Protease activity of enzyme extracts from tamarillo fruit and their specific hydrolysis of bovine caseins.

The characterisation of a serine protease isolated from tamarillo (Solanum betaceum) fruit and its milk casein hydrolysis activity were investigated. Compared with calf rennet, a crude extract from tamarillo exhibited wider caseinolytic activity on sodium caseinate. The purified protease was named "tamarillin" and revealed proteolytic activity toward purified α-, ß- and κ-casein. Similar to calf rennet, tamarillin preferably hydrolysed κ-casein, but, unlike calf rennet, it also displayed high proteolytic activity toward both α- and ß-casein. The major peptide generated from κ-casein by tamarillin was analysed by gel electrophoresis and liquid chromatography mass spectrometry to confirm its molecular mass as 14,290 Da. The cleavage site was confirmed by in-gel tryptic digestion and time-of-flight mass spectrometry analysis to be at Asn123-Thr124. This was in contrast to the Phe105-Met106 cleavage site of rennet hydrolysis.

Purification and characterisation of a protease (tamarillin) from tamarillo fruit.

A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60 °C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

Rapid Screening of Bovine Milk Oligosaccharides in a Whey Permeate Product and Domestic Animal Milks by Accurate Mass Database and Tandem Mass Spectral Library.

A bovine milk oligosaccharide (BMO) library, prepared from cow colostrum, with 34 structures was generated and used to rapidly screen oligosaccharides in domestic animal milks and a whey permeate powder. The novel library was entered into a custom Personal Compound Database and Library (PCDL) and included accurate mass, retention time, and tandem mass spectra. Oligosaccharides in minute-sized samples were separated using nanoliquid chromatography (nanoLC) coupled to a high resolution and sensitive quadrupole-Time of Flight (Q-ToF) MS system. Using the PCDL, 18 oligosaccharides were found in a BMO-enriched product obtained from whey permeate processing. The usefulness of the analytical system and BMO library was further validated using milks from domestic sheep and buffaloes. Through BMO PCDL searching, 15 and 13 oligosaccharides in the BMO library were assigned in sheep and buffalo milks, respectively, thus demonstrating significant overlap between oligosaccharides in bovine (cow and buffalo) and ovine (sheep) milks. This method was shown to be an efficient, reliable, and rapid tool to identify oligosaccharide structures using automated spectral matching.

Analysis of low molecular weight metabolites in tea using mass spectrometry-based analytical methods.

Tea is the second most consumed beverage in the world after water and there are numerous reported health benefits as a result of consuming tea, such as reducing the risk of cardiovascular disease and many types of cancer. Thus, there is much interest in the chemical composition of teas, for example; defining components responsible for contributing to reported health benefits; defining quality characteristics such as product flavor; and monitoring for pesticide residues to comply with food safety import/export requirements. Covered in this review are some of the latest developments in mass spectrometry-based analytical techniques for measuring and characterizing low molecular weight components of tea, in particular primary and secondary metabolites. The methodology; more specifically the chromatography and detection mechanisms used in both targeted and non-targeted studies, and their main advantages and disadvantages are discussed. Finally, we comment on the latest techniques that are likely to have significant benefit to analysts in the future, not merely in the area of tea research, but in the analytical chemistry of low molecular weight compounds in general.

Non-targeted analysis by LC-MS of major metabolite changes during the oolong tea manufacturing in New Zealand.

Oolong tea is a semi-fermented tea that is partially oxidised during the manufacturing process to create a product unique in composition. In this study, we investigated the potential of non-targeted LC-MS with two complementary chromatographic modes to provide a "comprehensive and unbiased" view of biochemical compositional changes occurring during oolong tea manufacturing in New Zealand. Tea leaf samples from throughout the manufacturing/fermentation process during three different harvest periods (spring, summer and autumn) were analysed by four different LC-MS streams. Principal component analysis revealed the de-greening stage of the manufacturing process was responsible for major changes in the biochemical profile, with the methodology detecting changes in a wide range of metabolites of differing polarities, such as flavonoids, nucleosides and primeverosides. Changes during the fermentation phase of the manufacturing process were less marked, however significant increases in levels of free amino acids, a hydroxyjasmonic acid and related metabolites were observed.

Monitoring tea fermentation/manufacturing by direct analysis in real time (DART) mass spectrometry.

Factors such as fermentation methods, geographical origin and season can affect the biochemical composition of tea leaves (Camellia sinensis L.). In this study, the biochemical composition of oolong tea during the manufacturing and fermentation process was studied using a non-targeted method utilising ambient ionisation with a direct analysis in real time (DART) ion source and mass spectrometry (MS). Caffeine dominated the positive ionisation spectra throughout the manufacturing process, while the negative ion spectra collected during manufacturing were rich in ions likely to be surface lipids. Correlation analyses on the spectra revealed two volatile compounds tentatively identified as indole and geranic acid, along with ammonium and caffeine clusters/adducts with geranic acid that increased in concentration during the fermentation stages of the process. The tentative identifications were assigned using a combination of DART-ion-trap MS(n) and DART-accurate mass MS(1) and MS(2) on tea samples and standard compounds. This study highlights the potential of DART-MS to rapidly monitor the progress of complex manufacturing processes such as tea fermentation.

