Albumin is a crucial component of serum-free media, playing a significant role in ex vivo cell culture as a lipid carrier and antioxidant. However, purified albumin contains undefined substances, making it challenging to achieve clinical application standards for effector cell culture. This study used natural killer (NK)-92 cells as a model to investigate the effects of the albumin substitute replacing bovine serum albumin (BSA) on cell expansion and metabolism in an in-house-designed, chemically defined, serum-free medium. We selected polyvinyl alcohol (PVA), N-acetylcysteine (NAC), and methyl-β-cyclodextrin (M-β-CD) as an albumin substitute combination and optimized their concentrations by using response surface methodology. The optimized albumin substitute was named PVA-NAC-M-β-CD (PNM). After 8 days of culture, NK-92 cells cultured with the PNM exhibited phenotype and cytotoxic function comparable to cells cultured with different concentrations of BSA. The expansion fold was 89.22 ± 3.55, significantly higher than the 51.23 ± 6.57 observed in the 0.75 g/L BSA group (p < 0.05). Further verification of functions of PNM showed that intracellular fatty acid levels, cholesterol consumption rates, and the pSTAT5 level in the PNM group were significantly higher than those in the 0.75 g/L BSA group (p < 0.05). Reactive oxygen species levels remained controlled, and mitochondrial membrane potential was similar. These findings suggested that the PNM can effectively replace the functions of BSA as a fatty acid carrier, antioxidant, and, to some extent, a cholesterol carrier. This study provides insights for developing chemically defined media to prepare clinical-grade NK cells efficiently.

Budding yeast, Saccharomyces cerevisiae, adapts to low-temperature stress by altering the amounts and proportions of phospholipids, as well as the structures of acyl chains. To better understand this process, a data-independent acquisition-based lipidomics using liquid chromatography-quadrupole time-of-flight mass spectrometry method was developed to quantify acyl chain isomers in glycerolipids and phospholipids. This method distinguished lipid molecular species, such as phosphatidylcholine (PC) 16:1_18:0 and PC16:0_18:1, at the level of acyl chain isomers. These species were previously identified only at the total acyl chain level as PC 34:1 using the conventional data-dependent acquisition (DDA) method. Using this approach, 145 diacyl lipid molecular species were identified in S. cerevisiae at 30 °C, compared to only 101 identified using the conventional DDA method. Analysis of S. cerevisiae at 10 °C revealed 121 phospholipid and diacylglycerol (DG) molecular species and 260 triacylglycerol (TG) features as well as other 51 sphingolipids and sterol lipids. The results demonstrated unique increases in phosphatidylinositol (PI), phosphatidylethanolamine (PE), monomethyl (MM) PE, and TG containing both 16:1 and medium-chain fatty acids (MCFAs). The knockout strains ole1Δ and slc1Δ, lacking the desaturase that produces 16:1 and the acyltransferase favoring MCFAs, respectively, exhibited growth defects exclusively while growing at 10 °C. These findings suggest that S. cerevisiae regulates its lipid profiles to address low-temperature stress.

Oxidative stress, caused by excessive production of reactive oxygen species (ROS), plays a crucial role in the occurrence and development of various diseases. Monascin can scavenge ROS and alleviate oxidative stress but with a low fermentation rate and bioavailability. Here, we optimized the fermentation process to increase the production of monascin (508.6 U/mL), and then systematically characterized its structure via HPLC, HPLC-MS, 1H NMR, and 13C NMR. Additionally, we innovatively modified carboxymethylcellulose sodium with selenium (CMC-Se) to encapsulate monascin (monascin@CMC-Se), which can sensitively respond to ROS and release monascin to effectively scavenge excessive ROS. Besides, the monascin@CMC-Se can significantly increase the bioaccessibility of monascin and alleviate cellular oxidative stress by enhancing its cellular uptake rate. Collectively, our work provides proof-of-principle evidence that the CMC-Se can precise delivery of monascin to an oxidatively stressed environment with high resistance to gastric fluids, laying a foundation to overcome inflammation-related diseases in the colon.

