Peyer's patch-mediated intestinal immune system modulating activity of pectic-type polysaccharide from peel of Citrus unshiu.

An intestinal immune system modulating polysaccharide (CUI-3IIb-3-2, 18kDa) was purified from Citrus unshiu peel. CUI-3IIb-3-2 mainly comprised GalA, GlcA, Ara, Gal and Rha, and it consisted of 4-linked GalA, terminal Araf, 4- or 5-linked/3,4- or 3,5-branched Ara, terminal Gal, and 2-linked/2,4-branched Rha. After CUI-3IIb-3-2 digestion by endo-α-d-(1→4)-polygalacturonase, its hydrolysate was fractionated into PG-1 and PG-2. Methylation analyses of PG-1 and PG-2 using base-catalysed ß-elimination suggested that CUI-3IIb-3-2 be assumed as pectic-type polysaccharide. Since the activities of PG-1 and PG-2 were potently decreased, the whole polysaccharide structure of CUI-3IIb-3-2 would be essential to maintain the activity. Meanwhile, when CUI-3IIb was orally administered in mice, bone marrow cell proliferation and GM-CSF/IL-6 production from Peyer's patch cell were significantly higher (1.76- and 2.03/2.51-fold, respectively) than a saline. Therefore, a pectic-type polysaccharide from citrus peel could stimulate Peyer's patches and produce hematopoietic growth factors resulted in bone marrow cell proliferation.

Anti-complementary activity of enzyme-treated traditional Korean rice wine (Makgeolli) hydrolysates.

BACKGROUND: Makgeolli brewed from rice contains about 150 g kg(-1) alcohol and has a fragrance as well as an acidic and sweet taste. During the brewing process, by-products such as rice bran and brewery cake are produced. At the end of fermentation the matured mash is transferred to a filter cloth and the Makgeolli is squeezed out from the cake, leaving the lees of the mash. These by-products have continued to increase every year, resulting in an ecological problem. It is therefore important to develop new uses for them. The objective of this study was to use the by-products from the brewing of Makgeolli as a valuable functional food or nutraceutical. RESULTS: The anti-complementary activities of crude polysaccharides isolated from Cytolase hydrolysates of Makgeolli lees at concentrations of 1000 and 500 µg mL(-1) were 84.15 and 78.70% respectively. The activity of polysaccharide krestin (PSK) was 60.00% at 1000 µg mL(-1). The active polysaccharide obtained with Cytolase comprised mainly glucose and mannose (molar ratio 1.00:0.62). CONCLUSION: Glucose- and mannose-rich crude polysaccharides were isolated from the Cytolase hydrolysate of Makgeolli lees. The polysaccharides retain anti-complementary activity to enhance the immune system as a functional food or nutraceutical.

Novel tripeptides with α-glucosidase inhibitory activity isolated from silk cocoon hydrolysate.

Active compounds with antidiabetic potential were isolated from silk peptide E5K6 by consecutive ultrafiltration and gel filtration using Biogel P-2 and RS-HPLC using a YMC-Pack Pro C18 column. The highest α-glucosidase inhibitory activity of silk peptide E5K6 resulted from fractions with MW <1 kDa. The activities of gel-filtered fractions from silk peptide E5K6 of <1 kDa were assayed in vitro, demonstrating that the fourth peak (F4) had the highest α-glucosidase inhibitory activity (IC(50) = 37.1 mg/mL). F4 of silk peptide E5K6 was separated by HPLC into two peaks. Moreover, the purified compounds were identified as Gly-Glu-Tyr (GEY, MW = 367 Da) and Gly-Tyr-Gly (GYG, MW = 295 Da) according to amino acid sequences, and their α-glucosidase inhibitory activities (IC(50)) were 2.7 and 1.5 mg/mL, respectively.

Fermentation of Korean red ginseng by Lactobacillus plantarum M-2 and its immunological activities.

We investigated ginsenoside transformation by fermentation of red ginseng with Lactobacillus plantarum M-2. We also examined the anti-metastasis and immune-stimulating activities of EtOH extracts of fermented red ginseng (FRG-E) in animal and human subjects. Total sugar decreased from 85.5 mg mL(-1) to 44.1 mg mL(-1) with increasing culture time during the fermentation with L. plantarum M-2. Uronic acid content reached a maximum level (534.3 µg mL(-1)) at 3 days of fermentation and decreased thereafter. Ginsenoside metabolites increased from 4,637.0 to 7,581.1 µg mL(-1) after 4 days. The prophylactic intraperitoneal injection of FRG-E (500 µg mouse(-1)) inhibited lung metastasis about 81.1%, while the inhibitory effect against tumor metastasis by treatment of EtOH extract from non-fermented red ginseng (NFRG-E) was 66.9%. Immunoglobulin A (IgA) and G (IgG) levels in the serum of healthy subjects were higher after FRG-E administration than at baseline, whereas NFRG-E induced reductions of these variables related to immunity. At 1 week, the change in IgA level by FRG-E (5.14 mg mL(-1)) was significantly higher than that by NFRG-E (-14.50 mg mL(-1); p < 0.05). It was concluded that the immunological activities of FRG-E were higher than those of NFRG-E, indicating that fermentation helped enhance the immunological activities of red ginseng.

Glucose tolerance and antioxidant activity of spent brewer's yeast hydrolysate with a high content of Cyclo-His-Pro (CHP).

