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

well. The grid feeding layer could greatly improve the 3.4. Metabolic profile analysis of EF-EBN and EBN
survival rate of loading cells, and improve the nutrition To comprehensively examine whether the cell-produced
exchange of cells in the center, which is more conducive metabolites in TeeBN could, in full or part, resemble the
to the release of active ingredients. More importantly, bioactive ingredients of natural EBN, we performed an
the 0.4-μm pore membrane could effectively inhibit cells’ ultra-performance liquid chromatography-quadrupole
absconding from feeding layer into receiving layer. time-of-flight mass spectrometry (UPLC-QTOFMS)-
based metabolomics analysis. The outcomes identified
Next, we characterized the receiving layer, which 601 metabolites in the engineered TeeBN and the high-
would become the final and edible product of TeeBN, quality natural EBN-I (Venn diagram, Figure 4A). Among
in comparison to natural EBN. Scanning electron them, 265 metabolites were shared between two groups,
microscopy (SEM) revealed both had a porous network demonstrating most EBN ingredients (85.76%) being
structure (Figure 3D and E), which is a key indicator in produced in TeeBN. A PCA further revealed the differences
EBN authentication. Living cells were not present in the (Figure 4B). The distribution areas of TeeBN and EBN
receiving layer, and no nucleic acid was found in the were separated, indicating significant differences in the
receiving layer (Figure S1E), both confirming there was compositions. The PLS-DA model further highlighted the
no cell leaking from the feeding layer, thus eliminating the difference (Figure S2A), followed by a permutation test
concern of exogenous animal cells in the edible product. preventing the model from overfitting (Figure S2B). Based
We determined the main nutritional substances and on the available model, the PLS-DA S-plot identified the
potentially toxic substances in TeeBN against two major, characteristic metabolites between TeeBN and the natural
high-quality natural EBN products from Indonesia (EBN-I) EBN-I, located in the lower-left and upper-right corner
and Vietnam (EBN-V), respectively. TeeBN contained and in total 222 (Figure 4C).
more total proteins (65.32 ± 1.47%) than EBN-V (60.32 ± Further analysis into the top 19 discriminating
2.57%, P < 0.01) and EBN-I (56.68 ± 1.21%, P < 0.0001; metabolites between TeeBN and EBN-I demonstrated
Figure 3F). Also, TeeBN had a significantly higher content that 18 were higher in the former (Figure 4D and
of EGF (6.82 ± 0.17 mg/g) than EBN-V and EBN-I (2.03 ± Figure S2C). Notably, two discriminating metabolites,
0.11 mg/g and 3.84 ± 0.05 mg/g, P < 0.0001, respectively; N-Acetyl-9-O-acetylneuraminic acid and N-Acetyl-7-
Figure 3G), which is both a main nutritional factor and a O-acetylneuraminic acid, belong to sialic acids, which
key marker in industrial EBN quality control. Further, the echoed with the above data on determining sialic acids,
level of sialic acid in TeeBN (61.43 ± 1.23 mg/g) was higher which is a key quantitative index for assessing EBN quality
than in EBN-V (45.28 ± 2.20 mg/g, P < 0.0001) and lower (Figure 4E–I). Finally, a full picture of the metabolic
than in EBN-I (97.21 ± 1.15 mg/g, P < 0.0001; Figure 3H). pathways of TeeBN and natural EBN-I was analyzed. As
For TeeBN, the concentration of the active ingredients shown in Figure 4J, TeeBN mainly influenced the pathways
could be further adjusted by changing the cell number of metabolism, organismal system, and environmental
in the feeding layer, providing extra controllability and information processing pathway (P < 0.05 and pathway
convenience for product design.
impact >0), with significant influences on amino acid
Natural EBNs are inevitably contaminated with fungi, metabolism, the digestive system, biosynthesis of other
spores, nitrites, and heavy metals, and these substances secondary metabolites, and membrane transport. In
are often detected in marketed products, including nitrite summary, TeeBN shared many nutritional factors including
content 100-fold exceeding the safety standard in the vitamins, amino acids, monosaccharides, cofactors, fatty
so-called “rarest” blood EBN . Fabricating TeeBN can acids, phospholipids, nucleosides, and carboxylic acids .
[36]
[8]
avoid these risks completely. Nitrate was non-detectable Its similarity with natural EBN-I in metabolite patterns
in TeeBN, in sharp contrast to 5.45 ± 0.21 mg and 4.82 ± suggested that TE-based production by epithelial cells
0.29 mg per kg in EBN-V and EBN-I, respectively resembled the natural process.
(Figure 3I). Almost no or only trace amounts of As, Pb,
and Cu (all <0.5 mg/kg) were found in TeeBN, but were 3.5. Blood serum metabolomics
also present in considerable levels in both EBN-V and Finally, we tested in mice how TeeBN as a potential food
EBN-I. Notably, among them, the content of As and Pb in was metabolized in vivo by examining the blood serum
EBN-V reached 2.75 ± 0.03 mg/kg and 2.43 ± 0.21 mg/kg, metabolomics in mice (Figure 5A). In total, 10,625 peaks
respectively, which were 174.8% and 21.2% higher than were detected in the control, TeeBN and natural EBN groups.
the Bird’s nest product verification implementation rules Among them, 185 metabolites were identified in comparison
(CAIQ-RZ-2015002-7) issued by the China Academy of to established databases (Figure 5B). PCA revealed high
inspection and quarantine (Figure 3J–L) . similarity among the profiles of serum metabolites of these
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Volume 9 Issue 5 (2023) 9 https://doi.org/10.18063/ijb.691