Evaluating the Nutritional Composition of Unripe Citrus and Its Effect on Inhibiting Adipogenesis and Adipocyte Differentiation.

Citrus fruits offer a range of health benefits due to their rich nutritional profile, including vitamin C, flavonoids, carotenoids, and fiber. It is known that unripe citrus has higher levels of vitamin C, dietary fiber, polyphenols, and flavonoids compared to mature fruits. In this study, we assessed the nutritional components of unripe citrus peel and pressed juices, as well as their anti-obesity potential through the modulation of adipocyte differentiation and the expression of adipogenesis-related genes, specifically PPARγ and C/EBPα, in 3T3-L1 preadipocytes. Our analysis revealed that unripe citrus peel exhibited elevated levels of fiber and protein compared to pressed juice, with markedly low levels of free sugar, particularly sucrose. The content of hesperidin, a representative flavonoid in citrus fruits, was 3,157.6 mg/kg in unripe citrus peel and 455.5 mg/kg in pressed juice, indicating that it was approximately seven times higher in unripe citrus peel compared to pressed juice. Moreover, we observed that the peel had a dose-dependently inhibitory effect on adipocyte differentiation, which was linked to a significant downregulation of adipogenesis-related gene expression. Thus, our findings suggest that unripe citrus possesses anti-obesity effects by impeding adipogenesis and adipocyte differentiation, with the peel demonstrating a more pronounced effect compared to pressed juice.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Characterization and structural analysis of the endo-1,4-ß-xylanase GH11 from the hemicellulose-degrading Thermoanaerobacterium saccharolyticum useful for lignocellulose saccharification.

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-ß-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg-1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL-1 and 34,015.3 s-1, respectively, resulting in kcat/Km value of 2658.7 mL mg-1 s-1, higher by 102-103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a ß-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation.

Physicochemical and Functional Properties of Yeast-Fermented Cabbage.

Microbial fermentation is often used to improve the functionality of plant-based food materials. Herein, we investigated changes in the physicochemical and functional properties of cabbage during yeast fermentation to develop new products using fermented cabbage. Among the 8 types of food-grade yeast, both Saccharomyces cerevisiae and Saccharomyces boulardii fermented 10% cabbage powder solution (w/w) the most effectively, leaving no soluble sugars after 12 h of fermentation. In addition, the yeast fermentation of cabbage resulted in functionally positive outcomes in terms of sulforaphane content, antioxidant properties, and anti-inflammatory activity. Specifically, the yeast-fermented cabbages contained about 500% more sulforaphane. The soluble fraction (5 µg/ml) of yeast-fermented cabbage had no cytotoxicity in murine RAW 264.7 cells, and the radical-scavenging capacity was equivalent to 1 µg/ml of ascorbic acid. Moreover, cabbage fermented with S. boulardii significantly suppressed both lipopolysaccharides (LPS)-induced nitric oxide production and LPS-induced reactive oxygen species production in RAW 264.7 cells, suggesting a potential anti-inflammatory effect. These results support the idea that yeast fermentation is promising for developing functionally improved cabbage products.

Dietary protocatechuic acid redistributes tight junction proteins by targeting Rho-associated protein kinase to improve intestinal barrier function.

Inflammatory bowel disease (IBD) is continuously increasing globally and caused by intestinal barrier dysfunction. Although protocatechuic acid (PCA) has a protective effect on colitis, the molecular mechanisms underlying its contribution to intestinal barrier function remain unknown. Transepithelial electrical resistance (TEER) and FITC-dextran permeability measurements reveled that PCA suppresses lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α-induced increase in intestinal permeability; zonula occludens (ZO)-1 and claudin-2 redistribution was also suppressed in the epithelial cell membranes of differentiated Caco-2 cells. PCA was found to directly bind Rho-associated coiled-coil containing protein kinase (ROCK), subsequently suppressing myosin light chain (MLC) phosphorylation. Notably, PCA binds ROCK to a similar degree as Y27632, a selective ROCK inhibitor. Orally administering PCA (5 or 25 mg per kg per day) to C57BL/6 mice alleviated the 3% dextran sulfate sodium (DSS)-induced colitis symptoms including reduced colon length, disrupted intestinal barrier structure, and increased proinflammatory cytokines expressions, such as interleukin (IL)-1ß, TNF-α, and IL-6. Furthermore, orally administering PCA suppressed DSS-induced ZO-1 and claudin-2/4 redistribution in mice colon membrane fractions. Therefore, PCA may serve as a promising nutraceutical to improve gut health and alleviate IBD by maintaining intestinal barrier function in vitro and in vivo.

