Stable isotope analysis confirms substantial changes in the fatty acid composition of bacteria treated with antimicrobial random peptide mixtures (RPMs).

Resistance of plant-pathogenic bacteria to classic antibiotics has prompted the search for suitable alternative antimicrobial substances. One promising strategy could be the use of purposely synthesized random peptide mixtures (RPMs). Six plant-pathogenic bacteria were cultivated and treated with two RPMs previously found to show antimicrobial activity mainly by bacterial membrane disruption. Here, we show that bacteria treated with RPMs showed partly remarkable changes in the fatty acid pattern while those unaffected did not. Quantitative changes could be verified by compound specific isotope analysis of δ13C values (‰). This technique was employed due to the characteristic feature of stronger bonds between heavier isotopes in (bio)chemical reactions. As a proof of concept, the increase in abundance of a fatty acid group after RPM treatment was accompanied with a decrease in the 13C content and vice versa. We propose that our findings will help designing and synthesizing more selective antimicrobial peptides.

Patterns and compound specific stable carbon isotope analysis (δ13 C) of capsaicinoids in Cayenne chilli fruits of different ripening stages.

INTRODUCTION: Capsaicinoids are alkaloids of high pungency which are exclusively formed by fruits of the genus Capsicum. Capsaicinoid content and composition of Capsicum fruits are influenced by ripening. OBJECTIVE: Determination of changes in content and pattern of individual capsaicinoids in chilli pods with fruit ripening. Compound specific stable carbon isotope analysis (δ13 C values (‰), CSIA) was used for a better understanding of capsaicinoid development during fruit ripening. METHODOLOGY: Cayenne chillies (Capsicum annuum) were grown in a glasshouse and harvested from different plants at four ripening stages (unripe, semi-ripe, ripe, overripe). Nine capsaicinoids (one verified by synthesis) were quantified by gas chromatography with mass spectrometry (GC-MS). For CSIA, an acetylation method for derivatising capsaicinoids was developed. RESULTS: Variations in the patterns of the nine capsaicinoids were not relatable to a distinct ripening stage and also total contents varied extensively from plant to plant. However, changes in total capsaicinoid concentrations were systematic. In almost all plants, maximum values were reached in unripe fruits, then decreased to semi-ripe samples and increased again in the following ripening process. Likewise, δ13 C values of individual capsaicinoids were always by ~2‰ heavier in unripe than in semi-ripe or ripe fruits. However, direction of changes in sum-δ13 C values (‰) (taking contributions of all capsaicinoids together) could not be explained by corresponding variations in capsaicinoid concentrations. CONCLUSIONS: Both quantification and δ13 C values (‰) verified the presence of ripening-related changes in the capsaicinoid content which may be caused by simultaneously proceeding reactions like synthesis, storage and degradation of capsaicinoids.

Differentiation of European and Chinese Truffle (Tuber sp.) Species by Means of Sterol Fingerprints.

The increasing demand of valuable truffles (Tuber sp.) has prompted new areas of naturally growing truffles entering the market. Hence, the identification of valueless Tuber species is an important task to prevent food fraud. Here, we show that sterol patterns are suited to differentiate five Tuber species (Tuber magnatum, Tuber melanosporum, Tuber aestivum, Tuber albidum, and Tuber indicum varieties) from each other. Next to the known main sterols of Tuber, ergosterol and brassicasterol, occurrence of minor sterols in differing shares resulted in characteristic fingerprints in the five Tuber species, irrespective of the country of origin. A total of 27 sterols were evaluated, and we proposed assignment criteria of main sterol relations as well as eight distinct biomarkers within the minor compounds for the differentiation of European and Chinese truffles.

Fate of free and bound phytol and tocopherols during fruit ripening of two Capsicum cultivars.

Phytol and tocopherols and their fatty acid esters (PFAE and TFAE) are isoprenoid lipid components which can be found for instance in vegetables. Their behavior during maturation of fruits and vegetables could reveal valuable information on their biosynthetic formation and biological function. As pods of the genus Capsicum contain considerable amounts of both PFAE and TFAE, two cultivars (i.e. Capsicum annuum var. Forajido and Capsicum chinense var. Habanero) were grown in a greenhouse project. The date of flowering and fruit formation of each blossom was noted and fruits were harvested in four specific periods which corresponded with different stages of ripening, i.e. unripe, semi-ripe, ripe and overripe. Quantification by means of gas chromatography mass spectrometry and creation of development profiles strongly supported the suggestion that PFAE and TFAE were formed as storage molecules during fruit ripening and parallel degradation of chlorophyll. Additionally, compound-specific carbon isotope ratios (δ13C values (‰)) of originally in PFAE and chlorophyll bound phytol ultimately proved that PFAE, besides tocopherols, serve as sink for the cytotoxic phytol moiety released from chlorophyll degradation during fruit ripening. Furthermore, color measurements were successfully implemented to simplify the usually cumbersome separation of chili fruits into different ripening degrees.

