Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Disruption of UDP-galactopyranose mutase expression: A novel strategy for regulation of galactomannan biosynthesis and monascus pigments secretion in Monascus purpureus M9.

The impact of the cell wall structure of Monascus purpureus M9 on the secretion of extracellular monascus pigments (exMPs) was investigated. To modify the cell wall structure, UDP-galactopyranose mutase (GlfA) was knocked out using Agrobacterium-mediated transformation method, leading to a significant reduction in the Galf-based polysaccharide within the cell wall. Changes in mycelium morphology, sporogenesis, and the expression of relevant genes in M9 were also observed following the mutation. Regarding MPs secretion, a notable increase was observed in six types of exMPs (R1, R2, Y1, Y2, O1 and O2). Specifically, these exMPs exhibited enhancement of 1.33, 1.59, 0.8, 2.45, 2.89 and 4.03 times, respectively, compared to the wild-type strain. These findings suggest that the alteration of the cell wall structure could selectively influence the secretion of MPs in M9. The underlying mechanisms were also discussed. This research contributes new insights into the regulation of the synthesis and secretion of MPs in Monascus spp..

Therapeutic potential of non-starch polysaccharides on type 2 diabetes: from hypoglycemic mechanism to clinical trials.

Non-starch polysaccharides (NSPs) have been reported to exert therapeutic potential on managing type 2 diabetes mellitus (T2DM). Various mechanisms have been proposed; however, several studies have not considered the correlations between the anti-T2DM activity of NSPs and their molecular structure. Moreover, the current understanding of the role of NSPs in T2DM treatment is mainly based on in vitro and in vivo data, and more human clinical trials are required to verify the actual efficacy in treating T2DM. The related anti-T2DM mechanisms of NSPs, including regulating insulin action, promoting glucose metabolism and regulating postprandial blood glucose level, anti-inflammatory and regulating gut microbiota (GM), are reviewed. The structure-function relationships are summarized, and the relationships between NSPs structure and anti-T2DM activity from clinical trials are highlighted. The development of anti-T2DM medication or dietary supplements of NSPs could be promoted with an in-depth understanding of the multiple regulatory effects in the treatment/intervention of T2DM.

Conformational Properties of Flaxseed Rhamnogalacturonan-I and Correlation between Primary Structure and Conformation.

The pectic polysaccharides extracted from flaxseed (Linum usitatissiumum L.) mucilage and kernel were characterized as rhamnogalacturonan-I (RG-I). In this study, the conformational characteristics of RG-I fractions from flaxseed mucilage and kernel were investigated, using a Brookhaven multi-angle light scattering instrument (batch mode) and a high-performance size exclusion chromatography (HPSEC) system coupled with Viscotek tetra-detectors (flow mode). The Mw of flaxseed mucilage RG-I (FM-R) was 285 kDa, and the structure-sensitive parameter (ρ) value of FM-R was calculated as 1.3, suggesting that the FM-R molecule had a star-like conformation. The Mw of flaxseed kernel RG-I (FK-R) was 550 kDa, and the structure-sensitive parameter (ρ) values ranged from 0.90 to 1.21, suggesting a sphere to star-like conformation with relatively higher segment density. The correlation between the primary structure and conformation of RG-I was further discussed to better understand the structure-function relationship, which helps the scale-up applications of pectins in food, pharmaceutical, or cosmetic industries.

In Vitro Fermentability of Soybean Oligosaccharides from Wastewater of Tofu Production.