The Ingestion of Dietary Prebiotic Alternatives during Lactation Promotes Intestinal Health by Modulation of Gut Microbiota.

Palm kernel expeller (PKE), a by-product of palm oil extraction, contains higher amounts of fiber than corn and soybean meal, but offers low energy density, protein value, and amino acid (AA) composition, limiting its use for swine. Recently however, it was reported that dietary fiber has a positive effect on the gut microbiota of the host, and therefore it is necessary to study the effect of PKE feeding on the intestinal microbiota of swine. In this study, we investigated the effects of supplementation with PKE in lactation diets on the gut microbiota composition of lactating sows and their litters. A total of 12 sows were randomly assigned to two dietary treatment groups in a completely randomized design. The treatments were a diet based on corn-soybean meal (CON) and CON supplemented with 20% of PKE. Sow and piglet fecal samples were collected before farrowing, on days 7 and 28 (weaning) after farrowing, and on days 7 and 28 (weaning) after farrowing, respectively, to verify gut microbiota composition by pyrosequencing analysis. The beta-diversity result showed a significant difference only in weaning-stage piglets, but dietary PKE altered the gut microbiota in sows by increasing the abundance of Lactobacillus compared with CON. In piglets, dietary PKE decreased the abundance of opportunistic pathogen Proteus and increased the abundance of potentially beneficial bacteria, such as Prevotellaceae and Prevotella. Our results can be helpful in developing feeding strategies and support the beneficial effects of dietary PKE to improve the gut health of animals.

Upstream processes of citrus fruit waste biorefinery for complete valorization.

Citrus fruit waste (CW) is a useful biomass and its valorization into fuels and biochemicals has received much attention. For economic feasibility, increased efficiency of the preceding extraction and enzyme saccharification processes is necessary. However, at present, there is a lack of systematic reviews addressing these two integral upstream processes in concert for CW biorefinery. Here, the state-of-the-art advancements in enzyme extraction and saccharification processes-using which relevant essential oils, flavonoids, and sugars can be obtained-are reviewed. Specifically, the extraction options for two commercially available CW-derived products, essential oils and pectin, are discussed. With respect to enzyme saccharification, the use of an undefined commercial mixture routinely results in suboptimal sugar production. In this respect, applicable strategies for enzyme mixture customization are suggested for maximizing the hydrolytic efficiency of CW. The enzyme degradation system for CW-derived carbohydrates and its extensive application for sugar production are also discussed.

Characterizing the effects of different chemicals on stem bending of cut snapdragon flower.

BACKGROUND: This study investigated the effects of ethylene release compounds (ethephon), ethylene-action inhibitors (silver thiosulfate: STS), and nitric oxide donor (sodium nitroprusside: SNP) on stem bending of snapdragon flowers. Moreover, the effects of plant growth supplements [6-benzyladenine (BA), gibberellic acid 3 (GA3), and calcium chloride (CaCl2)] on the stem bending were also extensively investigated. RESULTS: Ethephon completely prevented stem bending until 9 days after treatment (9 DAT). STS exhibited the highest bending rate, while SNP did not significantly affect the bending compared to the controls. The bending results were associated with the results of stem curvature, relative shoot elongation, ethylene production, and lignin content, that are involved in the stem bending mechanism. This was proven by the expression analysis of genes involved in ethylene and lignin biosynthetic pathways. The addition of plant growth supplements slightly or significantly delayed stem bending in the treatments (control, SNP, and STS) and significantly reduced petal senescence in ethephon at 9 DAT. CONCLUSION: These results show the preventive role of ethephon in the stem bending of cut snapdragon. Moreover, the combination of ethephon with supplements also provided information that could guide the development of strategies to delay stem bending in other cut flowers that undergo serious bending during a short vase life.

l-Lactic Acid Production Using Engineered Saccharomyces cerevisiae with Improved Organic Acid Tolerance.

Lactic acid is mainly used to produce bio-based, bio-degradable polylactic acid. For industrial production of lactic acid, engineered Saccharomyces cerevisiae can be used. To avoid cellular toxicity caused by lactic acid accumulation, pH-neutralizing agents are used, leading to increased production costs. In this study, lactic acid-producing S. cerevisiae BK01 was developed with improved lactic acid tolerance through adaptive laboratory evolution (ALE) on 8% lactic acid. The genetic basis of BK01 could not be determined, suggesting complex mechanisms associated with lactic acid tolerance. However, BK01 had distinctive metabolomic traits clearly separated from the parental strain, and lactic acid production was improved by 17% (from 102 g/L to 119 g/L). To the best of our knowledge, this is the highest lactic acid titer produced by engineered S. cerevisiae without the use of pH neutralizers. Moreover, cellulosic lactic acid production by BK01 was demonstrated using acetate-rich buckwheat husk hydrolysates. Particularly, BK01 revealed improved tolerance against acetic acid of the hydrolysates, a major fermentation inhibitor of lignocellulosic biomass. In short, ALE with a high concentration of lactic acid improved lactic acid production as well as acetic acid tolerance of BK01, suggesting a potential for economically viable cellulosic lactic acid production.

