Determination of ovine casein heterogeneity using gel electrophoresis and immunochemical techniques.

Discontinuous PAGE at alkaline and acid pH, polyacrylamide gel isoelectric focusing, two dimensional electrophoresis and immunoblotting have been used to study the heterogeneity of sheep caseins. Three main phenotypes were selected either because of mobility at alkaline and acid pH of the individual alpha s components or because of their relative intensity. On electrophoresis at alkaline pH, one phenotype showed two distinct bands of lower electrophoretic mobility than beta 1- and beta 2-caseins. A detailed study of these components using immunospecific detection with beta-casein antiserum showed that these minor components of ovine casein may have a polypeptide chain similar to that of beta 1- and beta 2-caseins. Complete electrophoretic patterns of the casein components in some individual milks are also presented.     

Mass spectrometry-based analysis of whole-grain phytochemicals

Whole grains are a rich source of several classes of phytochemicals, such as alkylresorcinols, benzoxazinoids, flavonoids, lignans, and phytosterols. A high intake of whole grains has been linked to a reduced risk of some major noncommunicable diseases, and it has been postulated that a complex mixture of phytochemicals works in synergy to generate beneficial health effects. Mass spectrometry, especially when coupled with liquid chromatography, is a widely used method for the analysis of phytochemicals owing to its high sensitivity and dynamic range. In this review, the current knowledge of the mass spectral properties of the most important classes of phytochemicals found in cereals of common wheat, barley, oats, and rye is discussed.     

Antioxidant activity of ovine casein hydrolysates: identification of active peptides by HPLC–MS/MS

This paper shows the potential role of different ovine casein fractions and their hydrolysates to exert antioxidant activity. ABTS^·+ decolorization assay was used to evaluate the antioxidant activity of the casein fractions (β-, κ- and α_s-caseins) before and after their hydrolysis by pepsin, trypsin and chymotrypsin. Although the antioxidant activity increased in all the fractions after hydrolysis, the effect was particularly remarkable in the κ-casein fraction, which increased its antioxidant activity almost threefold. Further assays in a linoleic acid oxidation system showed that κ-casein hydrolysate inhibited lipid peroxidation. Analysis of the ovine κ-casein hydrolysate by RP-HPLC–MS/MS allowed the identification of 12 peptide sequences with potential antioxidant properties. One of the most abundant peptides, the fragment HPHPHLSF [f(98–105)] was chemically synthesized. Results showed that this κ-casein-derived peptide was a potent inhibitor of linoleic acid oxidation with an activity similar to that obtained with the synthetic antioxidant BHT. Although other peptides might also contribute, HPHPHLSF was the one most likely to be responsible for the activity found in the κ-casein hydrolysate.     

Mass spectrometry-based approaches to assess the botanical authenticity of dietary supplements

Dietary supplements are legally considered foods despite frequently including medicinal plants as ingredients. Currently, the consumption of herbal dietary supplements, also known as plant food supplements (PFS), is increasing worldwide and some raw botanicals, highly demanded due to their popularity, extensive use, and/or well-established pharmacological effects, have been attaining high prices in the international markets. Therefore, botanical adulteration for profit increase can occur along the whole PFS industry chain, from raw botanicals to plant extracts, until final PFS. Besides the substitution of high-value species, unintentional mislabeling can happen in morphologically similar species. Both cases represent a health risk for consumers, prompting the development of numerous works to access botanical adulterations in PFS. Among different approaches proposed for this purpose, mass spectrometry (MS)-based techniques have often been reported as the most promising, particularly when hyphenated with chromatographic techniques. Thus, this review aims at describing an overview of the developments in this field, focusing on the applications of MS-based techniques to targeted and untargeted analysis to detect botanical adulterations in plant materials, extracts, and PFS.     

An improved procedure for identifying fiber types in ovine muscles

An improved histochemical procedure has been developed for the identification of three muscle fiber types (βR, αR, αW) in two ovine muscles. This procedure can be used in lieu of the traditional reciprocal procedures of using an oxidative enzyme and ATPase to identify fiber types. The advantage of this simultaneous staining procedure is that it eliminates duplication of effort and time plus it distinguishes the αR (intermediate) fibers more clearly than the traditional methods. For best results, muscle samples must be evaluated within 2h of slaughtering the animal and care must be taken to monitor the pH at regular intervals. More research is needed to determine specific conditions, i.e. sample time, pH, enzyme activity, etc., before this method can be recommended as the most appropriate procedure for determining fiber type in muscles of other species.     

