Furthermore, a stronger fluorescence was observed in the cells treated with Zn(II)− FNs than in the cells treated with FNs. This means that Zn(II)−FN complexes are more easily internalized into the cell cytoplasm.40 The distribution of FNs and Zn(II)−FNs in NRK cells can be clearly observed in the overlay of FNs and Zn(II)−FN images, which suggests that the Zn(II)−FNs can be easily taken up by the live cells. The cytotoxicity of FNs and Zn(II)−FNs was evaluated by measuring the relative viabilities of NRK cells treated with different concentrations of FNs and Zn(II)−FNs by the MTT assay. The results of the cell viability assay after treatment of the cells with FNs and Zn(II)−FNs for 24 h are shown in Figure 9. The cytotoxicity results of cells treated with FNs (Figure 9a) show that cell viability was significantly increased with an increasing FN concentration in the range from 0.31 to 2.5 mg/mL. Cell viability reached its maximum level at 2.5 mg/mL but slightly declined with a further increase of the FN concentration in the range from 5 to 10 mg/mL. According to the results presented in Figure 9b, the cell viability of the tested samples remained higher than 80% for the cells treated with Zn(II)−FNs, while the cell viability of cells treated with ZnSO4 was maintained around 60%, and the cell viability of

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01372.

■

In vitro digestion of Zn(II)−FNs, unloading of zinc through the digestive tract, and SEM and EDX analyses (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-411-86318657. E-mail: [email protected]. ORCID

Mingqian Tan: 0000-0002-7535-0035 Funding

This work was supported by the National Key Research and Development Program of China (2018YFD0901106) and the National Natural Science Foundation of China (31872915). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Bozalioğlu, S.; Ö zkan, Y.; Turan, M.; Ş imşek, B. Prevalence of zinc deficiency and immune response in short-term hemodialysis. J. Trace Elem. Med. Biol. 2005, 18 (3), 243−249. (2) Maret, W.; Sandstead, H. H. Zinc requirements and the risks and benefits of zinc supplementation. J. Trace Elem. Med. Biol. 2006, 20 (1), 3−18. (3) Shenkin, A. Basics in clinical nutrition: Physiological function and deficiency states of trace elements. E Spen Eur. E J. Clin. Nutr. Metab. 2008, 3 (6), e275−e280. (4) Keen, C. L.; Gershwin, M. E. Zinc deficiency and immune function. Annu. Rev. Nutr. 1990, 10 (10), 415−431. (5) Roohani, N.; Hurrell, R. F.; Kelishadi, R.; Schulin, R. Zinc and its importance for human health: An integrative review. J. Res. Med. Sci. 2013, 18 (2), 144−157. (6) Sahin, K.; Smith, M. O.; Onderci, M.; Sahin, N.; Gursu, M. F.; Kucuk, O. Supplementation of zinc from organic or inorganic source improves performance and antioxidant status of heat-distressed quail. Poult. Sci. 2005, 84 (6), 882−887. (7) García-Nebot, M. J.; Barbera, R.; Alegria, A. Iron and zinc bioavailability in caco-2 cells: Influence of caseinophosphopeptides. Food Chem. 2013, 138 (2), 1298−1303. 7003


