Ratko M. Jankov

A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro

Background It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low‐pH conditions.

Oxidation products of hyperforin from Hypericum perforatum

The isolation of two oxidation products of hyperforin from the aerial parts of Hypericum perforatum and their structure determination by means of 2D NMR methods is reported. The products had the same 1-(2-methyl-1-oxopropyl)-2,12-dioxo-3,10 beta-bis(3-methyl-2-butenyl)-11 beta-methyl-11 alpha-(4-methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0(4,8)]-3-dodec ene skeleton. In addition, one of them, with the same number of carbons as hyperforin (C35H52O5), contained a 1-methyl-l-hydroxyethyl group in the 6 beta-position, whereas the other compound (a hemiacetal, C32H46O5), presumably a degradation product of hyperforin, exhibited a 6-hydroxy function. The latter was an inseparable mixture of 6 alpha- and 6 beta-hydroxy epimers undergoing (according to phase sensitive NOESY) mutual interconversion.

Allergenic potency of kiwi fruit during fruit development

Abstract Food allergies, including kiwi fruit allergy, have been the subject of extensive research in the last few years. The aim of this study was to examine a possible relationship between the developmental stage of kiwi fruit and its allergenic potency. The protein and allergen patterns of kiwi fruit extracts in September, October, November and December fruit in the period from 2000–2002 were analysed. One of the factors that may contribute to the difficulties in proposing well-defined and standardized fruit extracts should also be the time of fruit harvesting. In this particular case, when the kiwi fruit was edible throughout November and December, we showed discrepancies in allergen content and potencies both in qualitative and quantitative terms. Two major allergens of kiwi fruit, Act c 1 and Act c 2, mainly accounted for the highest allergenic potential of November kiwi extract in vivo and in vitro. Not only the content of major allergens, but also the ratio of different proteins and even isoforms of the same allergen (Act c 2) change with fruit ripening. These findings should be taken into account during preparation of extracts for allergy diagnosis.

The influence of a residual group in low-molecular-weight allergoids of Artemisia vulgaris pollen on their allergenicity, IgE- and IgG-binding properties

Background: Reaction of ε‐amino groups of lysine with potassium cyanate, maleic, or succinic anhydride leads to allergoids of low molecular weight. No study has been performed to compare their properties and investigate the influence of a residual group on allergenicity and human IgE‐ and IgG‐binding of these derivatives.

The identification of a low molecular mass bacteriocin, rhamnosin A, produced by Lactobacillus rhamnosus strain 68.

Aims:  This study focuses on the isolation and characterization of a peptide with bacteriocin‐like properties isolated from Lactobacillus rhamnosus strain 68, previously identified by 16S rRNA gene sequencing and originating from human gastrointestinal flora.

Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.

Carnosic acid 12-methyl ether-γ-lactone, a ferruginol-type diterpene from Salvia officinalis

Abstract A new oxygenated diterpene γ-lactone, 12-methoxy-8,11,13-abietatrien-20,11-olide, a derivative of carnosic acid (carnosic acid 12-methyl ether-γ-lactone), has been isolated from the aerial parts of Salvia officinalis , along with three other known terpenoids: manool, rosmanol 7-ethyl ether and oleanic acid. The structures of all isolated compounds were established by spectroscopic data, some chemical reactions and were substantiated by X-ray diffraction.

In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+)) and stimulated by 50mM Fe(2+). Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.

Development of Sandwich Enzyme‐Linked Immunosorbent Assay for Determination of Tetanus Toxoid Concentration

Abstract According to the recommendation of the World Health Organization (WHO), the use of an in vivo test for measuring of the potency of tetanus toxoid vaccine (TTdV) is still unavoidable, but the establishment of a convenient in vitro test would significantly improve the work in this field. A sandwich enzyme‐linked immunosorbent assay (sELISA) was developed for a rapid and sensitive quantification of tetanus toxoid (TTd). We produced four monoclonal antibodies (MAbs) designated 41, 51, 62, and 71 that reacted with TTd and recognized different antigenic determinants on TTd. We also used two of these antibodies for developing a sELISA, with MoAb 71 as an immobilized and MoAb 51 as a capture antibody. The measurement range of this assay was from 31–1000 ng/mL and the minimum detection limit for TTd was 31 ng/mL. This high sensitivity of this sELISA and its good reproducibility suggest that the developed method could be reliably used to estimate the concentration of TTd, which could be easily extrapolated to the estimation of vaccine potency.