Occurrence and fate studies (sunlight exposure and stable carbon isotope analysis) of the halogenated natural product MHC-1 and its producer Plocamium cartilagineum.

MHC-1 is a halogenated natural product (HNP) produced by the red seaweed Plocamium cartilagineum. MHC-1 concentrations of 550-2700 µg/g dry weight were found in Plocamium collected by divers at Heligoland (Germany). Compared to that MHC-1 concentrations were much lower in samples collected on beaches in Ireland and Portugal. Exposure of leaves of Plocamium to sunlight showed that MHC-1 was readily transformed by hydrodebromination. At Heligoland in March, MHC-1 (δ13C value -45.2‰) was lighter in carbon by ~15‰ compared to the bulk δ13C value (‰) of Plocamium (-30.7‰). Collected at the same time and location at Heligoland, samples of Halichondria and Mastocarpus sp. were richer in carbon (by ~10‰) as Plocamium. However, the δ13C value of MHC-1 in Halichondria (-44.6‰) and Mastocarpus sp. (-42.1‰) was as negative as in Plocamium. This was indirect proof that MHC-1 was produced by Plocamium and then released into the water phase from where it then was bioconcentrated by Halichondria and Mastocarpus sp. In agreement with that, concentrations of MHC-1 in Halichondria and Mastocarpus sp. were much lower than in Plocamium. In addition, a potential isomer of MHC-1 (compound X) was detected in all samples from Heligoland at ~2% of the MHC-1 level.

Geographical and Species Differentiation of Truffles (Tuber spp.) by Means of Stable Isotope Ratio Analysis of Light Elements (H, C, and N).

Truffles (Tuber spp.) are considered the most expensive edible fungi and, therefore, are highly prone to food fraud. In this study, authentic truffles from different countries of origin and species were characterized by the determination of their stable carbon, nitrogen, and hydrogen isotope ratios (bulk δ2H, δ13C, and δ15N values). Chinese truffles from Yunnan or related provinces (n = 19) could be well-separated from all European samples (eight countries; n = 105) by means of their significantly (p < 0.05) more negative δ2H values. Furthermore, samples of the second most expensive European species Tuber magnatum were both more enriched in 15N and more depleted in 13C compared to the remaining samples, which allowed for an unequivocal differentiation. Hence, stable isotope ratio analysis could be of high value in terms of authentication of truffles.

Stable isotope signatures (δ2 H-, δ13 C-, δ15 N-values) of walnuts (Juglans regia L.) from different regions in Germany.

BACKGROUND: Walnuts (Juglans regia L.) are considered of high value for human nutrition and are cultivated worldwide. With globalization, however, the demand for regional products has increased. Isotopic fingerprints (δ2 H-, δ15 N- and δ13 C-values (‰)) created by means of stable isotope ratio mass spectrometry provide the possibility of tracing the geographical provenance of plant foods. RESULTS: Authentic walnut samples from different geographical regions in Germany could be separated from each other by means of their bulk δ2 H-values (‰) with samples from northern regions being more enriched in 2 H than samples collected from southern Germany. Next to precipitation data, the topography of the surrounding landscape of the sampling sites was taken into account for data interpretation by the creation of approximate elevation profiles. Moreover, German walnuts could be separated from French walnuts which showed significantly higher δ2 H-values (‰). Variations in annual total precipitation was reflected by the shift in δ2 H-ranges (‰) depending on the harvesting year. Additionally, organic walnuts could be well separated from conventionally produced walnuts due to their distinct δ15 N-values (‰) which could be ascribed to a cultivation practice common for organic farming. CONCLUSIONS: The data presented here outlined the potential of determining isotopic fingerprints of walnuts from different sites within one country for tracing their regional origin and revealing their cultivation conditions. Including topographic characteristics between the sampling locations as well as precipitation data helped to even differentiate walnuts from two adjoining countries. © 2019 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Stable Carbon and Nitrogen Isotope Ratios of Red Bell Pepper Samples from Germany, The Netherlands, and Spain.

