Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs.

Although soybean protein is the major component in livestock feeds, its effect on pigs' appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorectic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannulated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be attenuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study's result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/TRPM5 pathway.

Separation of Storage Proteins (7S and 11S) from Soybean Seed, Meals and Protein Isolate Using an Optimized Method Via Comparison of Yield and Purity.

The primary purpose of this study was to extract ß-conglycinin (7S) and glycinin (11S) from soybean seed, soybean meals and soybean protein isolate and compare their yield and purity. The previous methods were modified for the extraction and isolation of 7S and 11S globulins. The adjustment mainly included sample to solution ratio of 1:10 (previously 1:15). Comparing the yield of 11S fraction in Tris-HCl and water as extractable solutions, it was almost doubled in soybean seed (16.97% to 32.41%) with purity from 96 to 98% respectively. In case of soybean meal, samples yield increased from 45.46 to 61.86% with purity from 94 to 98%. On contrary, 7S yield was significantly improved in soybean protein isolate sample from 30.33 to 53.81% along with no contamination in its purity while soybean seed and soybean meal samples had less increase in both yield and purity in Tris-HCl and water as extractable solutions. Results of this study will bring new insights into soybean 7S and 11S separation and purification techniques as well as pave the way for their application in food industry.

Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Structural characterization and in-silico analysis of Momordica charantia 7S globulin for stability and ACE inhibition.

Momordica charantia (Mc) seeds are widely used edible crop with high nutritional quality. The food and pharmaceutical industries use it as a natural anti-oxygenic agent. Herein, a ~52 kDa protein, which is a major part of seed proteome has been purified, biochemically characterized and structure has been determined. MALDI-ESI-MS identified peptide fragments and contig-deduced sequence suggested the protein to be homologous to 7S globulins. The crystal structure shows that protein has a bicupin fold similar to 7S globulins and the electron density for a copper and acetate ligand were observed in the C-terminal barrel domain. In silico study reveals that a tripeptide (VFK) from Mc7S possess a higher binding affinity for angiotensin converting enzyme (ACE) than already reported drug Lisinopril (LPR). The protein is a glycoprotein and highly stable under varying thermal and pH conditions due to its secondary structures. The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay showed the protein to have an anti-oxygenic nature and can aid in scavenging free radical from sample. The protein can assist to enhance the nutritional and functional value of food by acting as a food antioxidant. Further, characterization of Mc7S required which might add in importance of Mc7S as antioxidant, anti-diabetic and anti-hypertensive.

Effects of Ultrasonic-Assisted Extraction on the Physicochemical Properties of Different Walnut Proteins.

The effects of ultrasonic-assisted extraction (UAE, 200 W, 20 min) on the yield and physicochemical properties of different walnut proteins (WNPs, including albumin, globulin, and glutelin) were investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that UAE could result in protein molecular fragmentation of albumin, but did not affect the major bands of globulin and glutelin. The CD spectra demonstrated that different WNPs obtained by UAE had different changes in their secondary structure. Under UAE, there was an increase in surface hydrophobicity (H0) of albumin and gluten and no change in the fluorescence intensity, while decreases were observed in the H0 and fluorescence intensity of globulin; and the contents of total and surface free sulfhydryl in albumin dramatically decreased. UAE reduced the size of the particles and the dimension of the microstructures in albumin and gluten, indicating that ultrasound could unfold protein aggregates. In addition, UAE increased the solubility, emulsifying activity (EA), foaming capacity (FC), and foam stability (FS) of the obtained proteins. The above results indicate that ultrasound extraction is a promising approach to improve the extraction yield and properties of walnut proteins.

Proteinaceous Residue Removal from Oat ß-Glucan Extracts Obtained by Alkaline Water Extraction.

Background: Wet methods of 1-3, 1-4 -ß-D-glucan isolation from cereals differ mainly in the type of grain fraction used as raw material, the solid-liquid ratio of ß-glucan in raw material vs. solvent used, and the type of aqueous solvent modification (alkali, neutral or acidic). All these factors impact the characterization of the residues finally found in extracts. Oat bran is a rich source of globulin fraction which can be transferred into the extracts, especially when a high pH is employed. Methods: A multi-stage (enzymatic and acidic) purification procedure was performed to remove the residues, especially starch and protein, from ß-glucan isolates from oat of different molar mass. Pancreatin, thermostable α-amylase, amyloglucosidase, and papain were used for consecutive residue removal. Three levels of low pH = 4.5, 3.5 and 3.0 were also tested for effective protein precipitation. Results: The starch hydrolysis and liquefaction significantly facilitate the proteinaceous matter removal although papain usage showed an intensive unfavorable impact on ß-glucan molar mass. Soluble protein content was significantly decreased after pancreatin and α-amylase treatment, while the significant reduction of amine nitrogen was noted after complete starch hydrolysis and a second acidification step. Conclusions: A complex procedure employing different enzymes is needed to successfully reduce the possibly bioactive residues in isolated oat ß-glucan fractions.