Bowel microbiota moderate host physiological responses to dietary konjac in weanling rats.

Diets rich in complex carbohydrates that resist digestion in the small bowel can alter large bowel ecology and microbiota biochemistry because the carbohydrates become substrates for bacterial growth and metabolism. Conventional or germ-free weanling rats were fed a control diet or diets containing 1.25, 2.5, or 5% konjac (KJ), a commonly used ingredient in Asian foods, for 28 d. In the absence of bowel microbiota, 5% KJ elicited a significant increase in colonic goblet cell numbers and increased expression of mast cell protease genes and of genes that were overrepresented in the KEGG pathway "Metabolism of xenobiotics by cytochrome P450" relative to the control diet. In contrast, feeding 5% KJ caused few changes in mucosal gene expression in conventional rats. Analysis of the colonic microbiota of conventional rats fed KJ showed modest increases in the proportions of Actinobacteria and Bacteroidetes relative to rats fed the control diet, with a concomitant reduction in Firmicutes, which included a 50% reduction in Lactobacillus abundance. Colonic concentrations of short-chain fatty acids and colonic crypt lengths were increased by feeding KJ. Goblet cell numbers were greater in conventional rats fed KJ relative to the control diet but were lower compared with germ-free animals. Serum metabolite profiles were different in germ-free and conventional rats. Metabolites that differed in concentration included several phospholipids, a bile acid metabolite, and an intermediate product of tryptophan metabolism. Overall, KJ in the diet was potentially damaging to the bowel mucosa and produced a protective response from the host. This response was reduced by the presence of the bowel microbiota, which therefore ameliorated potentially detrimental effects of dietary KJ.

Standardised methods for amino acid analysis of food.

Amino acids (AA) are essential nutritional components of a balanced diet and occur in foods in either the free AA form or as the building blocks of proteins. The analysis of AAs in foods is composed of a number of unit operations; the release of the AAs from the food matrix, the separation of the individual AAs and their quantification using calibration standards. Each of these steps has their own idiosyncrasies, e.g. different hydrolysis conditions are required for the optimal release of different AAs and there are a diverse number and type of food matrices, such that most laboratories adapt methods to best suit their applications. There is currently no official standardised method for AA analysis, although the Association of Analytical Communities (AOAC) has validated methods for a number of individual AA components. The established analytical techniques of HPLC (ion exchange or reversed phase) and GC-MS have recently been supplemented by a number of new methods. These include capillary electrophoresis MS and Ultra HPLC-MS, and LC with other detectors. This review will address the intricacies and concerns of the protein hydrolysis step, discuss what specifications or prerequisites need to be placed on the existing and new methods and laboratories using these methods, comment on whether one method can successfully satisfy the exacting requirements of the various unit operations, and finally pose the question 'Is there any merit in 'developing' a validated (e.g. AOAC) official method of analysis for AAs in food?'

Non-targeted analysis of tea by hydrophilic interaction liquid chromatography and high resolution mass spectrometry.

Tea is the second most consumed beverage in the world and its consumption has been associated with numerous potential health benefits. Factors such as fermentation methods, geographical origin and season can affect the primary and secondary metabolite composition of tea. In this study, a hydrophilic interaction liquid chromatography (HILIC) method coupled to high resolution mass spectrometry in both positive and negative ionisation modes was developed and optimised. The method when combined with principal component analysis to analyse three different types of tea, successfully distinguished samples into different categories, and provided evidence of the metabolites which differed between them. The accurate mass and high resolution attributes of the mass spectrometric data were utilised and relative quantification data were extracted post-data acquisition on 18 amino acids, showing significant differences in amino acid concentrations between tea types and countries. This study highlights the potential of HILIC chromatography combined with non-targeted mass spectrometric methods to provide a comprehensive understanding of polar metabolites in plant extracts.

Determination of immunoglobulin G in bovine colostrum and milk powders, and in dietary supplements of bovine origin by protein G affinity liquid chromatography: collaborative study.

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

Analysis of bovine immunoglobulin G in milk, colostrum and dietary supplements: a review.

The immunoprotective properties of bovine milk immunoglobulin G (IgG) have led to a recent proliferation of nutritional products incorporating this protein. It has therefore become critical that reliable analytical techniques for the measurement of the IgG content in such products are available. This literature review surveys current methods of analysis for IgG, including separation-based or immuno-based concentration analysis. The review also discusses nutraceutical applications, regulatory issues, stability of IgG and the significance of primary reference material in IgG analysis.

Affinity liquid chromatography method for the quantification of immunoglobulin G in bovine colostrum powders.

An affinity liquid chromatography (LC) method for the determination of bovine immunoglobulin G (IgG), using protein G coupled to an agarose support, was modified to permit the quantification of IgG in colostrum-based powders. Sample preparation included pH adjustment to 4.6 to precipitate casein and denatured whey protein. The method was applied to a range of colostrum powders and was compared with the alternative independent methods of surface plasmon resonance immunoassay, radial immunodiffusion, and reversed-phase LC. The method was rapid, and performance parameters included a working range of 10-150 microg IgG and precision relative standard deviation values of <10%.