Article


(27) Cai, X.; Zhao, L.; Wang, S.; Rao, P. Fabrication and characterization of the nano-composite of whey protein hydrolysate chelated with calcium. Food Funct. 2015, 6 (3), 816−823. (28) Blat, D.; Weiner, L.; Youdim, M. B. H.; Fridkin, M. A novel iron-chelating derivative of the neuroprotective peptide NAPVSIPQ shows superior antioxidant and antineurodegenerative capabilities. J. Med. Chem. 2008, 51 (1), 126−134. (29) Salehi, R.; Arami, M.; Mahmoodi, N. M.; Bahrami, H.; Khorramfar, S. Novel biocompatible composite (Chitosan−zinc oxide nanoparticle): Preparation, characterization and dye adsorption properties. Colloids Surf., B 2010, 80 (1), 86−93. (30) Perelshtein, I.; Ruderman, E.; Perkas, N.; Tzanov, T.; Beddow, J.; Joyce, E.; Mason, T. J.; Blanes, M.; Mollá, K.; Patlolla, A.; Frenkel, A. I.; Gedanken, A. Chitosan and chitosan−ZnO-based complex nanoparticles: Formation, characterization, and antibacterial activity. J. Mater. Chem. B 2013, 1 (14), 1968−1976. (31) Zhang, Z.; Zhou, F.; Liu, X.; Zhao, M. Particulate nanocomposite from oyster (Crassostrea rivularis) hydrolysates via zinc chelation improves zinc solubility and peptide activity. Food Chem. 2018, 258, 269−277. (32) Liu, F.; Wang, L.; Wang, R.; Chen, Z. Calcium-binding capacity of wheat germ protein hydrolysate and characterization of Peptidecalcium complex. J. Agric. Food Chem. 2013, 61 (31), 7537−7544. (33) Sharma, A. K.; Pavlova, S. T.; Kim, J.; Kim, J.; Mirica, L. M. The effect of Cu2+ and Zn2+ on the Aβ42 peptide aggregation and cellular toxicity. Metallomics 2013, 5 (11), 1529−1536. (34) Jiao, S. J.; Zhang, Z. Z.; Lu, Y. M.; Shen, D. Z.; Yao, B.; Zhang, J. Y.; Li, B. H.; Zhao, D. X.; Fan, X. W.; Tang, Z. K. ZnO p−n junction light-emitting diodes fabricated on sapphire substrates. Appl. Phys. Lett. 2006, 88 (3), 031911. (35) Gu, Z.-B.; Lu, M.-H.; Wang, J.; Wu, D.; Zhang, S.-T.; Meng, X.K.; Zhu, Y.-Y.; Zhu, S.-N.; Chen, Y.-F.; Pan, X.-Q. Structure, optical, and magnetic properties of sputtered manganese and nitrogencodoped ZnO films. Appl. Phys. Lett. 2006, 88 (8), 082111. (36) Wang, H.; Ho, H. P.; Lo, K. C.; Cheah, K. W. Preparation of ptype ZnO films with (N,Ga) co-doping by MOVPE. Mater. Chem. Phys. 2008, 107 (2), 244−247. (37) Ameen, S.; Akhtar, M. S.; Kim, Y. S.; Yang, O. B.; Shin, H. S. Polyaniline/gallium doped ZnO heterostructure device via plasma enhanced polymerization technique: Preparation, characterization and electrical properties. Microchim. Acta 2011, 172 (3−4), 471−478. (38) Arafat, Y.; Ali, S.; Shahzadi, S.; Shahid, M. Preparation, characterization, and antimicrobial activities of bimetallic complexes of sarcosine with Zn(II) and Sn(IV). Bioinorg. Chem. Appl. 2013, 2013 (1), 351262. (39) Sun, N.; Jin, Z.; Li, D.; Yin, H.; Lin, S. An exploration of the calcium-binding mode of egg white peptide, Asp-His-Thr-Lys-Glu, and in vitro calcium absorption studies of peptide-calcium complex. J. Agric. Food Chem. 2017, 65, 9782−9789. (40) Choi, S.; Cui, C.; Luo, Y.; Kim, S. H.; Ko, J. K.; Huo, X.; Ma, J.; Fu, L. W.; Souza, R. F.; Korichneva, I.; Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1. FASEB J. 2018, 32 (1), 404−416.