Soybean oligosaccharides (SBOS) isolated from wastewater of tofu production were studied in terms of their structural characteristics and in vitro fermentation by human fecal inocula. Three sub-fractions named Z1 (14%), Z2 (13%), and Z3 (17%) were obtained by Sephadex G-15 column separation. Z1 contained mainly stachyose; Z2 and Z3 contained stachyose, raffinose, and sucrose with different relative percentages. The in vitro batch fermentation model of human intestinal bacteria including 0, 12, 24, and 48 h was used to investigate the fermentation characteristics of SBOS. According to the results, during the fermentation process, the molecular weight of oligosaccharides decreased significantly with increasing fermentation time, indicating that oligosaccharides could be utilized and degraded by the colonic microbiota. Furthermore, SBOS could significantly promote the production of short-chain fatty acids (SCFAs), especially acetic, propionic, and butyric acids. SBOS increased the abundance of Firmicutes, while that of Proteobacteria was decreased. Additionally, SBOS could promote the proliferation of Dialister, Bacteroides, and Akkermansia at the genus level. Therefore, SBOS can be potentially used as prebiotic promoting gut health.

Fungus Polygalacturonase-Generated Oligogalacturonide Restrains Fruit Softening in Ripening Tomato.

Fruit softening exacerbates mechanical damage incurred during shipping and handling and the increase in pathogen susceptibility. Here, oligogalacturonides (OGs) produced by fungal polygalacturonase (PG) delayed fruit softening in tomato and maintained fruit firmness at 8.37 ± 0.45 N at 13 d of storage, which was consistent with the fruit firmness level of 5 d in the control groups. From RNA sequencing data in line production of phytohormones, we confirmed ethylene and jasmonic acid signals, the MAPK signaling cascade, and calmodulin involved in the OG-mediated firmness response of whole fruit. SlPG2, SlPL3, and SlPL5 were the major contributing factors for fruit softening, and their expression decreased continuously upon OG application. Suppression of the expression of ethylene response factors using a virus-induced gene-silencing strategy revealed that SlERF6 was negatively involved in OG-restrained fruit softening. Taken together, these results indicated that fungal PG-generated OGs have potential application value in controlling tomato fruit softening.

Structural characterization and immunomodulatory activity of mycelium polysaccharide from liquid fermentation of Monascus purpureus (Hong Qu).

Alkaline extracted endopolysaccharides (MPS) from Monascus purpureus (Hong Qu) mycelium were successfully separated into four sub-fractions, namely MPS-1 (18.0 %), MPS-2 (27.1 %), MPS-3 (12.6 %) and MPS-4 (14.7 %), by DEAE-Cellulose column chromatography. By combining monosaccharide composition analysis, methylation analysis and 1D & 2D NMR, the structure of sub-fractions was systematically characterized. Both MPS-1 and MPS-2 were comprised of mannose, glucose and galactose in the molar ratio of 1.5:1.6:1.0 and 10.6:1.0:13.8, respectively. The backbone of them both consisted of 2-α-Manp with several different branched chains. However, MPS-1 contained glucose based sugar residues such as 3-Glcp and 4-Glcp which were not shown on MPS-2. The proposed structures of MPS-3 and MPS-4 were not obtained due to the fairly complex molecular structure and relatively low yield. Moreover, based on the RAW 264.7 cells model, MPS-2 could significantly promote cytokines secretion including IL-6, TNF-α, and IL-10 and improve expression levels of the related mRNA.

Fractionation, structural characteristics and immunomodulatory activity of polysaccharide fractions from asparagus (Asparagus officinalis L.) skin.

The physicochemical properties, structural features and structure-immunomodulatory activity relationship of pectic polysaccharides from the white asparagus (Asparagus officinalis L.) skin were systematically studied. Using sequential ethanol precipitation, five sub-fractions namely WASP-40, WASP-50, WASP-60, WASP-70 and WASP-80 with distinct degree of esterification (DE) and molecular weight (Mw) were obtained. The Mw and DE values were decreased with the increase of the ethanol concentrations. Structurally, although 4-α-D-GalpA was the dominant sugar residue in all fractions, the molar ratios were decreased, whereas other sugar residues including arabinose- and mannose-based sugar residues overall increased with the increase of ethanol concentration. In addition, the effects of sub-fractions on the RAW 264.7 cells indicated that pectic polysaccharides with the higher DE value showed a stronger immunomodulatory activity. Moreover, the structure-activity relationship was also discussed in this study, which extends the value-added application of asparagus and its processing by-products.

