Fuu Sheu

BACKGROUND: Microbiota succession determines the flavor and quality of fermented foods. Quantitative PCR‐based quantitative microbiome profiling (QMP) has been applied broadly for microbial analysis from absolute abundance perspectives, transforming microbiota ratios into counts by normalizing 16S ribosomal RNA (16S rRNA) gene sequencing data with gene copies quantified by quantitative PCR. However, the application of QMP in fermented foods is still limited.RESULTS: QMP elucidated microbial succession of Taiwanese pickled cabbage. In the spontaneous first‐round fermentation (FR), the 16S rRNA gene copies of total bacteria increased from 6.1 to 10 log copies mL⁻¹. The dominant lactic acid bacteria genera were successively Lactococcus, Leuconostoc and Lactiplantibacillus. Despite the decrease in the proportion of Lactococcus during the succession, the absolute abundance of Lactococcus still increased. In the backslopping second‐round fermentation (SR), the total bacteria 16S rRNA gene copies increased from 7.6 to 9.9 log copies mL⁻¹. The addition of backslopping starter and vinegar rapidly led to a homogenous microbial community dominated by Lactiplantibacillus. The proportion of Lactiplantibacillus remained consistently around 90% during SR, whereas its absolute abundance exhibited a continuous increase. In SR without vinegar, Leuconostoc consistently dominated the fermentation.CONCLUSION: The present study highlights that compositional analysis would misinterpret microbial dynamics, whereas QMP reflected the real succession profiles and unveiled the essential role of vinegar in promoting Lactiplantibacillus dominance in backslopping fermentation of Taiwanese pickled cabbage. Quantitative microbiome profiling (QMP) was found to be a more promising approach for the detailed observation of microbiome succession in food fermentation compared to compositional analysis. © 2024 Society of Chemical Industry.

A cost-effective method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with stable-isotope dimethyl labelling was used for the determination of Gly m 6. The validation results revealed that the recoveries and precisions obtained from five spiked levels were in the ranges of 88.8–113.0% and 8.3–22.0%, respectively. The content and stability of the major soybean allergen Gly m 6 in various food processing procedures were evaluated by the quantification results of its surrogate signature peptide. The Gly m 6 content in soybean decreased by 42% after natto fermentation, and by 31% and 35% in pasteurised soymilk and sterilised soymilk, respectively, relative to the raw soymilk. Only 19% of Gly m 6 in raw soymilk was retained in the soymilk film. This study extended the feasibility of dimethyl labelling to soy-based food samples and examined the proteolysis of Gly m 6 in natto fermentation and its thermal instability.

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography– tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5–109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86–9.92%. The limit of quantification (LOQ) was set at 8 pg mL⁻¹ for SPA1-H and at 8 ng mL⁻¹ for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175–0.0515 mg g⁻¹ of Act d 1 and 0.0252–0.0556 mg g⁻¹ of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.

Cordyceps militaris is a dietary therapeutic fungus that is an important model species in Cordyceps research. In this study, we purified a novel protein from the fruit bodies of C. militaris and designated it as Cordyceps militaris protein (CMP). CMP has a molecular mass of 18.0 kDa and is not glycosylated. Interestingly, CMP inhibited cell viability in murine primary cells and other cell lines in a time- and dose-dependent manner. Using trypan blue staining and a lactate dehydrogenase release assay, we showed that CMP caused cell death in the murine hepatoma cell line BNL 1MEA.7R.1. Furthermore, the frequency of BNL 1MEA.7R.1 cells at the sub-G1 stage was increased by CMP. Apoptosis, as determined by Annexin V and propidium iodide analysis, indicated that CMP could mediate BNL 1MEA.7R.1 apoptosis, but not necrosis. After coincubation with CMP, a decrease in mitochondria potential was detected using 3,3'-dihexyloxacarbocyanine iodide. These results suggest that CMP is a harmful protein that induces apoptosis through a mitochondrion-dependent pathway. Stability experiments demonstrated that heat treatment and alkalization degraded CMP and further destroyed its cell-death-inducing ability, implying that cooking is necessary for food containing C. militaris.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4^−/− mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94–C111 and C95–C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Summary: T helper type 2 (Th2) cells, which produce interleukin‐4 (IL‐4), IL‐5 and IL‐13, control immunity to all forms of allergic inflammatory responses. Interleukin‐21 (IL‐21) reduces allergic symptoms in murine models and inhibits IL‐4‐induced IgE secretion by B cells. However, whether or not IL‐21 directly affects Th2 cells, which leads to reduced allergic symptoms, is unclear. In this study, we investigated the effects of IL‐21 on the differentiation and effector functions of Th2 cells. We found that IL‐21 reduced the number of differentiated Th2 cells and these Th2 cells showed a diminished Th2 cytokine production. Interleukin‐21 suppressed Th2 cytokine production of already polarized Th2 cells by down‐regulation of transcription factor GATA‐3. It also induced apoptosis of Th2 cells with decreased anti‐apoptotic factor Bcl‐2. Intranasal administration of IL‐21 at the beginning of ovalbumin (OVA) sensitization or before OVA challenge decreased Th2 cytokines in the bronchoalveolar lavage fluid of OVA/alum‐immunized allergic mice. In addition, the inhibitory effects of IL‐21 on Th2 effector functions can also be found in allergic patients. Our results demonstrate that IL‐21 suppresses the development of Th2 cells and functions of polarized Th2 cells. Hence, the administration of IL‐21 may be considered for use as a preventive and therapeutic approach when dealing with Th2‐mediated allergic diseases.

Edible fungus Poria cocos (Schw.) Wolf is a cooking material that has myriad health benefits. However, its active constituents have not been well-defined. We previously purified an immunomodulatory protein, PCP, from P. cocos and described its biochemical features and its ability to activate primary macrophage via TLR4. In this study, we cloned the gene of PCP and demonstrated its ability to activate Th1 response in cell cultures and in mice. The complete cDNA sequence of PCP consisted of 807 bp, which included a 579 bp coding sequence that encoded 194 amino acids. With the addition of co-stimulatory CD3/CD28 signals, PCP significantly increased the surface expression of CD44 and CD69 on effector T cells. PCP could also up-regulate T-bet and STAT4 expressions and IFN-γ and IL-2 secretions. Oral administration of PCP suppressed the production of both total and OVA-specific IgG1 in serum and enhanced the amounts of serum and OVA-specific IgG2a and Th1-related cytokine production in BALB/c splenocytes. In addition, oral administration of PCP significantly reduced IL-4 and IgE expressions in a murine model of atopic dermatitis. In conclusion, these results provide evidence that PCP could regulate mammalian immune cells and reveal their pharmaceutical potential in developing therapeutic strategies against Th2-mediated immune disorders.