Mass spectrometry-based electric nose system for assessing rice quality during storage at different temperatures

A mass spectrometry-based electronic nose (MS e-nose) was used to measure changes in rice quality during storage at different storage conditions. Rice was stored for 4 months at four different temperatures (0 °C, 20 °C, 30 °C, and 40 °C) and tested for fat acidity, sensory characteristics, and flavor pattern analysis using the MS e-nose. When the rice was stored for long durations at higher temperatures, fat acidity increased and sensory quality was low. Flavor volatile profiles of the rice determined by MS e-nose revealed a tendency for the results to separate into three groups (months 1 + 2, 3, and 4). Volatile profile changes in rice during storage depended on the storage time, regardless of storage temperature. It is likely that the fat acidity and sensory evaluation results, which were related, could be distinguished by their volatile-producing metabolic activities. Accordingly, MS e-nose system was successfully used to screen and qualitatively evaluate stored rice.     

Mass spectrometry-based profiling of phospholipids and sphingolipids in extracts from Saccharomyces cerevisiae

Lipids are rapidly moving to centre stage in many fields of biological sciences. Lipidomics, the systems-level scale analysis of lipids and their interacting factors, is thus an emerging field which holds great promise for drug and biomarker discovery. Here we present a mass spectrometry-based approach for profiling of polar lipids, in particular phospholipids and sphingolipids, in Saccharomyces cerevisiae. The first step includes semi-quantitative surveys of lipids in an untargeted fashion, which is particularly powerful for detection of changes that cannot easily be anticipated. This leads to the identification of ions with increased or decreased signal intensities. Comprehensive theoretical calculation of the masses of yeast phospholipid and sphingolipid molecular species, based on fatty acyl and headgroup heterogeneity, is next used to tentatively assign ions of interest. Subsequent targeted analysis using tandem mass spectrometry allows for characterization and quantification of phospholipids and sphingolipids. Given the high degree of conservation in pathways of lipid metabolism between different organisms, it can be expected that this method will lead to the discovery of novel enzymatic activities and modulators of known ones, particularly when used in combination with genetic and chemogenetic libraries and screens. We validated the method using the EUROSCARF library of non-essential deletion mutants. Mutants of SCS7, a lipid hydroxylase, and SLC1, a putative acyl transferase with unknown substrate specificity, were profiled for their phospholipid and sphingolipid content. The observed changes in lipid profiles are consistent with previous observations and extend our knowledge on in vivo substrate use under permissive growth conditions.     

Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk

A simple immunomagnetic procedure was developed to select macrophages from ovine milk by using a non-specific magnetic positive separation technique. Samples of ewe bulk milk were collected during early, mid, and late lactation; milk samples were centrifuged at 2,000 × g for 30 min at 4°C; the fatty fraction and supernatant were removed, and each pellet was dissolved in 500 μL of pH 7.4 phosphate buffered saline + 0.02% NaN₃. Cells were targeted for selection by using mouse-IgG anti-ovine macrophages. Several trials, testing 2 different fluorochrome-conjugated antibodies [i.e., mouse anti-human CD14:R-Phycoerythrin (RPE) (MCA1568PE, Serotec) and F(ab′)2 rabbit anti-mouse IgG:RPE (STAR12A, Serotec)] and 3 different labeling procedures, were performed to evaluate the purity of samples by flow cytometry. A morphological test was carried out by direct microscopic count in enriched fraction smears stained with May-Grünwald-Giemsa stain to confirm the presence of macrophages. The method described in the present technical note can be considered an innovative application to obtain a single-cell population of high purity selected from all the somatic cells in milk.     

Single nucleotide polymorphisms in the ovine casein genes detected by polymerase chain reaction-single strand conformation polymorphism

Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.     

