Effects of NaCl Concentration on Salting-in and Dilution During Salting-out on Soy Protein Fractionation

ABSTRACT:  Glycinin and β-conglycinin are the main storage proteins in soybeans that can be fractionated by using alkali extraction, SO₂, salting-in with NaCl, salting-out by dilution and pH adjustment to produce a glycinin-rich fraction, a β-conglycinin–rich fraction, and an intermediate fraction, which is a mixture of the two proteins. Two different strategies were employed to optimize the procedure to achieve high efficiency in recovering the β-conglycinin–rich fraction. The first strategy was to optimize salting-in effects of NaCl, and the effects of NaCl concentration on the yields and purities of the protein fractions were investigated. The maximum protein yield of the β-conglycinin–rich fraction was obtained at 500 mM NaCl, but at the expense of purity. The optimum NaCl concentration was 250 mM, at which good protein yield (18.5%) and purity (84.5%) were achieved. At higher NaCl concentrations, the protein yields of the intermediate fractions were significantly lower, and the protein loss in the whey fraction increased. The second strategy was to improve the salting-out step for the β-conglycinin–rich fraction. At 0- and 0.5-fold dilution, the purities and yields of the β-conglycinin–rich fractions were significantly lower than at 1.0- and 2.0-fold dilution. There were no differences in protein yields or purities when using 1.0- or 2.0-fold dilution. According to these results, the recommended NaCl concentration for the salting-in step is 250 mM and the dilution factor for salting-out is 1.0.     

Extraction of phospholipids from a dairy by-product (whey protein phospholipid concentrate) using ethanol

There has been a great interest in the phospholipids (PL) found in dairy products because of their health and functional properties. In this study, a technology that was originally developed for egg yolk PL extraction was applied to whey protein phospholipid concentrate (WPPC). This method successfully precipitated the proteins present in WPPC and extracted the lipids with a renewable alcoholic solvent, ethanol. The effect of ethanol concentration, extraction temperature, and extraction number on the recovery of total lipid, total PL, and individual PL class was evaluated. The optimum processing conditions for a combined 5-stage sequential extraction for producing a PL-enriched lipid fraction were determined to be 70% ethanol at 70°C, and the total lipid recovery, total PL recovery, and PL content achieved were 40.7, 58.1, and 45.8%, respectively. A lipid fraction with high nutritional value (high content of sphingomyelin or phosphatidylserine) can also be obtained by adjusting extraction conditions and collecting specific fractions, although the yield may decrease. Overall, producing a PL-rich lipid fraction from WPPC using ethanol extraction is feasible and scalable, and different processing conditions can be used depending on the type of lipid product desired.     

Original article: Extraction of lipid from scallop (Patinopecten yessoensis) viscera by enzyme‐assisted solvent and supercritical carbon dioxide methods

In the present study, lipid was extracted from scallop (Patinopecten yessoensis) viscera by using the enzyme‐assisted solvent method and the supercritical carbon dioxide (SC‐CO₂) method. Soxhlet extraction with ethyl ether produced a yield of 23.7 ± 0.6 g of lipid 100 g^?1 of dry matter. Enzyme‐assisted solvent extraction allowed recovering 60.6 ± 1.5% of P. yessoensis viscera lipid from the samples treated with papain, whereas a lipid recovery rate of 78.3 ± 0.6% was achieved by SC‐CO₂ extraction. The lipid extracted was divided into the unsaponifiable fraction (sterol) and the saponifiable fraction (fatty acid) and analysed by gas chromatography mass spectrometry. Results indicated that the fatty acid composition and sterol composition for lipids extracted by different methods were slightly different. Eicosapentaenoic acid and docosahexaenoic acid were dominant polyunsaturated fatty acids accounting for 35–40% of the total fatty acid.     

