Page 13 - IJB-9-5

International Journal of Bioprinting 3D printed edible bird’s nests

potassium iodide was added and reacted for 4 min, sodium of 2 μL. The column temperature was maintained at 40°C.
thiosulfate was dropped until the color turned from yellow All samples were stored at 4°C during the analysis period.
to light yellow, then 4 mL of 0.5 % starch solution was The mass spectrometer was performed in negative
added dropwise, and sodium hyposulfite was continuously mode with an electrospray ionization (ESI) ion source.
added until the blue color turned to colorless. Finally, the The optimal methods were as follows: heater temperature,
nitrite content in the sample was calculated and obtained. 425°C; sheath gas flow rate, 50 arb (arbitrary unit); aux

2.12. Determination of sialic acid, EGF, and gas flow rate, 13 arb; ion-spray voltage floating (ISVF),
total proteins -3,500 V in negative mode and 3,500 V in positive mode;
EBN and TeeBN with a dry weight of 1 g was weighed normalized collision energy, 20 to 40 to 60 V rolling for
and dissolved in 1 mL of deionized water. Sialic acid MS/MS. Data acquisition was performed from the scan 70
quantitation was performed by Sialic Acid Assay Kit to 1050 m/z.
(Sigma Aldrich, Burlington, USA). Briefly, following the The UPLC-MS data were analyzed using progenesis
improved Warren’s method, sialic acid was calculated by QI software (Waters, Milford, USA), with the positive
colorimetric (549 nm)/ fluorometric (λex = 555 nm/ λem = and negative data combined. Metabolites were identified
585 nm) . based on the Human Metabolome Database (HMDB)
[22]
Mouse EGF concentrations were determined by the and progenesis QI. Venn plots and heatmaps were used to
enzyme-linked immunosorbent assay kit (ELISA; Cusabio, visualize the significantly changed (Student’s t-test, P < 0.05)
Wuhan, China). Briefly, culture supernatants of the feeding metabolites (SCMs) between the EBN and TeeBN groups.
layer or EBN and TeeBN were collected and transferred Principal component analysis (PCA) and partial least
to anti-EGF-coated wells. Working buffer was added squares-discriminate analysis (PLS-DA) were conducted
according to the protocol and absorbance (450 nm) was to identify the SCMs among different groups, with a false
measured using the microplate reader (Molecular Devices, discovery rate (FDR) of <0.01 and variable influence on
San Jose, USA). projection (VIP) of >1. Pathway analysis was performed
using the Kyoto Encyclopedia of Gene Genotype (KEGG).
Total protein was quantitated by the BCA protein
assay kit (Beyotime, Shanghai, China) . Each sample 2.14. Experimental animals and treatment
[16]
was treated with BCA working buffer for 2 h at room procedures
temperature and absorbance (562 nm) was measured using This study was reviewed and approved by the Ethics
the microplate reader (Molecular Devices, San Jose, USA). Committee of the University of Macau (UMARE-03-2017
and UMARE-006-2022). All protocols met the requirements
2.13. Metabolomics of the National Institutes of Health (NIH) Guide for the
EBN and TeeBN samples for metabolomics analysis were Care and Use of Laboratory Animals. Eighteen C57BL/6
sourced from Indonesia [23,24] . Methanol-water solution mice (8 weeks, male) were randomly divided into three
(400 μL) was added to a 100 μL of EBN or TeeBN sample. groups (n = 6/group): (i) control group, treated by gavage
The mixture then went through a vortex for 30 s and with 0.2 mL of physiological saline; (ii) EBN group, treated
was sonicated at 40 kHz for 30 min at 5°C. Next, the by gavage with 0.2 mL of EBN solution; (iii) TeeBN group,
samples were placed at -20°C for 30 min to precipitate treated by gavage with 0.2 mL of TeeBN solution. All mice
proteins. After being centrifuged at 13,000 × g and 4°C had regular food and water. All mice received oral gavage
for 15 min, the supernatant was transferred for ultra-high for 7 consecutive days. Two hours after the last oral gavage,
performance liquid chromatography-MS/MS (UHPLC- all mice were anaesthetized and the blood was collected
MS/MS) analysis. A Thermo UHPLC system equipped from the eyes. The serum was obtained by centrifugation
with an ACQUITY BEH C18 column (length: 100 mm; (3500 × g, 10 min) at 4°C and stored at -80°C for use. The
inner diameter: 2.1 mm; grain diameter: 1.7 μm; Waters, preparation and analysis of serum samples for LC-MS were
Milford, USA) was used to perform chromatographic the same as in the EBN and TeeBN metabolomics.
separation of the metabolites. The mobile phase consisted
of 0.1 % (v/v) formic acid in water (phase A) and 0.1% (v/v) 2.15. Statistical analysis
formic acid in a mixture of acetonitrile and isopropanol Data are expressed as the mean ± standard deviation (SD).
(1:1, v/v) (phase B) at a flow rate of 0.40 mL/min. The All the experiments were conducted at least three times
elution gradient was used as: 0 to 3.0 min, 5% to 20% phase independently. Statistical significance was determined
B; 3.0 to 9.0 min, 20% to 95% phase B; 9.0 to 13.0 min, 95% using one-way and two-way analysis of variance (ANOVA)
phase B; 13.0 to 13.1 min, 95% to 5% phase B; 13.1 to 16 with Tukey’s post hoc multiple comparisons tests in
min, 5% phase B. The samples were injected at a volume GraphPad Prism 8 (San Diego, USA). In the metabolomics

Volume 9 Issue 5 (2023) 5 https://doi.org/10.18063/ijb.691