Two-Steps Procedure for the Purification of Corn Flour Hydrolysate

When corn flour hydrolysate filtrate (CFHF) was treated with 100% (w/v) (NH₄)₂SO₄ for purification, 80.5% of the protein was removed. Among the pH values studied, at pH 1.5, 90.2% of the total protein was precipitated from CFHF and supernatant was removed by decantation. The corn flour hydrolysate was purified with Amberlite and DEAE-cellulose after precipitating the protein at pH 1.5. Charcoal was better than cation- and anion- exchange resins for the removal of proteins, amino acids and pigments from the hydrolysate.     

Fractionation of Water-Soluble and -Insoluble Components from Egg Yolk with Minimum Use of Organic Solvents

A process was developed to fractionate both water-soluble and water-insoluble components from egg yolk with maximum retention of biological and functional properties. About 60–90% of the immunological activity of yolk was recovered in a supernatant after centrifugation of yolk diluted six- to tenfold with water. The remaining yolk pellet had excellent emulsifying properties for mayonnaise preparation. It was further fractionated by deoxycholate treatment to separate triglycerides (80% recovery). Ultrafiltration of the deoxycholate supernatant yielded protein (74% recovery) in the retentate, and phospholipids and cholesterol in the permeate. Phospholipids were then purified by dich-loromethane:ethanol (3:1) extraction, zinc chloride precipitation and acetone washing.     

黑曲霉YY-22产酸性果胶酶的分离纯化

本文研究了从黑曲霉YY-22发酵产酸性果胶酶粗酶液中分离出较高纯度果胶裂解酶(PL)、聚半乳糖醛酸酶(PG)、果胶酯酶(PE)的方法,同时考察了主要组分PL在各分离阶段的纯化效果。依次采用硫酸铵盐析、疏水相互作用层析及离子交换层析对果胶酶PG、PE、PL进行分离。结果表明:果胶酶粗酶液中硫酸铵饱和浓度为65%时,沉淀中PL回收率最大,部分杂蛋白在盐析过程中被分离出来;沉淀复溶后经Phenyl-Sepharose FF疏水相互作用层析首先分离出PE活性组分,又经Q-Sepharose HP强阴离子交换层析分别得到PG和PL活性组分。果胶酶粗酶液中主要组分PL经三步纯化后的比活力达到79.37 U/mg蛋白,纯化倍数为13.30,活力回收为33.05%。较高纯度PL、PG、PE的获得,为进一步研究酶的基本性质及其在果蔬加工和葡萄酒中的应用奠定基础。     

Immunochemical Properties of Native and Heat Denatured Ovalbumin

Affinity antibodies purified against native ovalbumin were found more reactive (higher avidity) against heat-denatured ovalbumin than against the native molecule by three different immunochemical methods. Quantitative immunoprecipitation in soluble phase revealed that more antigen-antibody complexes were insoluble with native ovalbumin than with heat-denatured ovalbumin; 25% of antibodies were still present in supernatant at equivalence as measured by ELISA. At the same conditions with heat-denatured ovalbumin, very small amounts of antibodies were precipitated while almost no activity was found in the supernatant. Classical ELISA or competitive ELISA test allowed detection down to 100 ng/mL of native ovalbumin and 10 ng/mL of denatured ovalbumin.     

Flocculation-enhanced protein recovery from fish processing by-products by isoelectric solubilization/precipitation

Muscle proteins were recovered from rainbow trout processing by-products (fish meat leftover on bones, head, skin, and etc.) by isoelectric solubilization/precipitation. Muscle proteins precipitated at pH 5.5 are typically recovered by high-speed centrifugation at a laboratory scale, which appears to impede process scale-up. Our objective was to investigate the effect of flocculants on separation of precipitated proteins from process water (supernatant). Flocculants with different surface charge properties and molecular weights (M_w) were added to precipitated proteins. Protein separation was evaluated by determining optical density (OD) of the supernatant using Bradford dye-binding method. A high M_w anionic flocculent at 100 mg/L resulted in excellent protein separation following 10 min reaction. The OD of the supernatant was comparable to that of clear water, suggesting that even water-soluble fish muscle proteins were removed from the process water. Freeze-thaw cycles, commonly encountered in the fish processing industry, resulted in even more rapid flocculation reaction. This flocculent could be added to a bio-reactor that precipitates muscle proteins at pH 5.5 in a continuous isoelectric solubilization/precipitation system. However, effects of the flocculants on human and animal health should be determined and appropriate approvals obtained before the recovered muscle proteins can be used in human food products and/or animal feeds.     