Compositional Characteristics of Materials Recovered from Headed Gutted Silver Carp (Hypophthalmichthys molitrix) by Isoelectric Solubilization and Precipitation Using Organic Acids

Protein was recovered from headed gutted silver carp by isoelectric solubilization at pH 2.5, 3.0, 11.5, or 12.0 and precipitation (ISP) at pH 5.5 using acetic (AA) or a 30% formic and lactic acid combination (F&L) and 10 N sodium hydroxide. Total protein and fat recovery yields, proximate composition and mineral analyses of fractions were determined. Protein and lipid recovery yields when solubilized under basic conditions were comparable to yields reported from other studies using hydrochloric acid; however, the recovered fractions were less pure. Processing at basic pH using AA was more effective than F&L at removing impurities (P < 0.05) from the recovered protein fraction and impurities were effectively removed from recovered lipids regardless of processing pH or acid type (P > 0.05). For the most part, sodium was greater (P < 0.05) and there was less calcium, phosphorus, magnesium, and iron (P < 0.05) in the recovered protein regardless of acid used when compared to the initial paste. This research shows that organic acids have the potential to recover protein and lipid by ISP processing. Practical Application: This research presents a reliable method for extracting nutritionally valuable fish protein and oils from otherwise hard to process fish and its byproducts. Replacing the traditionally used strong acids with organic acids might further accomplish bacterial load reduction while resulting in similar to or improved protein recovery yields. Therefore, this technology may increase the commercial viability of hard to process fish.     

Walnuts (Juglans regia L): proximate composition,protein solubility,protein amino acid composition and protein in vitro digestibility

Walnuts contained 16.66% protein and 66.90% lipids on a dry weight basis. Non‐protein nitrogen values ranged from 6.24 to 8.45% of the total nitrogen when the trichloroacetic acid concentration was varied within the range 0.25–1.0 M . Albumin, globulin, prolamin and glutelin respectively accounted for 6.81, 17.57, 5.33 and 70.11% of the total walnut proteins. Walnut proteins were minimally soluble at pH 4.0. The majority of total walnut protein polypeptides had estimated molecular weights in the range 12 000–67 000. The Stokes radius of the major protein in walnuts (glutelin fraction) was 66.44 ± 1.39 Å. Lysine was the first limiting essential amino acid in total walnut proteins as well as in the globulin and glutelin fractions. Leucine and methionine plus cysteine were the second limiting essential amino acids respectively for the prolamin and albumin fractions. Hydrophobic and acidic amino acids dominated the amino acid composition in all protein fractions. Native and heat‐denatured walnut glutelins were easily hydrolysed by trypsin, chymotrypsin and pepsin in vitro. © 2000 Society of Chemical Industry     

Partial Characterization of Ultrafiltrated Soy Protein Hydrolysates with Antioxidant and Free Radical Scavenging Activities

Soy protein isolate (SPI) was hydrolyzed with Flavourzyme® (SHF) or chymotrypsin (SHC). Hydrolysates were sequencially fractionated by ultrafiltration using different membrane pore sizes (50, 10, and 3 kDa). The antioxidant ability of each hydrolysate protein fraction was tested in a liposome oxidizing system and their free radical scavenging activity (FRSA) was evaluated with the DPPH method (diphenylpicrilhydrazine radical). Molecular weight (MW) distribution, solubility, surface hydrophobicity, and amino acid composition of each SPI hydrolysate fraction were measured and their effect on antioxidant and scavenging activities was established by multivariate correlation. The most active ultrafiltrated peptide fractions (P < 0.05), from SHF and SHC, had of MW of <3 kDa (F3 and C3, respectively). These fractions decreased liposome oxidation by 83.2% and 84.5%, respectively, and also showed the highest FRSA (F3: 21.3% and C3: 24.4%). In addition to molecular size, the antioxidant activity and FRSA of soy protein fractions were related to their amino acid composition, especially to an increased content of Phe and a lowered content of Lys. Also, hydrophobicity of ultrafiltrated peptide fractions was an important characteristic (P < 0.001) associated with their ability to trap free radicals. Ultrafiltered peptide fractions with low MW have a high potential to be used as natural alternatives to prevent lipid oxidation in foods.     