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

Assessing the Sensitizing and Allergenic Potential of the Albumin and Globulin Fractions from Amaranth (Amaranthus hypochondriacus) Grains before and after an Extrusion Process.

Background: The first cases of food allergy to amaranth grain have recently been published. This pseudocereal is considered hypoallergenic, and there is scarce information about the allergenic potential of amaranth proteins, either before or after food processing. Objective: To evaluate, in a mouse model of food allergy, the sensitizing and allergenic potential of extruded and non-extruded albumin and globulin fractions from amaranth grains. Materials and Methods: Amaranth (Amaranthus hypochondriacus) flour was obtained and the albumin and globulin fractions isolated. These protein fractions were also obtained after flour extrusion. An intraperitoneal 28-day protocol was carried out to evaluate the sensitizing and allergenic potential of the proteins. The common and rarely allergenic proteins ovalbumin and potato acidic phosphatase were utilized as reference. Specific IgE and IgG antibodies were evaluated for all the proteins tested. Mast cell protease-1 (mMCP-1) responses were evaluated in serum samples collected after intragastric challenges with the proteins of interest. All serological evaluations were carried out using ELISA. Results: Mice were sensitized to the non-extruded albumin fraction from amaranth grains and to ovalbumin (p = 0.0045). The extrusion process of amaranth proteins abrogated the IgE responses triggered under non-extruded conditions (p = 0.0147). mMCP-1 responses were significantly detected in the group of mice sensitized to ovalbumin (p = 0.0138), but not in others. Conclusions: The non-extruded albumin fraction from amaranth has the potential to sensitize BALB/c mice, but this sensitizing potential fails to induce detectable serum levels of the mast cell degranulation marker mMCP-1 after intragastric challenges. Furthermore, the extrusion process abolished the sensitization potential of the amaranth albumins.

Protein analysis reveals differential accumulation of late embryogenesis abundant and storage proteins in seeds of wild and cultivated amaranth species.

BACKGROUND: Amaranth is a plant naturally resistant to various types of stresses that produces seeds of excellent nutritional quality, so amaranth is a promising system for food production. Amaranth wild relatives have survived climate changes and grow under harsh conditions, however no studies about morphological and molecular characteristics of their seeds are known. Therefore, we carried out a detailed morphological and molecular characterization of wild species A. powellii and A. hybridus, and compared them with the cultivated amaranth species A. hypochondriacus (waxy and non-waxy seeds) and A. cruentus. RESULTS: Seed proteins were fractionated according to their polarity properties and were analysed in one-dimensional gel electrophoresis (1-DE) followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 34 differentially accumulated protein bands were detected and 105 proteins were successfully identified. Late embryogenesis abundant proteins were detected as species-specific. Oleosins and oil bodies associated proteins were observed preferentially in A. cruentus. Different isoforms of the granule-bound starch synthase I, and several paralogs of 7S and 11S globulins were also identified. The in silico structural analysis from different isoforms of 11S globulins was carried out, including new types of 11S globulin not reported so far. CONCLUSIONS: The results provide novel information about 11S globulins and proteins related in seed protection, which could play important roles in the nutritional value and adaptive tolerance to stress in amaranth species.

Characterizing the Structural and Functional Properties of Soybean Protein Extracted from Full-Fat Soybean Flakes after Low-Temperature Dry Extrusion.

The soy protein isolates (SPI) extracted from different extruded full-fat soybean flakes (FFSF), and their conformational and functional properties were characterized. Overall, the free thiol (SH) content of SPI increased when the extrusion temperature was below 80 °C and decreased at higher temperatures. Soy glycinin (11S) showed higher stability than ß-conglycinin (7S) during extrusion. Results also indicated that the increase in some hydrophobic groups was due to the movement of hydrophobic groups from the interior to the surface of the SPI molecules at extrusion temperatures from 60 to 80 °C. However, the aggregation of SPI molecules occurred at extrusion temperatures of 90 and 100 °C, with decreasing levels of hydrophobic groups. The extrusion temperature negatively affected the emulsifying activity index (EAI); on the other side, it positively affected the emulsifying stability index (ESI), compared to unextruded SPI.

Report- Immunological study of different fraction of wheat proteins.

Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.

Molecular and Functional Properties of Protein Fractions and Isolate from Cashew Nut (Anacardium occidentale L.).

