Elucidating the interaction mechanism of rice glutelin and soybean 11S globulin using multi-spectroscopy and molecular dynamics simulation methods.

Rice gluten, as the hydrophobic protein, exhibits restricted application value in hydrophilic food, which may be enhanced through interaction with soybean 11S globulin, characterized by favorable functional properties. This study aims at revealing their interaction mechanism via multi-spectroscopy and molecular dynamics simulation. The formation and structural change of rice glutelin-soybean 11S globulin complexes were detected using fluorescence, ultra-violet and circular dichroism spectra. The addition of 11S globulin increased the contents of α-helix, ß-turn and random coil, but decreased ß-sheet content, and the change in secondary structure was correlated with particle size. Moreover, exposure of hydrophobic groups and formation of disulfide bonds occurred in the complexes. Molecular dynamics simulation verified these experimental results through analyses of root mean square deviation and fluctuation, hydrogen bond, secondary structure, and binding free energy analysis. This study contributes to expounding the interaction mechanism of protein and protein from the molecular level.

Characterization of the structure, antioxidant activity and hypoglycemic activity of soy (Glycine max L.) protein hydrolysates.

This study aimed to hydrolyze soy isolate protein (SPI) using five enzymes (alcalase, pepsin, trypsin, papain, and bromelain) in order to obtain five enzymatic hydrolysates and to elucidate the effect of enzymes on structural and biological activities of the resulting hydrolysates. The antioxidant and hypoglycemic activities of the soy protein isolate hydrolysates (SPIEHs) were evaluated through in silico analysis, revealing that the alcalase hydrolysate exhibited the highest potential, followed by the papain and bromelain hydrolysates. Subsequently, the degree of hydrolysis (DH), molecular weight distribution (MWD), amino acid composition, structure, antioxidant activities, and hypoglycemic activity in vitro of SPIEHs were analyzed. After enzymatic treatment, the particle size, polymer dispersity index (PDI), ζ-potentials, ß-sheet content and α-helix content of SPIEHs was decreased, and the maximum emission wavelength of all SPIEHs exhibited red-shifted, which all suggesting the structure of SPIEHs was unfolded. More total amino acids (TAAs), aromatic amino acids (AAAs), and hydrophobic amino acids (HAAs) were found in alcalase hydrolysate. For 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal ion chelating activity, α-glucosidase inhibitory activity and α-amylase inhibitory activity, alcalase hydrolysate had the lowest IC50; alcalase hydrolysate and papain hydrolysate had the lowest IC50 for hydroxyl radical scavenging activity. Physiological activity of SPIEHs was evaluated thoroughly by 5-Axe cobweb charts, and the results revealed that alcalase hydrolysate exhibited the greatest biological activities.

A randomized double blind comparison of atosiban in patients with recurrent implantation failure undergoing IVF treatment.

BACKGROUND: Patients with recurrent implantation failure (RIF) may have more uterine contractions. Several observational studies suggested that atosiban administration around embryo transfer resulted in higher pregnancy rates in RIF patients. This study aimed to evaluate the effect of atosiban given before fresh embryo transfer on pregnancy outcomes of women with RIF. METHODS: A prospective, randomized, double-blind controlled clinical trial was performed in IVF center of Shanghai First Maternity and Infant Hospital. According to a computer-generated randomization list, 194 infertile women with RIF received fresh embryo transfer between July 2017 and December 2019 were randomly allocated into the atosiban (n = 97) and the placebo (n = 97) groups. Women in the treatment group received atosiban intravenously about 30 min before embryo transfer with a bolus dose of 6.75 mg over one minute. Those in the placebo group received only normal saline infusion for the same duration. RESULTS: There was no significant difference in the live birth rate between the atosiban and placebo groups (42.3% vs 35.1%, P = 0.302, RR = 1.206 (0.844-1.723)). No significant differences were found between the two groups in the positive pregnancy test, clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy and implantation rates. Similar results were found when stratified by the number of embryos previously transferred, number of previous failed embryo transfers, frequency of endometrial peristalsis on embryo transfer day (≥ 3 waves/min) or serum estradiol (E2) on the day of hCG above the median level. And, there was no correlation between the serum E2 level on the day of hCG and the frequency of endometrial peristalsis on embryo transfer day. The frequency of endometrial peristalsis on embryo transfer day, total FSH/HMG dosage and duration were the significant factors which independently predicted the likelihood of a live birth. CONCLUSIONS: These results suggested that atosiban treatment before fresh embryo transfer might not improve the live birth rate in RIF patients. TRIAL REGISTRATION: The study had been approved by the Institutional Review Board of the hospital (2017 ethics No.43) and was registered under Clinicaltrials.gov with an identifier NCT02893722.