Influence of Pine Nut (Pinus koraiensis) Oil Fractions on Physicochemical Properties of Rice Starch Solutions

The physicochemical properties of rice starch suspensions effected by interactions with pine nut oil fractions were investigated. Oil fractions (crude pine nut oil, glycolipid, nonpolar lipid, gums, and degummed oil) from pine nut (Pinus koraiensis) were prepared by solvent extraction and addition of boiling water. The influence of the fractions on the physicochemical properties of rice amylose solution (5%, w/w) was determined by measuring the complexing index, absorbance ratio (630/520 nm), colour, rheological characteristics, and microscopic structure at various concentrations. Their effect on rice starch swelling power was also determined. The glycolipid fraction had the highest complexing index with rice amylose. Rice amylose solutions containing the glycolipid fraction exhibited greater pseudoplastic fluid (n<0.7) behaviour, higher colour difference values, and lower absorption ratios (1.64‐1.48) than did those containing other fractions. Hydrated oil fraction (gums) complexed with amylose only poorly, if at all, but amylose solutions containing them had rheological properties similar to those containing the glycolipid fraction. The addition of oil fractions increased the swelling power value compared to the control.     

酸沉除镉制备米糠蛋白乳液稳定性的研究

采用粒径分布、微观结构、Zeta电位和分层系数研究米糠蛋白质量分数和预热处理对盐酸、酒石酸、苹果酸和柠檬酸酸沉除镉制备米糠蛋白乳液稳定性的影响。结果表明:当米糠蛋白质量分数为0.5%时,4种酸酸沉除镉制备米糠蛋白的乳液稳定性无显著性差异(P0.05),并且乳液稳定性均较差;当米糠蛋白质量分数为1.0%和2.0%时,酒石酸、苹果酸、柠檬酸酸沉除镉制备米糠蛋白乳液的粒径较小,显示出较好的乳液稳定性;4种酸酸沉除镉制备的米糠蛋白经过预热处理之后,乳液粒径减小,乳液稳定性提高,并且乳液稳定性无显著性差异(P0.05)。     

Selective extraction of phospholipids from whey protein phospholipid concentrate using supercritical carbon dioxide and ethanol as a co-solvent

In recent years, using dairy phospholipids (PL) as functional ingredients has increased because PL have nutritional benefits and functional properties. In this study, a novel 2-step supercritical fluid extraction (SFE) process was used to extract whey protein phospholipid concentrate (WPPC), a dairy co-product obtained during the manufacture of whey protein isolate, for PL enrichment. In the first step, nonpolar lipids in WPPC were removed using neat supercritical carbon dioxide (S-CO₂) at 41.4 MPa and 60°C. In the second stage, the feasibility of using the polar solvent ethanol as a co-solvent to increase the solubility of S-CO₂ extraction solvent was explored. A 3 × 3 × 2 factorial design with extraction pressure (35.0, 41.4, and 55.0 MPa), temperature (40 and 60°C), and concentration of ethanol (10, 15, and 20%) as independent factors was used to evaluate the extraction efficiency providing the most total PL, and the best proportion of each individual PL from the spent solids collected during S-CO₂ SFE. All lipid fractions were analyzed using thin-layer chromatography and high-performance lipid chromatography. The total amount of PL extracted from WPPC was significantly affected by ethanol concentration; the extraction pressure and temperature were nonsignificant. The optimal SFE condition for generating a concentrated PL lipid fraction was 35.0 MPa, 40°C, and 15% ethanol concentration; the highest amount of extracted PL averaged 26.26 g/100 g of fat. Moreover, adjusting SFE condition allowed successful recovery of a high concentration of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, giving averages of 11.07, 10.07, and 7.2 g/100 g of fat, respectively, 2 to 3 times more than conventional solvent extraction. In addition, exhausted solids obtained after the SFE process were enriched with denatured proteins (72% on dry basis) with significantly more water-holding capacity and emulsifying capacity than untreated WPPC. Overall, this 2-stage SFE process using neat S-CO₂ and ethanol has the greatest potential to produce a PL-rich lipid fraction from WPPC.     