(8) Chen, Q.; Guo, L.; Du, F.; Chen, T.; Hou, H.; Li, B. The chelating peptide (GPAGPHGPPG) derived from Alaska pollock skin enhances calcium, zinc and iron transport in Caco-2 cells. Int. J. Food Sci. Technol. 2017, 52 (5), 1283−1290. (9) Udechukwu, M. C.; Collins, S. A.; Udenigwe, C. C. Prospects of enhancing dietary zinc bioavailability with food-derived zinc-chelating peptides. Food Funct. 2016, 7 (10), 4137−4144. (10) Martins, S. I. F. S.; Jongen, W. M. F.; van Boekel, M. A. J. S. A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci. Technol. 2000, 11 (9−10), 364−373. (11) Liu, Z.; Zhou, X.; Qian, Y. Synthetic methodologies for carbon nanomaterials. Adv. Mater. 2010, 22 (17), 1963−1966. (12) Sk, M. P.; Jaiswal, A.; Paul, A.; Ghosh, S. S.; Chattopadhyay, A. Presence of amorphous carbon nanoparticles in food caramels. Sci. Rep. 2012, 2 (1), 383. (13) Jiang, C.; Wu, H.; Song, X.; Ma, X.; Wang, J.; Tan, M. Presence of photoluminescent carbon dots in Nescafé® original instant coffee: Applications to bioimaging. Talanta 2014, 127, 68−74. (14) Liao, H.; Jiang, C.; Liu, W.; Vera, J. M.; Seni, O. D.; Demera, K.; Yu, C.; Tan, M. Fluorescent nanoparticles from several commercial beverages: Their properties and potential application for bioimaging. J. Agric. Food Chem. 2015, 63 (38), 8527−8533. (15) Li, Y.; Bi, J.; Liu, S.; Wang, H.; Yu, C.; Li, D.; Zhu, B. W.; Tan, M. Presence and formation of fluorescence carbon dots in a grilled hamburger. Food Funct. 2017, 8 (7), 2558. (16) Song, X.; Cao, L.; Cong, S.; Song, Y.; Tan, M. Characterization of endogenous nanoparticles from roasted chicken breasts. J. Agric. Food Chem. 2018, 66 (28), 7522−7530. (17) Li, D.; Na, X.; Wang, H.; Xie, Y.; Cong, S.; Song, Y.; Xu, X.; Zhu, B.-W.; Tan, M. Fluorescent carbon dots derived from maillard reaction products: Their properties, biodistribution, cytotoxicity, and antioxidant activity. J. Agric. Food Chem. 2018, 66 (6), 1569−1575. (18) Sun, N.; Cui, P.; Jin, Z.; Wu, H.; Wang, Y.; Lin, S. Contributions of molecular size, charge distribution, and specific amino acids to the iron-binding capacity of sea cucumber (Stichopus japonicus) ovum hydrolysates. Food Chem. 2017, 230, 627−636. (19) Hola, K.; Zhang, Y.; Wang, Y.; Giannelis, E. P.; Zboril, R.; Rogach, A. L. Carbon dotsEmerging light emitters for bioimaging, cancer therapy and optoelectronics. Nano Today 2014, 9 (5), 590− 603. (20) Hill, S. A.; Benito-Alifonso, D.; Morgan, D. J.; Davis, S. A.; Berry, M.; Galan, M. C. Three-minute synthesis of sp3 nanocrystalline carbon dots as non-toxic fluorescent platforms for intracellular delivery. Nanoscale 2016, 8 (44), 18630−18634. (21) Liu, S.; Tian, J.; Wang, L.; Zhang, Y.; Qin, X.; Luo, Y.; Asiri, A. M.; Al-Youbi, A. O.; Sun, X. Hydrothermal treatment of grass: A lowcost, green route to nitrogen-doped, carbon-rich, photoluminescent polymer nanodots as an effective fluorescent sensing platform for label-free detection of Cu(II) ions. Adv. Mater. 2012, 24 (15), 2037− 2041. (22) Tan, J.; Zhang, J.; Li, W.; Zhang, L.; Yue, D. Synthesis of amphiphilic carbon quantum dots with phosphorescence properties and their multifunctional applications. J. Mater. Chem. C 2016, 4 (42), 10146−10153. (23) Wang, H.; Xie, Y.; Liu, S.; Cong, S.; Song, Y.; Xu, X.; Tan, M. Presence of Fluorescent Carbon Nanoparticles in Baked Lamb: Their Properties and Potential Application for Sensors. J. Agric. Food Chem. 2017, 65 (34), 7553−7559. (24) Cao, L.; Song, X.; Song, Y.; Bi, J.; Cong, S.; Yu, C.; Tan, M. Fluorescent nanoparticles from mature vinegar: Their properties and interaction with dopamine. Food Funct. 2017, 8 (12), 4744−4751. (25) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M. Full-color lightemitting carbon dots with a surface-state-controlled luminescence mechanism. ACS Nano 2016, 10 (1), 484−491. (26) Houser, R. P.; Fitzsimons, M. P.; Barton, J. K. Metal-dependent intramolecular chiral induction: The Zn2+ complex of an ethidiumpeptide conjugate. Inorg. Chem. 1999, 38 (6), 1368−1370. 7004


Hydrophilic Food-Borne Nanoparticles from Beef Broth as Novel

Recommend Documents