As an important crop, bananas still encounter fruit quality and shelf-life problems that are affected by the ripening process. Improving postharvest technologies may effectively address these challenges, such as by studying the ripening mechanism of banana cultivars with a slow ripening process. A banana cultivar that exhibits this characteristic is Musa balbisiana cv. 'Pisang Klutuk Wulung' (BB Group) or Pisang Klutuk Wulung (PKW), which has a ripening duration of 14-28 days. However, the metabolomics study on the ripening mechanism of this banana is still limited. This study aimed to analyze metabolite changes in ethylene-treated Pisang Klutuk Wulung during ripening in comparison to commercial bananas (Cavendish). Both bananas were subjected to exogenous ethylene treatment and analyzed using gas chromatography-mass spectrometry to perform metabolite profiling throughout the ripening process. The principal component analysis showed sample separation based on the ripening stages and banana species in pulp and peel. Orthogonal projection to latent structure analysis suggested that metabolite changes accompanied the ripening stages. Potential metabolite markers that distinguished the ripening of PKW and Cavendish were found, such as quinic acid, inositol, and 2-aminoethanol. This study shows differences in metabolite profiles between these bananas, especially the metabolites involved in sugar metabolism, cell wall metabolism, stress response, and biosynthesis of aromatic compounds. This study provides novel insights into the metabolic changes occurring during PKW ripening, contributing to the improvement of banana postharvest strategies.

Mathematical modeling of complex multi-catalytic reaction pathways is a powerful tool not only for determining optimal operational conditions but also for gaining insights into the kinetic behavior of efficient yet product-sensitive enzymes. This approach was applied to the biooxidation of glycerol using oxidases, aided by hematin, a natural mimic of heme peroxidases and catalases, which acts as a scavenger for enzymatically produced H2O2. The bi-catalytic reaction system was coupled with the formation of a colored imine product from phenol and 4-aminoantipyrine, enabling spectrophotometric monitoring of reaction kinetics. Alcohol oxidase (AOX) from Komagataella pastoris (formerly Pichia pastoris) was submitted to this strategy to overcome some kinetic drawbacks previously observed with galactose oxidase (GAO) from Dactylium dendroides. Although both enzymes exhibited comparable oxidation yields, computer-aided kinetic analyses revealed significant differences in efficiency and sensitivity to H2O2. AOX demonstrated two orders of magnitude greater efficiency and a similarly higher affinity for glycerol. However, AOX showed a stronger tendency to bind H2O2 compared to hematin or GAO, as suggested by the ratio of the inhibition constant (kinh) to the activation constant (k1) for hematin. The kinetic model was also used to simulate optimal conditions for enhancing the stability of AOX and hematin. Reducing glycerol concentration or increasing hematin concentration improved AOX stability, while the addition of phenol helped preserve hematin. The predicted stabilities turn the AOX/hematin/phenol system promising for biosensing or analytical applications.

Sake brewed using the kimoto-style exhibits high antioxidant capacity and is expected to inhibit the deterioration of sake quality due to oxidation. However, the antioxidant capacity of the added lactic acid bacteria has not been explored. We aimed to screen the lactic acid bacterium, Leuconostoc mesenteroides, with excellent brewing and antioxidant capacity, to develop sake with high antioxidant capacity. Three Le. mesenteroides strains (19-2, 19-5, and 19-23) were selected from eight screened strains based on their alcohol intolerance, growth performance in koji extract, aroma compound, and low biogenic amine production. Among these, Le. mesenteroides 19-23 exhibited a significantly higher hydrophilic-oxygen radical absorbance capacity (H-ORAC) in the culture medium than the control strain, Le. mesenteroides NBRC102481. In a medium-scale sake brewing test, sake prepared with Le. mesenteroides 19-23 had a significantly higher H-ORAC value than that prepared using the sokujo-style (without Le. mesenteroides). Additionally, metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry identified ferulic acid, p-coumaric acid, 3-hydroxyanthranilic acid (3-HAA), and 6,8-thioctic acid in the main mash. High-performance liquid chromatography-based quantification revealed that antioxidants in the unrefined filtrates of the main mash prepared using the kimoto-style tended to be more abundant than in those prepared using the sokujo-style. Specifically, 3-HAA and ferulic acid were more concentrated in some unrefined filtrates of the kimoto-style than sokujo-style. In conclusion, the screened Le. mesenteroides strain demonstrated potential for brewing sake with high antioxidant capacity using the kimoto-style, offering a promising method for antioxidant-rich sake production.