Optimization of ultrasound-assisted extraction of defatted wheat germ proteins by reverse micelles

In this work, an ultrasound-assisted procedure for the extraction of defatted wheat germ proteins by reverse micelles was established. Response surface methodology (RSM) was used to optimize the ultrasound-assisted extraction (UAE) parameters (ultrasonic output power, ultrasonic time and pulse mode) for enhancing the forward extraction efficiency of defatted wheat germ proteins by implementing a three-level, three-variable Box–Behnken experimental design. The independent variable with the largest effect on response was X₂ (ultrasonic time), followed by X₁ (ultrasonic output power) and X₃ (pulse mode). The optimum extraction conditions were found to be ultrasonic output power 363 W, ultrasonic time 24 min and pulse mode 2.4 s on and 2 s off (2.4 s:2 s). Under these conditions, the forward extraction efficiency of defatted wheat germ proteins can increase from 37% to 57%, and the final protein extraction efficiency by reverse micelles can reach 45.6%, making the advantage of reverse micelles much more obvious than alkaline extraction and isoelectric precipitation on the separation of protein.     

Protein extraction from defatted wheat germ by reverse micelles: Optimization of the forward extraction

In this work, the forward extraction of defatted wheat germ protein (DWGP) by reverse micelles was studied. The reverse micellar systems were formed by sulphosuccinic acid bis (2-ethylhexyl) ester sodium salt (AOT), isooctane and KCl solution. The effects of AOT concentration, pH, KCl concentration, extraction time, the amounts of defatted wheat germ flour (DWGF), W₀ (the molar ratio of water to surfactant, i.e. W₀ = [H₂O]/[AOT]) and temperature on the forward extraction efficiency of DWGP were tested. On the basis of single-factor experiments, the optimum extraction was achieved by response surface methodology (RSM). The experimental results lead to the conclusion that the highest forward extraction efficiency of DWGP was reached at the AOT concentration 0.06 g/mL, pH 8, KCl concentration 0.1 mol/L, time 30 min, the amounts of DWGF 0.500 g, W₀ 25 and temperature 36 °C. Under these conditions, the forward extraction efficiency of DWGP achieved 37%.     

Isolation and properties of lipolysis inhibitory proteins from wheat germ and wheat bran

Proteins inhibiting pancreatic lipasein vitro have been isolated from wheat germ and wheat bran, with relative molecular mass ranging from 24,400 to 27,500. Inhibition of pancreatic lipase by the wheat germ proteins is related to their ability to interact with the emulsified substrate and to hinder the adsorption of the enzyme on the interface. The extent of inhibition depends on the amount of substrate and is independent of the enzyme concentration. Bile salts forming micelles in the concentration range used are able to partially reverse the inhibition of pancreatic lipase by the wheat germ proteins. The nutritional significance of the data obtained is discussed.     

超声处理对碱法制备蚕蛹蛋白条件的优化

通过单因素分析及响应面优化,研究超声处理对碱法制备蚕蛹蛋白得率的影响,得到了蚕蛹蛋白得率的数学模型,优化了制备条件,提高了碱法制备蚕蛹蛋白的得率。结果表明:NaOH浓度和液固比(V/m)对蚕蛹蛋白得率影响显著(p0.05)。蚕蛹蛋白浸提的最佳工艺条件为:超声功率400 W,超声时间20 min,浸提液固比(V/m)60∶1,NaOH浓度0.3%。在此工艺条件下,蚕蛹蛋白的得率为88.14%。制备的蚕蛹蛋白中的必需氨基酸含量符合FAO/WHO推荐标准,可以作为食品基料使用。     

Modification of aqueous enzymatic oil extraction to increase the yield of corn oil from dry fractionated corn germ

In previous aqueous enzymatic oil extraction (AEOE) experiments we reported a best free oil yield of 49% of the hexane extracted yield of dry fractionated corn germ. In the current experiments, a dispersion of 10% cooked, dry-fractionated germ in water was treated with α-amylase, glucoamylase and a cellulase complex. Free oil was collected by centrifuging a foam fraction of the dispersion. Several dispersion treatments were tried to evaluate their release of free oil and effect on the production of foam. The foam contained up to 8% free oil (dispersed from germ containing 26% oil) and fines oil (not centrifugally separable), protein and germ particles. Treatment with α-amylase and glucoamylase prior to treatment with the commercial cellulase used in previous AEOE studies increased the free oil yields about 25-61% of the hexane-extractable yield. A preliminary cost analysis indicates that oil separation with the amylase enhanced AEOE appears to be preferable to AEOE alone and profitable if crude corn oil cost exceeds $1.1/kg ($0.50/lb).     