Purification and some properties of wheat germ lipoxygenase

Lipoxygenase from the germ of bread wheat was purified to near homogeneity by a classical scheme. After extraction at pH 4.5 from defatted germ, lipoxygenase activity was precipitated by 40% saturation (NH₄)₂SO₄ from a 25% saturation supernatant. After dissolution in a phosphate buffer at pH 7 and extensive dialysis against this buffer, the extract was submitted to gel filtration on Ultrogel AcA 34. The final step of DEAE Sephadex A50 chromatography gave three peaks (L₁, L₂ and L₃) with lipoxygenase activity. The total yield of the purification procedure was close to 30% and the degree of purification varied from 200 to 300 depending on the fraction considered. The three isoenzymes were also detected by disc electrophoresis using a specific staining method and were isolated by isoelectric focusing in a liquid medium. The molecular weight for each isoenzyme was determined to be 90 000–95 000 by gel filtration and 110 000 by electrophoresis in a gradient of acrylamide concentration. The stability, pH optimum and calcium effect have been studied. L₂ and L₃ were less stable than L₁. Optimum pH ranged between 6–6.5 and neither isoenzyme activity was affected by calcium ions. L₁ was twice as active towards β-carotene as L₂ and L₃ for the same level of oxygen uptake, but in comparison to lipoxygenase from horsebean the co-oxidising power of wheat lipoxygenase was very poor.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAE

The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE‐cellulose column chromatography techniques. Trehalase was precipitated between 35–50% ammonium sulfate saturation and approximately 5–8 fold purification was achieved. The yeast cAMP‐dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE‐cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, “cryptic trehalase” (tre‐c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE‐ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE‐cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6–6.8 and 30C, respectively. The kinetic parameters, K_m and V_max, were estimated as 11.78 mM trehalose and 12.47 μmole glucose/min‐mg protein, respectively.     

Recovery of Protein and Microorganisms From Shrimp Peeler Effluent

A study was conducted to determine the quantity and quality of proteinaceous solids recoverable from mechanical shrimp peeler effluent. Solids were recovered by HCl precipitation and centrifugation. Recovery of solids from untreated effluent was 1%-2% by weight (ca. 10% protein) and was predictable by turbidity. The precipitation/centrifugation process reduced supernatant total organic nitrogen and biochemical oxygen demand approximately 50% and turbidity by over 90% compared with untreated effluent. Total aerobic plate counts (APC) of bacteria recovered from unprocessed shrimp and precipitated solids were 10⁵-10⁶ CFU/g, approximately 1.5 log units greater than from peeled shrimp or untreated shrimp effluent. Total APC of bacteria recovered from clarified effluent was 3.2 × 10¹ CFU/mL.     

IgG Antibody from Hen Egg Yolks: Purification by Ethanol Fractionation

A procedure was developed for large-scale preparation of IgG antibodies from egg yolks. The supernatant from egg yolks was obtained after an initial 9–fold dilution with water. The lipids in the supernatant were then almost completely eliminated from the water-soluble protein fraction containing the antibody, by precipitation with 60% ethanol and filtration. Yolk antibody was purified from the lipid-free water-soluble protein fraction by ethanol fractionation at final concentration 30% (pH unadjusted), and again at 25% (pH 7.4). The purified fraction was composed of >99% pure IgG. Recovery of antibody was calculated as 40%.     

PREPARATION, PURIFICATION AND IDENTIFICATION OF 10-OXO-TRANS-8-DECENOIC ACID FROM THE CULTIVATED MUSHROOM, AGARICUS BISPORUS

ABSTRACT A procedure was developed to isolate 10-oxo-trans-8-decenoic acid (ODA) from mushrooms. ODA was produced by homogenizing mushrooms in phosphate buffer with added linoleic acid, extracted from the supernatant after centrifugation, and purified using column and thin-layer chromatography. The purified compound was then characterized using ultraviolet, infrared and mass spectrometry, and nuclear magnetic resonance spectroscopy. The purified compound, containing 97.5% ODA, was a white, waxy solid with a pK_a of 4.68. ODA was soluble in acetone, chloroform, ethanol, ethyl ether, methanol, methylene chloride and water, and slightly soluble in pentane, hexane, heptane and benzene. The TBA test was found to be a viable method for the quantification of ODA.     

Quantitative composition of the lipids of cucumber fruit (Cucumis sativus)

Cucumber flesh contains lipids which are qualitatively similar to those of other non-photosynthetic plant tissues. Phospholipids account for nearly half the weight of lipids. The neutral lipids are characterised by a high proportion of 1,2- and 1,3-diacylglycerols relative to triacylglycerol. The sterol composition is similar to that reported for cucumber seedlings; β-sitosterol together with C₂₉ unsaturated sterols being the major components. Linolenic acid is the major fatty acid component of the neutral, glycolipid and phospholipid fractions and this is reflected in the high levels (> 40%) present in all major lipid classes except phosphatidylethanolamine (25%).     

