Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.     

The degradation of 14C-labelled phosphatidyl choline in soil

When phosphatidyl [N-methyl-¹⁴CO]choline or phosphatidyl choline di[l-¹⁴C]palmitoyl were incubated in a low phosphorus status soil there was an early and rapid release of CO₂ and a concurrent increase in NaHCO₃-extractable inorganic phosphorus, indicating mineralization of the added organic phosphorus. Mineraiization slowed dramatically and by 20 days only 50% of the carbon from the molecule was accounted for as microbial biomass or respiration. The rates of release of ¹⁴CO₂ from the two labelled substrates indicated that ¹⁴CO₂ measured as respiration initially arose more swiftly from the carbon portion of the molecules with easiest access to enzymic degradation.     

Nondestructive quantification of organic compounds in whole milk without pretreatment by two-dimensional NMR spectroscopy

In this study, various organic compounds in commercial whole milk were quantified simultaneously by 1H 1D and 1H - 13C HSQC 2D NMR spectra without any pretreatment. 2D NMR spectroscopy was applied to quantification of milk compounds for the first time. Milk fat content was easily determined to be 3.6 +/- 0.1%, and the lactose content was 47.8 +/- 1.0 mg/mL by 1H NMR spectra. From 1H-13C HSQC spectra, the concentrations of citrate, N-acetylcarbohydrates, and trimethylamine were determined to be 3.2 +/- 0.2, 2.9 +/- 0.1, and 4.0 +/- 0.6 mM, respectively. The latter two compounds were quantified in milk for the first time. Butyric acid, total monounsaturated fatty acids, and total polyunsaturated fatty acids of triacylglycerols were 6.2 +/- 0.5, 9.1 +/- 0.9, and 2.9 +/- 0.3 mM, respectively. The fatty acid compositions (mol %) of triacylglycerols were then calculated and were observed to be in good agreement with reference values. The results indicated that 1H 1D and 1H-13C HSQC 2D NMR spectroscopy is useful for the rapid and nondestructive determination of various compounds in milk.     

Rapid assay of choline in foods using microwave hydrolysis and a choline biosensor

A fast procedure for the determination of choline in food was developed by coupling a microwave hydrolysis procedure with an O(2)/choline oxidase-based electrochemical biosensor. Time and temperature were varied to select the best conditions for the microwave hydrolysis. Results have been compared with those found by the traditional method, constituted by hydrolysis at 70 degrees C followed by enzymatic-colorimetric assay. Data obtained by the biosensor method correlated well with the enzymatic-colorimetric assay (R(2) = 0.998). Microwave versus traditional hydrolysis gave a good correlation both with the colorimetric and with biosensor procedures with a relative error below 6%. The method is sensitive and selective enough to be used for a wide variety of food items reducing remarkably the analysis time.     

Application of bubble separation for quantitative analysis of choline in Dioscorea (yam) tubers

A modified assay based on the AACC official method 86-45 (AACC, 2000) for the determination of choline in three cereals and three varieties of Dioscorea (yam) tubers was developed. When tested in wheat, rice, and oat flour, choline estimated by the modified method was 34.0-45.3% higher than that of the original AACC method. In a system with higher contents of starch and mucilage, such as Dioscorea (yam) tubers, extra procedures in sample preparation needed to be carried out to separate starch and mucilage. The choline contents of the following Dioscorea (yam) tubers using the original AACC method and the present modified AACC method through coupling an additional bubble separation procedure, respectively, were (mean +/- SD, mg/g solid) Keelung yam (D. pseudojaponicaY.) 0.92 +/- 0.09 and 2.21 +/- 0.12, Yangmingshan yam (D. alata L.) 0.77 +/- 0.09 and 1.78 +/- 0.28, and Ming-Chien yam (D. purpurea) 0.44 +/- 0.09 and 1.35 +/- 0.19. Choline was 231-306% higher than when the original AACC method was used. Dioscorea (yam) tubers were much higher in choline content than they were in cereals. Bubble separation is an appropriate procedure in the practice for the maximum assay of choline in yams. It is accurate, rapid, easy to handle, and especially good for recovering choline from a starch and polysaccharide-protein-containing system.     