蕹菜梗叶绿素浸提工艺的优化及其理化性质

利用单因子试验和正交试验探讨了蕹菜梗叶绿素的最佳浸提工艺,并对其理化性质进行分析。结果表明,蕹菜梗叶绿索的最佳浸提工艺为：乙醇浓度85％、料液比1：50、提取温度70℃、提取时间4h,其提取率为0．1957mg·g^-1;蕹菜梗叶绿素易溶于有机溶剂,但不溶于水,其可见光区的最大吸收波长为410nm,且具有较好的耐光和耐热性。     

Nutritive value of protein fractions extracted from soybean,rapeseed and wheat flours in the rat

The nutritive value of various protein fractions was studied. Fractions 2S and 12S from rapeseed, 2S, 7S and 11S from soybean were obtained by dissolution in ammonium sulfate solutions. Albumin-globulin, gluten, glutenin and gliadin fractions from wheat were obtained by dissolution in salted water (albumin-globulin), acetic acid (glutenin) and alcohol (gliadin). Liveweight gains, protein efficiency ratio (PER) and apparent digestibility coefficient (ADC) were used as measures of the nutritive value. The protein fractions had a lower nutritive value than the unfractionated proteins except for the albumin-globulin fraction of wheat which had a nutritive value higher than that of the unfractionated wheat protein. PER obtained with the rapeseed 2S and 12S fractions were 2.49 and 2.21, respectively, as compared to 2.64 for unfractionated rapeseed. With soybean fractions, PER were 0.92 for 2S, — 0.007 for 7S and 1.47 for 11S, as compared to 2.19 for the original protein. The wheat albumin-globulin fraction gave a PER of 2.78, as compared to 1.45 for the unfractionated wheat protein. Gluten, glutenin and gliadin fractions had a lower PER than that of unfractionated wheat protein. ADC of all fractions were higher than those of the original proteins. The difference in liveweight gains and PER observed between protein fractions can be partially explained on the basis of the essential amino acid content.     

Optimisation of the Selective Extraction of (Glucurono)arabinoxylans from Wheat Bran: Use of Barium and Calcium Hydroxide Solution at Elevated Temperatures

Two approaches were investigated in attempts to obtain a high extraction yield of (glucurono) arabinoxylans from water-unextractable cell wall material (WUS) of wheat bran using saturated barium hydroxide containing 0·26 M sodium borohydride. First, the effect of three pretreatments (autoclave treatment, alkaline peroxide and chlorite delignification) of the WUS prior to extraction appeared to have no effect on the extraction yield. Moreover, modifications to the composition and molecular weight distribution of the (glucurono)arabinoxylans occurred when such treatments were used. Second, the effect of an increasing extraction temperature and concentration of alkali was investigated. Increasing the extraction temperature improved the extraction yield of (glucurono)arabinoxylans from 29% at 20°C to 50% at 95°C. Increasing the barium hydroxide concentration with the temperature resulted in no further improvements in extraction yield up to 70°C. Above this temperature the extraction yield decreased. Substitution of barium hydroxide by calcium hydroxide resulted in lower yields and a lower selectivity; the lower solubility of calcium hydroxide may have been responsible for this. Supplementary experiments to investigate the mechanism of the selectivity of the bivalent hydroxide extraction with addition of sodium borohydride indicated a possible role for borate, derived from borohydride.     

响应面法优化金柚皮渣中果胶的提取工艺

以硫酸-咔唑法为测定果胶方法,采用超声波辅助酸提果胶,并对提取果胶工艺进行优化,包括超声辅助酸提前处理及提取温度、提取时间、料液比和p H等提取条件对果胶提取效果的影响。结果表明,在单因素试验的基础上,采取响应面试验设计,确定优化参数为:提取时间88.7 min、提取温度75.5℃和p H1.44。在此条件下,从金柚果皮中提取果胶的得率为26.8%。     

Proteolytic extraction enhances specific detection of the novel ‘S’-type low molecular weight glutenin subunit in wheat by monoclonal antibody

Specificity of monoclonal antibodies (MAbs) is required in diagnostic immunoassays. However, structural homology between a target antigen and other cellular proteins often causes promiscuous cross-reactivity of a MAb to proteins other than its target antigen, thereby hindering specificity. It is proposed in this study that specific proteolytic enzymes could be useful in the reduction or elimination of cross-reactive epitopes while retaining target epitopes for certain MAbs. The hypothesis was tested using a novel ‘S’-type low molecular weight glutenin subunit (LMW-GS-‘S’) and its antibodies. The results demonstrated that the protease, chymotrypsin, markedly reduced the cross-reactivity in samples that would otherwise obscure the specific detection of LMW-GS-‘S’ by MAb F8-14E6 in an enzyme-linked immunosorbent assay (ELISA). Further investigations revealed that the working mechanism for this proteolytic treatment was indeed due to the selective cleavage that destroyed cross-reactive epitopes whilst retaining the intact specific epitope. As more proteases are being discovered or engineered, the successful utilization of protease in immunoassays as reported here will benefit general immunodiagnostic assay development in both medicine and agriculture by targeting specific epitopes with tailored proteolytic treatment of antigens.     