酪蛋白沉淀检测方法及其在牛乳经济掺假鉴定中的应用

试验表明酪蛋白等电点沉淀检验方法的最适检测条件为:10%乙酸溶液,磁力搅拌,pH4.4～4.8,3000 r/min离心15 min,102℃直接干燥法。通过检测,生牛乳、银桥纯牛奶、市售便宜奶粉、乳清粉、食用明胶、三聚氰胺、大豆分离蛋白粉的酪蛋白质量分数(g/g蛋白质)分别为79.8%、78.7%、0、0、0、0和90.3%。结果表明,本检测方法能够很好的检测正常牛乳的酪蛋白含量,并能对市售便宜奶粉、乳清粉、食用明胶和三聚氰胺进行经济掺假鉴定,结合对产品豆腥味的感官评价和对假酪蛋白色泽偏暗的颜色测定的方法,可以对大豆分离蛋白粉进行掺假鉴定,验证了纯牛乳制品掺假检验的定量指标:酪蛋白质量分数(g/g蛋白质)≥73%。     

Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress

Acrolein (ACR) exposure leads to the formation of protein-ACR adducts. Protein modification by ACR has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here, we report an analytical strategy that enables the quantification of Michael-type protein adducts of ACR in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein-ACR adducts in relation to the unmodified protein thiol pool. Treatment of 3-month-old rats with CCl(4) , an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct ACR-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by ACR were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by ACR has potential value as an index of mitochondrial oxidative stress.     

Hydration of casein micelles and caseinates: Implications for casein micelle structure

By studying the hydration of casein micelles using a variety of techniques, a distinction could be made between water that appeared bound by the protein (∼0.5 g g⁻¹ protein), water associated with the κ-casein brush (∼1.0 g g⁻¹ protein) and water entrapped in the casein micelles (∼1.8 g g⁻¹ protein), yielding a total micellar hydration of ∼3.3 g g⁻¹ protein, in line with casein micelle voluminosity derived from intrinsic viscosity measurements. For caseinate particles, however, the main contributor to intrinsic viscosity was not protein hydration but the non-spherical particle shape. These non-spherical particles in caseinate are likely to be naturally present as primary casein particles (PCP) in casein micelles. PCP could be used to build casein micelles by controlled introduction of micellar salts. Based on the findings of this study, casein micelles could be described as a porous network of non-spherical PCP linked by calcium phosphate nanoclusters.     

Sensory properties of cheddar type cheese produced from recombined milk fat and casein fractions of bovine and ovine origin

The sensory properties of cheddar cheese produced from ovine and bovine milk and recombined mixtures of the fat and casein from the two species have been monitored at intervals over a nine month maturation period. Seven aroma, eleven flavour and five texture attributes were evaluated by an experienced sensory panel. To facilitate interpretation of sensory data, Factor Rotation was applied to the scores obtained by Principal Component Analysis. The sensory space maps resulting from this analysis clearly indicated that cheddar aroma and flavour originate predominantly in the protein fraction of bovine milk. The protein fraction is also dominant in the development of texture. Cheese produced from ovine protein is crumbly throughout maturation, whereas that produced from bovine protein becomes more crumbly with age.     

凝乳酶干酪素生产设备选型的探讨

目的:研究适合凝乳酶干酪素的生产设备;方法:以凝乳酶干酪素产品的技术指标为标准,提出凝乳酶干酪素生产设备选型的基本工艺要求及设备选型方案;结果:干酪槽能满足凝乳酶生产工艺要求,且凝块切割均匀,产品灰分低,是优选方案;结论:优选方案可为凝乳酶干酪素生产厂家的设备选型提供依据.     

Novel immunofluorescent technique for identification of ovine immunoglobulins and other potential opsonins binding to live Staphylococcus aureus

A qualitative in vitro technique has been developed for identification of potential opsonins (immunoglobulins G1, G2, A, M; the third component of ovine complement C3, and fibronectin) of sheep origin, binding to cell walls of viable Staphylococcus aureus. The technique uses monospecific antisera to these ovine proteins. The antisera are conjugated to FITC-protein A such that the protein A-binding sites in the Fc region of the immunoglobulin molecules are occupied with FITC-protein A complexes and are prevented, therefore, from binding to protein A in the staphylococcal cell wall. The technique is highly specific and sensitive, and once conjugated monospecific antisera are prepared, many tests can be done in a short time. Staphylococcus aureus incubated in samples of milk whey showed variable binding of immunoglobulins G1, G2, and M; immunoglobulin A was bound from only one sample of milk whey, and the third component of ovine complement C3- and fibronectin binding were not detected. Most samples of blood serum and colostral whey produced binding of immunoglobulins G1, G2, A, and M, and the third component of ovine complement C3 to the Staphylococcus aureus cell surface; fibronectin binding was found in serum but not in colostral whey. Washings from involuted mammary glands invariably produced binding of all immunoglobulins but not of the third component of ovine complement C3 or fibronectin.     

