Yuji Miyaguchi

Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats

Oyster (Crassostrea talienwhanensis Crosse) proteins were produced from fresh oyster and subsequently digested with pepsin. The separations were performed with a Sephadex LH-20 gel filtration chromatography and a RP-HPLC. A purified peptide with sequence Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (VVYPWTQRF) was firstly isolated and characterized from oyster protein hydrolysate and its ACE inhibitory activity was determined with IC50 value of 66μmol/L in vitro. Stability study for ACE inhibitory activity showed that the isolated nonapeptide had the good heat and pH stability and strong enzyme-resistant properties against gastrointestinal proteases. Kinetic experiments demonstrated that inhibitory kinetic mechanism of this peptide was non-competitive and its Km and Ki values were calculated. The yield of this peptide from oyster proteins was 8.5%. Furthermore, the oyster protein hydrolysate (fraction II), prepared by pepsin treatment firstly exhibited antihypertensive activity when it was orally administered to spontaneously hypertensive rat (SHR) at a dose of 20mg/kg. These results demonstrated that the hydrolysate from oyster proteins prepared by pepsin treatment could serve as a source of peptides with antihypertensive activity.

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.

Simple method for isolation of glyceraldehyde 3‐phosphate dehydrogenase and the improvement of myofibril gel properties

Porcine glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (G3PD) was prepared effectively by a combination of ethylene diamine tetra-acetate (EDTA) pretreatment and affinity purification. After salting out of porcine sarcoplasmic proteins (SP) with ammonium sulfate at 75% saturation, the obtained supernatant (SP-f3) was treated with EDTA, leaving G3PD in the supernatant (G3PD-E) and most other SPs in the precipitate. At that time, the separation of G3PD-E required more than 20 mmol/L EDTA. G3PD-E was then subjected to affinity purification by batchwise method using blue-sepharose CL-6B, and purified G3PD (G3PD-AP) was obtained using 2 mol/L potassium chloride (KCl) as an eluent. Texture analysis showed that the hardness, adhesiveness and gumminess of the myofibril gel at 0.2-mol/L NaCl increased with the addition of G3PD-AP. Scanning electron microscopy revealed that the G3PD-AP reinforced the gel network of the myofibril. However, scanning electron micrograph analysis showed that the network-structure of the gel by the addition of G3PD-AP developed in a different manner from that by adding 0.6 mol/L NaCl. These results showed that glycolytic enzyme, G3PD, contributes to the improvement of the rheological properties of meat products.

Metabolomic analyses of plasma and liver of mice fed with immature Citrus tumida peel

Supplementing food with functional small molecules has been shown to prevent diseases and improve the quality of life, especially in elderly people. Citrus fruits and citrus fruit-products are popular food supplements across the world. In this study, we focused on a Japanese citrus fruit, Citrus tumida hort. ex Tanaka (C. tumida), and elucidated the effects of supplementation of the peels of immature C. tumida (PIC) on food intake, body and fat tissue weights, and metabolic profiles of plasma and liver in mice. Supplementation with 5% (w/w) PIC for 4 weeks significantly suppressed body weight gain and decreased adipose tissue weight, including that of the epididymal, perirenal, and subcutaneous fats. Metabolome analyses using capillary electrophoresis time-of-flight mass spectrometry showed that the level of 2-hydroxyvaleric acid was reduced in the blood plasma of mice fed with PIC. Supplementation with PIC significantly elevated the levels of dipeptides (Thr-Asp, Ser-Glu, and Ala-Ala), glucuronic acid (and/or galacturonic acid-2), and S-methylglutathione, and significantly reduced the levels of betaine aldehyde in the liver. In conclusion, PIC supplementation affects the metabolism of fatty acids, pectin, glutathione, and choline. Our study demonstrates the potential beneficial effects of PIC, especially in metabolic syndrome and obesity. PIC may be developed as a functional food and used in the treatment of these diseases. Nutritional and metabolome studies are effective in studying the effects of specific dietary supplements and will contribute to the development of functional foods.

Effects of combined treatment with fermented soybean (natto) intake and exercise on bone metabolism in ovariectomized rats

Objectives: Using ovariectomized rats, we examined the influence of combined exercise tolerance and natto intake on the bone loss inhibitory effect. Methods: We divided female Wistar rats into the following groups: Ovariectomy, Ovariectomy + Exercise, Ovariectomy + Natto Intake, Ovariectomy + Exercise + Natto Intake, and Pseudo-operative (Sham group). After conducting experiments on each group, we collected the tissues and performed morphological and molecular biological analyses. Results: In comparison with the Ovariectomy group, only in the Ovariectomy + Exercise group was there a significant bone loss inhibitory effect in the femoral cancellous bone. Although there was a tendency toward this trend seen in the Natto Intake and Exercise + Natto Intake groups, these differences were not significant. The increase in messenger RNA expression levels of alkaline phosphatase (osteoblast marker) in the bone marrow caused by ovariectomy was suppressed by individual factors, and by those in combination. However, messenger RNA expression levels of estrogen receptor alpha in the bone marrow showed a decreasing tendency with each factor, and decreased significantly with the combination, similar to the Sham group. Conclusion: This suggests that natto intake and exercise maintain bone mass by different molecular mechanisms and that these two factors do not simply act synergistically in combination to maintain bone mass.

Combined Effects of Protamine and Monocaprin on Growth Inhibition of Escherichia coli.

大腸菌に対するプロタミンとモノカプリンとの併用による相乗抗菌効果について検討したところ,以下に示す結果が得られた.(1)大腸菌に対する抗菌力はプロタミン濃度に依存し,0.05%の場合,30℃,40時間培養後も菌の増殖がほとんどみられなかった.(2)プロタミンと各種薬剤を併用したところ,モノカプリンとの併用で強い抗菌力の発現が認められた.(3)プロタミンとモノカプリンを併用した場合,抗菌力は弱酸性から中性域(pH5-7)でより強くみられた.(4)大腸菌のコロニー形成阻害能が,プロタミン単独またはプロタミンとモノカプリンの併用区で強く認められた.(5)プロタミンは菌表層部の疎水性を増大させ,また,菌体内の紫外吸収物質の漏洩はモノカプリンによって促進された.(6)菌体膜のコハク酸脱水素酵素の活性はプロタミン処理により低下し,モノカプリンを併用することで,さらに同活性の低下がみられた.(7)両薬剤の併用効果はプロタミンにより薬剤感受性が増大し,それによりモノカプリンの外膜透過性が高まり,その作用点である細胞膜に到達し,紫外吸収物質やSDHの漏洩など膜機能に障害を与えたためと推定された.

Improvement of Emulsifying Properties of Globin (Part I). Preparation of Globin-Fatty Acid Complexes and their Emulsifying Properties.

グロビン-脂肪酸複合体を調製し,得られた複合体の乳化性について検討した.脂肪酸としては,ステアリン酸(C18),ミリスチン酸(C14)およびデカン酸(C10)を用いた.1. グロビンをエタノール,尿素およびβ-メルカプトエタノールで前処理すると,グロビンの上記脂肪酸に対する親和性は大きく変化した.2. グロビンエマルションの安定性は,pH 7.0で最低であったが,尿素加熱処理グロビンあるいはβ-メルカプトエタノール処理グロビンのエマルションは,pH 7.0でも高い安定性を示した.3. 中性付近でのグロビンの乳化性は,脂肪酸との複合体を形成させることによって改善された.特に尿素加熱処理グロビンと脂肪酸との複合体は,pH 7.0で,優れた乳化性を示した.