Immunomodulatory activities of non-prolamin proteins in wheat germ and gluten

Albumin (Alb), globulin (Glo), glutelin (Gll) and glutenin (Gln) were separately extracted from wheat germ and wheat gluten. Amino acisd composition, molecular weight distribution, solubility, in vitro digestibility, and immunomodulatory activities were all analyzed. Gll and Gln have similar molecular weight distributions, which differed from those of Alb and Glo. Alb showed the highest solubility at various pH values (except pH 4.0), whereas Glo showed the highest in vitro digestibility. Glo and Gll have the highest proportion of essential to total amino acids, while Alb and Gll have the highest protein digestibility-corrected amino acid scores. Gll had the strongest immunomodulatory effects in terms of stimulation of RAW 264.7 cells to produce IL-6, TNF-α, and IL-10, and good stimulatory effects on splenocyte proliferation, production of IL-2, phagocytosis, and secretion of nitric oxide in RAW 264.7 cells. Gll can be considered a good protein source for use in health foods.     

不同沉淀剂对酚抽法提取植物蛋白质效果比较分析

酚抽法已经逐渐成为植物蛋白质组研究中通用的蛋白质提取方法,但在各种改进酚抽法中,应用到的沉淀试剂各不相同,致使蛋白沉淀得率、纯度和沉淀时间也各异,本研究以热带植物橡胶树叶片、胶乳和海马齿叶片为研究材料,在过饱和硫酸铵甲醇溶液、醋酸氨甲醇溶液和丙酮溶液沉淀剂下对比蛋白提取的效果,通过单向电泳检测、胶图质量分析、质谱鉴定、蛋白沉淀时间和得率比较,发现3种沉淀剂提取的蛋白样品1-DE图谱显示的蛋白条带数量均较多,但醋酸铵沉淀法提取蛋白图谱有条纹不清晰现象。得出结论：丙酮沉淀法对橡胶树叶片和胶乳蛋白质的提取率较高,硫酸铵沉淀法对海马齿叶片蛋白质提取率较高。过饱和硫酸铵甲醇溶液和丙酮沉淀剂提取的蛋白质质量较高,所得蛋白图谱背景清晰,质谱鉴定蛋白质信息量大。研究结果可优化提取高质量植物蛋白质的方法,并有望为顽拗类植物蛋白质组学研究提供参考。     

Optimization of extraction techniques and quantification of Betulinic Acid (BA) by RP-HPLC method from Ancistrocladus heyneanus Wall. Ex Grah.

Simple, cost effective, quick and sustainable technique was investigated for determining Betulinic acid (BA) from samples of Ancistrocladus heyneanus. The methods comprised of continuous shaking, Soxhlet, ultra sonication and microwave assisted extraction techniques. RP-HPLC technique was used to quantify BA from varied samples and confirmation of the samples was done using TLC, FT-IR and FT-Raman methods. The study also evaluated productivity of operational parameters such as solvent composition and extraction time for three distinctive parts of the plant (green leaves, brown leaves and stem). Brown leaves showed response to continuous shaking extraction and ultrasonic extraction techniques with 95% Aq. MeOH as a good extraction solvent. BA was detected and quantified in continuous shaking method with 15, 30 and 45 min of exposure time. Comparison of 6 min with 12 min of ultra sonication, showed longer sonication diminished extraction efficiencies. Concluding, brown leaves in 95% MeOH and ultrasonic extraction technique to be the fastest, easiest and best method for detection and screening of BA from A. heyneanus.     