Polysaccharide from Pleurotus nebrodensis: Physicochemical, structural characterization and in vitro fermentation characteristics.

A high Mw (5012 kDa) polysaccharide (PNPS) from the fruiting body of Pleurotus nebrodensis was isolated using water extraction followed by ethanol precipitation. The structural characteristics and in vitro fermentation behaviors of this polysaccharide was investigated. Chemical composition analysis showed the total sugar content of PNPS was up to 97.20 ± 1.80 wt%. Monosaccharide composition analysis showed PNPS contained mainly glucose (89.22 ± 5.70 mol%) while small percentage of mannose (5.60 ± 0.74 mol%) and galactose (5.18 ± 0.33 mol%) were also detected. According to the linkage pattern analysis (methylation analysis), PNPS comprised mainly 4-ß-D-Glcp (58.90 mol%), while other residues including α-D-Glcp, 6-α-D-Galp, 3,6-α-D-Manp, 3-ß-D-Glcp and 6-α-D-Glcp were detected with a comparable amount. Combined with results from 1D and 2D NMR spectrum, a proposed structure of PNPS was presented. In vitro fermentation of PNPS by gut microbiota showed total SCFA production of all treatment groups was higher than negative control group (NC) significantly (p < 0.05) after 48 h of fermentation. The formation of SCFAs was mainly acetic acid, followed by propionic acid and butyric acid, and the pH was decreased from 6.95 to 4.70. After 72 h, the total sugar content decreased from 5.813 ± 0.87 mg/L to 0.23 ± 0.065 mg/L, and the molecular weight of PNPS decreased.

Structural characterisation and immunomodulatory activity of polysaccharides from white asparagus skin.

The physicochemical properties, structural features and immunomodulatory effects of the white asparagus (Asparagus officinalis L.) skin polysaccharides (WASP) were systematically studied. WASP showed a pectic-like structure with a relatively low degree of esterification (DE, 18%); the weight-average molecular weight (Mw) and intrinsic viscosity were 76.1 kDa and 13 mL/g, respectively. Structurally, the dominated sugar residue of WASP was 4-α-D-GalpA (39.7 mol%), while other residues including α-L-Araf, 3-α-L-Rhap, 2,4-α-L-Rhap, and 4-ß-D-Galp were also detected with a comparable amount. A proposed structure of WASP was also presented. Physiologically, WASP could modulate the immune response of RAW 264.7 macrophages through increasing the release of immune factors (IL-6, TNF-α and IL-10) and improving the expression of mRNA. To conclude, the pectic-like polysaccharides from white asparagus (Asparagus officinalis L.) skin could be potentially used as an immunomodulatory agent in functional food.

RQ3, A Natural Rebaudioside D Isomer, Was Obtained from Glucosylation of Rebaudioside A Catalyzed by the CGTase Toruzyme 3.0 L.

In this study, a monoglucosyl rebaudioside A product was isolated from the mixture of glucosylated rebaudioside A obtained from the most reported and industrial used cyclodextrin glycosyl transferase, Toruzyme 3.0 L (CGTase, Toruzyme 3.0 L). The molecular structure of the monoglucosyl rebaudioside A was characterized using LC-MS/MS and methylation analysis combined with 1D and 2D NMR, indicating that it is 13-[(2-O-(3-α-O-D-glucopyranosyl)-ß-D-glucopyranosyl-3-O-ß-D-glucopyranosyl-ß-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid ß-D-glucopyranosyl ester (also known as RQ3, which naturally exists in Stevia extract as an isomer of rebaudioside D). This study may help to further understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L in the aspect of molecule linkage pattern, and also benefit the application of the glucosylated rebaudiosides.