Influence of particle size on protein extractability from soybean and okara

Nitrogen Solubility Index (NSI) of okara was 38 which was only about half of soybean (71). Maximum protein recovery of 97.0% and 93.4% was achieved with soybean and okara flour from their respective fine fractions (<75 μm) in a three-step-sequential extraction. Recovery of protein from unclassified primary ground flour of soy and okara was much lower compared to their corresponding fine fractions (particle size <75 μm). Secondary grinding of coarse fraction improved the overall protein recovery to an extent of 3.3% in okara and to a much larger extent of 6.8% in soybean. Results showed that a two-step sequential extraction with respective solid-to-solvent (w/v) ratios of 1:20 and 1:10 was suitable in terms of protein recovery. Protein recovery from soy granules and okara flakes improved by 30.6% and 6.9%, respectively with the introduction of primary and secondary grinding steps indicating the benefits of the proposed approach for practical applications.     

Effect of stabilization of rice bran on the extractability and recovery of proteins

The extractability profile of rice bran proteins under different conditions were studied to determine optimal conditions of extraction. Milling and differential sieving of rice bran increased the protein content in fine fraction by 7%. Maximum extractability of protein from defatted rice bran at pH 11.0 was 72%, out of which 70% could be precipitated at isoelectric pH 4.0. Increasing the time of extraction or using sodium chloride, sodium dodecyl sulfate or sodium hexametaphosphate were not effective in extracting more protein. The extraction and precipitation profiles of proteins from acid stabilized rice bran were comparable to untreated rice bran. However, it was very low for heat stabilized and parboiled rice bran. Denaturation of protein due to heat treatment affected the extractability of protein. Protein contents of protein concentrates obtained from untreated rice bran and acid stabilized rice bran were in the range of 71–73% whereas protein concentrates from heat stabilized rice bran and parboiled rice bran had lower protein contents of 39.5 and 54.5%, respectively.     

Extracts of Mexican Oregano (<Emphasis Type="Italic">Lippia berlandieri</Emphasis> Schauer) with Antioxidant and Antimicrobial Activity

Antimicrobial activity of fractions obtained from Mexican oregano (Lippia berlandieri Schauer) chloroform extract was tested by growth inhibition against Escherichia coli, Staphylococcus aureus and Bacillus cereus, and antioxidant capacity was tested by inhibition of linoleic acid oxidation. Fractions were obtained by differences in polarity or structure (phenolic and non-phenolic fraction). Gram-positive organisms were more susceptible to Mexican oregano extracts. Fraction 3 (by polarity) and phenolic fractions I, II, III, IV and V were the extracts with higher antimicrobial activity. The non-phenolic fraction had effect against B. cereus. Polarity fraction 5 and phenolic Fraction II had a high antioxidant capacity; a 0.08% concentration of fraction 5 had a similar effect as butylated hydroxytoluene at 0.01% concentration. Fractions of Mexican oregano with different polarity and functional groups had antioxidant and antimicrobial activity and can be used in a variety of applications.     

Protein Extraction from Conophor (Tetracarpidium conophorum) Seed

The extraction procedure and the effect of dispersing agents, pH and molar concentrations on the proteins of conophor seeds which are rich in proteins (21-23%) and edible oil (56%) were evaluated. The extraction was found to be pH dependent. Excellent extraction of proteins was found to occur at acid pH (2.0) and alkaline pH (12.0). The minimum point of nitrogen dispersion occurred at pH 4. The type and concentration of the dispersing agent affected the amount of protein extracted.     