Proliferation of Bifidobacterium L80 under different proportions of milk protein hydrolysate.

The intestinal microecological environment is critical to an infant's growth. For those infants consuming milk power, it is very important to improve the intestinal microecological environment to promote the healthy growth of infants. In this paper, Milk protein hydrolysate (MPH), consisting of different proportions of proteins and small molecule peptides (5:5, 4:6, 3:7, 2:8, 1:9) were added to infant formula powder (IFP). The effects of MFP-enriched IFP addition on proliferation and metabolism of Bifidobacterium L80 were studied. Compared with MPH-free IFP, MFP-enriched IFP with 1:9 of proteins to small molecule peptides significantly enhanced the proliferation of Bifidobacterium L80, resulting in higher cell density, greater viable counts and higher titratable acidity. MFP-enriched IFP increased the content of seven organic acids and H2O2 in the system, and improved the antibacterial activity to E. coli BL21. This study suggested that MPH could be an effective addition to infant formula powder to promote the growth of Bifidobacterium, so to improve the intestinal health of infants.

[Antidepressant mechanism of Shugan Lipi recipe in regulating tryptophan metabolism and its common pharmacodynamic substances].

This article is to explore the antidepressant mechanism of Shugan Lipi recipe in regulating tryptophan metabolism,and to find out their common pharmacodynamic substances. UPLC-Q-TOF-MS technology was used to establish fingerprints of Shugan Lipi recipe,and 124 components were identified. The depressed mouse model was replicated by triple-one multiple stress method. Chaihu Shugan Powder,Sini Powder and Xiaoyao Powder were administered in groups to observe the changes in body weight and behavior of the mice. The results showed that compared with the model group,the body weight,sucrose preference percentage and autonomous activity behavior of each administration group were improved. Among them,the effect of Chaihu Shugan Powder was better than that of Sini Powder and Xiaoyao Powder. LC-MS/MS method was used to determine the contents of 5-hydroxytryptamine( 5-HT),kynurenine( KYN) and tryptophan( TPP) in blood,liver,brain,colon and other tissues,as well as TDO enzyme activity in liver. Western blot and RT-PCR were used to detect the protein and gene expression of TDO enzyme,respectively. It was found that the three prescriptions increased the ratio of 5-HT/KYN in different degrees,decreased the ratio of KYN/TRP in liver,colon and brain,and decreased the expression level and activity of TDO enzyme in liver. The order of their ability to regulate tryptophan metabolism was Chaihu Shugan Powder>Sini Powder>Xiaoyao Powder. In addition,the correlation between the chromatographic peaks in the fingerprints of Shugan Lipi recipes and the pharmacodynamic indexes of tryptophan metabolism was analyzed by the grey relation analysis. The grey relation analysis found that the chemical components with the highest correlation with tryptophan metabolism were mainly from Paeoniae Radix Alba,Citri Reticulatae Pericarpium,Aurantii Fructus Immaturus and Aurantii Fructus. UPLC-Q-TOF-MS was used to analyze the migration components in the plasma of mice after administration of Shugan Lipi recipe,and to verify the common pharmacodynamic substances of Shugan Lipi recipe. The migration of these detected components in plasma was studied,and a total of 18 prototype components and 36 metabolites were identified. Therefore,it was believed that Chaihu Shugan Powder,Sini Powder and Xiaoyao Powder could play an antidepressant role by reducing the expression of TDO enzyme in the liver and regulating the metabolism of tryptophan.The components contained in Paeoniae Radix Alba,Citri Reticulatae Pericarpium,Aurantii Fructus Immaturus and Aurantii Fructus were the common pharmacodynamic substances of Shugan Lipi recipe,which played an important role in regulating tryptophan metabolism.