Comparative Metabolite Profiling of Traditional and Commercial Vinegars in Korea.

Vinegar, composed of various organic acids, amino acids, and volatile compounds, has been newly recognized as a functional food with health benefits. Vinegar is produced through alcoholic fermentation of various raw materials followed by acetic acid fermentation, and detailed processes greatly vary between different vinegar products. This study performed metabolite profiling of various vinegar products using gas chromatography-mass spectrometry to identify metabolites that are specific to vinegar production processes. In particular, seven traditional vinegars that underwent spontaneous and slow alcoholic and acetic acid fermentations were compared to four commercial vinegars that were produced through fast acetic acid fermentation using distilled ethanol. A total of 102 volatile and 78 nonvolatile compounds were detected, and the principal component analysis of metabolites clearly distinguished between the traditional and commercial vinegars. Ten metabolites were identified as specific or significantly different compounds depending on vinegar production processes, most of which had originated from complex microbial metabolism during traditional vinegar fermentation. These process-specific compounds of vinegars may serve as potential biomarkers for fermentation process controls as well as authenticity and quality evaluation.

Application of plant mucilage polysaccharides and their techno-functional properties' modification for fresh produce preservation.

The use of edible coating/film to improve fresh produce's quality and shelf life is an old but reliable and popular method of preservation. Recently, plant-derived mucilages have been extensively used to prepare edible packages (MEPs). This review focuses on recent studies that characterize mucilages from different plants, and examine their specific applications as edible packages in preserving fruits and vegetables. Structure-function relations and corresponding influence on film-forming properties are discussed. This review also surveys the additive-modifications of MEPs techno-functional properties. MEPs from a range of plant sources are effective in preventing quality loss and improving the storability of various fruits and vegetables. The preservative mechanisms and essential techno-functional properties of MEPs required for fruit and vegetable packaging were summarized. The key findings summarized in this study will help promote the utilization of mucilages and draw attention to other novel applications of this valuable polymer.

Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast.

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

Characterization of Cold-Tolerant Saccharomyces cerevisiae Cheongdo Using Phenotype Microarray.

The cold-tolerant yeast Saccharomyces cerevisiae is industrially useful for lager fermentation, high-quality wine, and frozen dough production. S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiae EC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlighted that the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4 gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrial fermentation rich in galactose, such as lactose and agarose.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Enhancement of catabolite regulatory genes in Saccharomyces cerevisiae to increase ethanol production using hydrolysate from red seaweed Gloiopeltis furcata.

Glucose and galactose are monosaccharides obtained from Gloiopeltis furcata (Red algae). A total monosaccharide yield of 62.3 g/L was obtained from G. furcata using thermal acid hydrolysis and enzymatic saccharification. Activated carbon was used to eliminate hydroxymethylfurfural (HMF) from the hydrolysate. Previously obtained monosaccharides are used for ethanol production by Saccharomyces cerevisiae. S. cerevisiae consumes glucose first, then galactose. The methods for reducing fermentation time and increasing the ethanol yield coefficient using the simultaneous consumption of glucose and galactose have been evaluated. Gal3p and Gal80p of S. cerevisiae act as signal transducers that govern the galactose inducer Gal4p mediated transcriptional activation of the Gal gene family. Gal80p binds to Gal4p for transcription deactivation. Therefore, Gal80p was deleted for Gal4p expression without interruption.

Recent advances in the biological valorization of citrus peel waste into fuels and chemicals.

In the quest to reduce global food loss and waste, fruit processing wastes, particularly citrus peel waste (CPW), have emerged as a promising and sustainable option for biorefinery without competing with human foods and animal feeds. CPW is largely produced and, as recent studies suggest, has the industrial potential of biological valorization into fuels and chemicals. In this review, the promising aspects of CPW as an alternative biomass were highlighted, focusing on its low lignin content. In addition, specific technical difficulties in fermenting CPW are described, highlighting that citrus peel is high in pectin that consist of non-fermentable sugars, mainly galacturonic acid. Last, recent advances in the metabolic engineering of yeast and other microbial strains that ferment CPW-derived sugars to produce value-added products, such as ethanol and mucic acid, are summarized. For industrially viable CPW-based biorefinery, more studies are needed to improve fermentation efficiency and to diversify product profiles.