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.     

Selective Concentration of Bovine Immunoglobulins and α-Lactalbumin from Acid Whey using FeCl3

Immunoglobulins and α-lactalbumin of acid whey were concentrated in supernatant and precipitate when FeCl₃ was added at pH 4.2 and 2.8, respectively. Optimized conditions of pH 4.2 were preferable because of higher retention of immunochemical activity of immunoglobulins. In acid whey treated with 7.5 mM FeCl₃ at pH 4.2 and 4°C, 90% of β-lactoglobulin coprecipitated with serum albumin while 70% of immunoglobulins (92% immunochemically active IgG) and 95% of α-lactalbumin were retained in the supernatant. More than 98% of added iron was subsequently eliminated as precipitate by holding the treated whey at pH 8-9 and 4°C, without losing immunochemical activity of immunoglobulin G, in addition to retained activity of immunoglobulins A and M.     

Adsorption of Shiga Toxin to Poly‐γ‐Glutamate Precipitated

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx‐adsorbing substance from natto by extraction with H₂O, acid treatment, Proteinase K treatment, and an ion exchange chromatography. The purified substance showed an average molecular mass of about 600 kDa. We identified it as poly‐γ‐glutamate (PGA) by amino acid analysis of its hydrolysate and peptide analysis after its treatment with Proteinase K. Purified PGA (MW: molecular weight = about 600 kDa) was considered to adsorb both Stx1 and Stx2 when we separated adsorbed and unadsorbed Stxs (MW = about 72 kDa) by an ultrafiltration method with a centrifugal filter unit (MWCO: molecular weight cut‐off = 100 K). However, PGA with the ability to adsorb Stx was an insoluble form precipitated in the filter unit during centrifugation. PGA precipitated beyond the saturated density was also confirmed to well adsorb both Stx1 and Stx2 by an equilibrated dialysis method. To the best of our knowledge, this is the 1st report on food‐adsorbing Stx.     

米酒乳杆菌素分离纯化条件研究

目的：对米酒乳杆菌产生的广谱细菌素进行分离纯化技术研究。方法和结果：采用活性炭脱色、冷乙醇沉淀、Sephadex G50 葡聚糖凝胶层析和高效液相色谱技术对米酒乳杆菌产生的细菌素进行分离纯化。结果表明,发酵浓缩液脱色的最佳工艺条件为活性炭加量3.5%、温度40℃、脱色时间20h,脱色率达64.5%。冷乙醇沉淀的最佳乙醇终浓度为80%,葡聚糖凝胶层析的洗脱液流速为0.5ml/min,上样量3ml,高效液相色谱纯化的流动相为90% 的甲醇水溶液,在此条件下可得到电泳纯的细菌素。结论：确定了米酒乳杆菌素的分离纯化条件,为细菌素的理化性质和抑菌机理的研究提供依据。     

Properties of the peptides liberated from rice protein in sokujo-moto

In the supernatant of sokujo-moto, a high level of acid carboxypeptidase (ACP) activity and a large amount of peptides were observed, however, the amount of free amino acids liberated was small. In order to determine why these peptides were not hydrolyzed to any significant degree by the ACP, the properties of the peptides in sokujo-moto were investigated in this study. Peptides were fractionated from sokujo-moto by ion exchange column chromatography. ACP purified from rice-koji (rice overgrown with Aspergillus oryzae) was allowed to react with the peptides, and it was found that they were not hydrolyzed to any significant degree by the enzyme. Gel filtration chromatography was performed to ascertain the molecular size distribution of the peptides in sokujo-moto, and it was revealed that they were of low molecular sizes; molecular size: mainly in the range of 200-400, and chain length: 2-3. ACP purified from rice-koji was also allowed to react with various synthetic peptides, and it was found that ACP of rice-koji could not rapidly hydrolyze low-molecular-size peptides, such as dipeptides or tripeptides. Acid protease (AP) purified from rice-koji released peptides of molecular sizes mainly in the range of 300-600 or above from rice protein under acidic conditions (pH 3.6; the pH of sokujomoto). When AP and ACP were allowed to act at the same time on rice protein, mainly low-molecular-size peptides (molecular sizes mainly in the range of 200-400) were produced. From these results, it was estimated that AP released peptides with molecular sizes mainly in the range of 300-600 or above from rice protein and ACP degraded the relatively higher molecular size peptides among them to lower molecular size peptides; consequently only low-molecular-size peptides with molecular sizes mainly in the range of 200-400 were released in the supernatant of sokujo-moto.     