Structural characterization and functional properties of CNPP, a byproduct formed during CPP preparation.

Casein nonphosphopeptide (CNPP), a byproduct formed during the preparation of casein phosphopeptide (CPP), is often discarded on a large scale. Although our previous studies have demonstrated the ameliorative effect of CNPP on muscle wasting disorders, its structure-function mechanism is still unclear. Therefore, considering the great influence of structural characteristics on function, this study aims to explain the potential mechanism by characterizing the physicochemical and functional properties of CNPP. The results of structural characterization indicated that CNPP was of low molecular weight and composed of the complete range of amino acids; it was particularly rich in leucine. Compared with casein, CNPP had a lower molecular size and total/free sulfhydryl content (reduced 2.44 and 2.02 µmol/g in CNPP, respectively). Additionally, Fourier transform infrared spectroscopic analysis revealed that enzymatic hydrolysis caused protein unfolding, and the content of ß-turns and random coils reached 50.20% and 10.67%, respectively. Fluorescence-dependent detection of CNPP indicated a reduction of spectral intensity and the occurrence of a red shift. The changes in the structure of CNPP significantly affected its functional characteristics. CNPP has better solubility, foaming, and digestion properties than those of casein and whey protein. Specifically, the foam stability and emulsification properties decreased in the order of casein > CNPP > whey protein. The present study can provide a substantial basis for future application of CNPP as a functional ingredient against sarcopenia.

HS-GC-IMS with PCA to analyze volatile flavor compounds across different production stages of fermented soybean whey tofu.

The variations in flavor substances across the different stages of fermented soybean whey tofu (FSWT) production were analyzed by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) combined with principal component analysis (PCA). The results revealed 24 representative flavor compounds in the samples across all production stages. After heating, the signal intensity of hexanal, 1-octen-3-ol, heptanal, and (E)-2-hexenol, which are unpleasant flavor substances found in raw soymilk, weakened, whereas those of some aroma substances increased. Furthermore, fermented flavor compounds, namely, 2-heptanone, 2-pentylfuran, pentanal, and 2,3-butanedione, were produced after the addition of fermented soybean whey as a coagulant. A PCA based on the signal intensity of the detected volatile compounds revealed effective differentiation of samples from different stages into comparatively independent spaces. These results showed that the flavor fingerprints of the samples from different stages of FSWT production can be successfully built using HS-GC-IMS and PCA based on the detected volatile compounds.

[Optimal expression and activity detection of recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies in P.pastoris].

AIM: To express the recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) using yeast cells secreting type carrier pPIC9K, and screen for optimal conditions of engineered yeast cells expressing cRFB4FC and cRFB4CH3. METHODS: The genes of cRFB4FC and cRFB4CH3 were cloned into P. pastoris eukaryotic expression vector pPIC9K to construct the recombinant plasmids pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3, then identified by DNA sequencing. The recombinant plasmid pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were transfected into P. pastoris SMD1163. The recombinant yeast cell line chosen by G418 resistance was identified by PCR. After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blotting, and biologic activity was identified by ELISA. Finally, orthogonal test was conducted to optimize the expression conditions of the recombinant yeast cell lines so as to increase the expression level of the antibodies. RESULTS: The secretory type yeast carriers pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were successfully constructed and chosen by G418 as well as identified by PCR to obtain the highly copied and integrated recombinant yeast cell line. The cRFB4FC and cRFB4CH3 proteins were expressed by yeast cells containing pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 induced by methanol. The relative molecular mass (M(r);) of cRFB4FC and cRFB4CH3 were about 326 000 and 295 000 Da. They could be identified by goat anti-human IgG(Fc) monoclonal antibody with Western blotting on SDS-PAGE, and higher biologic activity was confirmed by ELISA. Under the optimized expression conditions, the mean expression levels of cRFB4FC and cRFB4CH3 in recombinant yeast increased by 196.4% and 151.7%. CONCLUSION: The recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) proteins can be highly expressed in the P.pastoris SMD1163 expression system, and possess immunological activity.