UNLABELLED: To elevate the Cyclo-His-Pro (CHP) content in yeast, the yeast hydrolysate that was obtained from enzymatic hydrolysis was subjected to various treatments. Flavourzyme-treated hydrolysate showed the highest CHP content (674.0 µg/g) among the various proteases treatments. Ultrafiltration was selected as the best method for concentrating CHP in yeast hydrolysate, based on the yields and CHP contents. In addition, we evaluated the radical scavenge and glucose tolerance of yeast hydrolysate with a high content of CHP. Yeast hydrolysate showed intense scavenging abilities of both 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals. The IC(50) values of yeast hydrolysate on DPPH and ABTS radicals were 1.9 and 0.9 mg/mL, respectively. There were significant differences in glucose level between the diabetes-control and yeast hydrolysate group at 30, 60, 90, and 120 min after injection in a type 1 diabetes model (P < 0.01). Also, there were significant differences in blood glucose levels between the 2 groups at 30, 60, and 100 min after injection in the type 2 diabetes group (P < 0.05). Therefore, it is possible to use the yeast hydrolysate with high levels of CHP as an antioxidative and/or antidiabetic material for the preparation of functional foods. PRACTICAL APPLICATION: This study tried to develop a material containing a high content of CHP using yeast for possible applications of this cyclic dipeptide in the therapy of metabolic disorders. The yeast hydrolysate prepared with Flavourzyme showed a high level of CHP. The hydrolysate with a high content of CHP showed high levels of radical scavenging activities and oral glucose tolerance activity. Therefore, it is possible to use the yeast hydrolysate with high levels of CHP as an antioxidative and/or antidiabetic material for the preparation of functional foods.

Protective effect of cyclo(his-pro) on streptozotocin-induced cytotoxicity and apoptosis in vitro.

Cyclo(His-Pro) (CHP) is a naturally occurring, cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). CHP was efficiently obtained from soybean meal by hydrolysis with flavourzyme and alcalase. In this study, the effects of CHP on streptozotocin (STZ)-induced beta-cell dysfunction and apoptosis were investigated in rat insulinoma cells (RINm5F) secreting insulin. When the RINm5F cells were treated with 2mM STZ, insulin secretion decreased to approximately 54% that of control cells. However, CHP treatment restored the insulin-secreting activity of RINm5F cells to approximately 71% that of the untreated control cells. Moreover, CHP significantly protected the cells from STZ-mediated cytotoxicity via reduction of nitric oxide (NO) production (2.3-fold) and lipid peroxidation (1.9-fold), which were induced by STZ. Moreover, CHP treatment also attenuated STZ-induced apoptotic events, such as activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in RINm5F cells, indicating that CHP could protect the cells from apoptotic cell death induced by oxidative stress of STZ by increasing the expression of an anti-apoptotic protein, Bcl-2. These results suggest that CHP could be a candidate material for a protective and therapeutic agent against STZ-mediated cytotoxicity and apoptosis.

Enhancement of anti-complementary and radical scavenging activities in the submerged culture of Cordyceps sinensis by addition of citrus peel.

To investigate the optimal conditions for the production of Cordyceps sinensis by the submerged culture method, glucosamine and exopolysaccharide (EPS) productivities were determined in culture broth containing different carbon sources, principally rice bran and citrus peel. An optimal medium composition (1.5% rice bran, 0.5% molasses, 3% CSL, 0.1% KH(2)PO(4), and 0.05% MgSO(4)) and the optimal condition (25 degrees C and 5-6 d culture time) for high EPS productivity with potent immune-stimulating activities were obtained. The addition of citrus peel to the culture of C. sinensis under the optimized conditions improved EPS productivity and glucosamine content. Furthermore, anti-complementary activity was higher (58.0-80.8%) using citrus peel as compared to no addition of citrus peel (48.2-68.7%). Antioxidant activity (AEAC value) of the citrus peel culture was high (284.3-384.6 mg/100g) compared to that of the culture without citrus peel (142.8-219.5mg/100g), indicating that the citrus peel helped enhance the anti-complementary and antioxidant activities of C. sinensis.

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556.

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.

The influence of the nucleotide sequences of random Shine-Dalgarno and spacer region on bovine growth hormone gene expression.

To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5'-coding regions and were induced in E. coli BL21(DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of 6.9% to 8.5% of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below -26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5'-end region of the bGH gene should be disrupted for the effective expression of bGH.

Purification and characterization of complement-activating acidic polysaccharides from the fruits of Capsicum annuum.

Hot water-soluble crude polysaccharide (HCAP-0) that was obtained from the fruits of Capsicum annuum showed potent anti-complementary activity. The activity was unchanged by pronase digestion, but decreased by periodate oxidation. The HCAP-0 was fractionated by DEAE ion-exchange chromatography to give two major fractions, HCAP-II and III. These two fractions were finally purified by gel filtration to give HCAP-IIa, HCAPIIIa1, and IIIa2 fractions that had high anti-complementary activities. The HCAP-IIIa1 and IIIa2 consisted of homogeneous polysaccharides. The anti-complementary activities were unaffected by treatment with polymyxin B, indicating that the modes of complement activation were not due to preexisting lipopolysaccharide. The molecular weight and sugar content of HCAP-IIIa2 had potent anti-complementary activity. The highest yields were 55 kDa and 75.9%, and the molar ratio of galactose (Ara:Gal, 1.0:4.6) was higher than other sugars. The crossed immuno-electrophoresis showed that both classical and alternative pathways were activated by HCAP-IIIa2.