毛榛榛仁肽的氨基酸营养组成与总还原能力评价

以超临界CO2脱脂制备出的毛榛榛仁粕为原料,经Alcalase碱性蛋白酶水解榛仁粕蛋白提取液后,采用葡聚糖凝胶G-25对水解液进行分离纯化,得到Ⅰ和Ⅱ2种榛仁多肽组分,测定其氨基酸组成,并对其营养和总还原能力进行了评价。研究结果表明:对于毛榛榛仁多肽组分Ⅰ和Ⅱ,其氨基酸比值系数分值分别为36.15和41.11,营养价值较低,限制氨基酸分别是赖氨酸和亮氨酸。抗氧化实验结果表明,在相同质量浓度条件下,多肽组分Ⅱ的总还原能力要高于多肽组分Ⅰ,但均小于VC。     

Physicochemical and functional properties of albumin,globulin and glutelin fractions of green lentil seed

Defatted lentil seed flour proteins were separated into their constituent albumin (ALB), globulin (GLB) and glutelin (GLT) fractions followed by determination of their structural and functional properties. The GLB fraction demonstrated superior solubility (84%–100%) at acidic and alkaline pH values when compared to the lower values for ALB and GLT. Amino acid composition analysis showed lower contents of hydrophobic and sulphur-containing residues for GLB. However, GLB had the highest in vitro protein digestibility, which may be due to lower contents of rigid secondary structure fractions like the β-sheet and β-turns. In contrast, water and oil holding capacities as well as gelling ability were better for GLT and ALB than GLB. The GLT fraction formed very poor emulsions at pH 3 and 5 but emulsification was significantly (p < 0.05) improved (smaller oil droplets) at pH 7 and 9. Foaming capacity was strongest for GLB, especially at pH 5, 7 and 9 where increase in protein concentration had a negative effect on foam formation. Overall, the protein type and pH of the environment had stronger effects on emulsion and foaming properties than the protein concentration.     

The condensed tannin content of vegetative Lotus pedunculatus,its regulation by fertiliser application,and effect upon protein solubility

The condensed tannin concentration in Lotus pedunculatus (cv. Grasslands ‘Maku’) was 8–11% DM when grown in acid soils without fertiliser application and 2–3% DM when grown in high fertility soils. Application of P & S fertiliser to the acid soils increased DM yield and reduced condensed tannin content to 4–5% DM, with over 88% of the variation in condensed tannin content being explained by variation in DM yield; it also increased plant total N concentration and halved the molar ratio of condensed tannin: protein (MR). Condensed tannins quantitatively precipitated soluble protein in lotus and also effectively precipitated protein in mixtures of lotus with white clover. The minimum concentration of condensed tannin necessary to precipitate protein was 2–4% DM, corresponding to MR values of 6–13. However, protein from such mixtures could be deaminated by fermentations with rumen fluid in vitro, but potentially soluble protein in pure lotus (MR 29) was protected from deamination by condensed tannins. A portion of lotus tannin could not be bound by plant constituents in finely ground fresh herbage and was designated ‘free’ tannin. This fraction was linearly related to total plant tannin content, was predicted to be zero at 1.8% DM total tannin, and increased at 0.15 units per unit increase in total tannin content above this figure. Maximum ‘free’ tannin comprised 17% of total condensed tannin. Effects of total and ‘free’ tannin content upon the intake and digestion of lotus by sheep are discussed.     

Japanese barnyard millet (Echinochloa frumentacea): Protein content,quality and SDS–PAGE of protein fractions

Total protein from five varieties of Japanese barnyard millet (Echinochloa frumentacea,) was separated into albumin/globulin, prolamin and glutelin fractions. The protein fractions were examined by sodium dodecylsulphate polyacrylamide gel electrophoresis. Total protein of the varieties ranged from 110·5 to 139·3 mg g^?1 of which 11·3–17·2% was albumin/globulins, 6·8–9·3% prolamins, 7·5–11·6% prolamin–like, 5·9–9·1% glutelin-like and 39·3–54·4% true glutelins. Amino acid analyses of the total protein showed that the varieties had essentially the same ammo acid composition. With the exception of lysine the amino acid levels adequately matched the provisional FAO scoring pattern. The amino acid composition of the protein fractions was also very similar. Electrophoretic analysis showed that the albumin/globulin fraction contained three or four components; the prolamin and glutelin fractions each had five components. The glutelin fraction had higher molecular weight components than the other two fractions.     