Extraction of Triglycerides and Phospholipids from Canola with Supercritical Carbon Dioxide and Ethanol

Phospholipids used as emulsifiers were sparingly soluble in supercritical CO₂ (SC-CO₂), but could be recovered with the addition of ethanol as an entrainer. Triglycerides and lipids containing phospholipids were extracted with SC-CO₂ in two subsequent steps from both canola flakes and press cake containing 0 and 5% ethanol, respectively. 70°C, 62.0 MPa and addition of ethanol at an initial level of 5% gave maximum yield of lipids. TLC showed the presence of phospholipids in SC-CO₂-ethanol extracts. SC-CO₂ extracts of flakes did not contain long chain fatty acids (C20:0, C22:0, C22:1), but they were extracted when ethanol was added to flakes and when press cake was the raw material.     

Rheological properties of African yam bean (Sphenostylis stenocarpa Hochst. Ex A. Rich.) calcium proteinate and isoelectric protein isolates

The rheological characterizations of African yam bean (Sphenostylis stenocarpa) protein dispersions were investigated. Isoelectrically precipitated protein-IP_alk and IP_salt isolates obtained from alkaline and salt extractions respectively were more soluble than calcium precipitated proteins (CaP_alk and CaP_salt) at pH 3, 7 and 8. Regression analysis showed that Power law, Casson and Bingham rheological models adequately described rheological behaviors of S. stenocarpa protein dispersion. However, Power law gave the best fit. The flow behavior indices (n), at different ionic strength, pH, and temperature media were less than unity, indicating that S. stenocarpa protein dispersion exhibited pseudoplastic behaviors under the conditions tested. Salt extracted proteins were more pseudoplastic than alkali extracted counterpart with n for salt extracted proteins (IP_salt & CaP_salt) lower than that of alkali extracted protein (CaP_alk & CaP_salt). This is a numerical indication that salt extracted S. stenocarpa proteins were of larger shear-thinning tendency than the alkali extracted proteins. The consistency coefficients, k of isoelectrically precipitated protein (0.305-0.327 Pasⁿ) were significantly (P < 0.05) higher than that of calcium proteinates in the range ranged 0.167-0.180 Pasⁿ. Both isoelectrically precipitated proteins and calcium proteinates exhibited yield stress, however, isoelectrically precipitated S. stenocarpa protein exhibited significantly (P < 0.05) higher yield stress (0.275-0.308 Pa) than the calcium proteinates (0.148-0.165 Pa). The effect of temperature on apparent viscosity of the proteins was evaluated using an Arrhenius-type equation. The activation energies (E_a) obtained were in the range 33-51.2 and 42.6-55.5 Jmol⁻¹ for calcium proteinate and isoelectrically precipitated protein respectively.     

底物亲和法分离纯化蕲蛇胃蛋白酶

利用酶(胃蛋白酶)与其底物(酪蛋白)的亲和性,从蕲蛇胃中提取得到一种胃蛋白酶.让底物和酶在pH 2.5的乳酸缓冲液中充分结合,然后调pH至4.0(底物的等电点)沉淀底物和酶的结合物,随后让沉淀物再溶解于乳酸缓冲液中,添加低浓度的SDS将底物和酶分离,最后用DEAE离子交换色谱柱进行纯化,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,分离得到的胃蛋白酶为电泳纯,其分子量为33KD.酶的最适pH值小于3.0,最适温度为50℃.该结果证明胃蛋白酶能利用其与酪蛋白的亲合性及SDS沉淀作用有效地分离,这种胃蛋白酶能在食品生产的加热、强酸处理过程中应用.     

婴幼儿配方粉中香兰素和乙基香兰素的检测方法比较

目的 建立婴幼儿配方粉中香兰素、乙基香兰素的液相色谱法和同位素稀释液相色谱质谱联用两种测定方法。方法 液相色谱法: 样品经乙酸锌沉淀蛋白、Oasis MAX SPE柱净化, HSS T3色谱柱分离后, 在279 nm波长处进行检测, 外标法定量。同位素稀释液质联用法: 样品采用乙腈沉淀蛋白稀释净化后, 采用Waters BEH C18柱分离, 在质谱检测器多反应监测(UPLC-MS/MS)模式下, 用同位素稀释液质联用法进行检测。结果 液相色谱法: 香兰素、乙基香兰素的定量下限(LOQ)均为0.1 mg/kg。香兰素的方法回收率为88%~90.8 %, 相对标准偏差(RSD)小于5.2 %; 乙基香兰素的方法回收率为84%~93.3 %, RSD小于6.2 %。同位素稀释液质联用法香兰素、乙基香兰素的LOQ分别为香兰素1、0.5 mg/kg。香兰素、乙基香兰素的方法回收率分别为82.1%~91.2 %、90.2%~97.6 %, RSD分别小于6.1 %、5.8 %。结论 本文建立的两种方法对同一样品的检测结果一致, 为婴幼儿配方粉中香兰素、乙基香兰素的定量检测提供了较为理想的方法。