High performance liquid chromatography of phenolic choline ester fragments derived by chemical and enzymatic fragmentation processes: analysis of sinapine in rape seed

High-performance liquid chromatography methods based on reversed-phase chromatography (RPC) and normal phase chromatography (NPC) were introduced for the separation of some representative phenolic acids, choline and betaine, which are the fragments of phenolic choline esters. Sinapine, which is the major phenolic choline ester found in rape seed, was quantitatively hydrolyzed to choline and sinapic acid upon treatment with a solution of sodium hydroxide at room temperature. Choline was further converted to betaine by incubating the base hydrolyzate with choline oxidase. Both sinapic acid and betaine formed the basis for the quantitative determination of sinapine in rape seed by RPC and NPC, respectively. The amounts of sinapine found in rape seed via either of the two fragments (i.e., sinapic acid or betaine) were in very close agreement.     

Composition and Regiodistribution of Fatty Acids in Triacylglycerols and Phospholipids from Red and Black Rices

Regiospecific profiles of fatty acids (FA) of triacylglycerols (TAG) and phospholipids (PL) isolated from red and black rices were investigated. The lipids extracted from red and black rices were separated by thin‐layer chromatography (TLC) into eight subfractions, respectively. With a few exceptions, the major lipid components were TAG (76.4–80.5%), free FA (7.2–9.8%), and phospholipids (3.5–3.6%), while hydrocarbons, steryl esters, diacylglycerols (1,3‐DAG and 1,2‐DAG), and monoacylglycerols were present in minor proportions (0.1–4.1%). The PL components isolated from the two cultivars were phosphatidyl choline (52.3–53.7%), phosphatidyl ethanolamine (22.3–23.1%), phosphatidyl inositol (20.6–21.3%), and others (<3.4%). No significant differences (P < 0.05) in FA distribution were found when these cultivars were compared. The principal characteristics of the FA distribution in the TAG and PL were evident in the rices between the two cultivars: unsaturated FA were predominantly located at the sn‐2 position (77.3–96.8%), while saturated FA primary occupied the sn‐1 or the sn‐3 position (35.0–78.7%) in these lipids. The results of this study could provide useful information to both consumers and producers during manufacture of traditional rice foods in Japan.     

Effect of malondialdehyde on the determination of furosine in milk-based products

For nutritional purposes the recombination of milk or milk proteins with polyunsaturated fatty acids had long been considered in newly designed products, such as functional foods. Four milk-resembling model systems were prepared having malondialdehyde content ups to 1 mM. Systems were heated between 110 and 150 degrees C for up to 30 min. The effect of the presence of bifunctional aldehydes on the determination of furosine as a heat-induced marker was investigated. Levels of 0.01 mM MAD in the reaction mixture could affect significantly the determination of furosine in a milk-based system. Impairment of lysyl residues through the analysis of furosine could be underestimated in the presence of malondialdehyde.     

Combination of total solids determined by oven drying and fat determined by Mojonnier extraction for measurement of solids-not-fat content of raw milk: collaborative study

Each of 9 laboratories analyzed 9 pairs of blind duplicate raw milk samples for fat, using the Mojonnier ether extraction method (16.E13-16.E17), and for total solids, using a new direct forced air oven method. Solids-not-fat was determined by subtracting percent fat from percent total solids. The solids-not-fat content of the samples tested in the collaborative study ranged from 8.48 to 9.36%. The average repeatability standard deviation (sr) and the average reproducibility standard deviation (SR) for the solids-not-fat method were 0.019 and 0.041, respectively. Average repeatability (RSDr) and reproducibility (RSDR) relative standard deviations were 0.218 and 0.466%, respectively. The mean repeatability value (r) was 0.055; the mean reproducibility value (R) was 0.117. The difference between milk total solids determined by direct forced air oven drying and milk fat determined by Mojonnier ether extraction has been approved for interim official first action for determination of solids-not-fat content of milk.     