青刺果抗菌肽组分的分离及其稳定性分析

旨在高值化利用青刺果榨油后副产物青刺果粕中的蛋白质，以青刺果粕为原料，提取青刺果蛋白并制备蛋白酶解物，采用活菌吸附法联合反相高效液相色谱（RP-HPLC）从蛋白酶解物中筛选出抗菌肽组分，评价其抑菌活性，并对抑菌活性最好的组分进行稳定性分析。结果表明：筛选出的3个抗菌肽组分（F1、F2和F3）中，F2组分抑菌活性最好，当其质量浓度为10 mg/mL时对金黄色葡萄球菌和大肠杆菌的抑菌活性分别达到45.5%和39.1%；抗菌肽组分F2抑菌活性随温度升高和盐溶液浓度增加均降低，最适pH为6.0，在酸性环境中极不稳定，对酸性蛋白酶、碱性蛋白酶、胃蛋白酶和胰蛋白酶的耐受性均较差，对中性蛋白酶耐受性较好。综上，青刺果粕中的蛋白酶解物具有抑菌活性，具有开发青刺果抗菌肽的潜力。     

Antioxidant properties of protein hydrolysate from Douchi by membrane ultrafiltration

Protein extracted from Douchi was hydrolyzed by alcalase based on a single-factor experiment and response surface methodology was employed to optimize hydrolysis conditions. The optimum conditions were determined to be: 63℃ extraction temperature, 1.4% quantities of enzyme and substrate, and 1.7 h extraction time. The optimal protein hydrolysate was subsequently subjected to ultrafiltration, and the fraction of molecular weight 10–50 kDa was identified as the most potent contributor to antioxidant activity. Amino acid analysis indicated that acidic amino acids were the most abundant in all fractions of the protein hydrolysate. Infrared spectroscopy showed there were no significant differences in secondary structure between fractions.     

The effect of distillation conditions and alcohol content in ‘heart’ fractions on the concentration of aroma volatiles and undesirable compounds in plum brandies

This study investigates the effect of double‐ or single‐stage distillation and different alcohol content in ‘hearts’ (middle fractions) on the distribution of aroma volatiles and undesirable compounds (methanol, hydrocyanic acid, ethyl carbamate) during distillation of plum brandies. Irrespective of the distillation method used, the first fractions (‘heads’) included mainly aliphatic aldehydes, acetals and esters as well as higher alcohols (1‐propanol, 2‐methyl‐1‐propanol, 1‐butanol, 2‐methyl‐1‐butanol and 3‐methyl‐1‐butanol). Furfural, 1‐hexanol, benzyl alcohol, 2‐phenylethanol and ethyl carbamate occurred in relatively high concentrations in the ‘tail’ fractions. Increasing the concentration of alcohol in the heart fractions from 70 to 90% v /v resulted in a gradual decrease in the concentration of all detected volatile compounds. Compared with single‐stage distillation, double distillation produced heart fractions with lower concentration of acetaldehyde and benzaldehyde and with higher contents of furfural and esters, such as isobutyl acetate and isoamyl acetate. There was a statistically significant increase in the amounts of methanol and ethyl carbamate obtained from double distillation compared with similar fractions derived from the single‐stage process. However, in all fractions these compounds occurred in concentrations much lower than the limits specified by EU regulations. The heart fraction from the double‐stage process with 83% v /v alcohol content received the best scores for aroma and flavour. Copyright © 2017 The Institute of Brewing & Distilling