Development of Antioxidant Protein Extracts from Gilthead Sea Bream (Sparus aurata) Side Streams Assisted by Pressurized Liquid Extraction (PLE)

The pressurized liquid extraction (PLE) technique was used, for the first time, to obtain protein extracts with antioxidant activity from side streams (muscle, heads, viscera, skin, and tailfins) of gilthead sea bream (Sparus aurata) in order to give added value to these underutilized matrices. Extraction conditions previously optimized for sea bass (Dicentrarchus labrax) side streams were applied. Protein recovery percentages were 22% (muscle), 33% (heads), 78% (viscera), 24% (skin), and 26% (tailfins), which represented an increase of 1.2–4.5-fold compared to control samples (extraction by stirring). The SDS-PAGE profiles revealed that PLE-assisted extraction influenced protein molecular weight distribution of the obtained extracts. PLE conditions also allowed increasing the antioxidant capacity measured by both Trolox equivalent antioxidant capacity (TEAC; 1.3–2.4 fold) and oxygen radical absorbance capacity (ORAC; 1.9–6.4) assays for all fish extracts. Inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF-MS) were used to investigate the presence of toxic metals and mycotoxins in sea bream side streams. The levels of As, Hg, Cd, and Pb were below those established by authorities for fish muscle for human consumption (except for Cd in viscera samples). Through a nontargeted screening approach, no mycotoxins or related metabolites were detected for all sea bream side streams. This study contributes to the research on the valorization of fish processing side streams using environmentally friendly technology.     

Optimization of the ultrasound-assisted extraction of tryptophan and its derivatives from rice (Oryza sativa) grains through a response surface methodology

An analytical ultrasound-assisted extraction (UAE) technique has been optimized and validated for the extraction of tryptophan and its derivatives from rice grains. A Box–Behnken design in conjunction with a response surface methodology based on six factors and three levels was used to evaluate the effects of the studied factors prior to optimizing the UAE conditions. The significant (p < 0.05) response surface models with high coefficients of determination were fitted to the experimental data. The most significant (p < 0.0001) effect is the solvent-to-sample ratio while quadratic effects caused by temperature and solvent-to-sample ratio were of moderate importance (p < 0.05). The optimal UAE conditions were as follows: extraction time of 5 min, ultrasound amplitude of 30%, cycle of 0.7 s⁻¹, extraction temperature of 30 °C, 8% methanol in water as the extraction solvent at pH 3 and a solvent/solid ratio 5:1. The method validation ensured that appropriate values were obtained for the LOD, LOQ, precision and recovery. Furthermore, the method was successfully applied to the analysis of a number of rice samples of different varieties. It was demonstrated that this particular UAE method is an interesting tool for the determination of tryptophan and tryptophan derivatives in rice grain samples.     

Characterization of Agro-diversity by Seed Storage Protein Electrophoresis： Focus on Rice Germplasm from Uttarakhand Himalaya, India

The characteristics of 48 rice varieties from Uttarakhand Himalaya,India were detected by morphological and biochemical markers.The grains of the selected rice varieties varied in their morphological(grain length,grain width and grain weight)and biochemical characters(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE).Based on the presence of 70,65,60,57,37-39,22-23,13 and 10 kDa protein bands in the 48 rice varieties,seven types of profiles were identified.An unweighted pair group average ...     

A New Member of the TBC1D15 Family from Chiloscyllium plagiosum: Rab GTPase-Activating Protein Based on Rab7 as a Substrate

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources.     

In Vitro Bioactivity of Astaxanthin and Peptides from Hydrolisates of Shrimp (Parapenaeus longirostris) By-Products: From the Extraction Process to Biological Effect Evaluation,as Pilot Actions for the Strategy “From Waste to Profit”

Non-edible parts of crustaceans could be a rich source of valuable bioactive compounds such as the carotenoid astaxanthin and peptides, which have well-recognized beneficial effects. These compounds are widely used in nutraceuticals and pharmaceuticals, and their market is rapidly growing, suggesting the need to find alternative sources. The aim of this work was to set up a pilot-scale protocol for the reutilization of by-products of processed shrimp, in order to address the utilization of this valuable biomass for nutraceutical and pharmaceuticals application, through the extraction of astaxanthin-enriched oil and antioxidant-rich protein hydrolysates. Astaxanthin (AST) was obtained using “green extraction methods,” such as using fish oil and different fatty acid ethyl esters as solvents and through supercritical fluid extraction (SFE), whereas bioactive peptides were obtained by protease hydrolysis. Both astaxanthin and bioactive peptides exhibited bioactive properties in vitro in cellular model systems, such as antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities (IA). The results show higher astaxanthin yields in ethyl esters fatty acids (TFA) extraction and significant enrichment by short-path distillation (SPD) up to 114.80 ± 1.23 µg/mL. Peptide fractions of <3 kDa and 3–5 kDa exhibited greater antioxidant activity while the fraction 5–10 kDa exhibited a better ACE-IA. Lower-molecular-weight bioactive peptides and astaxanthin extracted using supercritical fluids showed protective effects against oxidative damage in 142BR and in 3T3 cell lines. These results suggest that “green” extraction methods allow us to obtain high-quality bioactive compounds from large volumes of shrimp waste for nutraceutical and pharmaceutical applications.