Optical sensor for sulfur dioxide determination in wines

A method for the determination of free and total sulfur dioxide in wines, based on the use of an optical sensor that employs a dichlorobis(diphenylphosphino)methane dipalladium I complex [Pd(2)(dppm)(2)Cl(2)] immobilized in a PVC membrane plasticized with o-nitrophenyloctylether (o-NPOE) is described. A sensing membrane [4.2% Pd(2)(dppm)(2)Cl(2), 20.8% PVC, and 75% o-NPOE] was adapted to the tip of a bifurcated optical fiber bundle to perform reflectance measurements at 550 nm. The detection system consisted of two cells (40 mL), which hold the sample solution (plus reagents) and the optical sensor, respectively. For the determination of free SO(2), a wine sample was mixed with H(2)SO(4) solution in the sample cell, into which N(2) was bubbled, providing mixing of the solutions and conducting the SO(2) formed toward the detection cell. For determination of total SO(2), a KOH solution was mixed with the wine in the sample cell. Afterward, an H(2)SO(4) solution was added to the cell, and then N(2) was bubbled to conclude the measurement. Linear responses up to 50 and 150 mg L(-1) were obtained for free and total SO(2), with detection limits of 0.37 and 0.70 mg L(-1), respectively. The repeatability of the method was evaluated by carrying out 10 measurements using a single wine sample, providing relative standard deviation values of 2.2 and 2.5% for free and total SO(2), respectively. The sensing membrane prepared from 10 muL of the cocktail solution lasted for 80 measurements, whereas those prepared from 200 muL can be used for 250 measurements. The method was applied to free and total SO(2) determination in wines, and the results did not show significant difference from those obtained with the Ripper reference method at a confidence level of 95%.     

Einfluß von Ca2+ und pH auf das Fettsäuremuster von Phospholipiden der Rapswurzel (Brassica napus L.)

Effect of Ca²⁺ and pH upon the fatty acid composition of phospholipids from roots of rape plants (Brassica napus L.) The influence of Ca²⁺ and pH upon the dry matter of the shoot and the root of rape plants (Brassica napus L.) as well as upon the accumulation of nutrients in the shoot and the fatty acid composition of phosphatidyl ethanolamine (PÄ) and phosphatidyl choline (PC) from rape plant roots was tested by means of a water culture experiment. The experiment was designed with two concentrations of Ca-nutrition (3 mM and 0,03 mM CaCl₂ · 6 H₂O) and with three levels of pH (3,5; 5; 8) on the basis of four replications. The amounts and percentages of macro nutrients in the shoot indicated a specific effect on ion uptake by the treatments. K and Mg absorbed as cations were accumulated most intensively in the shoot at pH 5 whereas P absorbed as an anion was accumulated independent of pH at the same Ca-concentration. The fatty acid composition of PÄ and PC was distinctly dependent on the treatments. With regard to linolenic acid it appeared that Ca-nutrition may soften the harmful effect of a high H⁺-concentration. The results were discussed in relation to membrane functions.     

Quantitation of silibinin, a putative cancer chemopreventive agent derived from milk thistle (Silybum marianum), in human plasma by high-performance liquid chromatography and identification of possible metabolites

Silibinin has recently received attention as a potential cancer chemopreventive agent because of its antiproliferative and anticarcinogenic effects. A simple and specific reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of silibinin in human plasma. Sample preparation involved simple protein precipitation, and separation was achieved on a Waters Atlantis C18 column with flow rate of 1.0 mL/min at 40 degrees C and UV detection at 290 nm. Silibinin was detected as two peaks corresponding to trans-diastereoisomers. The peak area was linear over the investigated concentration range (0-5000 ng/mL). The limits of detection were 2 and 1 ng/mL for the two diastereoisomers (d1 and d2), with a recovery of 53-58%. This method was utilized to detect silibinin in plasma of colorectal patients after 7 days of treatment with silipide (silibinin formulated with phosphatidyl choline).     

Isolation and gas chromatographic determination of chlorsulfuron in milk

A method for the isolation and gas chromatographic determination of chlorsulfuron in milk is presented. Blank or chlorsulfuron-spiked milk samples were blended into C-18 (octadecylsilyl derivatized silica, ODS) packing material. A column made from the C-18/milk matrix was washed with hexane after which chlorsulfuron was eluted with dichloromethane (DCM). The DCM eluate contained chlorsulfuron which was free from interfering co-extractants when analyzed by gas chromatography utilizing a nitrogen/phosphorus detector. Chlorsulfuron was found to undergo a thermally induced decomposition to give 2-amino-4-methoxy-6-methyl-1,3,5-triazine, which was detected and quantitated by this method. Standard curves for these analyses were linear (r = 0.992 +/- 0.004, n = 5), with an average percentage recovery of 91.6 +/- 10.8%, over the concentration range examined (62.5-2000 ng/mL). The inter- and intra-assay variabilities were 11.6 +/- 7.5% and 6.2%, respectively.     

Development of a simple method for the determination of genistein, daidzein, biochanin A, and formononetin (biochanin B) in human urine.