Overcoming therapeutic efficiency limitations against TRAIL-resistant tumors using re-sensitizing agent-loaded trimeric TRAIL-presenting nanocages.

Tumor-specific apoptosis-inducing ligands have attracted considerable attention in cancer therapy. But, the evasion of apoptosis by tumors can cause acquired resistance to the therapy. TNF-related apoptosis-inducing ligand (TRAIL) has been investigated as an ideal antitumor agent owing to its inherent tumor cell-specific apoptotic activity. However, there are several barriers to its wider application, including the inability for stable formation of the trimeric structure, poor stability and pharmacokinetics, and differences in the sensitivity of different tumor types. Especially, almost 70% of tumor cells have acquired resistance to TRAIL, leading to failure of TRAIL-based therapeutics in clinical trials. To overcome therapeutic efficiency limitations against TRAIL-resistant tumors, we exploited the characteristic of a naturally derived nanocage that not only delivers TRAIL in its native-like trimeric structure, but also delivers a drug (doxorubicin [DOX]) that re-sensitizes TRAIL-resistant tumor cells. These TRAIL-presenting nanocages (TTPNs) showed high loading efficiency, pH-dependent release profiles, and effective intracellular delivery of the re-sensitizing agent DOX. As a result, DOX-TTPNs efficiently re-sensitized TRAIL-resistant tumor cells to TRAIL-mediated apoptosis in vitro by regulating levels of the TRAIL receptor, DR5, and anti- and pro-apoptotic proteins involved in extrinsic and intrinsic apoptosis pathways. We further demonstrated the antitumor efficacy of DOX-TTPNs in vivo, showing that even at a very low dose, the incorporated DOX successfully re-sensitized tumors to the apoptotic effects of TRAIL, underscoring the potential of this platform as an antitumor agent. Given that other homotrimeric TNF superfamily ligands and immunotherapeutic agents can be substituted for TRAIL ligand and re-sensitizing drugs on the surface and in the inner cavity of the nanocage, respectively, this platform is potentially suitable for development of a broad range of anticancer or immunotherapeutic combinations.

Designed protein- and peptide-based hydrogels for biomedical sciences.

Proteins are fundamentally the most important macromolecules for biochemical, mechanical, and structural functions in living organisms. Therefore, they provide us with diverse structural building blocks for constructing various types of biomaterials, including an important class of such materials, hydrogels. Since natural peptides and proteins are biocompatible and biodegradable, they have features advantageous for their use as the building blocks of hydrogels for biomedical applications. They display constitutional and mechanical similarities with the native extracellular matrix (ECM), and can be easily bio-functionalized via genetic and chemical engineering with features such as bio-recognition, specific stimulus-reactivity, and controlled degradation. This review aims to give an overview of hydrogels made up of recombinant proteins or synthetic peptides as the structural elements building the polymer network. A wide variety of hydrogels composed of protein or peptide building blocks with different origins and compositions - including ß-hairpin peptides, α-helical coiled coil peptides, elastin-like peptides, silk fibroin, and resilin - have been designed to date. In this review, the structures and characteristics of these natural proteins and peptides, with each of their gelation mechanisms, and the physical, chemical, and mechanical properties as well as biocompatibility of the resulting hydrogels are described. In addition, this review discusses the potential of using protein- or peptide-based hydrogels in the field of biomedical sciences, especially tissue engineering.

Characterization of an Antibacterial Agent Targeting Ferrous Iron Transport Protein FeoB against Staphylococcus aureus and Gram-Positive Bacteria.

The emergence of multidrug-resistant Staphylococcus aureus strains has become a serious clinical problem. Iron is absolutely required for the bacterial growth, virulence associated with colonization, and survival from the host immune system. The FeoB protein is a major iron permease in bacterial ferrous iron transport systems (Feo) that has been shown to play a crucial role in virulence of some pathogenic bacteria. However, FeoB is still uncharacterized in Gram-positive pathogens, and its effects on S. aureus pathogenesis are unknown. In this study, we identified a novel inhibitor, GW3965·HCl, that targets FeoB in S. aureus. The molecule effectively inhibited FeoB in vitro enzyme activity, bacterial growth, and virulence factor expression. Genome-editing and metabolomic analyses revealed that GW3965·HCl inhibited FeoB function and affected the associated mechanisms with reduced iron availability in S. aureus. Gentamicin resistance and Caenorhabditis elegans infection assays further demonstrated the power of GW3965·HCl as a safe and efficient antibacterial agent. In addition to S. aureus, GW3965·HCl also presented its effectiveness on inhibition of the FeoB activity and growth of Gram-positive bacteria. This novel inhibitor will provide new insight for developing a next-generation antibacterial therapy.