Mass spectroscopy-gas cromatographic identification of chlorinated hydrocarbons in trout

The PCB-content and the content of chlorinated hydrocarbons in trout was examined by GC-MS. PCBs ranging from trichlorobiphenyls to hexachlorbiphenyls, chlordan, DDE, DDD, DDT and dieldrine were found and identified by mass spectrometry. We suppose that less chlorinated PCBs in trout are not deposed or will be excreted very quickly.     

酪蛋白酶解特性的研究

用胰蛋白酶酶解酪蛋白,用正交实验得出的最佳酶解条件进行酶解。取不同酶解时间的酶解物,分析酶解物的水解度、氨基氮含量、可溶性氮含量、肽氮含量及pH值随时间的变化趋势,水解度和氨基氮含量在12h内出现显著性增长,可溶性氮含量和肽氮含量在1h内出现及其显著的增加,pH值在10h内表现出明显的下降趋势。从不同角度分析和推测酪蛋白酶解过程。     

Zinc-binding capacity of yak casein hydrolysate and the zinc-releasing characteristics of casein hydrolysate-zinc complexes

Many factors affect the bioavailability of dietary Zn, which leads to its low availability in some food systems and Zn nutrient deficiency. However, some proteins or peptides can form complexes with Zn and increase its absorption and bioavailability in intestinal conditions. The purpose of this work was to determine the Zn-binding activity of yak casein hydrolysate (YCH) and examine its stability, solubility, and dialyzability in a simulated intestinal environment. The Zn-binding activity of YCH, prepared using alcalase, pepsin, trypsin, Flavozyme (Novo Nordisk Biochem Inc., Franklinton, NC), or papain, was investigated. Evidence for the formation of complexes between Zn and YCH also were detected by UV-visible spectroscopy and Fourier transform infrared spectroscopy. Results were that YCH prepared with alcalase and trypsin possessed the highest Zn-binding capacity compared with YCH prepared with pepsin, Flavozyme, or papain. The 6-h YCH obtained with alcalase showed the highest Zn-binding capacity. Compared with native yak casein, the Zn-binding activity of YCH was significantly lower, but its solubility and dialyzability were markedly higher under intestinal basic pH ranges. This is important because high solubility and dialyzability is associated with better bioavailability. Both UV-visible spectroscopy and Fourier transform infrared spectroscopy spectra indicated that some sites of YCH can bind with Zn ions and form complexes that make Zn more soluble and dialyzable under simulated intestinal conditions. Therefore, YCH-Zn complexes may have potential to improve Zn bioavailability.     

夏黑葡萄附生酵母菌分离及多相分类鉴定

葡萄野生酵母是酿造优质葡萄酒的重要菌种来源,为发现有价值的野生葡萄酵母,本研究以夏黑葡萄为研究对象,采用平板划线分离的方法得到3株葡萄野生酵母YYMPT-1、YYMPT-2和YYMPT-3,利用扫描电子显微镜（SEM）进行形态观察,PCR扩增26S核糖体DNA的D1/D2区（26S r DNA D1/D2）、核糖体内转录间隔区（ITS1-5.8SITS2）和肌动蛋白基因（Actin gene）区域,并构建系统发育树,将3株酵母菌分别鉴定为有孢汉逊酵母（Hanseniaspora uvarum）、近玫色锁掷酵母（Sporidiobolus pararoseus）和假丝酵母（Candida zemplinina）。本研究结果为优质葡萄酒酿造和改善葡萄酒风味提供了3株具有潜在工业化应用价值的资源菌。      

Yak milk casein as a functional ingredient: preparation and identification of angiotensin-I-converting enzyme inhibitory peptides

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.