Some molecular and functional properties of albumin (83.6% protein), globulin (95.5% protein), glutelin (81.3% protein) as well as protein isolate (80.7% protein) from cashew nut were investigated. These proteins were subjected to molecular (circular dichroism, gel electrophoresis, scanning electron microscopy) and functional (solubility, emulsification, foaming, water/oil holding capacity) tests. Cashew nut proteins represent an abundant nutrient with well-balanced amino acid composition and could meet the requirements recommended by FAO/WHO. SDS-PAGE pattern indicated cashew nut proteins were mainly composed of a polypeptide with molecular weight (MW) of 53 kDa, which presented two bands with MW of 32 and 21 kDa under reducing conditions. The far-UV CD spectra indicated that cashew proteins were rich in ß-sheets. The surface hydrophobicity of the protein isolate was higher than that of the protein fractions. In pH 7.0, the solubility of protein fractions was above 70%, which was higher than protein isolate at any pH. Glutelin had the highest water/oil holding capacity and foaming properties. Protein isolate displayed better emulsifying properties than protein fractions. In summary, cashew nut kernel proteins have potential as valuable nutrition sources and could be used effectively in the food industry.

ß-Conglycinin enhances autophagy in porcine enterocytes.

ß-Conglycinin (ß-CG) is well known for inducing intestinal allergies and dysfunction in neonates and young pigs. However, the underlying mechanisms are largely unknown. In this study, to clarify the role of autophagy in ß-CG-induced cytotoxicity, we investigated the effects of ß-CG on cell viability and autophagy activity in porcine enterocytes (IPEC-1 cells). The results indicated that the cell viability was decreased with the increasing levels of ß-CG. ß-CG treatment enhanced the eGFP-LC3 puncta per cells and LC3-II/LC3-I, and the latter was further increased in IPEC-1 cells cultured with bafilomycin A1. We conclude that ß-CG enhances autophagy activity in enterocytes.

Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds.

Chan (Hyptis suaveolens) is a Mesoamerican crop highly appreciated since the pre-Hispanic cultures. Its proteins are a good source of essential amino acids; however, there are no reports on the properties of its individual proteins. In this study, the 11S globulin (Hs11S) was purified and biochemically characterized. The molecular weight of native Hs11S was about 150-300 kDa with isoelectric points of 5.0-5.3, composed by four monomers of 53.5, 52, 51.1 and 49.5 kDa, each formed by one acidic subunit and one basic subunit linked by a disulfide bond. Dynamic light scattering, size exclusion chromatography and native PAGE show that Hs11S is assembled in different oligomeric forms. LC-MS/MS analysis confirmed its identity. Hs11S presents antigenic determinants in common with lupin 11S globulin. Carbohydrate moieties or phosphate groups linked to Hs11S were not detected. This information is very useful in order to exploit and utilize rationally chan 11S globulin in food systems.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Impact of extraction and elution media on non-size effects in size exclusion chromatography of proteins.

Size exclusion chromatography is extensively used to separate proteins and to determine their apparent molecular weights. It separates proteins based on hydrodynamic volume, but interactions between the chromatography resin and proteins lead to non-size effects. This report discusses the impact of co-solvents [salt, urea, sodium dodecyl sulfate (SDS), dithiothreitol] in extraction media when separating wheat gluten proteins, soy glycinin, bovine serum albumin and ovalbumin on a Biosep-SEC-S4000 column. With acetonitrile/water (1:1, v/v) containing 0.05% (v/v) trifluoroacetic acid as eluent, salts and SDS in the extraction media increase while urea decreases non-size effects. Most gluten and globular proteins are extractable in sodium phosphate buffer (0.050M; pH 6.8) containing 2.0% (w/v) SDS. This chromatographic medium allows analyzing mixtures of various proteins without any non-size effects.

Structural analysis and binding properties of isoforms of tarin, the GNA-related lectin from Colocasia esculenta.

The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro (Colocasia esculenta). The results showed that tarin is a glycoprotein with 2-3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9kDa and large subunits of 12.6kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response.

Effects of protein in wheat flour on retrogradation of wheat starch.

Albumins, globulins, gliadins, and glutenins were isolated from wheat flour and the effects of those proteins on retrogradation of wheat starch were investigated. The results showed that only glutenins retarded retrogradation of wheat starch and other 3 proteins promoted it. The results of IR spectra proved that no S-S linkage formed during retrogradation of wheat starch blended with wheat proteins. Combination of wheat starch and globulins or gliadins through glucosidic bonds hindered the hydrolysis of wheat starch by α-amylase. The melting peak temperatures of retrograded wheat starch attached to different proteins were 128.46, 126.14, 132.03, 121.65, and 134.84 °C for the control with no protein, albumins, glutenins, globulins, gliadins groups, respectively, and there was no second melting temperature for albumins group. Interaction of wheat proteins and starch in retrograded wheat starch greatly decreased the endothermic enthalpy (△H) of retrograded wheat starch. Retrograded wheat starch bound to gliadins might be a new kind of resistant starch based on glycosidic bond between starch and protein.

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins.

Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein.

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K(m), V(max), K(cat), and K(cat)/K(m) of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg(-1) s(-1), 65,655 ± 10.92 s(-1), and 328.3 ± 3.12 µM(-1) s(-1), respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.