A simple method was developed for the determination of free and/or total isoflavones daidzein, genistein, and their respective 4'-methoxy derivatives biochanin A and formononetin (biochanin B) at low levels in human urine. A solid-phase extraction on octadecyl silica (C(18)) columns was used for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElut 1010 column connected on-line to a Florisil cartridge by a Teflon stopcock was used for effective eluate purification. A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers using GC/MS-SIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, whereas robigenin was used in the case of total isoflavone determination. Recoveries for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL(-)(1) level and from 63.5 to 89. 2% at the 5 ng mL(-)(1) level in urine. Analytical curves were linear between 5 and 25 ng mL(-)(1). Detection limits varied from 1 ng mL(-)(1) for formononetin to 2.3 ng mL(-)(1) for daidzein. Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL(-1) level.     

超高效液相色谱-串联质谱法测定牛奶和奶粉中五种苯并咪唑类药物

为建立牛奶及奶粉中5种苯并咪唑类药物(阿苯达唑、芬苯达唑、奥芬达唑、噻苯达唑、苯硫氨酯)简便、准确的分析检测方法,以固相萃取为净化手段,采用Acquity UPLC^® BEH C₁₈(100 mm×2.1 mm,1.7 μm)色谱柱进行分离,电喷雾离子源(ESI⁺),多反应监测(MRM)模式检测。结果表明,5种苯并咪唑类药物在1~500 μg·L^-1范围内呈线性相关,相关系数(r)均大于0.996,加标平均回收率介于91.7%~97.8%之间,相对标准偏差(RSD)在1.4%~6.5%之间,基质效应均小于15%。5种药物的检出限均在0.1~3 μg·kg^-1范围内,定量限均在0.5~10 μg·kg^-1范围内。该方法操作简单、快速且灵敏度高,重现性好,能够用于牛奶及奶粉5种苯并咪唑类药物的同时测定。     

异位酸饲料添加剂对奶牛产奶的作用

50头经产的黑白花奶牛200天的试验表明,在基础日粮中添加异位酸的试验牛,平均每头每日产奶量比喂基础日粮的对照牛多1.01公斤,增加5.14%(P<0.01);折算成标准奶,每头每日比对照牛多产1.41公斤,增加7.84%(P<0.01)。乳脂、乳蛋白和乳中干物质的含量分别提高4.43%,1.31%和2.68%。试验牛产1公斤标准奶所需要的奶牛能量单位和粗蛋白质比对照牛低。两组牛的健康状况和繁殖性能相似。     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.     

Biochemical fluorometric method for the determination of riboflavin in milk

Methods of analysis of vitamin B2 in foods generally consist of the extraction of the sample, followed by enzymatic hydrolysis and quantitative measurement of the analyte, typically through RP-HPLC. The scope of our work here is to present a soft method to measure the free riboflavin content of a nontransparent and nonhomogeneous matrix such as milk, avoiding any extraction and separation of phases that are required in any published method for determination of the free RBF content in foods. We combine the front-face (FF) measurement of the light emission of milk with the ability of the apo-form of the riboflavin-binding protein (RBP) from chicken egg white to quench the riboflavin fluorescence. Thus, we titrate the RBF present in milk by gradually adding a solution of RBP to the milk sample and measuring, upon each addition, the FF residual emission due to uncomplexed RBF. The RBP binding capability has been measured in the same matrix of the analyte. Our results indicate a concentration of free RBF practically co-incident with the certified value for total B2 vitamin content in reference milk CRM 421. Keywords: Front-face fluorescence; riboflavin; apo-riboflavin-binding protein; milk fluorescence.     

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.     

A novel method to analyze betaine in chicken liver: effect of dietary betaine and choline supplementation on the hepatic betaine concentration in broiler chicks

Betaine was measured from liver tissue by a high-performance liquid chromatographic (HPLC) method developed for this study. The method involves homogenization of liver in acetate buffer at pH 6 and precipitation of protein with trichloroacetic acid, which was removed by diethyl ether extraction. Betaine was separated using a cation exchange column in Ca(2+) form and detected with a refractive index detector. This method also allows the determination of S-adenosylmethionine (S-AM) from the same liver extract but with different HPLC conditions. Broiler chicks were fed with experimental diets supplemented with four different doses of betaine or choline ranging from 0 to 5 mol equiv. After a 3 week feeding period, the livers were analyzed for betaine and S-AM. Dietary betaine was twice as efficient in increasing the hepatic betaine concentration as dietary choline. The hepatic S-AM concentrations were similar in all dietary treatments.