Bell pepper is one of the most cultivated crops in the world. By means of δ13C and δ15N values (‰), red bell peppers from Germany, The Netherlands, and Spain could be distinguished from each other. German fruits cultivated under greenhouse conditions were extremely depleted in 13C with δ13C values for fatty acids of partly less than -50‰. The strong depletion in 13C was in accordance with the use of biomethane-derived CO2 as growth regulator in the greenhouses. Seasonal variations in the δ13C values (‰) of German samples were tentatively assigned to varying CO2 enrichment during the annual production cycle. δ13C values (‰) of Dutch samples also correlated with greenhouse production, whereas Spanish samples most likely originated from open field cultivation. Additionally, the use of color measurement as a tool for presorting the samples was investigated in order to slim the scattering ranges of δ13C and δ15N values (‰) for clearer differentiation.

Occurrence of tocopheryl fatty acid esters in vegetables and their non-digestibility by artificial digestion juices.

Tocopheryl fatty acid esters (TFAE) consist of tocopherols esterified to fatty acids, but only little is known about this substance class. In this study, twelve vegetable groups were screened on TFAE and contents of (free) tocopherols and TFAE were determined in red bell pepper, red chili pepper, cucumber and walnut (n = 5, respectively). Intact TFAE were separated by solid phase extraction from free tocopherols and analyzed by GC/MS. Highest TFAE values were determined in chili pepper (4.0-16 mg/100 g fresh weight, FW) and walnut (4.1-12 mg/100 g FW), followed by bell pepper (1.3-1.5 mg/100 g FW) and cucumber (0.06-0.2 mg/100 g FW). Contribution of TFAE to the total tocopherol content ranged from 7-84%. Neither the treatment with artificial digestion juices nor exposure to sunlight showed degradation of TFAE. This substance class might represent a hitherto overlooked storage form for free tocopherols in plants as they occur to be more stable. But as the ester bond in medium chain TFAE seems not to be fissile in the human body, they might not contribute in the same way as free tocopherols to the vitamin E activity of vegetables and might have to be determined separately.

Phytyl fatty acid esters in vegetables pose a risk for patients suffering from Refsum's disease.

Patients suffering from Refsum's disease show mutations in the enzyme necessary for the degradation of phytanic acid. Accumulation of this tetramethyl-branched fatty acid in inner organs leads to severe neurological and cardiac dysfunctions which can even result in death. Thus, patients with Refsum's disease have to follow a specific diet resigning foods with high levels of phytanic acid and trans-phytol like products from ruminant animals with a tolerable daily intake (TDI) of ≤ 10 mg/d. We recently reported the occurrence of phytyl fatty acid esters (PFAE, trans-phytol esterified with a fatty acid) in bell pepper with trans-phytol amounts of up to 5.4 mg/100 g fresh weight (FW). In this study we carried out in vitro-digestion experiments of PFAE with artificial digestion fluids. Our results demonstrate that PFAE actually are a source for bioavailable trans-phytol and thus add to the TDI. Eating only one portion of bell pepper (∼150 g) could therefore lead to exploitation of the TDI of up to 81%. Analysis of additional vegetable matrices showed that also rocket salad with up to 4.2 mg/100 g FW trans-phytol bound in PFAE represents a risk-relevant food for patients with Refsum's disease and should therefore be taken into account.

Phytyl Fatty Acid Esters in the Pulp of Bell Pepper (Capsicum annuum).

Phytyl fatty acid esters (PFAE) are esters of fatty acids with the isoprenoid alcohol phytol (3,7R,11R,15-tetramethylhexadec-2E-enol). In this study, PFAE were identified and quantified in bell pepper using gas chromatography with mass spectrometry (GC-MS). All red (n = 14) and yellow (n = 6) samples contained six or seven PFAE at 0.9-11.2 mg/100 g fresh weight. By contrast, PFAE were not detected in green bell pepper samples (n = 3). PFAE might eventually be a source for bioavailable phytol, which can be transformed into phytanic acid by humans. Phytanic acid cannot be properly degraded by patients who suffer from Refsum's disease (tolerable daily intake (TDI) ≤ 10 mg of phytanic acid). The phytol moiety of the PFAE (0.4-5.4 mg/100 g fresh weight) would contribute up to ∼50% to the TDI with the consumption of only one portion of bell pepper fruit pulp.